Abstract
RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre–mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding the molecular mechanisms of RNA splicing and the biology of numerous RNA splicing-related diseases. We previously isolated a Caenorhabditis elegans mutant defective in an essential gene from a genetic screen for suppressors of the rubberband Unc phenotype of unc-93(e1500) animals. This mutant contains missense mutations in two adjacent codons of the C. elegans microfibrillar-associated protein 1 gene mfap-1. mfap-1(n4564 n5214) suppresses the Unc phenotypes of different rubberband Unc mutants in a pattern similar to that of mutations in the splicing factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). We used the endogenous gene tos-1 as a reporter for splicing and detected increased intron 1 retention and exon 3 skipping of tos-1 transcripts in mfap-1(n4564 n5214) animals. Using a yeast two-hybrid screen, we isolated splicing factors as potential MFAP-1 interactors. Our studies indicate that C. elegans mfap-1 encodes a splicing factor that can affect alternative splicing.
Highlights
RNA splicing removes non-coding introns and joins adjacent coding exons from pre-mRNAs to generate functional coding mRNAs
From a genetic screen for suppressors of the rubberband Unc phenotype caused by the Caenorhabditis elegans unc-93(e1500) mutation, we isolated a mutation that affects a highly conserved essential gene, mfap-1
MFAP-1 can affect the alternative splicing of tos-1, an endogenous reporter gene for splicing, and is required for the altered splicing at a cryptic 39 splice site of tos-1. mfap-1 enhances the effects of the gene uaf-1 in suppressing the rubberband Unc phenotype of unc-93(e1500) animals
Summary
RNA splicing removes non-coding introns and joins adjacent coding exons from pre-mRNAs to generate functional coding mRNAs. Alternative splicing can generate numerous splice isoforms from the same pre-mRNA [1]. Numerous proteins and non-coding RNAs regulate RNA splicing [3]. The U1 snRNP complex and the SF1/U2AF65/U2AF35 protein complex recognize the 59 and 39 splice sites of an intron, respectively [4,5,6,7,8,9,10,11], and the U2 and U4/U5/U6 snRNP complexes assemble in a step-wise manner and undergo compositional and conformational rearrangements to drive the two steps of the trans-esterification reaction in RNA splicing [7,12]. Mutations in trans-splicing factors or cis-regulatory splicing elements cause numerous diseases [13,14]
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