trans splicing of polycistronic Caenorhabditis elegans Pre-mRNAs: Analysisof the SL2 RNA

Genes in Caenorhabditis elegans operons are transcribed as polycistronic pr e-mRNAs in which downstream gene products are trans spliced to a specialize d spliced leader, SL2. SL2 is donated by a 110-nucleotide RNA, SL2 RNA, pre sent in the cell as an Sm-bound snRNP. SL2 RNA can be conceptually folded i nto a phylogenetically conserved three-stem-loop secondary structure. Here we report an in vivo mutational analysis of the SL2 RNA. Some sequences can be changed without consequence, while other changes result in a substantia l loss of trans splicing. Interestingly, the spliced leader itself can be d ramatically altered, such that the first stem-loop cannot form, with only a relatively small loss in trans-splicing efficiency. However, the primary s equence of stem II is crucial for SL2 trans splicing. Similarly, the conser ved primary sequence of the third stem-loop plays a key role in trans splic ing. While mutations in stem-loop III allow snRNP formation, a single nucle otide substitution in the loop prevents trans splicing. In contrast, the an alogous region of SL1 RNA is not highly conserved, and its mutation does no t abrogate function. Thus, stem-loop III appears to confer a specific funct ion to SL2 RNA. Finally, an upstream sequence, previously predicted to be a proximal sequence element, is shown to be required for SL2 RNA expression.