Characterization of U2 snRNA associated proteins:
Functional Analysis of C. merolae Hsh49 Protein
by
Mona Aminorroayaee
B.Sc., Tabriz University of Medical Science, 2009

Thesis Submitted in Partial Fulfillment of
The Requirements for the Degree of
Master of Science
In
Mathematical, Computer, and Physical Sciences
(Biochemistry)

The University of Northern British Columbia
January 2018
©Mona Aminorroayaee, 2018

i

Abstract
Pre-mRNA splicing is an important process involving the removal of non-coding regions
(introns) from messenger RNA. Disruption of this mechanism can cause diseases such as cancer.
A particle known as the spliceosome is responsible for recognition and removal of introns. This
complex machinery consists of five small nuclear RNAs, U1, U2, U4, U5, and U6, and many
proteins. Each snRNA associates with proteins to form small nuclear ribonucleo-proteins or
snRNPs. Due to the considerable number of proteins involved in this process in humans, the
study of splicing has been a struggle. Cyanidioschyzon merolae (C. merolae) is a unicellular
acidophilic red alga that we hope will be a good model for studying splicing factors due to its
greatly reduced complexity (only ~70 splicing proteins, compared to ~300 in humans). Thus, we
are investigating the interactions of snRNAs with their specific proteins by expressing the entire
snRNP particles recombinantly and investigating their function.
I sought to use this system to investigate the interactions between the U2 snRNA and its proteins.
U2 snRNA associates with two multi-protein complexes, SF3a and SF3b. The SF3b complex

is required for the recognition of the intron’s branch point and precise excision of introns
from pre-mRNA. I attempted to express all of these proteins simultaneously as well as
individually, with my efforts ultimately focused on Hsh49. This is one of five subunits of the
SF3b protein complex in C. merolae. I was not able to detect any interaction between U2
snRNA and the Hsh49 protein by Electrophoretic mobility shift assay (EMSA), Fluorescence
polarization (FP), or immunoprecipitation analysis. Nevertheless, Circular Dichroism (CD)
showed that Hsh49 folded properly.

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TABLE OF CONTENTS
ABSTRACT ....................................................................................................................................................... II
TABLE OF CONTENTS ..................................................................................................................................... III
LIST OF FIGURES ............................................................................................................................................. V
LIST OF TABLES ............................................................................................................................................ VIII
CHAPTER 1 INTRODUCTION ............................................................................................................................ 1
1.1 PRE-MRNA SPLICING............................................................................................................................................. 2
1.2 SPLICEOSOME ASSEMBLY ........................................................................................................................................ 3
1.3 SPLICING AND DISEASE ........................................................................................................................................... 4
1.4 ROLE OF U2 SNRNP IN YEAST AND HUMANS.............................................................................................................. 5
1.4.1 Structure of U2 snRNA ................................................................................................................................... 6
1.5 U2 SNRNP IN C. MEROLAE ..................................................................................................................................... 8
1.5.1 U2 snRNA proteins ........................................................................................................................................ 9
1.6 PREVIOUS EXPRESSION OF U2-ASSOCIATED PROTEINS ................................................................................................. 15
1.7 OVERALL RESEARCH OBJECTIVE .............................................................................................................................. 16
CHAPTER 2 CO-EXPRESSION OF ALL U2 SNRNPS AND SF3A PROTEINS .......................................................... 18
2.1 MATERIALS AND METHODS ................................................................................................................................... 20
2.1.1 Preparation of C. merolae genomic DNA .................................................................................................... 20
2.1.2 Construction of Co-expression Vectors containing U2 snRNP genes and SF3a genes ................................. 20
2.1.3 Co-expression and Purification of the U2 snRNPs: ...................................................................................... 27
2.2 RESULTS AND DISCUSSION..................................................................................................................................... 29
CHAPTER 3 INDIVIDUAL EXPRESSION AND PURIFICATION OF PRP9, PRP11, PRP21 AND RDS3 PROTEINS ..... 38
3.1 MATERIAL AND METHODS..................................................................................................................................... 39
3.1.1 Preparation of C. merolae genomic DNA .................................................................................................... 39
3.1.2 Construction of expression Vectors containing Prp9, Prp11, Prp21, Rds3 and Hsh49 genes ...................... 39
3.1.3 Expression and purification of Prp9, Prp11, Prp21 and Rds3 proteins ........................................................ 41
3.2 RESULTS AND DISCUSSION..................................................................................................................................... 46
CHAPTER 4 FUNCTIONAL ANALYSIS OF C. MEROLAE HSH49 PROTEIN ........................................................... 61
4.1.1 Fluorescence Anisotropy Assay ................................................................................................................... 63
4.2 MATERIALS AND METHODS ................................................................................................................................... 63
4.2.1 Circular Dichroism Spectropolarimetry........................................................................................................ 63
4.2.2 RNA-Protein Binding Assays ........................................................................................................................ 65
4.2.2.1 Fluorescence Anisotropy Assay ...................................................................................................................................65
4.2.2.2 Fluorescence-based electrophoretic mobility shift assay (F-EMSA) ............................................................................69
4.2.2.3 Electrophoretic mobility shift assay (EMSA) ................................................................................................................70
4.2.2.4 Generation of [32p]-labelled U2 snRNA by IVT............................................................................................................70

4.2.3 Co-immunoprecipitation of Hsh49 .............................................................................................................. 73
4.2.3.1 Dot blot assay ..............................................................................................................................................................73
4.2.3.2 Western blot assay ......................................................................................................................................................73
4.2.3.3 Preparation of C. merolae whole-cell extract ..............................................................................................................74
4.2.3.4 Generating anti-CmHsh49 antibody ............................................................................................................................74
4.2.3.5 Hsh49 co-immunoprecipitation ...................................................................................................................................74
4.2.3.6 Northern blot analysis .................................................................................................................................................74

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4.2.4 Alignment of Hsh49 amino acid and U2 snRNA sequences ......................................................................... 75
4.3 RESULTS ............................................................................................................................................................ 76
4.3.1 Expression and Purification of C. merolae Hsh49 ........................................................................................ 76
4.3.2 Sequence alignment of C. merolae U2 snRNA and Hsh49 with S. cerevisiae and Homo sapiens U2 snRNA
and Hsh49 ............................................................................................................................................................ 80
4.3.3 Circular Dichroism Spectropolarimetry........................................................................................................ 86
4.3.4 Fluorescence Anisotropy (FA) Assay ............................................................................................................ 87
4.3.5 Fluorescence-based electrophoretic mobility shift (F-EMSA) Assay ............................................................ 92
4.3.6 EMSA for detecting the Hsh49 protein-U2 snRNA interaction .................................................................... 93
4.3.7 Co-Immunoprecipitation ............................................................................................................................. 98
4.3.7.1 Hsh49 Dot Blot Assay ...................................................................................................................................................98
4.3.7.2 Western Blot Assay ......................................................................................................................................................99
4.3.7.3 Hsh49 Co-Immunoprecipitation ................................................................................................................................100
4.3.7.4 Northern Blot Analysis ...............................................................................................................................................101

4.4 DISCUSSION ..................................................................................................................................................... 102
CHAPTER 5 GENERAL DISCUSSION ...............................................................................................................108
5.1 FUTURE DIRECTIONS .......................................................................................................................................... 111
LITERATURE CITED .......................................................................................................................................112

iv

List of Figures
Figure 1................................................................................................................................................... 2
Figure 2. The pathway of spliceosome assembly during pre-mRNA splicing......................................... 4
Figure 3. Secondary structures of free U2 snRNA and base pairing interaction of U2 snRNA to the
branch point and a comparison of yeast and human sequences. ......................................................... 6
Figure 4. Secondary structure of the U2 and U6 snRNA complex in humans and yeast. ...................... 8
Figure 5. Model for Rds3 function. ...................................................................................................... 10
Figure 6. The structure of the SF3a core. ............................................................................................. 13
Figure 7. Interaction network between U2 snRNA proteins. ............................................................... 15
Figure 9. Construction of the U2 snRNP co-expression vectors. Each gene of the U2 snRNP was first
introduced into a single plasmid and then all genes were combined serially into plasmids until they
were present in one final plasmid. Plasmids were digested with SwaI (S) or PacI (P), followed by heat
inactivation, T4 DNA polymerase treatment and annealing................................................................ 26
Figure 10. Construction of the SF3a co-expression vectors. Individual SF3a genes were first
introduced into a single plasmid and then all genes were combined serially into plasmids until they
were present in one final plasmid. Plasmids were digested with SwaI (S) or PacI (P), followed by heat
inactivation, T4 DNA polymerase treatment and annealing................................................................ 27
Figure 11. Determining the presence of Prp11, Prp21, Prp9, Prp5 and Prp43 genes in pQlink by PCR.
.............................................................................................................................................................. 30
Figure 12. Determining the presence of U2 snRNA, Prp11 and Rse1 genes in pQlink by PCR. ........... 30
Figure 13. Determining the presence of Hsh155 and Cus1 genes in pQlink by PCR. ........................... 31
Figure 14. Determining the presence of Rds3, Prp11, Rse1 and Hsh49 genes in pQlink by PCR. ........ 32
Figure 15. Determining the presence of each SF3a gene in pQlink. .................................................... 33
Figure 16. Small scale co-expression and purification of Sms-U2 snRNA and Sms-U2 snRNP
constructs. ............................................................................................................................................ 34
Figure 17. Large-scale co-expression and purification of Sms, Sms-U2 snRNA and Sms-U2 snRNP
constructs. ............................................................................................................................................ 35
Figure 18. Co-expression of the SF3a from pQlink. .............................................................................. 36
Figure 19. Determining the presence of each SF3a gene in PMCSG23................................................ 47
Figure 20. SF3a protein expression from pQlink. ................................................................................. 48
Figure 21. SF3a protein expression from PMCSG23 expression vector. .............................................. 49
Figure 22. Expression of Rds3 from pQlink. ......................................................................................... 50
Figure 23. Expression of Rds3 from PMCSG23 expression vector. ...................................................... 51
Figure 24. Solubility test of the Rds3. .................................................................................................. 51
Figure 25. Purification of Rds3. ............................................................................................................ 53
Figure 26. TEV protease cleavage and dialysis of the Rds3. ................................................................ 54
Figure 27. Purification of Rds3 after TEV protease cleavage and dialysis............................................ 55
Figure 28. Expression of the Hsh49 protein from PMCSG23 expression vector. ................................. 57
Figure 29. Purification of the Hsh49 was analyzed on 15% high TEMED SDS gel. ............................... 59
Figure 30. HumanU2 snRNA-protein crosslinks (Dybkov et al. 2006). ................................................. 62
Figure 31. The basic principle of fluorescence polarization (Pagano et al. 2011). .............................. 63
Figure 32. Expression of the Hsh49 protein from PMCSG23 expression vector. ................................. 77
Figure 33. Purification of the Hsh49 was analyzed on 15% high TEMED SDS gel. ............................... 79
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Figure 34. Sequence alignment of C. merolae (C.m) U2 snRNA with S. cerevisiae (S.c) U2 snRNA. .... 81
Figure 35. Sequence alignment of C. merolae (C.m) U2 snRNA with Homo sapiens (H.S) U2 snRNA. 82
Figure 36. Sequence alignment of C. merolae (C.m) Hsh49 protein with S. cerevisiae (S.C) Hsh49
protein. ................................................................................................................................................. 83
Figure 37. Sequence alignment of C. merolae (C.m) Hsh49 protein with Homo sapiens (H.S) Hsh49
protein. ................................................................................................................................................. 84
Figure 38. A Multiple sequence alignment of C. merolae (C.m) Hsh49 with S. cerevisiae (S.C) and
Homo sapiens Hsh49 (H.S) protein. ..................................................................................................... 85
Figure 39. A Multiple sequence alignment of C. merolae (C.m) Hsh49 with S. cerevisiae (S.C) and
Homo sapiens Hsh49 (H.S) protein. ..................................................................................................... 85
Figure 40. A Multiple sequence alignment of C. merolae (C.m) Hsh49 with S. cerevisiae (S.C) and
Homo sapiens Hsh49 (H.S) protein. ..................................................................................................... 85
Figure 41. CD spectra of the Hsh49 secondary structure. ................................................................... 86
Figure 42. Melting temperature (Tm) of the recombinant Hsh49 protein. ......................................... 87
Figure 43. The result of the fluorescence anisotropy experiment of the U4 snRNA-Snu13 interaction.
The concentration of the Snu13 was between 0 to 400 nM. .............................................................. 88
Figure 44. The results of the fluorescence anisotropy of the recombinant Hsh49 and fluorescein
labelled of the U2 snRNA. .................................................................................................................... 89
Figure 45. The results of the fluorescence anisotropy experiments of the U2 snRNA-Hsh49
interaction. ........................................................................................................................................... 90
Figure 46. A comparison of the conformational changes of the fluorescein U2 snRNA oligonucleotide
in three different types of binding buffers by using the fluorescence anisotropy assay. ⁎⁎⁎ ₌ p<
0.001..................................................................................................................................................... 92
Figure 47. Fluorescence-based electrophoretic mobility shift assay (F-EMSA) with recombinant
Hsh49 and fluorescent labelled U2 snRNA. ......................................................................................... 93
Figure 48. Electrophoretic mobility shift assay with recombinant Hsh49 and radioactive [32p]labelled U2 snRNA. ............................................................................................................................... 94
Figure 49. Electrophoretic mobility shift assay with recombinant Hsh49 and radioactive [32p]labelled U2 snRNA. ............................................................................................................................... 95
Figure 50. Electrophoretic mobility shift assay with recombinant Hsh49 and radioactive [32p]labelled U2 snRNA. ............................................................................................................................... 96
Figure 51. Electrophoretic mobility shift assay with cleaved and uncleaved recombinant Hsh49 and
radioactive [32p]-labelled U2 snRNA. .................................................................................................. 97
Figure 52. Electrophoretic mobility shift assay of uncleaved recombinant Hsh49 and His-MBP tags
(negative control) with radioactive [32p]-labelled U2 snRNA. ............................................................ 98
Figure 53. Dot blot test. Determining the sensitivity of the anti-Hsh49 antiserum generated against
recombinant C. merolae Hsh49 protein. .............................................................................................. 99
Figure 54. Specificity of the anti-Hsh49 antiserum via Western blot analysis using different
concentrations of the recombinant C. merolae Hsh49. ..................................................................... 100
Figure 55. Western blot of Hsh49 C. merolae whole cell extract. ..................................................... 101
Figure 56. Northern blot analysis of the immunoprecipitated RNA probed for U2 snRNA. .............. 102
Figure 57. The crystal structure of the first RRM of Homo sapiens Hsh49 (PDB file 5GVQ; Kuwasako et
al. 2017). ............................................................................................................................................ 104

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Figure 58. The secondary structure of the first RRM of Homo sapiens Hsh49 (PDB file 5GVQ;
Kuwasako et al. 2017). ....................................................................................................................... 104
Figure 59. The crystal structure of the second RRM of Homo sapiens Hsh49 (PDB file 1X5T; Sato et
al., to be published). .......................................................................................................................... 105
Figure 60. The secondary structure of the second RRM of Homo sapiens Hsh49 (PDB file 1X5T; Sato
et al., to be published). ...................................................................................................................... 105
Figure 61. The Crystal structure of the S. cerevisiae Hsh49 protein (PDB file 5LSB; vanRoon et al.
2017). ................................................................................................................................................. 106
Figure 62. The secondary structure of the S. cerevisiae Hsh49 (PDB file 5LSB; vanRoon et al. 2017).
............................................................................................................................................................ 106

vii

List of Tables
Table 1. Comparison of all U2 snRNA proteins in humans, yeast, and C. merolae a ........................... 14
Table 2. The U2 snRNA and all U2 snRNP genes with their associated accession numbers, masses,
and lengths (Stark et al. 2015). ............................................................................................................ 19
Table 3. DNA oligonucleotides which were used to amplify and sequence the desired gene. The first
DNA oligonucleotide for each gene is the forward and the second DNA oligonucleotide in the reverse
primer. . ................................................................................................................................................ 22
Table 4. U2snRNP and SF3a plasmids which were used to make the related co-expression vector... 24
Table 5. DNA oligonucleotides which were used to amplify and sequence the desired gene. . ......... 41
Table 7. MDG non-inducing media. ..................................................................................................... 43
Table 8. ZYM-5052 Auto-inducing media. ............................................................................................ 44
Table 9. Measurement parameters for the CD spectra of Hsh49. ....................................................... 64
Table 10. The fluorescence anisotropy reagent (Buffer 1) used for the detection of the U2 snRNAHsh49 interaction. (Hsh49 with benzonase and without benzonase). ................................................ 66
Table 11. The fluorescence anisotropy reagent (Buffer 2) used for the detection of the U2 snRNAHsh49 interaction. ................................................................................................................................ 66
Table 12. The fluorescence anisotropy reagent (Buffer 3) used for the detection of the U2 snRNAHsh49 interaction. ................................................................................................................................ 67
Table 13. The fluorescence anisotropy reagent (Buffer 4) used for the detection of the U2 snRNAHsh49 interaction. ................................................................................................................................ 67
Table 14. The fluorescence anisotropy reagent (Buffer A) used for detection of conformational
changes of the U2 snRNA. .................................................................................................................... 68
Table 15. The fluorescence anisotropy reagent (Buffer B) used for detection of conformational
changes of the U2 snRNA. .................................................................................................................... 68
Table 16. The fluorescence anisotropy reagent (Buffer C) used for detection of conformational
changes of the U2 snRNA. .................................................................................................................... 68
Table 17. The F-EMSA reagent used for the detection of the U2 snRNA-Hsh49 interaction. ............. 69
Table 18. The labelling reaction ........................................................................................................... 70
Table 19. EMSA reagents (Binding Buffer Reagent 1) used for the detection of the U2 snRNA-Hsh49
interaction. ........................................................................................................................................... 71
Table 20. EMSA reagents (Binding Buffer Reagent 2) used for the detection of the U2 snRNA-Hsh49
interaction. ........................................................................................................................................... 72

viii

Acknowledgments
I would like to thank first and foremost my supervisor, Dr. Stephen Rader, for
giving me the opportunity to conduct my research in his lab. Stephen provided a friendly
training environment that motivates me to be curious in each step of performing my research
and have a deep understating of the Pre-mRNA splicing process. I would also like to thank
Stephen for the time that he patiently spent to introduce and explain complicated concepts in
this area. I really enjoyed my studies during these years.
I would like to acknowledge the members of my supervisory committee, Dr. Kerry
Reimer, Dr. Brent Murray, and Dr. Keith Egger for their valuable questions and informative
comments on this thesis.
I am also so thankful to past and current members of the Rader lab: Dr. Liz Dunn,
Ambreen Siraj, Fatimat Shidi, Viktor Slat, Maya de Vos, Radu Pasca, Raliat Abioye, Kevin
Huolt, and Samantha Smith for their critique questions and insightful conversation that
helped me to develop my knowledge and I would like to thank Corbin Black for his
assistance to find the best possible sequence alignment data. I would also like to
acknowledge Sebastian J. Mackedenski for his help in circular dichroism spectropolarimetry
data collection.
I would particularly like to thank Dr. Martha Stark for introducing practical
techniques in the Lab and for all her helpful and informative advices through my project.
I am also grateful to Brooke Boswell and Luke Spooner for proofing and editing of
this thesis.
ix

I would like to thank UNBC for granting me the financial support that I was given
to pursue my studies.
My special thanks go to my husband, Rahim Pasha Khajei, for his ongoing support
and wise counsel over the years. I am so thankful to my parents, Yasamin Bozarjomehr and
Javad Aminorroayaee for their love and guidance, and my brothers Emia and Aria for making
me happy through passing though times.
Thank you very much, everyone!
Mona Aminorroayaee
March 22, 2018

x

Chapter 1
Introduction
Pre messenger RNA splicing is an important process involving the removal of noncoding regions (introns) from messenger RNA, which is carried out by a highly complex and
dynamic machine called the spliceosome. This complex machinery consists of five small
nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, and many proteins. Each snRNA
associates with proteins to form small nuclear ribonucleoproteins, (snRNPs). Due to the
considerable number of proteins involved in this process in humans, the study of splicing has
been a struggle. Cyanidioschyzon merolae (C. merolae) is a unicellular acidophilic red alga
that we hope will be a good model for studying splicing factors due to its greatly reduced
complexity (only ~70 splicing proteins, compared to ~300 in humans; Will et al. 2011).
Thus, we are investigating the interactions of snRNAs with their specific proteins by
expressing entire snRNP particles recombinantly in bacteria.
During spliceosome assembly, U1 snRNA recognizes and base-pairs with the 5’
splice site of the pre-mRNA. However, Stark et al. (2015) could not find any candidates for
the U1-associated proteins or U1 snRNA, hinting that C. merolae does not have a U1 snRNP.
Biochemical studies of the U2 snRNA would help to improve understanding of the initiation
of the spliceosome assembly in the absence of the U1 snRNA, and provide information about
the interaction of the U2 snRNA with its specific proteins.

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1.2 Spliceosome Assembly
The spliceosome is a large RNA-protein complex located in the nucleus, and it
identifies and interacts with the three reactive regions of substrate transcripts (5′SS, 3′SS,
BPS) in order to perform pre-mRNA splicing (Chiou et al. 2014). The spliceosome consists
of five small nuclear ribonucleoproteins (snRNPs) including U1, U2, U4, U6, and U5, which
are the major components of the spliceosome. Each snRNP contains a snRNA and associated
proteins (Wahl et al. 2009). The human spliceosome includes five small nuclear RNAs and
more than 300 proteins, while the spliceosome in Saccharomyces cerevisiae (S. cerevisiae)
contains five small nuclear RNAs and almost 100 different proteins (Jurica et al. 2003; Wahl
et al. 2009, Fabrizio et al. 2009).
The assembly of the spliceosome starts with recognition and base pairing of U1 and
U2 snRNPs to the 5′ splice site and branch site of the pre-mRNA respectively. The U2
snRNA binds to the branch site, from which the catalytic adenosine at the branch point
binding sequence bulges out (Figure 2, Commitment Complex). Single molecule analysis
posits that either U1 or U2 can bind first to the pre-mRNA during the early steps of
spliceosome assembly (Hoskins et al, 2016). The U6 snRNA associates with the U4 snRNA
to form the U4/U6 di-snRNA; then, the binding of U5 snRNA to U4/U6 di-snRNP results in
creation of the U4/U6.U5 tri-snRNP (Will et al. 2011). After association of tri-snRNP to
form the pre-spliceosome (Figure 2, left side), the interaction between U1 snRNA and the 5′
splice site is disrupted, as well as that between the U4 and U6 snRNAs. Next, U6 snRNA
associates with U2 snRNA and the 5′ splice site through base pairing interactions (Figure 2,
Active Spliceosome). The order and arrangement of the spliceosome is key to the
identification and excision of introns from the rest of the pre-mRNA during splicing (Wahl et
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Research indicates that approximately half of all genetic diseases are due to
disruptions in splicing (Lopez-Bigas et al. 2005). Examples of human genetic diseases
caused by mutations in splicing include: Retinitis pigmentosa, which leads to blindness;
spinal muscular atrophy, which causes mortality in childhood; myotonic dystrophy, the most
common muscular disorder in adults; Frasier syndrome; Parkinson’s disease; and Atypical
cystic fibrosis (Faustino and Cooper 2003). In addition, a wide range of cancers has been
linked to errors in splicing. Therefore, it seems that a better understanding of the splicing
reaction is an essential step to providing more research opportunities for treatment of human
genetic diseases.

1.4 Role of U2 snRNP in Yeast and Humans
Many of the interactions in the spliceosome seem to function in ensuring that
activation occurs at the right time and place. As mentioned above, U2 snRNA binds to the
branch point, thereby inducing the branch site A to bulge out in its active conformation. At
the same time, U2 components such as SF3b appear to sequester the bulged A to prevent it
from reacting prematurely (Lardelli et al. 2010). In addition, U6 snRNA associates with U2
snRNA and the 5′ splice site through base pairing interactions (Wahl et al. 2009). The
association of the U2 snRNP with the pre-mRNA branch site is one of the primary stages in
the pre-mRNA splicing process and plays a fundamental role during splicing assembly since
they directly participate in chemical catalysis (Nilsen 1998). Moreover, the U2 snRNP
undergoes several rearrangements along with the recognition of the branch point adenosine
and formation of base pair interaction between theU2 snRNA and “consensus sequence
within the intron” (Sashital et al. 2007).

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6

modifications that occur in Stem I of mammalian U2 snRNA (Massenet et al.1998), while
there is no post-transcriptional modification in yeast Stem I. NMR structures of the U2 stem I
from S. cerevisiae and the fully modified U2 stem I from humans reveal that there is a great
similarity in structure. Despite the fact that they include divergent tandem wobble pairs and
modified nucleotides found in human U2 stem I, this indicates that the overall folds of the
stem loops of U2 snRNA are similar in yeast and humans (Sashital et al. 2007, Figure 3a).
However, the U2 snRNA stem I is more stable in humans than yeast stem I (Sashital et al.
2007, Figure 3a).
Most binding of the U2 snRNP to the branch site in humans occurs through the
polypyrimidine tract (Figure 3b) in an ATP-dependent manner. It seems this base pairing
interaction increases the stability of the binding between U2 snRNA and the polypyrimidine
tract, and helps to specify the exact location of branch formation. With this in mind, it is clear
that without this interaction, the first step of the splicing pathway and spliceosome assembly
would be impossible (Kramer et al. 1988; Wu et al. 1989). However, in yeast, introns do not
contain a polypyrimidine tract (Rymond and Rosbash 1985; Rymond et al. 1987). Overall,
from yeast to mammals, U2 snRNA has been conserved evolutionarily (Ares et al. 1986; Igel
and Ares. 1988). The base paring interaction between the U2 snRNA and the branch point
with the intron and the pre-mRNA splicing mechanism is similar in both yeast and mammals
(Parker et al. 1987; Chabot et al. 1985; Ruskin et al. 1988, Zhuang et al. 1989).

7

Figure 4. Secondary structure of the U2 and U6 snRNA complex in humans and yeast. The secondary
structure of the U2 and U6 snRNA in humans was presented by Sun and Manley (Sun and Manley. 1995) The
NMR secondary structure of the U2 and U6 snRNA in yeast was proposed by Sashital (Sashital et al. 2004)

1.5 U2 snRNP in C. merolae
Cyanidioschyzon merolae (C. merolae) is a unicellular, acidophilic red alga. The
small size of the genome and reduced number of introns in C. merolae make the idea of
studying splicing in this system an interesting subject. C. merolae contains the smallest
genome size (16.5 Mb) among eukaryotes (Misumi et al. 2005; Suzuki et al. 1992). The
number of genes in the C. merolae genome is the same as S. cerevisiae (yeast), but 26 genes
contain introns (0.5% of the genome) in C. merolae (Matsuzaki et al. 2004) compared to 287
in S. cerevesiae (Juneau et al. 2007). It is worth mentioning that C. merolae grows in acidic
hot springs with a pH of ⁓1.5 and temperature of ⁓ 45 C (Matsuzaki et al. 2004), raising the
possibility that its snRNAs are intrinsically more stable. It is also the first alga in which the
8

genome was sequenced and the first whose sequence was 100% completed (Nozaki et al.
2007).
By considering such features, some interesting questions emerge – such as whether
or not proteins involved in the splicing process can remain stable in high temperatures and/or
how the process of splicing occurs in this organism.
A comprehensive bioinformatics study carried out by Stark et al. (2015) has
determined that the C. merolae spliceosome contains four snRNAs including U2, U4, U5,
and U6. This research also did not find any candidates for the U1-associated proteins or U1
snRNA, suggesting that C. merolae does not have a U1 snRNP. By considering this fact, it
would be interesting to know how splicing starts in the absence of the U1 snRNA and if it is
possible that the U2 snRNA starts the splicing process in C. merolae. These questions
motivated me to study the U2 snRNA in C. merolae. In this regard, it is imperative to know
the U2 snRNA’s components and understand the role and function of each particle during the
splicing process.
1.5.1 U2 snRNA proteins
Here I will cover all of the functions of the U2 snRNA proteins which have been
recognized either in yeast or humans. The U2 snRNA associates with ten specific proteins
and seven Sm proteins in C. merolae, making the U2 snRNP the most complex particle in
splicing (Table 1). The following are the proteins U2 snRNA associates with:
 SF3a and SF3b

9

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10

 U2AF
U2AF is one of the splicing factors that facilitates interaction of the U2 snRNP to
the branch point. It consists of a 65 kD subunit and a 35 kD subunit. The U2AF65 binds the
polypyrimidine tract, while U2AF35 recognizes the 3′ AG dinucleotide in humans (Wu et al.
1999; Wu and Fu. 2015).
Mud2
Since there is no polypyrimidine tract in C. merolae, Stark et al. (2015) suggest that
Mud2p interacts with the branch point sequences directly.
Msl5
Msl5, or branch point binding protein (BBP), binds 5′_UACUAAC sequences of
the branch point and Mud2p in S. cerevisiae. In yeast and mammals, Msl5 recognizes the
intron by interacting with the pre-mRNA branch point. Both Mud2 and Msl5 are
commitment complex components and play a critical role in the stability of U1 snRNA,
which occurs by interacting between these two proteins and U1 snRNP protein, Prp40 (Rutz
et al. 1999; Abovich et al, 1997; Chang et al. 2012).
Sub2
During pre-spliceosome assembly, two ATPases, Sub2 and Prp5, are necessary for
interactions between U2 snRNA and the branch point binding site (O’Day et al. 1996; Parker
et al. 1987; Kistler et al. 2001). It is assumed that Sub2p is involved in the removal of Mud2
and Msl5 from the branch site, so that U2 snRNA is exposed to the branch site (Kistler et al.
2001).
11

Prp5
This protein contributes to the remodeling of U2 snRNA to make it more accessible
to the branch point binding sequences. Prp5 activity occurs as a result of the association of
the three subunits of SF3a with the U2 snRNA. In this association, Prp21 acts as a bridge
between Prp9 and Prp11 (Wiest et al. 1996; Lin and Xu 2012; Figure 6).
Cus1
Assembly of U2 snRNP into the spliceosome to form the pre-spliceosome is the
earliest function of Cus1p. After this protein binds to U2 snRNA, an interaction with premRNA results. During the splicing process, Cus1p forms a complex with Hsh49 and Hsh155
and remains associated with the spliceosome (Pauling et al. 2000).
Prp43
Prp43 is one of the U2 snRNA proteins related to the DEXH/D-box RNA helicase
family. It functions in the early steps of spliceosome disassembly. Prp43 removes U2, U5,
and U6 snRNPs from the post-splicing lariat intron ribonucleoprotein complex in order to
release mature RNA (Arenas and Abelson. 1997; Martin et al. 2002, Combs et al. 2006). The
function of the Prp43 protein before spliceosome disassembly is not well understood.

12

Figure 6. The structure of the SF3a core. Prp11 and Prp9 do not bind each other; however, Prp21 acts as a
bridge between these two proteins (PDB file 4DGW; Lin & Xu. 2012).

Lea1, Msl1 and Cus2
Lea1, Msl1, and Cus2 are non-essential proteins in yeast. The Lea1 protein is a
specific particle in yeast U2 snRNP and interacts with Msl1. The association of Msl1 with
U2 snRNA is possible only in the presence of Lea1 (Caspary et al. 1998). Cus2 interacts with
Prp11, which is one of the subunits of SF3a, and there is evidence that Cus2 might has a role
in recruiting SF3a to the U2 snRNP (Yan et al. 1998).

13

Table 1. Comparison of all U2 snRNA proteins in humans, yeast, and C. merolae a
All of the proteins in the table have been found by mass spectrometry except for MSL5. Msl5 and Mud2 are the
commitment complex proteins that are identified with 10 U2 specific proteins in C. merolae (Stark et al. 2015).

CmSmB/B’
CmSmD1
CmSmD2
CmSmD3
CmSmE
CmSmF
CmSmG
CmPrp9
CmPrp11
CmPrp21

Saccharom
yces
cerevisiae
SmB/B’
SmD1
SmD2
SmD3
SmE
SmF
SmG
Prp9
Prp11
Prp21

CmHsh155

Hsh155

SF3b1

CmCus1
CmRse1
CmHsh49

Cus1
Rse1
Hsh49

SF3b2
SF3b3
SF3b4

CmRds3

Rds3

PHF5

C. merolae

Human
SmB/B’
SmD1
SmD2
SmD3
SmE
SmF
SmG
SF3a3
SF3a2
SF3a1

SF3b6
CmPrp43
CmPrp5
CmMud2

Prp43
Prp5
Mud2

hPrp43
hPRP5
U2AF2

CmMsl5

MSL5/BBP

SF1

Msl1
Ysf3
Lea1
RES
Complex:
Bud13,
Pml1, Ist3

a

U2AF1
SNRPB2
SF3B5
SNRPA1
RES Complex:
MGC13125, SNIP,
CGl-79

Aliases of human U2 snRNA protein

HsT2456, SMD1, SNRPD, Sm-D1
SMD3, Sm-D3
B-raf, HYPT11, SME, Sm-E
SMG, Sm-G
PRP9, PRPF9, SAP61, SF3a60
PRP11, PRPF11, SAP62, SF3a66
PRP21, PRPF21, SAP11420, SF3A1
Hsh155, MDS, PRP10, PRPF10, SAP155,
SF3b155
Cus1, SAP145, SF3B145, SF3b1, SF3b150
RSE1, SAP130, SF3b130, STAF130
AFD1, Hsh49, SAP49, SF3b49
SPOC1
CGI-110, HSPC175, Ht006, P14, SAP14,
SAP14a, SF3B14, SF3B14a
YGL120C
YBR237WRNA5
U2AF65
BBP, D11S636, MBBP, ZCCHC25, ZFM1,
ZNF162
FP793, RN, RNU2AF1, U2AF35, U2AFBP
SPBC8D2.09c
SF3b10, Ysf3
Lea1

U2 related: U2AF,
PUF60, SPF30,
SPF31, SPF45,
CHERP, SR140
Cvitkovic & Jurica; (2013) NCBI Gene.

14

Almost all U2 snRNA proteins interact with each other during pre-mRNA splicing.
In the SF3a complex, Prp21 has more interactions with other U2 snRNA proteins compared
with Prp9 and Prp11. Among the SF3b complex, Hsh155 mostly interacts with other U2
proteins while other proteins of the SF3b complex interact with each other rather than U2
snRNA proteins. Moreover, Sms interact with each other and other U2 snRNA proteins
(Figure 7). The diagram shows the physical interactions between U2 snRNA proteins. These
studies are curated by BioGRID and are listed in the Saccharomyces Genome Database.

Figure 7. Interaction network between U2 snRNA proteins. Physical interactions or protein-protein
interactions are based on the purification of proteins from Saccharomyces cerevisiae genome. The number of
lines connecting to each protein show how many other proteins it is able to interact with. The Prp5 and Prp43 do
not have any interaction with other U2 snRNA proteins in S. cerevisiae (Saccharomyces Genome Database).
Proteins labeled with the same colors form a complex.

1.6 Previous expression of U2-associated proteins
Production of recombinant proteins is a powerful technique to study the
biochemical characterization of a desired protein. The expression or co-expression of the U2

15

snRNA proteins has been done in different organisms either for functional or structural
analyses.
In one study, the human SF3b complex was co-expressed in insect cells to
investigate the structure of these proteins (Cretu et al. 2016). In another study, two subunits
of SF3b complex, yeast Hsh49p, and Cus1protein, were expressed individually in a strain of
Escherichia coli, BL21 (Igel et al. 1998; Pauling et al. 2000). Also, Rds3p was expressed
separately in yeast cells (Wang and Rymond. 2003). Yeast SF3a complex with full length
proteins has been co-expressed in BL21 (Lin et al. 2012). Rosetta cells, another strain of
E.coli, were used for expression of yeast Prp43 and Prp5 proteins (Tauchert et al. 2016;
Liang et al. 2015).

1.7 Overall Research Objective
Since the presence of the U1 snRNA or the U1-associated proteins have not been
recognized in C. merolae, a question that arises is: Does the U2 snRNA help initiate
spliceosome assembly? Additionally, how do U2 snRNA specific proteins act upon each
other during pre-mRNA splicing process?
Understanding the function and structure of the U2 snRNPs helps to address these
questions about initiation and regulation of spliceosome assembly, as well as the role of each
U2 snRNA protein in the whole process of pre-mRNA splicing. My overall research goal was
to generate plasmids for recombinant expression of the U2 snRNA proteins, assembling them
into a 35 kbp plasmid containing the genes for the U2 snRNA, seven Sm proteins and ten U2
associated proteins. I investigated expression conditions for the entire snRNP, as well as
tested expression of sub-complexes and individual proteins. This allowed me to use the
16

recombinantly expressed proteins to investigate C. merolae’s recognition of the branch-point
sequence, and to further understand the association of the U6 snRNA to the U2 snRNA.

17

Chapter 2
Co-expression of all U2 snRNPs and SF3a
proteins
U2 snRNA in C. merolae is associated with ten snRNP-specific proteins and seven
Sm proteins, making U2 snRNP the most complex particle for splicing (Table 2). SF3a is one
of the multi-protein complexes that associates with the U2 snRNA and contains three
subunits including Prp9, Prp11, and Prp21. The SF3a complex is crucial for initiation of the
first step of the splicing reaction that occurs when SF3a dissociates from the spliceosome.
Moreover, SF3a is required for the spliceosome assembly. X-ray crystallography of SF3a
revealed that there is no interaction between Prp9 and Prp11. In fact, Prp21 acts as a bridge to
connect Prp11 and Prp9 in Saccharomyces cerevisiae (Lin et al. 2012).
To characterize the function, structure, and interaction of the U2 snRNA proteins
and the SF3a protein complex in C. merolae, it is necessary to develop an in vitro
reconstitution system. A challenging but key step towards this approach is to express
seventeen recombinant proteins and purify all of them. No-one has ever biochemically
expressed or purified the U2 snRNPs from C. merolae with the purpose of reconstituting the
U2 snRNPs. In order to carry out biochemical experiments on the U2 snRNPs, I attempted to
express U2 snRNPs and SF3a recombinantly. Romier et al. (2006) state that co-expression of
protein complexes seems more stable and prevents proteins from degrading or aggregating
when compared to the expression of a single protein. Moreover, some proteins rely on each
other to function properly. For instance, in Saccharomyces cerevisiae, Cus2 protein, which
interacts with Prp11, has a role in recruiting SF3a to the U2 snRNP (Yan et al. 1998).
18

Two plasmids were generated; the first plasmid (35 kbp) contains the gene for the
U2 snRNA, ten U2-associated proteins and seven Sm proteins. The second plasmid (8.2 kbp)
is for recombinant expression of the SF3a multi-protein complexes. In order to do in vitro
reconstitution of the U2 snRNP proteins, their respective genes were obtained through a
polymerase chain reaction (PCR) using the C. merolae genomic DNA. Those genes were
introduced into an expression vector by a ligation independent cloning technique. I expect
these recombinant snRNPs to be useful for the mechanistic and structural studies of splicing
in C. merolae.
Table 2. The U2 snRNA and all U2 snRNP genes with their associated accession numbers, masses, and lengths
(Stark et al. 2015).

U2 snRNP
Prp9
Prp11
Prp21
Hsh155
Cus1
Rse1
Hsh49
Rds3
Prp5
Prp43
U2 snRNA
SmB/B’
SmD1
SmD2
SmD3
*SmE
SmF
SmG

Accession number
CMQ406C
CMN095C
CMJ300C
CMB002C
CMT357C
CML103C
CME063C
CMS014C
CMR433C
CMM048C
AP006493
CMK022C
CMF084C
CMN302C
CMM065C
CMM109C
CMH215C
CMQ171C
CMO342C

Mass (kDa)
60.28
19.09
50.36
104.04
29.09
179.36
14.2
14.04
112.57
83.04
8.85
15.22
36.29
18.83
11.93

Length (base pair)
1617
504
1361
2864
771
5036
399
393
3084
2178
131
243
405
999
513
318

10
10.68

273
303

*SmE proteins are encoded by CMM109C and CMH215C genes and the SmE proteins will vary by only one
amino acid.

19

2.1 Materials and Methods
2.1.1 Preparation of C. merolae genomic DNA
I used C. merolae 10D strain (NIES-1332) provided by the Microbial Culture
Collection at the National Institute for Environmental Studies in Tsukuba, Japan
(mcc.nies.go.jp/). C. merolae genomic DNA was prepared by Martha Stark as previously
described (Stark et al. 2015).
2.1.2 Construction of Co-expression Vectors containing U2 snRNP genes and SF3a
genes
Amplification of all U2 snRNP genes was performed by using a polymerase chain
reaction from one microgram of C. merolae genomic DNA. Gene specific primers
(synthesized by Eurofins Genomics), which were employed for amplification of these genes,
had a BamHI restriction site in the forward primer and a NotI restriction site in the reverse
primer (Table 3).
The ligation independent cloning technique was used for directional cloning of
amplified U2 snRNP (cloning of the Sms genes in the pQlink vector was performed by
Fatimat Shidi) and SF3a gene products. This method creates long cohesive sticky ends on
both the vector and the insert that results in the generation of desired molecules after
annealing the insert and the vector. All U2 snRNP and SF3a genes were combined into a
polycistronic system, which means each gene contains its own promoter, ribosome binding
site and stop codon.

20

After the target was amplified by PCR, the PCR products were treated with T4
DNA polymerase. First, 50 ng of an amplified gene were treated with 1 µL of T4 DNA
polymerase (3ʹ to 5ʹ exonuclease activity) in the presence of 25 mM dCTP (T4 DNA
polymerase degrades the DNA from the 3ʹ end until it encounters to the first dC residue). The
pQlinkNmod was digested with the EcoRI and HindIII site by using 10 U PmlI /µL (three
hours digestion without gel purification) and 60 ng of this linearized co-expression vector.
Next, the pQlinkNmod was treated with 1 µL of T4 DNA polymerase in the presence of 25
mM dGTP (T4 DNA polymerase removes nucleotides until it encounters to the first dG
residue) (Kim. 2007), 100 mM DTT, and 2 µL of 10 x T4 DNA polymerase buffer. The
pQlinkNmod was produced by Liz Dunn (fragment between EcoRI and BamHI was removed
from original pQlink) (Addgene plasmid 13670; Scheich et al. 2007) and cloned this part
with an oligo duplex, oSDR1084/1085. Each of the T4 DNA polymerase treatment reactions
was incubated at room temperature for 30 minutes separately. These two reactions were
combined together and heated at 70 C for 20 minutes, then allowed to sit at room temperature
for five minutes to anneal each U2 snRNP and SF3a gene into the co-expression pQlinkNmod
vector. Each of the U2 snRNP and SF3a genes was sequenced by the UNBC genetic facility
by using oSDR1084 and oSDR1085 primers. For better sequencing of extensive genes (more
than 800 nucleotides), in addition to using oSDR1084 and oSDR1085 primers, gene specific
primers were also used (Table 3). The 4Peaks software was used to confirm the results of
sequencing (http://nucleobytes.com/index.php/4peaks).

21

Table 3. DNA oligonucleotides which were used to amplify and sequence the desired gene. The first DNA
oligonucleotide for each gene is the forward and the second DNA oligonucleotide in the reverse primer.
TACTTCCAATCCCACGAGGAGAAATTAACT is the LIC site for the forward primer and
TTATCCACTTCCCACG is the LIC site for the reverse primer.
oSDR#
oSDR1105
oSDR1106
oSDR1107
oSDR1108
oSDR1109
oSDR1110
oSDR1111
oSDR1112
oSDR1113
oSDR1114
oSDR1115
oSDR1116
oSDR1117
oSDR1118
oSDR1119
oSDR1120
oSDR1082
oSDR1083
oSDR1246
oSDR1247
oSDR1084
oSDR1085

U2 snRNA Sequence
Genes
Cm Prp9
TACTTCCAATCCCACGAGGAGAAATTAACTATGAACGCGCTCACC
Cm Prp9
TTATCCACTTCCCACGTTACGTACGACTGGATCC
Cm Prp11 TACTTCCAATCCCACGAGGAGAAATTAACTATGGACGCCTACGCA
Cm Prp11 TTATCCACTTCCCACGCTAGTCTCTTCGCACATAC
Cm Hsh155 TACTTCCAATCCCACGAGGAGAAATTAACTATGGAAGCGTTGGAAAG
Cm Hsh155 TTATCCACTTCCCACGTTACAGAGTTGCGGC
Cm Cus1 TACTTCCAATCCCACGAGGAGAAATTAACTATGGTGGAAGATTCAGAC
Cm Cus1 TTA TCC ACT TCC CAC G TCA CGG GGG GTC TG
Cm Rse1 TACTTCCAATCCCACGAGGAGAAATTAACTATGGCGGAGCCGCTG
Cm Rse1 TTATCCACTTCCCACGTTACGTCGCATGCAACG
Cm Rds3 TACTTCCAATCCCACGAGGAGAAATTAACTATGTCCCTGAAACATACG
Cm Rds3 TTATCCACTTCCCACGTTACAAGTCACCAGCG
Cm Prp5
TACTTCCAATCCCACGAGGAGAAATTAACTATGGAGCAACTGGGAAT
Cm Prp5
TTATCCACTTCCCACGCTAGAACATCGTCTCTTCG
Cm Prp43 TACTTCCAATCCCACGAGGAGAAATTAACTATGACGCATGTACCAAGC
Cm Prp43 TTATCCACTTCCCACGTCACGGTGCCGCGGA
Cm Hsh49 TACTTCCAATCCCACGAGGAGAAATTAACTATGAACGGCTCTGGGCTAGG
Cm Hsh49 TTATCCACTTCCCACGTCACTTCTCTGAGAGCTGCTGCAC
Cm Prp21 TACTTCCAATCCCACGAGGAGAAATTAACTATGGAGCCGCAGACG
Cm Prp21 TTATCCACTTCCCACGCTATCGTGTGTGATGACCGTTAAAC
pQLink
CCATTTGTCG AGAAATCATA AAAAATTTAT TTGCTTTGTG
pQLink
CAACCG AGCGTTCTGA ACAAATCCAG

Ligation independent cloning was used to construct a co-expression vector
containing all the U2 snRNP and SF3a genes by using the pQlinkH (Addgene plasmid
13667; Scheich et al. 2007) and the pQlinkNmod vectors (Scheich et al. 2007; Figure 8).
Plasmid names are listed in Table 4. All the vectors containing single genes of the U2 snRNP
were combined into a single co-expression vector. In other words, individual genes were
combined serially until they were all present in a single plasmid (Figure 9 and Figure 10). For
this purpose, a pQlink vector with the first gene of interest was digested with the SwaI
restriction enzyme, while the PacI restriction enzyme was used for the digestion of the other
pQlink vector containing the second gene of interest. The pQlink consists of three restriction
sites, two for PacI and one for SwaI. The PacI restriction sites are located at the 3ʹ of the
22

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LVDWWKH‫މ‬RIWKHWUDQVFULSWLRQDOWHUPLQDWRUIRUWKHLQVHUWHGJHQH


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23

Table 4. U2snRNP and SF3a plasmids which were used to make the related co-expression vector.

Plasmid name
pSR648
pSR653
pSR658
pSR659
pSR635
pSR646
pSR652
pSR654
pSR657
pSR710
pSR730
pSR711
pSR713
pSR757
pSR737
pSR738
pSR760
pSR763

Backbone
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617
pQlinkN pSR617

pSR752
pSR754
pSR764

pQlinkH pSR627
pQlinkH pSR627
pQlinkH pSR627

pSR765

pQlinkH pSR627

pSR793
pSR795

pQlinkH pSR627
pQlinkH pSR627

Gene
Cm Rds3
Cm Prp11
Cm Prp9
Cm Rse1
Cm Hsh49
Cm Hsh155
Cm Cus1
Cm Prp5
Cm Prp43
Cm Prp9/Prp11
Cm Prp21
Cm Rse1/Hsh49
Cm Hsh155/Cus1
Cm Prp9/Prp11+ Prp21
Cm Hsh155/Cus1+ Rse1/Hsh49
Cm Prp43/Prp5
Cm Prp5/Prp43 + Prp9/Prp11/Prp21
Cm Hsh155/Cus1+ Rse1/Hsh49 + Cm
Prp5/Prp43 + Prp9/Prp11/Prp21
Cm His-SmF/E3/G/D3/B/D1/D2
Cm His-SmF/E3/G/D3/B/D1/D2 + U2
Cm Hsh155/Cus1/
Rse1/Hsh49/Prp5/Prp43/Prp9/Prp11/Prp21+ Cm
His-SmF/E3/G/D3/B/D1/D2/U2
Cm Hsh155/Cus1/
Rse1/Hsh49/Prp5/Prp43/Prp9/Prp11/Prp21+Rds3/
Cm His-SmF/E3/G/D3/B/D1/D2/U2
Cm Prp21
Cm Prp9/Prp11+Prp21

In summary, 500 ng of DNA plasmid was digested with 5 µL of SwaI or 5 µL of
PacI restriction enzyme. BSA was used in each of SwaI and PacI digestion reaction.
Incubation of PacI and SwaI digestion reaction was performed at 37 C and 25 C respectively
for 3 hours. Afterward, heat inactivation of the enzyme was carried out at 75 C for 20
minutes. Next, the digested products were treated with 0.5 µL of T4 DNA polymerase in a 20
µL mixture reaction containing 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 100 ng/µL BSA, 0.1
M DTT and 0.5 µL of each 50 mM dCTP and 50 mM dGTP for PacI and SwaI digests
respectively. SwaI and PacI plasmids were mixed and were heated at 65 C for five minutes
24

then the reaction was allowed to cool at room temperature. Two µL of 25 mM EDTA was
added and 5 µL of the reaction was transformed into 50 µL of RbCl2 competent DH5α cells.
Results of transformation were screened by performing colony PCR using gene specific
forward and reverse primers to confirm that all of the U2 snRNP genes from the PacI and the
SwaI-digested plasmid were present in the same pQlink expression vector. Restriction
enzymes were used to ensure that the SF3a construct contained all three genes (Prp9, Prp11
and Prp21) in the pQlink expression vector. The ligation independent cloning was repeated to
combine all individual genes into the same expression plasmid. This was done separately for
the U2 snRNP and SF3a genes (Figure 9 and Figure 10).

25

Figure 9. Construction of the U2 snRNP co-expression vectors. Each gene of the U2 snRNP was first
introduced into a single plasmid and then all genes were combined serially into plasmids until they were present
in one final plasmid. Plasmids were digested with SwaI (S) or PacI (P), followed by heat inactivation, T4 DNA
polymerase treatment and annealing.

26


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27

(Amresco; solon, Ohio) to the culture. The cells were left in the 16 C incubator for 12‒18
hours afterward. Cells were harvested and stored at -80 C for further analysis.
A 25 mL cell pellet was resuspended in 500 mL of lysis buffer (20 mM HEPES-NaOH pH
7.5, 500 mM NaCl, 5 mM β-Mercaptoethanol and 20 mM Imidazole) to lyse the cells. The
cell lysate was sonicated four times, in ten-second bursts, at five to seven Watts, and with ten
second pauses on ice in between. To separate the soluble and insoluble fractions, the lysate
was centrifuged at 13000 x g in the Beckman Coulter Allegra X-12R centrifuge for ten
minutes at 4 C. The cells which appeared in supernatant are soluble and the cells which
precipitated as a pellet are insoluble. The supernatant and pellet were run on a gel to check
the solubility of the protein expression.
In order to do small scale protein purification, the clear lysate was passed through a
Ni2+ resin which the histidine tagged protein will bind to. It is expected that other proteins of
interest would co-purify via the connection between the Ni2+ resin and the tagged SmF for
the U2 snRNP and Prp21 for the SF3a purification. Afterwards, a wash step was done to
remove all bacterial proteins in the expression system from the bound proteins. Finally, the
bound proteins were eluted.
For the purification of the U2 snRNA proteins and SF3a protein complex, a batch
method (Thermo Scientific) were used. The supernatant was loaded on Pierce spin cups
(Thermo Scientific #69700), which were equilibrated with two resin bed volumes of wash
buffer (20 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 5 mM β-Mercaptoethanol and 20 mM
Imidazole) by resuspending the resin with wash buffer followed by a two minute spin at 700
x g in the Eppendorf 5417c centrifuge. Excess buffer was discarded and the lysate was then

28

mixed with resin and incubated for 30 minutes on an end-over-end rotator. The lysate was
centrifuged as above and the lysate removed. The resin was washed twice with two resin-bed
volumes of wash buffer and centrifuged for two minutes at 700 x g, and the excess buffer
was removed. The U2 snRNA proteins and SF3a protein complex were eluted by using one
resin bed volume of elution buffer (20 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 5 mM βMercaptoethanol and 500 mM Imidazole) by repeating this step twice, and saving each
supernatant fraction in a separate tube. The eluted proteins were analyzed by SDS-PAGE
(12% high TEMED gel).

2.2 Results and Discussion
In order to develop in vitro reconstitution, all genes were cloned into the same
pQlink expression vector. Results of transformation were screened by performing colony
PCR using gene specific forward and reverse primers to confirm that all the U2 snRNP genes
from the PacI and the SwaI-digested plasmid were present in the same pQlink expression
vector (Table 4, Figure 11‒Figure 14). Restriction enzyme digestion showed that all three
genes of the SF3a construct were present in the pQlink expression vector as well. To see the
difference between the size of digested and undigested plasmids, the 0.7% agarose ethidium
bromide gel was run for different length of time (Figure 15).
In pQlink expression vectors, histidine tags were placed at the N-terminus of SmF
and Prp21 genes located in the U2 snRNP and SF3a constructs, respectively. The sixhistidine residues, were tagged with a protein of interest to facilitate the purification of the
protein (Schieich et al. 2007).

29


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36

A co-expression technique aids the protein partners to fold better and increase the
solubility of the recombinant expression protein (Romier et al. 2006). However, coexpression of the SF3a protein complex resulted in an insoluble particle. According to a
study by Lin and Xu, Saccharomyces cerevisiae SF3a complex co-expressed in E.coli (BL21
strain) successfully. GST tag was used as a fusion tag for the expression of Prp9, while Prp11
expressed as a poly-His-tagged fusion protein (Lin et al. 2012). The GST tag helps protein to
fold properly and increase the solubility of the recombinant proteins. It is possible that using
His-tag as a fusion tag with Prp21 could result in the insolubility of the SF3a complex in my
project. As co-expression and purification of the U2 snRNP and the SF3a complex proteins
were unsuccessful, I decided to express and purify the U2 snRNP proteins individually for
the biochemical and functional analyses.

37

Chapter 3
Attempted individual expression and
purification of Prp9, Prp11, Prp21 and
Rds3 proteins
As a result of the unsuccessful co-expression and purification of the U2 snRNP
proteins and the SF3a protein complex, as discussed in the previous chapter, I decided to
express the SF3a protein and Rds3 protein individually. Rds3 is a subunit of the SF3b protein
complex that interacts with the other four subunits of SF3b in yeast: Cus1p, Hsh49p,
Hsh155p, Rds3p, and Ist3p/Snu17p. Rds3p is an essential factor for the stability of the
spliceosome. The absence of Rds3p prevents spliceosome formation and blocks U2 snRNP
addition (Wang and Rymond. 2003).
I have generated separate plasmids for each gene of the SF3a proteins and Rds3
protein. A polymerase chain reaction was performed to obtain the genes of these proteins
from C. merolae genomic DNA. Using the ligation independent cloning method, those genes
were introduced in two different expression vectors: pQlink and PMCSG23 plasmids.
PMCSG23 was derived from the PMCSG21 backbone and this vector contains histidine tags
(His6-tag), a maltose-binding protein tag (MBP-tag), and sequences that encode the tobacco
etch virus (TEV) protease cleavage site. The MBP tag was used for further purification of
protein and it increases the solubility of the protein. The TEV cleavage site facilitates protein
purification. Since co-expression of the SF3a protein complex resulted in an insoluble
particle, having the MBP fusion protein may aid to obtain soluble protein expression. I tried
different conditions in order to express these proteins individually.
38

3.1 Material and Methods
3.1.1 Preparation of C. merolae genomic DNA
I used C. merolae 10D strain (NIES-1332) provided by the Microbial Culture
Collection at the National Institute for Environmental Studies in Tsukuba, Japan
(mcc.nies.go.jp/). C. merolae genomic DNA was prepared by Martha Stark as previously
described (Stark et al. 2015).
3.1.2 Construction of expression Vectors containing Prp9, Prp11, Prp21, Rds3 and
Hsh49 genes
Amplification of the SF3a, Rds3 and Hsh49 genes were performed by PCR from
one microgram of C. merolae genomic DNA. Gene specific primers (prepared by Eurofins
Genomics) were employed for amplification of these genes (Table 3).
In order to construct an expression vector, each gene of the SF3a subunits and Rds3
was cloned separately into two different expression vectors: pQlink and PMCSG23 (Table 6).
The PMCSG23 expression vector containing the Hsh49 gene was constructed using the same
procedure that was employed for Rds3.
This was accomplished using the ligation independent cloning into the PMCSG23
expression vector which will add a MBP and a six-histidine tag at the N-terminus of the SF3a
subunits, Rds3 and Hsh49 (Chapter 2). PMCSG23 was derived from the PMCSG21
backbone and this vector contains six histidine tags, maltose-binding protein (MBP), and
sequences that encode the tobacco etch virus (TEV) protease cleavage site. The MBP tag was

39

used for further protein purification, which increases the solubility of the protein, and the
TEV cleavage site facilitates protein purification.

Before cloning the genes, 10 µg of the PMCSG23 vector was digested with 5µL
SspI (100 U) in 100 µL volume by incubation at 37 C for six hours. Five µL of annealing
reaction was transformed into 50 µL of RbCl2 competent DH5α cells. Results of the
transformation were inspected via sequencing. Sequencing of each gene was done by the
UNBC genetic facility and the results of sequencing were analyzed by 4Peaks software
(http://nucleobytes.com/index.php/4peaks). Gene specific primers along with oSDR1283 and
oSDR1284 primers were used for the sequencing of the Prp9, Prp11, Prp21, Rds3 and Hsh49
in the PMCSG23 vector (Table 5). Gene specific primers and oSDR1084 and oSDR1085
primers were also recruited for the sequencing of these genes in the pQlink vector (Table 4).

40

Table 5. DNA oligonucleotides which were used to amplify and sequence the desired gene. The first DNA
oligonucleotide for each gene that are listed, is the forward and the second DNA oligonucleotide is the reverse
primer.
oSDR#

U2 snRNA
Genes

Sequence

oSDR1319

Cm Prp9

TACTTCCAATCCAATGCAAACGCGCTCACCGC

oSDR1320

Cm Prp9

TTATCCACTTCCAATGTTACGTACGACTGGATCCAG

oSDR1129

*Cm Prp9

CAGAGTGTTTAGATGCC

oSDR1321

Cm Prp11

TACTTCCAATCCAATGCAGACGCCTACGCACTCAAAAAC

oSDR1322

Cm Prp11

TTATCCACTTCCAATGCTAGTCTCTTCGCACATACAG

oSDR1323

Cm Prp21

TACTTCCAATCCAATGCAGAGCCGCAGACGGTTC

oSDR1324

Cm Prp21

TTATCCACTTCCAATGCTATCGTGTGTGATGACCG

oSDR1223

Cm Rds3

TACTTCCAATCCAATGCATCCCTGAAACATACCG

oSDR1224

Cm Rds3

TTATCCACTTCCAATGTTACAAGTCACCAGCG

oSDR1294

Cm Hsh49

TACTTCCAATCCAATGCAAACGGCTCTGGGCTG

oSDR1295

Cm Hsh49

TTATCCACTTCCAATGTCACTTCTCTGAGAGCTGC

oSDR1283

pMCSG23

TGATCAACGCCGCCAGC

oSDR1284

pMCSG23

GCAGCGGTTTCTTTACC

*is the sequencing primer to read the Prp9 gene.

Table 6. SF3a, Rds3 and Hsh49 plasmids that were used to make the related expression vector.

Plasmid name
pSR828
pSR827
pSR826
pSR766
pSR800

Backbone
pMCSG23 pSR614
pMCSG23 pSR614
pMCSG23 pSR614
pMCSG23 pSR614
pMCSG23 pSR614

Gene
Cm Prp21
Cm Prp11
Cm Prp9
Cm Rds3
Cm Hsh49

3.1.3 Expression and purification of Prp9, Prp11, Prp21 and Rds3 proteins
The Rosetta pLysS cell was used for expression of the Prp9, Prp11, Prp21, and
Rds3 by growing in two different media: Luria Bertani and 2XYT media. 50 mg/mL
ampicillin and 25 mg/mL chloramphenicol were used to select for the pQlink vectors and for
the Rosetta pLysS plasmids, respectively. 25 mg/mL spectinomycin was used to select for the
PMCSG23 vector as well. Rosetta pLysS cell was used for the expression of the Hsh49 by
41

growing into 2XYT media. 25 mg/mL chloramphenicol was used to select for the Rosetta
pLysS plasmids, and 25 mg/mL spectinomycin was used to select for the PMCSG23 vector.
The entire process of the expression of these proteins was performed as explained in
the materials and methods section of Chapter 2. However, different strategies were applied
for better expression of these proteins; for example, two different concentrations of isopropyl
1-thioβ-D-galactopyranoside (IPTG), 0.5 mM and 1 mM were used for induction, and
different temperatures (16 C, 25 C, 37 C) were applied after induction. Once any of the
proteins expressed well, I inoculated the starter culture to one liter and it was grown at 37 C,
at 200 rpm to reach the OD600 0.6‒0.8 range. At this point an induction was done by adding 1
mM or 0.5 mM IPTG (VWR) to the culture. The cells were left at different temperatures (16
C, 25 C, 37 C) for 12‒18 hours. Aside from using IPTG, auto-induction was performed for
the expression of the SF3a subunits in the PMCSG23 expression vector. After transformation
to the expression vector, a single colony was selected and grown in 5 mL MDG non-inducing
media (Table 7). 25 mg/mL spectinomycin and 25 mg/mL chloramphenicol were added to
the culture and it was incubated at 37 C for 24 hours. An overnight saturated culture
containing OD600 ⁓5‒10 was spun down and resuspended in 5 mL ZYM-5052 Auto-inducing
media (Table 8).

42

Table 7. MDG non-inducing media.

Ingredients

Final
Concentration

Na2HPO4

25 mM

KH2PO4

25 mM

NH4Cl

50 mM

Na2SO4

5 mM

MgSO4

2 mM

Glucose

50%

TRACE METALS (see below)

0.2x

Aspartic acid

0.25%

TRACE METALS

1000x

FeCl3

50 mM

CaCl2

20 mM

MnCl2

10 mM

ZnSO4

10 mM

CoCl2

2 mM

CuCl2

2 mM

NiCl2

2 mM

Na2MoO4

2 mM

NaSeO3

2 mM

H3BO3

2 mM

HCl

60 mM

43

Table 8. ZYM-5052 Auto-inducing media.

N-Z-amine
yeast extract
MgSO4
TRACE METALS
M stock solution (see below)
5052 stock solution (see below)

Final
Concentration
1%
0.5%
2 mM
1000x
50x
50x

M stock solution
Na2HPO4
KH2PO4
Na2SO4
NH4Cl

50x
1.25 M
1.25 M
0.25 M
2.5 M

5052 stock solution
Glycerol
Glucose
alpha-lactose monohydrate

50x
25%
2.5%
10%

Ingredients

The saturated culture was grown at 37 C for 24 hours. Cells were harvested and
stored at -80 C for further analysis. A one liter cell pellet was resuspended in 10 mL of lysis
buffer (20 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 5 mM β-Mercaptoethanol and 20 mM
Imidazole) containing half of a tablet of Roche Complete, EDTA-free protease inhibitor
cocktail. The cell lysate was sonicated four times, in ten-second bursts, at five to seven W,
and with ten second pauses on ice in between. The lysate was centrifuged at 25000 x g in a
JA25.50 rotor (Beckman Coulter Avanti HP-20 XPI) centrifuge for 30 minutes at 4 C. One
percent Streptomycin Sulfate was added to the lysate in order to precipitate some of the
genomic DNA. The lysate was centrifuged as above to get clear lysate. The supernatant was
filtered by passing through a 0.45 µm nylon syringe filter and was then passed over a 1 mL

44

His-Trap column. AKTA FPLC system with Unicorn software version 5.01 was used for the
protein purification. Rds3 bound to the His-Trap column was washed with ten column
volumes (10 mL) of buffer A (wash buffer: 20 mM HEPES-NaOH pH 7.5, 500 mM NaCl,
and 20 mM Imidazole). Then it was eluted with 5 column volumes (25 mL) of buffer B
(elution buffer: 20 mM HEPES-NaOH pH 7.5, 500 mM NaCl, and 500 mM Imidazole). The
flow rate for both the washed and the elution steps was 0.5 mL/min over the Ni-NTA Histrap column. The fraction that was collected was 5 mL and it was quantified with a Nanodrop
ND-1000 spectrophotometer. The eluted fraction was then passed over a 5 mL MBP Trap
column and washed with 3 column volumes (15 mL) of buffer A (wash buffer: 20 mM
HEPES-NaOH pH 7.5, and 500 mM NaCl). Then the fraction was eluted with 5 column
volumes (25 mL) of buffer B (elution buffer: 20 mM HEPES-NaOH pH 7.5, 500 mM NaCl
and 10 mM Maltose). This second purification step was performed for better purification of
the Rds3 and SF3a proteins. The flow rate for purification of Rds3 over the MBP-Trap
column was 1 mL/min. The purification steps were performed at 4 C in AKTA FPLC system.
The collected fraction was 2.5 mL and it was incubated with one milligram of TEV protease
per 50 mg of purified protein at 4 C for 18 hours. The TEV cleaved protein was dialyzed with
20 mM HEPES-NaOH pH 7.5, 250 mM NaCl. After this step, the removal of cleaved His
tags, His6-TEV Protease, or proteins of interest retaining His-tags was required. To do so, the
dialyzed protein was passed through Ni2+ resin in a batch bind purification. This time the
cleaved protein did not bind to the Ni2+ resin, but the His6 tag and TEV protease did bind. An
amylose-resin batch bind purification was carried out on the flow-through to get more
purified protein. The samples were analyzed by SDS-PAGE.

45

The Hsh49 protein was purified using the same protocol explained as the Rds3
protein, described in the material and methods of this chapter. It is necessary to mention that
both the expression and the purification were conducted at a large scale to be able to
complete the purification using the FPLC system. I used a 4 L culture for purification of
Hsh49 protein.
After performing FPLC using the AKTA FPLC system, the eluted fraction was
quantified with a Nanodrop ND-1000 spectrophotometer. The eluted fraction was passed
over a 5 mL MBP-trap column for better purification and was then incubated with one
milligram of TEV protease per 50 mg of purified protein at room temperature for 18 hours.
The TEV cleaved protein was dialyzed with 20 mM HEPES-NaOH pH 7.5, 300 mM NaCl.
After this step, the dialyzed protein was passed through a His-trap column purification to
remove cleaved His and MBP tags, His6-TEV protease, or any proteins that were still with
His or MBP tags. The Flow through was dialyzed with 20 mM HEPES-NaOH pH 7.5, 300
mM NaCl. The dialyzed Hsh49 sample was collected and then concentrated in a YM-3
centrifuge filter unit (Microcon Ultracel) to get the concentration of 10 mg/mL according to
the manufacture’s protocol. The concentrated flow through was quantified with the Nanodrop
ND-1000 spectrophotometer. The samples were analyzed by SDS-PAGE.

3.2 Results and Discussion
In this Chapter, I focused on expressing and purifying the SF3a protein subunits and
the Rds3 protein separately. Each of the Prp9, Prp11, Prp21 and Rds3 genes were cloned in
the PMCSG23 expression vector, and histidine and MBP tags were placed at the N-terminus
46

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DQGUHSUHVHQW 6 VROXELOLW\DQG , LQVROXELOLW\RIWKH5GVUHVSHFWLYHO\

51

Following the successful expression of the Rds3 protein, purification of this protein
was performed by FPLC. Since the Rds3 protein contains histidine and MBP tags, it was first
purified through the His-trap column (Figure 25a) and then the MBP trap column (Figure
25b).
To remove the His-MBP tags from the Rds3 protein, a TEV protease cleavage was
performed at 4 C since TEV protease cleavage at room temperature resulted in the Rds3
protein precipitation (Figure 26a). It was then dialyzed to reduce the concentration of salt and
imidazole which were present in the elution fraction following the second purification. A
small amount of the Rds3 protein was precipitated after dialysis as shown in Lane 5 in Figure
26b.

52



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55

The Hsh49 gene was cloned in the PMCSG23 expression vector, and histidine and MBP tags
were placed at the N-terminus. To confirm the presence of this gene in the PMCSG23 vector,
the gene was sequenced by the UNBC genetic facility.
After cloning the Hsh49 gene in the PMCSG23 expression vector, the target protein was
transformed into host cells and cells were grown in 2XYT media (37 C for pre- and postinduction). SDS-PAGE was used to analyze the expression and purification of the Hsh49 in
the PMCSG23 expression vector. Based on Figure 32a, Hsh49 protein was expressed well in
the 2XYT media. The Hsh49 protein appeared as a partially soluble protein and partially as
an insoluble protein based on Figure 32b. As was described in the materials and methods of
Chapter 2, to separate the soluble and insoluble fractions, the lysate was centrifuged at 13000
x g for ten minutes at 4 C. The proteins that appeared in the supernatant were soluble and the
proteins that precipitated as a pellet were insoluble. The supernatant and pellet were run on a
gel to check the solubility of the protein expression.

56

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57

Hsh49 protein. TEV protease cleavage was conducted at room temperature since TEV
protease is more active at room temperature (Figure 33b). It was then dialyzed to reduce the
concentration of salt and imidazole which were present in the elution fraction. As I
mentioned above, the second purification through the His-trap column was done to obtain
pure Hsh49 protein. This purification helps to eliminate His-MBP tags, His-TEV protease or
any Hsh49 protein that retained tags (Figure 33c). The flow through was dialyzed and then
concentrated. Finally, the concentrated Hsh49 was run on a 15% high TEMED SDS gel
which showed that the majority of tags were removed from this protein (Figure 33d).

58





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59

Based on the results, it was surprising that expression of proteins after cloning in
pQlinkNmod and PMCSG23 expression vectors was not successful. The reason that the
expression failed is hard to address but it is possible that the protein could be toxic for the
cell when expressed in a higher yield. Another possibility is that the protein might be
unstable due to the absence of some proteins with which it forms a complex (Howe. 2007). In
this project, the SF3a protein complex was co-expressed well but an individual expression of
each subunit of the SF3a failed. This might be due to the lack of one or two of the SF3a
subunits and could have led to protein degradation.
The Rds3 protein was expressed very well and purified through the His and MBP
trap column in several steps to isolate this protein and attain a pure result. Unfortunately, I
was not successful in removing the tags from the Rds3 protein. Potentially, it is possible that
the protein may have folded in a way that made the tags inaccessible to the Ni2+-NTA resin
or amylose resin columns. Removal of tags is necessary for the functional assays since tags
can affect the function of proteins. Moreover, tags may affect growing crystals for the
structural determination of proteins. Also during this time, expression and purification of the
Hsh49 protein was performed and the tags were removed from Hsh49 in order to carry out
the functional assays. In order to maintain the scope and timeline of the project, I did not
proceed with any further analysis of Rds3.

60

Chapter 4
Functional Analysis of C. merolae Hsh49
protein
Given the difficulty I had with the expression of the SF3a protein complex and
purification of the Rds3 protein (Chapter 3), I started to express and purify another U2
snRNA protein called Hsh49, to characterize the function and the interaction of this protein
with U2 snRNA. Hsh49 is one of the five subunits of the SF3b protein complex in C.
merolae. The SF3b complex is required for recognition of the intron’s branch point, and
precise excision of introns from pre-mRNA. In fact, SF3b plays an essential role during prespliceosome assembly (Golas et al. 2003).
Hsh49 consists of two RNA recognition motifs (RRMs): N-terminal RRM (RRM1)
and C-terminal RRM (RRM2). In yeast, RRM1 interacts with the Cus1 protein (another
subunit of the SF3b complex) and it seems that association of the Cus1 to the Hsh49 is
necessary for the interaction of the U2 snRNA to the branch site of the pre-mRNA (Igel et al.
1998; Gozani et al. 1996; Champion-Arnaud et al. 1994). In yeast, Hsh49 has been shown to
have RNA binding activity in which RRM2 associates with the pre-mRNA, and for this
association Cus1 protein is not required (Igel et al. 1998). As shown in Figure 28, human
Hsh49 interacts with several sites of the U2 snRNA, notably the 5ʹ end of the U2 snRNA and
nucleotides located in stem loop I and IIb (Dybkov et al. 2006). Moreover, human Hsh49
creates a stable connection with part of the 3ʹ end of the pre-mRNA (Staknis et al. 1994). In
this chapter, I describe the examination of the Hsh49 with U2 snRNA to determine whether

61

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63

CD spectra were performed to determine the secondary structure and stability of
recombinant expressed, purified Hsh49. Stability and conformational changes of Hsh49 were
assigned under thermal stress by using Jasco J-815 CD spectropolarimeter (Jasco Inc). The
Hsh49 protein first was dialyzed and diluted in NaF (250 mM NaF and 20mM HEPESNaOH pH 7.5) because chloride ions from NaCl have a high absorbance in the far UV
spectral region (260‒190 nm). Hsh49 protein samples were scanned in the far-UV region
using a 0.1 cm path length quartz cuvette. CD spectra were assessed at a constant temperature
of 25C which was controlled by a Peltier controller (Table 9). This wavelength was used to
estimate the fraction of the alpha helices and beta sheets in the Hsh49 protein. The K2D3
circular dichroism web server (Louis-Jeune et al. 2011) was used to estimate the secondary
structure of Hsh49 from its CD spectrum.

Table 9. Measurement parameters for the CD spectra of Hsh49.

Photometric mode:

CD,HT

Measure range:

260‒190 nm

Data pitch:

Scanning speed:

50 nm/minute

Baseline correction:

Baseline

0.1 nm

Shutter control:

Manual

Sensitivity:

Standard

CD detector:

PMT

D.I.T.:

2 Seconds

PMT voltage:

Auto

Bandwidth:

1.00 nm

Accumulations:

3

Start mode:

Immediately

B. Thermal Stability of Hsh49
The melting temperature was assessed by increasing the temperature to unfold this
protein. The concentration of Hsh49 was 1.6 mg/mL (⁓ 40µM) for CD scanning. Thermal
stability of the Hsh49 was measured in the range of 28.79‒84.44 C by using a thermal ramp
(Peltier temperature controller) based on the α-helix content at 222 nm as measured by CD
spectrum (Jasco J-815).
64

4.2.2 RNA-Protein Binding Assays
This part of Chapter 4 covers the experiments performed to determine the base
pairing interaction between the U2 snRNA and the Hsh49 protein. First, a fluorescence
polarization (FP) assay was done by using 3ʹ fluorescein labelled U2 snRNA. The in vitro
electrophoretic mobility shift assay (EMSA) experiment was then performed by recruiting
radiolabelled full-length U2 snRNA and recombinant Hsh49.
4.2.2.1 Fluorescence Anisotropy Assay
A. Generation of fluorescence labelled U2 snRNA
The U2 snRNA oligonucleotide (35 nucleotides) was synthesized with fluorescein
(6-FAM) at its 3ʹ end by Integrated DNA Technologies (IDT).
5ʹ-CUCAUGGUGUAUCGAGAGCUUCAAGCUUCGAUAUU-3ʹ-F
B. Analysis of the U2 snRNA-Hsh49 Protein Interaction using Fluorescence
Anisotropy Assay
In order to determine the U2 snRNA-Hsh49 interaction by fluorescence anisotropy,
different concentrations of the Hsh49 protein were incubated with 10 nM of fluorescein
labelled U2 snRNA oligo and different types of buffer to find out the optimal conditions for
this experiment (Table 10‒Table 16). Incubation was done in a 1.5 mL microcentrifuge tube
at room temperature (20 C ‒25 C) for 10 minutes. The trials were run in triplicates using the
excitation and emission filter settings of 485 and 521 respectively by Synergy 2 (BioTek

Instruments, Inc). Synergy 2 (Fluorescence Anisotropy) provided the values of free and
bound RNA and the data were analyzed on the basis of fluorescence anisotropy values. The
data were plotted using Prism 7 software.

65

Table 10. The fluorescence anisotropy reagent (Buffer 1) used for the detection of the U2 snRNA-Hsh49
interaction. (Hsh49 with benzonase and without benzonase).

FA binding reaction reagents (300 uL)
Binding Buffer (see below)
Hsh49

Volume
90 µL
-

Fluorescent labelled U2 RNA oligo (50 nM)
H2O

60 µL
To 300 µL

Binding Buffer 1 Reagents (per 1ml)
1 M HEPES-NaOH pH 7.5
3 M NaCl
H2O

20 µL
83.3 µL
896.7 mL

Final Concentration
*0 to 200000 nM
0 to 140000 nM
0 to 400 nM
10 nM

20 mM
250 mM

*Concentrations of Hsh49 used for fluorescence anisotropy experiments (Figure 44 and Figure 45d).
Table 11. The fluorescence anisotropy reagent (Buffer 2) used for the detection of the U2 snRNA-Hsh49
interaction.

FP binding reaction reagents (300 uL)
Binding Buffer (see below)
Hsh49
Fluorescent labelled U2 RNA oligo (50 nM)
H2O

Volume
90 µL
60 µL
To 300 µL

Final Concentration
0 to 40000 nM
10 nM

Binding Buffer 2 Reagents (1 mL)
1 M HEPES-NaOH pH 7.5
1 M MgCl2
H2O

20 µL
2.5 µL
977.5 µL

20 mM
2.5 mM

66

Table 12. The fluorescence anisotropy reagent (Buffer 3) used for the detection of the U2 snRNA-Hsh49
interaction.

FA binding reaction reagents (300 uL)
Binding Buffer (see below)
Hsh49
Fluorescent labelled U2 RNA oligo (50 nM)
H2O

Volume
90 µL
60 µL
To 300 µL

Final Concentration
0 to 40000 nM
10 nM

Binding Buffer 3 Reagents (1 mL)
1 M HEPES-NaOH pH 7.5
1 M MgCl2
10% Tritonx-100
H2O

20 µL
2.5 µL
10 µL
967.5 µL

20 mM
2.5 mM
0.1%

Table 13. The fluorescence anisotropy reagent (Buffer 4) used for the detection of the U2 snRNA-Hsh49
interaction.

FA binding reaction reagents (300 uL)
Binding Buffer (see below)
Hsh49
Fluorescent labelled U2 RNA oligo (50 nM)
H2O

Volume
90 µL
60 µL
To 300 µL

Final Concentration
0 to 40000 nM
10 nM

Binding Buffer 4 Reagents (1 mL)
1 M HEPES-NaOH pH 7.5
3 M NaCl
10% Tritonx-100
H2O

20 µL
83.3 µL
10 µL
886.7 µL

20 mM
250 mM
0.1%

67

Table 14. The fluorescence anisotropy reagent (Buffer A) used for detection of conformational changes of the
U2 snRNA.

FA binding reaction reagents (300 uL)
Binding Buffer A (see below)
Fluorescent labelled U2 RNA oligo (50 nM)
H2O

Volume
150 µL
60 µL
To 300 µL

Final Concentration
10 nM

Binding Buffer A Reagents (10ml)
100 mM MgCl2
3 M NaCl
50% glycerol
H2O

0.3 mL
0.33 mL
4 mL
5.37 mL

3 mM
100 mM
20%

Table 15. The fluorescence anisotropy reagent (Buffer B) used for detection of conformational changes of the
U2 snRNA.

FA binding reaction reagents (300 uL)
Binding Buffer B (see below)
Fluorescent labelled U2 RNA oligo (50 nM)
H2O
Binding Buffer B Reagents 2 (10ml) (Igel et
al. 1998)
40 mM HEPES, pH 7.9
250 mM KCl
2 mM DTT
10% glycerol
H2O

Volume
150 µL
60 µL
To 300 µL

Final Concentration
10 nM

0.4 mL
2.5 mL
0.02 mL
2 mL
5.08 mL

1.6 mM
62.5 mM
0.004 mM
2%

Table 16. The fluorescence anisotropy reagent (Buffer C) used for detection of conformational changes of the
U2 snRNA.

FA binding reaction reagents (300 uL)
Binding Buffer C (see below)
Fluorescent labelled U2 RNA oligo (50 nM)
H2O
Binding Buffer C Reagents (1ml)
1 M HEPES-NaOH pH 7.5
3 M NaCl
H2O

Volume
150 µL
60 µL
To 300 µL

Final Concentration
10 nM

20 µL
83.3 µL
896.7 mL

20 mM
250 mM

68

4.2.2.2 Fluorescence-based electrophoretic mobility shift assay (F-EMSA)
The binding reaction was prepared for the F-EMSA assay which contained the
fluorescein labelled U2 snRNA oligo (ro65) and Hsh49 in varying concentrations. F-EMSA
binding reaction reagents are listed in Table 17.

Table 17. The F-EMSA reagent used for the detection of the U2 snRNA-Hsh49 interaction.

F-EMSA binding reaction reagents (20 uL)

Volume

Final Concentration

Binding Buffer (see below)

10 µL

-

10 mg/mL Ecoli-tRNA

1 µL

0.5 nM

Hsh49

-

0 to 10000 nM

Fluorescent labelled U2 RNA oligo (100 nM)

2 µL

10 nM

H2O

To 20 µL

Binding buffer reagents 1 (10ml)
100 mM MgCl2

0.3 mL

3 mM

3 M NaCl

0.33 mL

100 mM

50% glycerol

4 mL

20%

10% Tritonx-100

0.2 mL

0.2%

H2O

5.17 mL

The reagents of the binding reaction were added to a 1.5 mL microcentrifuge tube
and mixed well. 3 µL of 6x native loading dye (50% glycerol, 1.5% bromophenol blue, 1.5%
xylene cyanol) was added to each sample. 20 µL of samples were resolved by loading on to a
prechilled, 6% polyacrylamide (29:1 Acrylamide/Bis, Bio-rad) non-denaturing gel in 1x TBE
running buffer. The gel was run at 200 V for 45 minutes in the cold room (4 C) and it was
exposed to a cyclone storage imager system (Packard Instrument Company, Inc.) for 18
hours at -80 C. Visualization and analysis of the gel image were performed by using software
version 0.4 of the Packard Instrument Company, Inc.
69

4.2.2.3 Electrophoretic mobility shift assay (EMSA)
4.2.2.4 Generation of [32p]-labelled U2 snRNA by IVT
In vitro transcription of the full length C. merolae U2 snRNA (131 nt) was a gift
from Martha Stark. Following this step, a phenol-chloroform extraction and an ethanol
precipitation were performed. For the U2 probe, the oligonucleotide oSDRro65 was 5ʹ end
labelled with γ-32p ATP (Table 18).

Table 18. The labelling reaction

16.5 µL
2.5 µL
2.5 µL
2.5 µL
1 µL
25 µL

H2O
PNK Buffer
10 uM oligo
γ-32P
PNK (NEB)
Total Reaction Volume

The first three components were prepared at the bench, while the last two
components were added in the hot room. The heat block was pre-heated to 37 C for ten
minutes and volume of the reaction was brought up to 50 µL by adding 25 µL of H2O. One
µL of the reaction was then counted by using a liquid scintillation counter. The rest of the
reaction was spun through a G-25 sized exclusion column using the refrigerated centrifuge.
The resin was re-suspended in the column by vortexing. The cap was loosened and the
bottom closure twisted off, and the column was then placed in the collection tube and spun
for one minute at 735 x g. After this step, the column was placed into a fresh DNase-free 1.5
mL microcentrifuge tube and an appropriate amount of the sample was applied to the topcentre of the resin. The sample was spun for two minutes at 735 x g. The purified sample was
collected in the bottom of the 1.5 mL microcentrifuge tube and quantified using a liquid
scintillation counter and stored at -20 C.
70

A. Analysis of the U2 snRNA-Hsh49 protein interaction using EMSA
The binding reaction which was prepared for the EMSA assay contained the [32p]labelled IVT U2 snRNA probe (ro65) (7000 cpm) and the Hsh49 in varying concentrations.
Two types of binding buffers were also used for EMSA binding reactions (Table 19 and
Table 20).

Table 19. EMSA reagents (Binding Buffer Reagent 1) used for the detection of the U2 snRNA-Hsh49
interaction.

EMSA binding reaction reagents (20 uL)

Volume

Final Concentration

Binding Buffer (see below)

10 µL

-

10 mg/mL Ecoli-tRNA

1 µL

0.5 nM

Hsh49

-

0 to 5000 nM

[32p]-labelled U2 RNA probe

2 µL

7000 cpm/reaction

H2O

To 20 µL

Binding Buffer Reagents 1 (10 mL)
100 mM MgCl2

0.3 mL

3 mM

3 M NaCl

0.33 mL

100 mM

50% glycerol

4 mL

20%

10% Tritonx-100

0.2 mL

0.2%

H2O

5.17 mL

71

Table 20. EMSA reagents (Binding Buffer Reagent 2) used for the detection of the U2 snRNA-Hsh49
interaction.

EMSA binding reaction reagents (20 µL)

Volume

Final Concentration

Binding Buffer (see below)

10 µL

-

10 mg/mL Ecoli-tRNA

1 µL

0.5 nM

Hsh49

-

100 to 5000 nM

[32p]-labelled U2 RNA probe

4 µL

7000 cpm/reaction

H2O

To 20 µL

Binding Buffer Reagents 2 (10 mL)
(Igel et al. 1998)
40 mM HEPES, pH 7.9

0.4 mL

1.6 mM

250 mM KCl

2.5 mL

62.5 mM

2 mM DTT

0.02 mL

0.004 mM

10% glycerol

2 mL

2%

0.2% Tritonx-100

0.2 mL

0.004%

2 mM EDTA

0.04 mL

0.008 mM

The reagents of the binding reaction were added to a 1.5 mL microcentrifuge tube
and mixed well. In order to determine the optimal incubation temperature, a range of
temperatures were tested for EMSA binding reactions. The samples were directly run onto a
prechilled, 6% polyacrylamide (29:1 Acrylamide/Bis, Bio-rad) non-denaturing gel in 1x TBE
running buffer.
The gel was run at 200 V for 90 minutes in the cold room and exposed to a cyclone
storage imager system (Packard Instrument Company, Inc.) for 18 hours at -80 C.
Visualization and analysis of the gel image were performed by using software version 0.4 of
the Packard Instrument Company, Inc.

72

4.2.3 Co-immunoprecipitation of Hsh49
4.2.3.1 Dot blot assay
Strips of nitrocellulose membrane were cut and several small circles were drawn on
these strips. Purified Hsh49 protein was serially diluted in 20 mM HEPES-NaOH pH 7.5 and
250 mM NaCl. In the center of each circle, 2 µL of each protein dilution was loaded. After
the membrane was dried, it was incubated in a blocking solution (5% W/V stain milk powder
in 0.1% PBS-Tween) for 1 hour at room temperature. The membrane was washed three times
briefly with 0.1% PBS-Tween before being incubated with rat anti-Hsh49 antibody (1:500,
1:1000 and 1:1500) for 1 hour at room temperature. The membrane was washed as before
and incubated with goat anti-rat-HRP antibody (diluted 1:1000) for 1 hour at room
temperature. The wash step was processed before using the SantaCruz ImmunoCruz Western
Blotting Luminol Reagent (SC-2048) for visualization.
4.2.3.2 Western blot assay
Different concentrations of purified Hsh49 protein, including 10 ng, 100 ng and 1
µg, was run on the 15% high TEMED SDS polyacrylamide gel. The gel was then transferred
to the nitrocellulose membrane and incubated with anti-Hsh49 (primary antibody, diluted
1:500) in PBST, then incubated with the goat anti-rat-HRP antibody (secondary antibody,
diluted 1:1500). To visualize the blot, Western Blotting Luminol Reagent (Santa Cruz
Biotechnology) was used according to the manufacturer's protocol. The procedure for
Western blot analysis was explained previously (Reimer et al. 2017).

73

4.2.3.3 Preparation of C. merolae whole-cell extract
C. merolae whole-cell extract was prepared from the 10D strain from (NIES-1332)
provided by the Microbial Culture Collection at the National Institute for Environmental
Studies in Tsukuba, Japan (mcc.nies.go.jp/) (Stark et al. 2015). The cells were harvested and
squeezed in liquid nitrogen. Cell pellets were grounded slowly in a mortar and pestle by
adding liquid nitrogen. Cells were thawed by the addition of cold lysis buffer. The lysate was
centrifuged to remove cell debris. Glycerol was added to the cell extract and stored at -80°C.
Detailed procedures were performed as previously described (Reimer et al. 2017).
4.2.3.4 Generating anti-CmHsh49 antibody
Polyclonal anti-serum against recombinant purified Hsh49 (see methods Chapter 3)
was generated from a rat following the standard protocol (Harlow and Lane. 1988).
4.2.3.5 Hsh49 co-immunoprecipitation
Immunoprecipitation procedures were performed as described previously (Reimer
et al. 2017). Briefly, in order to immunoprecipitate the Hsh49 from C. merolae whole cell
extract, anti-Hsh49 serum, and non-immune serum were coupled to protein G Sepharose 4
Fast Flow (GE Healthcare) or magnetic protein G Dynabeads according to the
manufacturer’s protocol. The Dynadeads-Ab-Ag complex was then incubated with C.
merolae extract. To eliminate nonspecific proteins, several wash steps were performed.
Proteins were eluted with laemmli buffer followed by Western blot analysis.
4.2.3.6 Northern blot analysis
To determine if Hsh49 binds to U2 snRNA, the immunoprecipitated sample was
analyzed via Northern blot. Proteinase K digestion and Phenol: Chloroform extraction were

74

performed to elute RNA from the beads, followed by EtOH-precipitation. The RNA was
resuspended and electrophoresed on a 6%, 7 M urea polyacrylamide gel. The RNA was
transferred onto a Hybond N+ nylon membrane in 1X TBE buffer on a semi-dry blotter
(Panther Semidry Electroblotter HEP-3 Owl). The membrane was hybridized to a 32Plabelled U2 oligonucleotide complementary to U2 snRNA. The blot was washed several
times and visualized on a phosphorimager with a Cyclone Phosphor Imager using OptiQuant
software (Packard Instruments) (Reimer et al. 2017).
4.2.4 Alignment of Hsh49 amino acid and U2 snRNA sequences
An amino acid sequence of Hsh49 and U2 snRNA sequence from Homo sapiens
and Saccharomyces cerevisiae were obtained through BLAST searches at the National
Centre for Biotechnology Information at the National Library of Medicine and at the
Universal Protein knowledgebase (http://www.ncbi.nlm.nih.gov/gene/). U2 snRNA and Hsh49
amino acid sequences of C. merolae were obtained from the C. merolae genome database
(http://merolae.biol.s.u-tokyo.ac.jp/). Sequences of Hsh49 and U2 snRNA were aligned using
EMBL (European Bioinformatics Institute) (http://www.ebi.ac.uk/Tools/psa/emboss_needle/).
The C. merolae Hsh49 aligned with Homo sapiens RRM1 AND RRM2 using PROMALS3D
(Pei et al. 2008).

75

4.3 Results
4.3.1 Expression and Purification of C. merolae Hsh49
The Hsh49 gene was cloned in the PMCSG23 expression vector, and histidine and
MBP tags were placed at the N-terminus. To confirm the presence of this gene in the
PMCSG23 vector, the gene was sequenced by the UNBC genetic facility.
After cloning the Hsh49 gene in the PMCSG23 expression vector, the target protein was
transformed into host cells and cells were grown in 2XYT media (37 C for pre- and postinduction). SDS-PAGE was used to analyze the expression and purification of the Hsh49 in
the PMCSG23 expression vector. Based on Figure 32a, Hsh49 protein was expressed well in
the 2XYT media. The Hsh49 protein appeared as a partially soluble protein and partially as
an insoluble protein based on Figure 32b. As was described in the materials and methods of
Chapter 2, to separate the soluble and insoluble fractions, the lysate was centrifuged at 13000
x g for ten minutes at 4 C. The proteins that appeared in the supernatant were soluble and the
proteins that precipitated as a pellet were insoluble. The supernatant and pellet were run on a
gel to check the solubility of the protein expression.

76

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77

Hsh49 protein. TEV protease cleavage was conducted at room temperature since TEV
protease is more active at room temperature (Figure 33b). It was then dialyzed to reduce the
concentration of salt and imidazole which were present in the elution fraction. As I
mentioned above, the second purification through the His-trap column was done to obtain
pure Hsh49 protein. This purification helps to eliminate His-MBP tags, His-TEV protease or
any Hsh49 protein that retained tags (Figure 33c). The flow through was dialyzed and then
concentrated. Finally, the concentrated Hsh49 was run on a 15% high TEMED SDS gel
which showed that the majority of tags were removed from this protein (Figure 33d).

78





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79

4.3.2 Sequence alignment of C. merolae U2 snRNA and Hsh49 with S. cerevisiae and
Homo sapiens U2 snRNA and Hsh49
Sequence alignment of C. merolae U2 snRNA with the U2 snRNA in S. cerevisiae
showed that the similarity of the U2 snRNA in these two organisms is 8% (Figure 34).
However, the similarity of the U2 snRNA in C. merolae and Homo sapiens is 23% (Figure
35). Hsh49 sequence alignment in C. merolae indicating 12% identical and 19% similarity to
the yeast Hsh49 (Figure 36). Moreover, Hsh49 in C. merolae is 9% identical and 13% similar
to the Homo sapien Hsh49 (Figure 37). Since there are some gaps in the C. merolae Hsh49
sequence alignment of Hsh49 from S. cerevisiae and Homo sapiens, those sequences were
trimmed to improve the alignment. The results showed that the similarity of the Hsh49 in C.
merolae and S. cerevisiae is 52%. However, the similarity of the Hsh49 in C. merolae and
Homo sapiens is 50%. In addition, the Hsh49 sequence alignment in C. merolae is 34%
identical to the yeast Hsh49 and 35% identical to the Homo sapien Hsh49.
In order to know if the C. merolae Hsh49 contains RRM domains, multiple
sequence alignments of Hsh49 coding regions were performed against S. cerevisiae and
Homo sapiens (Figure 38). I also manually aligned the C. merolae Hsh49 against each RRM
domain individually (Figure 39 and Figure 40). The results showed that the first sequences of
C. merolae Hsh49 contain RRM1 and the second half matches contain RRM2. In these three
sequence alignments, the predicted secondary structure follows the canonical RRM fold:
βαββαβ (Figure 38‒Figure 40). Moreover, half of the RRMs of C. merolae Hsh49 were
aligned against both RRMs of Homo sapiens Hsh49 that showed 48% similarity and 33%
identity. Each Homo sapiens Hsh49 RRM1 and RRM2 were separatly aligned against C.
merolae Hsh49 to compare the RRM similarity. The sequence alignment results demonstrate
80

that Homo sapiens Hsh49 RRM1 is more similar to C. merolae Hsh49 RRM1 than RRM2
with 50% similarity. However, the similarity of Hsh49 RRM2 between C. merolae and
Homo sapiens is 37%.

Figure 34. Sequence alignment of C. merolae (C.m) U2 snRNA with S. cerevisiae (S.c) U2 snRNA.

81

Figure 35. Sequence alignment of C. merolae (C.m) U2 snRNA with Homo sapiens (H.S) U2 snRNA.

82

Figure 36. Sequence alignment of C. merolae (C.m) Hsh49 protein with S. cerevisiae (S.C) Hsh49 protein.

83

Figure 37. Sequence alignment of C. merolae (C.m) Hsh49 protein with Homo sapiens (H.S) Hsh49
protein.

84

Figure 38. A Multiple sequence alignment of C. merolae (C.m) Hsh49 with S. cerevisiae (S.C) and Homo
sapiens Hsh49 (H.S) protein. Yellow and green highlighted sequences relate to RRM1 and RRM2
respectively.

Figure 39. A Multiple sequence alignment of C. merolae (C.m) Hsh49 with S. cerevisiae (S.C) and Homo
sapiens Hsh49 (H.S) protein. C. merolae Hsh49 manually aligned with the first RRM1 of Homo sapiens and S.
cerevisiae (excluding non-C. merolae regions).

Figure 40. A Multiple sequence alignment of C. merolae (C.m) Hsh49 with S. cerevisiae (S.C) and Homo
sapiens Hsh49 (H.S) protein. C. merolae Hsh49 manually aligned with the RRM2 of Homo sapiens and S.
cerevisiae (excluding non-C. merolae regions).

85

4.3.3 Circular Dichroism Spectropolarimetry
CD spectroscopy is a powerful technique to determine the secondary structure of
the protein. Far-UV spectral region (190‒260) of CD data showed that the secondary
structure of Hsh49 contains 91% α-helices, 0% β-sheets and 9% random coils (Figure 41).

Figure 41. CD spectra of the Hsh49 secondary structure. Circular dichroism spectra of the recombinant
Hsh49 protein. The protein was scanned in 250 mM NaF and 20 mM HEPES-NaOH (pH 7.5). The CD
spectrum was generated from 3 accumulations using a Jasco J-815.

In order to determine if the Hsh49 was folded, CD spectra was done by increasing
the temperature to unfold this protein. The melting temperature of the Hsh49 that was
obtained from CD was 76.48 C and it showed a very good transition between the folded and
unfolded state of the protein. The unfolding of Hsh49 was reversible and it showed
conformational stability in this range of temperatures (Figure 42).

86

Figure 42. Melting temperature (Tm) of the recombinant Hsh49 protein. Tm calculation and related
computed data were obtained from the thermal melting curve of Hsh49.

4.3.4 Fluorescence Anisotropy (FA) Assay
Fluorescence anisotropy was performed on the Snu13 protein with the fluorescein
labelled U4 snRNA oligo as a positive control and confirmed the interaction between the U4
snRNA and the Snu13 protein (Figure 43). The U4 oligo is a 5ʹ kink-turn of C. merolae U4
snRNA that was previously demonstrated to interact with the Snu13 protein (Black et al.
2016). The fluorescein U4 snRNA oligo is 29 nucleotides in length and fluorescein is
attached on the 5ʹ of this oligo. The concentration of Snu13 was between 0 to 400 nM. Snu13
was serially diluted into 30 mM HEPES-NaOH (pH 7.5) and 100 mM NaCl. The binding
buffers that were used for this experiment included HEPES-NaOH (pH 7.5) and NaCl (Table
10).

87

Figure 43. The result of the fluorescence anisotropy experiment of the U4 snRNA-Snu13 interaction. The
concentration of the Snu13 was between 0 to 400 nM.

The 35 nucleotide length, fluorescein U2 snRNA oligonucleotide was synthesized
to study the interaction of Hsh49 to this part of the U2 snRNA using a fluorescence
anisotropy assay. Different types of buffer were used for these FA assays to optimize the
binding conditions. Hsh49 was serially diluted into 20 mM HEPES-NaOH (pH 7.5) and 250
mM NaCl for each FA assay.
The fluorescence anisotropy (FA) assay (Figure 44a) was done with Hsh49 protein
(0 to 200,000 nM) which was treated with benzonase and reagents that are listed in Table 10.
Benzonase was added during the purification of the Hsh49 protein to remove all forms of
single stranded or double stranded DNA and RNA. Therefore, it prevents any non-specific
interactions that may occur between the Hsh49 and the bacterial RNA. No interaction was
observed between Hsh49 and U2 snRNA (Figure 44a). There is a trend in this anisotropy
assay, but we do not expect that the interaction is occurring as the trend is different from the
U4 snRNA-Snu13 interaction (Figure 43), specifically in that the polarization decreases, the
opposite of what we expect for a binding interaction.
88

There was potential that the benzonase with the Hsh49 may degrade the U2 snRNA,
so in this case, there would not be any RNA that could interact with the protein. So another
FA assay was performed on the Hsh49 protein (0 to 140,000 nM) samples treated without
benzonase to determine if there is any interaction between Hsh49-U2 snRNA. As shown in
Figure 44b, there was no interaction between Hsh49-U2 snRNA. The reagents were the same
as used in the first FA assay (Table 10).
a)

b)

Figure 44. The results of the fluorescence anisotropy of the recombinant Hsh49 and fluorescein labelled
of the U2 snRNA. In which Hsh49 was treated with benzonase during purification and the concentration of the
Hsh49 for this experiment was between 0 to 200,000 nM (a), also Hsh49 was treated without benzonase and
Hsh49 concentration for this experiment was between 0 to 140,000 nM (b).

I performed another FA assay with Hsh49 which was treated with no benzonase and
its concentration varied from 0 to 400 nM (Figure 45a‒d). To determine how MgCl2 and
detergent will affect the Hsh49-U2 snRNA interaction, I ran FA assays with different types
of binding buffers.

89

a)

b)

c)

d)

Figure 45. The results of the fluorescence anisotropy experiments of the U2 snRNA-Hsh49 interaction.
Using four different types of binding buffers (1‒4) (Table 10‒Table 13).

The first FA assay was performed (Figure 45a) using MgCl2 and HEPES-NaOH
(pH 7.5) as a binding buffer (Table 11), because MgCl2 increases the specificity of DNA and
protein interaction, I assumed it might increase the specificity of the RNA and protein
interaction. MgCl2 also increases the stability of the RNA (Moll et al. 2002; Misra and
Draper. 1998). For the second FA assays (Figure 45b), HEPES-NaOH (pH 7.5), MgCl2 and
Triton X-100 was used (Table 12). Triton X-100 is a detergent and inhibits nonspecific
binding. HEPES-NaOH (pH 7.5), NaCl and Triton X-100 (Table 13) were employed for the
90

third FA assay (Figure 45c). Finally, the fourth FA assay (Figure 45d) was done by utilizing
NaCl and HEPES-NaOH (pH 7.5) (Table 10). Upon comparing the results of these four FA
assays, no interactions were observed that indicated the Hsh49-U2 snRNA formed a
complex.
Due to buffer composition, the conformation of the RNA molecule can be varied
which might affect protein- nucleic acid interaction. The conformation of the RNA indicates
transition between open and folded configurations. For instance, the presence of salts such as
KCl and MgCl2 increase the RNA stability and MgCl2 affects proper RNA folding (Draper.
2004)
In order to compare how the conformation of the fluorescein U2 snRNA
oligonucleotide will change and how the U2 snRNA conformation will affect RNA-protein
interaction, three different types of binding buffers (A‒C) (Table 14‒Table 16) were used in
this study. Moreover, incubating of the fluorescein labelled oligonucleotide with binding
buffers was compared at two different temperatures, 65 C and 25 C. Therefore, there were
two mixtures for each binding buffer. The first mixture was incubated at room temperature
for two minutes and the second mixture was heated at 65 C for three minutes and slowly
cooled to room temperature for two minutes. According to the bar graph (Figure 46), the
labelled U2 snRNA oligo with buffer A at room temperature had the highest fluorescence
anisotropy value. The t-test results showed that the fluorescence anisotropy value of the U2
snRNA oligo in Buffer A was higher than the U2 snRNA oligo in Buffer B which was
significantly different with a p value of 6.3E-14. The higher anisotropy value of the U2
snRNA oligo in Buffer A is because the U2 snRNA tumbles slowly and the oligo is probably
in a more extended conformation in this buffer. Moreover, at 25 C the anisotropy value of the
91

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102

misfolding Hsh49 during the purification process. If Hsh49 misfolds or loses its secondary
structure, this can affect the formation of a complex between the Hsh49-U2 snRNA.
Based on the CD spectra result, recombinant Hsh49 contains 91% α-helices and 0%
β-sheets. Also, CD spectra showed the thermal stability of this protein over a temperature
range of 28.79 to 84.44 C.
I compared the α-helix and β-sheet content of C. merolae Hsh49 with that of Homo
sapien and S. cerevisiae Hsh49. The secondary structure of human Hsh49 consists of 19% αhelices and 23% β-sheets (Figure 57‒ Figure 60) while S. cerevisiae Hsh49 contains 23% αhelices and 26% β-sheets (Figure 61 and Figure 62). The content of α-helices and β-sheets in
the secondary structure of Hsh49 in yeast and humans are close to each other.
Based on the CD spectra results, the content of α-helices and β-sheets in C. merolae
is very different from yeast and humans. The sequence alignment of the Hsh49 protein in C.
merolae demonstrated that there is less similarity to the S. cerevisiae and Homo sapiens. This
result suggests that the Hsh49 does not have the same function in C. merolae as it has in S.
cerevisiae and Homo sapiens. Therefore, I decided to perform the functional analyses.
As shown previously, the secondary structure of Hsh49 in C. merolae consists of
91% α-helices and 0% β-sheets (Figure 41). Hsh49 sequence alignment in C. merolae
indicates it is 34% identical and 52% similar to the S. cerevisiae Hsh49. On the other hand,
Hsh49 in C. merolae is 35% identical and 50% similar to the human Hsh49. Sequence
alignment results indicate that the first sequences of C. merolae Hsh49 contain RRM1 and
the second half matches contain RRM2. In addition, the results show that Homo sapiens
Hsh49 RRM1 is more similar to C. merolae Hsh49 RRM1 than RRM2.
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Therefore, based on the stability and folding of the recombinant Hsh49 protein that
was obtained from CD spectra result, FA, EMSA and immunoprecipitation analysis were
performed to characterize the Hsh49-U2 snRNA interaction.

Figure 57. The crystal structure of the first RRM of Homo sapiens Hsh49 (PDB file 5GVQ; Kuwasako et
al. 2017).

Figure 58. The secondary structure of the first RRM of Homo sapiens Hsh49 (PDB file 5GVQ; Kuwasako
et al. 2017).

104

Figure 59. The crystal structure of the second RRM of Homo sapiens Hsh49 (PDB file 1X5T; Sato et al.,
to be published).

Figure 60. The secondary structure of the second RRM of Homo sapiens Hsh49 (PDB file 1X5T; Sato et
al., to be published).

105

Figure 61. The Crystal structure of the S. cerevisiae Hsh49 protein (PDB file 5LSB; vanRoon et al. 2017).

Figure 62. The secondary structure of the S. cerevisiae Hsh49 (PDB file 5LSB; vanRoon et al. 2017).

F-EMSA indicated that the fluorescent U2 oligo was not degraded; therefore,
fluorescence anisotropy was performed and the results did not show any interaction between
the Hsh49 and the fluorescein labelled 35 nucleotide U2 snRNA oligo in C. merolae. It is
possible that the Hsh49 interacts with different sites of the U2 snRNA which does not include
106

the 35 oligonucleotides that were used in the FA assays. Therefore, I decided to study the
interaction of the full-length U2 snRNA with the recombinant Hsh49 protein by EMSA to
determine if any interaction occurs.
Based on previous studies that were done on Hsh49 originating from Homo sapiens
and S. cerevisiae cells, it was shown that Hsh49 has an RNA binding activity and associates
to the U2 snRNA. Igel et al. (1998) found that the S. cerevisiae GST-Hsh49 protein interacts
with a 309-nt in vitro U2 snRNA transcript. This finding came from EMSA. Furthermore,
Homo sapiens Hsh49 interacts with several regions of the full-length U2 snRNA which
consists of the 5ʹ end of the U2 snRNA, and stem loops I and IIb (Dybkov et al. 2006). This
is contrasted with my EMSA findings which showed that there was no interaction between
the Hsh49-U2 snRNA in C. merolae. The FA and EMSA results confirm the Northern
analysis which indicated that no interaction occurs between Hsh49-U2 snRNA. In all FEMSA and EMSA results, free U2 snRNA oligo showed two bands in all lanes and these
bands could be related to the conformational changes of the U2 snRNA.
Western blot analysis showed that the anti-Hsh49 antiserum which was generated
against the recombinant Hsh49 could recognize and immunoprecipitate the Hsh49 from C.
merolae extract. This result supports the successful recombination of the Hsh49 protein.
It was demonstrated that Hsh49 in yeast interacts with the Cus1 but the presence of
the Cus1 is not essential for the U2 snRNA-Hsh49 interaction in yeast (Igel et al. 1998). It is
possible that the pre-mRNA splicing in C. merolae occurs differently between yeast and
human cells. Perhaps, the presence of Cus1 or some other factors are necessary for this
interaction in C. merolae.

107

Chapter 5
General Discussion
C. merolae has a small genome, containing only 26 introns and a small number of
core splicing proteins. In C. merolae, 43 splicing proteins are used compared to almost 90 in
budding yeast and around 140 in Homo sapiens. Furthermore, C. merolae only contains four
snRNAs including U2, U4, U5 and U6 snRNA, since no candidate for the U1- associated
proteins or U1 snRNA were found (Stark et al. 2015). The reduced number of splicing
proteins and introns enables C. merolae to be considered a simpler organism to study
splicing. The U2 snRNP, with ten specific proteins and seven Sm proteins, is one of the most
complex particle in C. merolae.
In order to better understand the molecular function and structural role of the U2
snRNP and SF3a complex, the first step is to generate an in vitro reconstitution system. The
prerequisite step towards this approach was the expression and purification of these small
nuclear ribonucleoproteins. In Chapter 2, my major attempt was to co-express and purify
seventeen recombinant proteins. Current studies suggest that the co-expression of proteins
results in stability of each protein and prevents protein degradation. In addition, protein coexpression increases the solubility and yield of protein (Romier et al. 2006; Stefan et al.
2015). Previous studies showed that both Lsm and Sm complexes co-expressed and
copurified successfully (Dunn, 2014, unpublished). However, co-expression of the U2
snRNP and SF3a protein complex was not successful. Not all of the U2 snRNA proteins
could be expressed and SF3a appeared as an insoluble particle.

108

As a result of the unsuccessful co-expression of the U2 snRNPs and SF3a protein
complex, I decided to express and purify some U2 snRNA proteins individually. Chapter 3 is
concerned with the expression of the Rds3 and SF3a protein to study the function and
structure of these proteins. Of the SF3a and Rds3 genes which were cloned individually into
the pQlink vector, only Prp9 and Prp21 were expressed. None of the SF3a genes which were
cloned in the PMCSG23 vector were expressed. The Rds3 protein was expressed and purified
well after its gene was cloned in the PMCSG23 vector. However, I could not remove the His
and MBP tags completely from this protein, and the tags could later interfere with the protein
interactions or with obtaining the crystal structure of the desired protein.
Chapter 4 describes the individual expression and purification of one of the U2
snRNA protein subunits, Hsh49. Since this protein could express and purify successfully, I
performed some functional assays to determine the interaction between the Hsh49 protein
and U2 snRNA. Before performing this step, I pursued CD spectra for two reasons: First to
determine if the recombinant Hsh49 is folded, which would help me to do functional assays,
and second, to assess the content of α-helices and β-sheets of the secondary structure of the
Hsh49. The CD spectrum results indicated that the recombinant Hsh49 is folded and the
secondary structure of the recombinant Hsh49 contains 91% α-helices, 0% β-sheets and 9%
random coils.
Unexpectedly, no interaction was observed between the portion of the U2 snRNA
oligonucleotide (35 nucleotide) and the recombinant Hsh49 via fluorescence anisotropy
assay. Electrophoretic mobility shift assay (EMSA) did not show any interaction between the
recombinant Hsh49 and full length IVT U2 snRNA oligonucleotide. The EMSA results were

109

confirmed by the northern blot analysis of the immunoprecipitated RNA, for which there was
no band observed indicating Hsh49-U2 snRNA interaction.
The question that arises: is it possible that the Hsh49 was misidentified and it is not
really the right protein? Sequence alignments of Hsh49 in C. merolae showed amino acids
sequence identity with their S.cerevisiae and Homo sapiens homologs, with 12% and 9%
respectively. Comprehensive bioinformatics survey of the C. merolae splicing machinery
recognized four snRNAs (U2, U4, U5 and U6) and 43 core splicing proteins. In addition,
BLAST searches using Reciprocal Best Hit methodology could identify all 10 U2 snRNA
associated proteins including Hsh49 with E-values below the cutoff of 1E-13 (Stark et al.
2015). Importantly, 2′O-methyl- Mass spectrometry data confirmed the association of Hsh49
protein with U2 snRNA and some U2 snRNA proteins. This was performed using 2′Omethyl antisense oligonucleotide pull-downs against U2 snRNA of C. merolae extract. This
experiment was followed by Northern analysis and mass spectrometry which showed that
Hsh49 copurified with U2 snRNA (Reimer et al. 2017). These results provides confirmatory
evidence that the Hsh49 is the right protein.
The data gathered in this study showed that the recombinant Hsh49 did not interact
with the U2 snRNA. It may be possible that the interaction between the Hsh49 and the U2
snRNA does not occur directly. More specifically, the presence of one or a group of the U2
snRNA proteins are necessary to mediate this interaction. Previous studies showed that the
Cus1 protein directly interacts with the Hsh49 protein in yeast through the first RRM and this
interaction is essential for the tethering of the U2 snRNA to the branch point of the premRNA (Igel et al. 1998; Champion-Arnaud et al. 1994).

110

Moreover, another subunit of the SF3b containing Hsh155 and Rse1 proteins
interact with one another through the protein-protein interaction. These two proteins also
associate with the Hsh49 and Cus1 protein complex. On the other hand, biochemical studies
indicate that the U2 snRNA interacts with the Hsh155, Hsh49 and Cus1 in yeast (ChampionArnaud et al. 1994).

5.1 Future Directions
In light of the fact that the interaction of Hsh49 to U2 snRNA might need other
proteins of the SF3b complex, assembling U2 snRNP could help to detect the interaction of
the Hsh49 protein with the U2 snRNA. One approach would be checking the interaction of
the U2 snRNA-Hsh49 in the presence of the Cus1 protein. It seems that a deeper
understanding of the U2 snRNA and Hsh49 protein is needed; therefore, other subunits of the
SF3b complex should be studied.

111

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