Towards identifying New Human Ribonucleases that Cleave microRNA Using a High-Throughput Method.

Endoribonucleases were once thought of as only being key enzymes responsible for the degradation of prokaryotic mRNAs. They are now believed to play critical role in initiating eukaryotic/mammalian RNA decay and hence RNA abundance. To date, only few mammalian endoribonucleases that cleaved mRNA have been identified and studied. It is unknown if mammalian endoribonucleases can control microRNA (miRNA) abundance. The major goal of this MSc thesis was to develop a high-throughput method to identify new human endoribonucleases that cleave miR155. The first objective of this thesis was to develop a high-throughput method to express and purify human recombinant proteins from the hEx1 human fetal brain library. This was followed by development of a high-throughput fluorescence-based assay to screen purified recombinant proteins for activity against fluorogenic miR155 substrate. Through a series of optimization experiments, we have successfully established a high-throughput procedure and the criteria in selecting a preliminary list of positive candidates. --P.i.