SPLICED LEADER RNA OF TRYPANOSOMES - IN-VIVO MUTATIONAL ANALYSIS REVEALS EXTENSIVE AND DISTINCT REQUIREMENTS FOR TRANS SPLICING AND CAP4 FORMATION

In trypanosomes mRNAs are generated through trans splicing, The splice d leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing p rocess. We have established iii vivo assays to study iu detail ti ans splicing, cap4 modification, and RNP assembly of the SL RNA in the try panosomatid species Leptomonas seymouri. First, me found that extensiv e sequences within the mini-exon are required for SL RNA function in v ivo, although a conserved length of 39 nt is not essential, In contras t, the intron sequence appears to be surprisingly tolerant to mutation ; only the stemloop LI structure is indispensable, The asymmetry of th e sequence requirements in the stem I region suggests that this domain may exist in different functional conformations, Second, distinct min i-exon sequences outside the modification site are important for effic ient cap4 formation. Third, all SL RNA mutations tested allowed core R NP assembly, suggesting flexible requirements for core protein binding , In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the stro ng phylogenetic conservation of the mini-exon sequence and of the over all SL RNA secondary structure; they also suggest that there may be ce rtain differences between trans splicing in nematodes and trypanosomes . This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.