trans splicing of polycistronic Caenorhabditis elegans pre-mRNAs: analysis of the SL2 RNA.
Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products are trans 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 prevents trans 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.
Evans
D
D
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver 80262, USA.
Blumenthal
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T
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D015870:Gene Expression; D017238:Genes, Helminth; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D018510:RNA, Helminth / Q000737:chemistry / Q000378:metabolism*; D020038:RNA, Spliced Leader / Q000378:metabolism; D013329:Structure-Activity Relationship; D020040:Trans-Splicing; D014158:Transcription, Genetic
D012322:RNA Precursors; D018510:RNA, Helminth; D020038:RNA, Spliced Leader