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CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.

Kuroyanagi H, Watanabe Y, Hagiwara M - PLoS Genet. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan. kuroyana.end@tmd.ac.jp

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.

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UNC-75 regulates the neuron-specific selection of unc-32 exon 4b.(A) RT-PCR analysis of endogenous unc-32 exon 4 in the synchronized L1 worms of N2 (wt; lane 1), asd-1 (yb978); fox-1 (e2643) (lane 2) and unc-75 (yb1701) (lane 3). (B) Fluorescence images of an L4 worm of the ybIs1891 reporter allele in the unc-75 (yb1701) background as in Figure 1F. Scale bar, 100 µm. (C) RT-PCR analyses of the partially spliced RNAs from the endogenous unc-32 gene in N2 and unc-75 (yb1701) as in Figure 7B. (D) Relative amounts of the six partially spliced RNAs normalized to the pre-mRNA analyzed in (C). (E) Schematic illustration of the neuron-specific selection of exon 4b by UNC-75. See Discussion for detail. (F) Microphotoimages of the unc-32 (e189) (left) and the unc-75 (yb1725); unc-32 (e189) (right) worm. Scale bar, 200 µm. Note that the unc-32 worm exhibits the coiler Unc phenotype.
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pgen-1003337-g008: UNC-75 regulates the neuron-specific selection of unc-32 exon 4b.(A) RT-PCR analysis of endogenous unc-32 exon 4 in the synchronized L1 worms of N2 (wt; lane 1), asd-1 (yb978); fox-1 (e2643) (lane 2) and unc-75 (yb1701) (lane 3). (B) Fluorescence images of an L4 worm of the ybIs1891 reporter allele in the unc-75 (yb1701) background as in Figure 1F. Scale bar, 100 µm. (C) RT-PCR analyses of the partially spliced RNAs from the endogenous unc-32 gene in N2 and unc-75 (yb1701) as in Figure 7B. (D) Relative amounts of the six partially spliced RNAs normalized to the pre-mRNA analyzed in (C). (E) Schematic illustration of the neuron-specific selection of exon 4b by UNC-75. See Discussion for detail. (F) Microphotoimages of the unc-32 (e189) (left) and the unc-75 (yb1725); unc-32 (e189) (right) worm. Scale bar, 200 µm. Note that the unc-32 worm exhibits the coiler Unc phenotype.

Mentions: As unc-32 exon 4b is also selected in a neuron-specific manner (Figure 1F), we tested whether the RBFOX family and UNC-75 are also involved in the regulation of the exon 4 cluster. Consistent with the absence of a (U)GCAUG stretch in the exon 4 cluster region, the asd-1; fox-1 double mutation did not affect the splicing patterns of exon 4 of the endogenous unc-32 gene (Figure 8A, lanes 1, 2). On the other hand, the unc-75 mutation caused marked reduction of the exon 4b isoform (lane 3). Furthermore, the neuron-specific expression of E4b-mRFP from the exon 4 reporter ybIs1891 was also abolished in the unc-75 mutant (Figure 8B, compare with Figure 1F). These results indicated that UNC-75 is required for the selection of exon 4b in the nervous system. We performed an EMSA to localize the UNC-75-binding site(s) with four overlapping probes in the exon 4 cluster region, but none of the probes were shifted as effectively as Probe 2 in Figure 5B by full-length UNC-75 (data not shown). We speculate that other cooperative factors may be required for the specific recognition of the exon 4 cluster region by UNC-75.


CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.

Kuroyanagi H, Watanabe Y, Hagiwara M - PLoS Genet. (2013)

UNC-75 regulates the neuron-specific selection of unc-32 exon 4b.(A) RT-PCR analysis of endogenous unc-32 exon 4 in the synchronized L1 worms of N2 (wt; lane 1), asd-1 (yb978); fox-1 (e2643) (lane 2) and unc-75 (yb1701) (lane 3). (B) Fluorescence images of an L4 worm of the ybIs1891 reporter allele in the unc-75 (yb1701) background as in Figure 1F. Scale bar, 100 µm. (C) RT-PCR analyses of the partially spliced RNAs from the endogenous unc-32 gene in N2 and unc-75 (yb1701) as in Figure 7B. (D) Relative amounts of the six partially spliced RNAs normalized to the pre-mRNA analyzed in (C). (E) Schematic illustration of the neuron-specific selection of exon 4b by UNC-75. See Discussion for detail. (F) Microphotoimages of the unc-32 (e189) (left) and the unc-75 (yb1725); unc-32 (e189) (right) worm. Scale bar, 200 µm. Note that the unc-32 worm exhibits the coiler Unc phenotype.
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pgen-1003337-g008: UNC-75 regulates the neuron-specific selection of unc-32 exon 4b.(A) RT-PCR analysis of endogenous unc-32 exon 4 in the synchronized L1 worms of N2 (wt; lane 1), asd-1 (yb978); fox-1 (e2643) (lane 2) and unc-75 (yb1701) (lane 3). (B) Fluorescence images of an L4 worm of the ybIs1891 reporter allele in the unc-75 (yb1701) background as in Figure 1F. Scale bar, 100 µm. (C) RT-PCR analyses of the partially spliced RNAs from the endogenous unc-32 gene in N2 and unc-75 (yb1701) as in Figure 7B. (D) Relative amounts of the six partially spliced RNAs normalized to the pre-mRNA analyzed in (C). (E) Schematic illustration of the neuron-specific selection of exon 4b by UNC-75. See Discussion for detail. (F) Microphotoimages of the unc-32 (e189) (left) and the unc-75 (yb1725); unc-32 (e189) (right) worm. Scale bar, 200 µm. Note that the unc-32 worm exhibits the coiler Unc phenotype.
Mentions: As unc-32 exon 4b is also selected in a neuron-specific manner (Figure 1F), we tested whether the RBFOX family and UNC-75 are also involved in the regulation of the exon 4 cluster. Consistent with the absence of a (U)GCAUG stretch in the exon 4 cluster region, the asd-1; fox-1 double mutation did not affect the splicing patterns of exon 4 of the endogenous unc-32 gene (Figure 8A, lanes 1, 2). On the other hand, the unc-75 mutation caused marked reduction of the exon 4b isoform (lane 3). Furthermore, the neuron-specific expression of E4b-mRFP from the exon 4 reporter ybIs1891 was also abolished in the unc-75 mutant (Figure 8B, compare with Figure 1F). These results indicated that UNC-75 is required for the selection of exon 4b in the nervous system. We performed an EMSA to localize the UNC-75-binding site(s) with four overlapping probes in the exon 4 cluster region, but none of the probes were shifted as effectively as Probe 2 in Figure 5B by full-length UNC-75 (data not shown). We speculate that other cooperative factors may be required for the specific recognition of the exon 4 cluster region by UNC-75.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan. kuroyana.end@tmd.ac.jp

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.

Show MeSH
Related in: MedlinePlus