THE STEREOCHEMICAL COURSE OF THE 1ST STEP OF PREMESSENGER RNA SPLICING

We have determined the effects on splicing of sulfur substitution of t he non-bridging oxygens in the phosphodiester bond at the 5' splice si te of a pre-mRNA intron. Pre-mRNAs containing stereochemically pure Rp and Sp phosphorothioate isomers were produced by ligation of a chemic ally synthesized modified RNA oligonucleotide to enzymatically synthes ized RAs. When these modified pre-mRNA substrates were tested for in v itro splicing activity in a HeLa cell nuclear extract system, the RNA with the Rp diastereomeric phosphorothioate was not spliced while the Sp diastereomeric RNA spliced readily. The sulfur-containing branched trinucleotide was purified from the splicing reaction of the Sp RNA an d analyzed by cleavage with a stereospecific nuclease. The results sho wed that the Sp phosphorothioate was inverted during the splicing reac tion to the Rp configuration; a finding previously obtained for a Grou p I self-splicing RNA. This inversion of configuration is consistent w ith a transesterification mechanism for pre-mRNA splicing. The lack of splicing of the Rp modified RNA also suggests that the pro-Rp oxygen at the 5' splice site is involved in a critical chemical contact in th e splicing mechanism. Additionally, we have found that the HeLa cell R NA debranching enzyme is inactive on branches containing an Rp phospho rothioate.