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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 directly and specifically binds to a short fragment in unc-32 intron 7a in vitro.(A) Top, schematic illustration of the radiolabelled RNA probes 1 to 4, 2-1 to -4 and 2-1-1 used in the EMSAs. Bottom, sequences of Probe 2-1-1 and its mutants 2-1-1a to -1e. The modified stretches are underlined and the mutant sequences are indicated. Lowercase indicates the sequence derived from the T7 promoter. (B) Left, EMSA using the probes 1 to 4 without (−) or with (+) FLAG-tagged recombinant UNC-75 (FLAG-UNC-75). Right, EMSA using the probes 2-1 to 2-4 without (−) or with 2-fold dilution series of FLAG-UNC-75. (C) EMSAs using Probe 2-1-1 and its mutants shown in (A) without (−) or with 4-fold dilution series of FLAG-UNC-75.
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pgen-1003337-g005: UNC-75 directly and specifically binds to a short fragment in unc-32 intron 7a in vitro.(A) Top, schematic illustration of the radiolabelled RNA probes 1 to 4, 2-1 to -4 and 2-1-1 used in the EMSAs. Bottom, sequences of Probe 2-1-1 and its mutants 2-1-1a to -1e. The modified stretches are underlined and the mutant sequences are indicated. Lowercase indicates the sequence derived from the T7 promoter. (B) Left, EMSA using the probes 1 to 4 without (−) or with (+) FLAG-tagged recombinant UNC-75 (FLAG-UNC-75). Right, EMSA using the probes 2-1 to 2-4 without (−) or with 2-fold dilution series of FLAG-UNC-75. (C) EMSAs using Probe 2-1-1 and its mutants shown in (A) without (−) or with 4-fold dilution series of FLAG-UNC-75.

Mentions: To determine the element(s) in the exon 7 cluster region that UNC-75 directly and specifically recognizes in vitro, we performed EMSAs with the radiolabelled RNA probes schematically illustrated in Figure 5A (top panel). Recombinant full-length UNC-75 protein shifted the mobility of Probe 2 (Figure 5B, lanes 3,4) and Probe 2-1 (lanes 9–12) and not of the other probes (Figure 5B). As more than half of Probe 2-1 overlapped with Probe 1 or Probe 2-2, we prepared a shorter probe 2-1-1 (Figure 5A) containing most of the sequence unique to Probe 2-1. UNC-75 shifted the mobility of Probe 2-2-1 (Figure 5C, lanes 1–4, 25–28), demonstrating that UNC-75 directly and specifically binds to the 2-1-1 fragment in this region.


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 directly and specifically binds to a short fragment in unc-32 intron 7a in vitro.(A) Top, schematic illustration of the radiolabelled RNA probes 1 to 4, 2-1 to -4 and 2-1-1 used in the EMSAs. Bottom, sequences of Probe 2-1-1 and its mutants 2-1-1a to -1e. The modified stretches are underlined and the mutant sequences are indicated. Lowercase indicates the sequence derived from the T7 promoter. (B) Left, EMSA using the probes 1 to 4 without (−) or with (+) FLAG-tagged recombinant UNC-75 (FLAG-UNC-75). Right, EMSA using the probes 2-1 to 2-4 without (−) or with 2-fold dilution series of FLAG-UNC-75. (C) EMSAs using Probe 2-1-1 and its mutants shown in (A) without (−) or with 4-fold dilution series of FLAG-UNC-75.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585155&req=5

pgen-1003337-g005: UNC-75 directly and specifically binds to a short fragment in unc-32 intron 7a in vitro.(A) Top, schematic illustration of the radiolabelled RNA probes 1 to 4, 2-1 to -4 and 2-1-1 used in the EMSAs. Bottom, sequences of Probe 2-1-1 and its mutants 2-1-1a to -1e. The modified stretches are underlined and the mutant sequences are indicated. Lowercase indicates the sequence derived from the T7 promoter. (B) Left, EMSA using the probes 1 to 4 without (−) or with (+) FLAG-tagged recombinant UNC-75 (FLAG-UNC-75). Right, EMSA using the probes 2-1 to 2-4 without (−) or with 2-fold dilution series of FLAG-UNC-75. (C) EMSAs using Probe 2-1-1 and its mutants shown in (A) without (−) or with 4-fold dilution series of FLAG-UNC-75.
Mentions: To determine the element(s) in the exon 7 cluster region that UNC-75 directly and specifically recognizes in vitro, we performed EMSAs with the radiolabelled RNA probes schematically illustrated in Figure 5A (top panel). Recombinant full-length UNC-75 protein shifted the mobility of Probe 2 (Figure 5B, lanes 3,4) and Probe 2-1 (lanes 9–12) and not of the other probes (Figure 5B). As more than half of Probe 2-1 overlapped with Probe 1 or Probe 2-2, we prepared a shorter probe 2-1-1 (Figure 5A) containing most of the sequence unique to Probe 2-1. UNC-75 shifted the mobility of Probe 2-2-1 (Figure 5C, lanes 1–4, 25–28), demonstrating that UNC-75 directly and specifically binds to the 2-1-1 fragment in this region.

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