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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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RRM3 of UNC-75 mediates specific binding to the UUGUUGUGUUGU stretch in unc-32 intron 7a.(A) Left, neutral PAGE and CBB staining of the recombinant FLAG-tagged wild-type (WT) UNC-75 protein and the mutant proteins UNC-75(G53S), UNC-75(G165E) and UNC-75(L431F). Right, EMSA using Probe 2-1-1 without or with 2-fold dilution series of the wild-type and mutant UNC-75 proteins. (B) Left, CBB staining of the recombinant FLAG-tagged full-length UNC-75 protein (WT; lane 1) and UNC-75(1–114) (RRM1; lane 2), UNC-75(121–213) (RRM2; lane 3) and UNC-75(417–514) (RRM3; lane 4) proteins. Right, EMSA using Probe 2-1-1 and 2-fold dilution series of the three UNC-75 RRM proteins and the full-length protein. (C) EMSA using Probe 2-2-1 (lanes 1–3) and five mutant probes (lanes 4–18) without (−) or with 2-fold dilution series of UNC-75 (417–514) protein. (D) Fluorescence images of the wild-type (ybIs1622; top) and M6 mutant (bottom) of the exon 7 reporter worms with a dual-bandpass filter. Scale bar, 200 µm.
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pgen-1003337-g006: RRM3 of UNC-75 mediates specific binding to the UUGUUGUGUUGU stretch in unc-32 intron 7a.(A) Left, neutral PAGE and CBB staining of the recombinant FLAG-tagged wild-type (WT) UNC-75 protein and the mutant proteins UNC-75(G53S), UNC-75(G165E) and UNC-75(L431F). Right, EMSA using Probe 2-1-1 without or with 2-fold dilution series of the wild-type and mutant UNC-75 proteins. (B) Left, CBB staining of the recombinant FLAG-tagged full-length UNC-75 protein (WT; lane 1) and UNC-75(1–114) (RRM1; lane 2), UNC-75(121–213) (RRM2; lane 3) and UNC-75(417–514) (RRM3; lane 4) proteins. Right, EMSA using Probe 2-1-1 and 2-fold dilution series of the three UNC-75 RRM proteins and the full-length protein. (C) EMSA using Probe 2-2-1 (lanes 1–3) and five mutant probes (lanes 4–18) without (−) or with 2-fold dilution series of UNC-75 (417–514) protein. (D) Fluorescence images of the wild-type (ybIs1622; top) and M6 mutant (bottom) of the exon 7 reporter worms with a dual-bandpass filter. Scale bar, 200 µm.

Mentions: To test whether all the three RRMs of UNC-75 are involved in the recognition of the 2-2-1 fragment, we performed EMSAs using three mutant recombinant proteins UNC-75 (G53S), UNC-75 (G165E) and UNC-75 (L431F) (Figure 6A, left), each of which had a single missense mutation in one of the three RRMs as found in the mutant alleles. UNC-75 (G53S) and UNC-75 (G165E) less efficiently shifted the mobility of Probe 2-1 and Probe 2-1-1 than wild-type UNC-75 (Figure S4, lanes 1–10; Figure 6A, right, lanes 1–13). UNC-75 (L431F) failed to shift the mobility of these probes (Figure S4, lanes 11–13; Figure 6A, right, lanes 14–17). These results indicated that the missense mutations affected the RNA-binding properties of UNC-75 in vitro and that all the three RRMs of UNC-75 are required for the specific recognition of unc-32 intron 7a.


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)

RRM3 of UNC-75 mediates specific binding to the UUGUUGUGUUGU stretch in unc-32 intron 7a.(A) Left, neutral PAGE and CBB staining of the recombinant FLAG-tagged wild-type (WT) UNC-75 protein and the mutant proteins UNC-75(G53S), UNC-75(G165E) and UNC-75(L431F). Right, EMSA using Probe 2-1-1 without or with 2-fold dilution series of the wild-type and mutant UNC-75 proteins. (B) Left, CBB staining of the recombinant FLAG-tagged full-length UNC-75 protein (WT; lane 1) and UNC-75(1–114) (RRM1; lane 2), UNC-75(121–213) (RRM2; lane 3) and UNC-75(417–514) (RRM3; lane 4) proteins. Right, EMSA using Probe 2-1-1 and 2-fold dilution series of the three UNC-75 RRM proteins and the full-length protein. (C) EMSA using Probe 2-2-1 (lanes 1–3) and five mutant probes (lanes 4–18) without (−) or with 2-fold dilution series of UNC-75 (417–514) protein. (D) Fluorescence images of the wild-type (ybIs1622; top) and M6 mutant (bottom) of the exon 7 reporter worms with a dual-bandpass filter. Scale bar, 200 µm.
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Related In: Results  -  Collection

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

pgen-1003337-g006: RRM3 of UNC-75 mediates specific binding to the UUGUUGUGUUGU stretch in unc-32 intron 7a.(A) Left, neutral PAGE and CBB staining of the recombinant FLAG-tagged wild-type (WT) UNC-75 protein and the mutant proteins UNC-75(G53S), UNC-75(G165E) and UNC-75(L431F). Right, EMSA using Probe 2-1-1 without or with 2-fold dilution series of the wild-type and mutant UNC-75 proteins. (B) Left, CBB staining of the recombinant FLAG-tagged full-length UNC-75 protein (WT; lane 1) and UNC-75(1–114) (RRM1; lane 2), UNC-75(121–213) (RRM2; lane 3) and UNC-75(417–514) (RRM3; lane 4) proteins. Right, EMSA using Probe 2-1-1 and 2-fold dilution series of the three UNC-75 RRM proteins and the full-length protein. (C) EMSA using Probe 2-2-1 (lanes 1–3) and five mutant probes (lanes 4–18) without (−) or with 2-fold dilution series of UNC-75 (417–514) protein. (D) Fluorescence images of the wild-type (ybIs1622; top) and M6 mutant (bottom) of the exon 7 reporter worms with a dual-bandpass filter. Scale bar, 200 µm.
Mentions: To test whether all the three RRMs of UNC-75 are involved in the recognition of the 2-2-1 fragment, we performed EMSAs using three mutant recombinant proteins UNC-75 (G53S), UNC-75 (G165E) and UNC-75 (L431F) (Figure 6A, left), each of which had a single missense mutation in one of the three RRMs as found in the mutant alleles. UNC-75 (G53S) and UNC-75 (G165E) less efficiently shifted the mobility of Probe 2-1 and Probe 2-1-1 than wild-type UNC-75 (Figure S4, lanes 1–10; Figure 6A, right, lanes 1–13). UNC-75 (L431F) failed to shift the mobility of these probes (Figure S4, lanes 11–13; Figure 6A, right, lanes 14–17). These results indicated that the missense mutations affected the RNA-binding properties of UNC-75 in vitro and that all the three RRMs of UNC-75 are required for the specific recognition of unc-32 intron 7a.

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