Structure Probing of U4 snRNA: Investigations into the Mechanism of U4/U6 di-snRNP Formation.

Precursor messenger RNA splicing is catalyzed by a dynamic complex termed the spliceosome. An essential step in spliceosome assembly occurs when U4 snRNA base pairs with U6 snRNA to form the U4/U6 di-snRNP. Although failure to form the di-snRNP is lethal, the mechanism by which di-snRNP formation occurs is still unknown, in part due to lack of information about the structure of free U4 snRNP prior to binding U6. To investigate the contribution of U4 in di-snRNP formation, I carried out a series of structure probing experiments to determine the secondary structure of a Brr2 released U4 snRNP. The structural model establishes the presence of four stem loops in yeast U4 snRNA, including a novel short stem loop at the extreme 5' end of the molecule. To determine which nucleotides of U4 are required for base pair formation, I carried out a modification/interference experiment. Modification of the 5' stem loop uridines (U5, U6, and U8) interfered with di-snRNP formation, while modification of uridines within the central and 3' regions of U4 snRNA did not inhibit di-snRNP formation. Based on these results, I propose that intermolecular base-pairing between U4 loop nucleotides (U6 - A11) of the novel stem loop and U6 snRNA nucleotide (U70 - A75) may initiate di-snRNA formation. The U6 specific protein Prp24 would catalyze the subsequent annealing and stabilization of the U4/U6 intermolecular helices I and II. --P. ii.