C. elegans utilizes a specialized spliced leader RNA, SL2 RNA, to process the products of downstream genes in operons. By comparative phylogenetic analysis of the 22 known SL2 RNA genes, we have identified conserved sequences: the SL 5' end, the trans -splice site, the Sm-binding site, and the top of the third stem-loop. To study the importance of these regions on expression and trans -splicing specificity, we expressed SL2 RNA variants in transgenic worms and measured trans -splicing to
gpd-3 mRNA. Unlike SL1 RNA, SL2 RNA has no promoter element located within the SL sequence. Rather, expression is dependent on a proximal sequence element, similar to those of the snRNA genes. It had been hypothesized that base-pairing within the SL2 RNA, between the SL and the trans -splice site and bases just downstream, mimics that found between the 5' splice site and U1 snRNA, which plays no role in trans -splicing. To test this idea in SL2 trans -splicing, we mutated the 20 nt at the 5' end of the 22 nt SL. Since the predicted secondary structure is conserved among the natural variants of SL2 RNA, we expected that this large mutation would prevent trans -splicing due to an RNA structural change. Therefore, we also made mutations with compensatory changes that would allow formation of this first stem. Interestingly, the first mutation, which mutates nearly the entire SL, was nevertheless trans -spliced. Even more striking was the trans -splicing of mutant SL2 RNAs with changes in most of the first stem and loop, demonstrating that large mutations to the 'intron' sequences of SL2 RNA are also tolerated. In contrast, mutations to the conserved top of the third stem-loop do prevent trans -splicing, even though the mutant SL2 RNA is expressed at a high level, demonstrating the importance of this conserved sequence to SL2 RNA function. The top of the third stem-loop of SL1 RNA is not conserved, suggesting that SL2 RNA may have evolved a key function at this location.