Thiophosphates in yeast U6 snRNA specifically affect pre-mRNA splicing in vitro

A thorough mutational analysis of U6 RNA in combination with a functional reconstitution assay, revealed that three domains are important for U6 function in pre-mRNA splicing. In order to further analyze why these regions are so critical for splicing, we make use of phosphorothioate substituted U6 RNAs. Wild-type U6 RNA was transcribed in vitro with T7 RNA polymerase in the presence of either phosphorothiate (alpha-S) ATP, GTP, UTP or CTP. The functionality of the transcripts was monitored by in vitro reconstitution. While substitution with alpha-S ATP, GTP or UTP blocked splicing, substitution with alpha-S CTP had little or no effect on splicing. We made use of this alpha-S CTP effect in an attempt to elucidate which phosphates in the U6 RNA molecule play a role in the first or in the second step of splicing. U6 mutants in which a change of an A, G or U to C does not have any significant effect on splicing were transcribed in the presence of alpha-S CTP. Observed effects on splicing thus have to be attributed to the presence of the thio-substituted phosphate group rather than the nucleotide change. The results of in vitro reconstitution give a clear answer for at least three phosphates; two of them play a role in the first step, while one of them is involved in the second step of splicing.