trans Splicing of PolycistronicCaenorhabditis elegans Pre-mRNAs: Analysis of the SL2 RNA

Abstract

Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products aretrans spliced to a specialized spliced leader, SL2. SL2 is donated by a 110-nucleotide RNA, SL2 RNA, present in the cell as an Sm-bound snRNP. SL2 RNA can be conceptually folded into 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 substantial loss of trans splicing. Interestingly, the spliced leader itself can be dramatically altered, such that the first stem-loop cannot form, with only a relatively small loss in trans-splicing efficiency. However, the primary sequence of stem II is crucial for SL2 trans splicing. Similarly, the conserved primary sequence of the third stem-loop plays a key role in trans splicing. While mutations in stem-loop III allow snRNP formation, a single nucleotide substitution in the loop preventstrans splicing. In contrast, the analogous region of SL1 RNA is not highly conserved, and its mutation does not abrogate function. Thus, stem-loop III appears to confer a specific function to SL2 RNA. Finally, an upstream sequence, previously predicted to be a proximal sequence element, is shown to be required for SL2 RNA expression.

ACKNOWLEDGMENTS

pSL2+, pASL2, and pSL15S were the kind gifts of Xin-Yun Huang and David Hirsh, Atul M. Deshpande, and Kimberly Ferguson and Joel Rothman, respectively. The human autoimmune anti-Sm serum and Y12 monoclonal antibodies were the kind gifts of Scott Kuersten and Iain Mattaj. We are grateful to Atul M. Deshpande, Diego A. R. Zorio, Devin Leake, Izzy Perez, David Caprio, and Margaret MacMorris for helpful comments on the manuscript. We thank Devin Leake for additional assistance with the figures.

This work was supported by grant GM42432 from the National Institute of General Medical Sciences.