Abstract
An enormous number of alternative pre–mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a–4c and 7a–7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA–binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of the unc-32 gene.
Highlights
Alternative splicing of pre-mRNAs is a major source of proteomic complexity in metazoans
We previously demonstrated that evolutionarily conserved RBFOX family RNA–binding proteins ASD-1 and FOX-1 and a muscle-specific RNA–binding protein SUP-12 cooperatively direct muscle-specific selection of exon 5B of the C. elegans egl-15 gene
We demonstrate that two sets of mutually exclusive exons, 4a– 4c and 7a–7b, of the unc-32 gene are regulated in tissuespecific manners and that ASD-1 and FOX-1, expressed in a variety of tissues, can regulate the neuron-specific selection of unc-32 exon 7a in combination with the neuronspecific CUG-BP and ETR-3-like factor (CELF) family RNA–binding protein UNC-75
Summary
Alternative splicing of pre-mRNAs is a major source of proteomic complexity in metazoans. More than 90% of human multi-exon genes undergo alternative pre-mRNA processing and many alternative splicing events are controlled in tissue- and cell-type dependent manners [1]. Mis-splicing of pre-mRNAs underlie many inherited diseases [2]. A variety of auxiliary trans-acting factors and cis-acting elements regulating alternative splicing have been identified [3,4,5,6]. Combinations of hundreds of RNA features were used to assemble ‘splicing codes’ to predict splicing patterns in four major tissues to a significant extent [8]. Much of our knowledge of splicing regulation relies on experiments utilizing cultured cells, and complex mechanisms of the tissue-specific regulation of pre-mRNA splicing by coordination of multiple trans-factors and cis-elements in living organisms remain less understood
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call