THE HUMAN U6 SNRNA INTRAMOLECULAR HELIX - STRUCTURAL CONSTRAINTS AND LACK OF SEQUENCE SPECIFICITY

Splicing of mRNA precursors occurs in a massive structure known as the spliceosome and requires the function of several small nuclear RNAs ( snRNAs). A number of studies have suggested potentially important role s for two snRNAs, U2 and U6, in splicing catalysis. These two RNAs int eract extensively with each other, as well as with the pre-mRNA, and p ossible similarities with catalytic RNAs have been noted. An important feature of the U2-U6 complex is an intramolecular helix in U6, which forms in conjunction with activation of the spliceosome. Here we descr ibe a detailed genetic analysis of residues that make up this helix in human U6 snRNA, using an in vivo assay in which splicing of a test pr e-mRNA is dependent on exogenous U6 snRNA. Our results show that many, but not all, positions tested are sensitive to mutation. Unexpectedly , base pairing is fully compatible with function at all positions, and at many is both necessary and sufficient. For example, conversion of two noncanonical A-C pairs to G-C pairs did not affect splicing, nor d id conversion of an A-G to C-G. Extension of the helix by a base pair was also tolerated, provided that base pairing was maintained. Most no table was the behavior of a bulged U (U74), which has been suggested p reviously to be of particular importance. Although U74 was sensitive t o substitution or deletion, incorporation into the helix by insertion of an A across from it was without effect, even in the context of a se cond helix-stabilizing mutation. We discuss these results in terms of possible mechanisms by which U6 snRNA might function in splicing catal ysis.