U6 SNRNA FUNCTION IN NUCLEAR PRE-MESSENGER-RNA SPLICING - A PHOSPHOROTHIOATE INTERFERENCE ANALYSIS OF THE U6 PHOSPHATE BACKBONE

U6 snRNA is essential for and may participate in the catalysis of pre- mRNA splicing. Extensive mutational analyses in several systems have i dentified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate back bone. We previously described a mutation in a nematode U6 snRNA that c auses it to be used as a splicing substrate within the spliceosome. Th is unusual reaction has made it possible to apply modification interfe rence analysis to U6 function. Here, we have used phosphorothioate sub stitution to identify pro-R oxygens throughout the U6 backbone that ar e necessary for the first and/or second catalytic steps of splicing. F our pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely require d for the second step. The two pro-R oxygens critical for the first st ep of splicing are in the helix 1b U2/U6 interaction region and the in tramolecular stem-loop of U6, respectively. A comparison of the positi ons of these two pro-R oxygens with those found to be critical for aut ocatalytic excision of a group II intron suggests a possible functiona l similarity between U6 snRNA and domain V of group II introns.