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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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The UGCAUG stretch and the RBFOX family proteins are involved in the neuron-specific selection of exon 7a from the unc-32 exon 7 reporter.(A) Fluorescence images of the exon 7 reporter worms ybIs1622 [rgef-1::unc-32E7a-EGFP rgef-1::unc-32E7b-mRFP] with dual-bandpass (Dual), green (GFP2) and red (DSR) filters and a bright field image (BF). Note that individual neurons differentially express E7a-EGFP and E7b-mRFP in a cell-type-specific pattern. (B) Nucleotide sequence alignment of intron 7b from C. briggsae, C. elegans and C. remanei. Residues shaded in black and gray are conserved among three and two species, respectively. Conserved stretches are boxed in red and the sequences of the M1 and M2 mutant pairs of the reporter minigenes are indicated. (C) Schematic illustrations of the mutated pairs of the exon 7 reporter minigenes. Red arrows indicate the positions of the modification. (D) A fluorescence image of the M1 mutant reporter worms with a dual-bandpass filter. (E) Fluorescence images of the ybIs1622 worms in the asd-1 (yb978) (top), fox-1 (e2643) (middle) and asd-1; fox-1 (bottom) backgrounds with a dual-bandpass filter. Scale bars, 200 µm in (A), (D) and (E). (F) Top, radiolabelled wild-type (WT) and mutant (Mut) intron 7b probes. The substituted bases are underlined. Lowercase indicates the sequence derived from the T7 promoter. Bottom left, Neutral PAGE and CBB staining of the recombinant FLAG-tagged ASD-1 and FOX-1 proteins. Bottom right, EMSA using the WT and Mut probes without (−) or with 4-fold dilution series of FLAG-ASD-1 and -FOX-1.
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pgen-1003337-g002: The UGCAUG stretch and the RBFOX family proteins are involved in the neuron-specific selection of exon 7a from the unc-32 exon 7 reporter.(A) Fluorescence images of the exon 7 reporter worms ybIs1622 [rgef-1::unc-32E7a-EGFP rgef-1::unc-32E7b-mRFP] with dual-bandpass (Dual), green (GFP2) and red (DSR) filters and a bright field image (BF). Note that individual neurons differentially express E7a-EGFP and E7b-mRFP in a cell-type-specific pattern. (B) Nucleotide sequence alignment of intron 7b from C. briggsae, C. elegans and C. remanei. Residues shaded in black and gray are conserved among three and two species, respectively. Conserved stretches are boxed in red and the sequences of the M1 and M2 mutant pairs of the reporter minigenes are indicated. (C) Schematic illustrations of the mutated pairs of the exon 7 reporter minigenes. Red arrows indicate the positions of the modification. (D) A fluorescence image of the M1 mutant reporter worms with a dual-bandpass filter. (E) Fluorescence images of the ybIs1622 worms in the asd-1 (yb978) (top), fox-1 (e2643) (middle) and asd-1; fox-1 (bottom) backgrounds with a dual-bandpass filter. Scale bars, 200 µm in (A), (D) and (E). (F) Top, radiolabelled wild-type (WT) and mutant (Mut) intron 7b probes. The substituted bases are underlined. Lowercase indicates the sequence derived from the T7 promoter. Bottom left, Neutral PAGE and CBB staining of the recombinant FLAG-tagged ASD-1 and FOX-1 proteins. Bottom right, EMSA using the WT and Mut probes without (−) or with 4-fold dilution series of FLAG-ASD-1 and -FOX-1.

Mentions: To focus on the neuron-specific selection of exon 7a, we utilized the rgef-1 (also known as F25B3.3) promoter to drive pan-neuronal expression of the exon 7 reporter. As expected, transgenic worms with an integrated reporter allele ybIs1622 [rgef-1::unc-32E7a-EGFP rgef-1::unc-32E7b-mRFP] predominantly expressed E7a-EGFP in the nervous system and appeared Green with a dual-bandpass filter (Figure 2A). We therefore used the rgef-1 promoter for further analyses described below.


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)

The UGCAUG stretch and the RBFOX family proteins are involved in the neuron-specific selection of exon 7a from the unc-32 exon 7 reporter.(A) Fluorescence images of the exon 7 reporter worms ybIs1622 [rgef-1::unc-32E7a-EGFP rgef-1::unc-32E7b-mRFP] with dual-bandpass (Dual), green (GFP2) and red (DSR) filters and a bright field image (BF). Note that individual neurons differentially express E7a-EGFP and E7b-mRFP in a cell-type-specific pattern. (B) Nucleotide sequence alignment of intron 7b from C. briggsae, C. elegans and C. remanei. Residues shaded in black and gray are conserved among three and two species, respectively. Conserved stretches are boxed in red and the sequences of the M1 and M2 mutant pairs of the reporter minigenes are indicated. (C) Schematic illustrations of the mutated pairs of the exon 7 reporter minigenes. Red arrows indicate the positions of the modification. (D) A fluorescence image of the M1 mutant reporter worms with a dual-bandpass filter. (E) Fluorescence images of the ybIs1622 worms in the asd-1 (yb978) (top), fox-1 (e2643) (middle) and asd-1; fox-1 (bottom) backgrounds with a dual-bandpass filter. Scale bars, 200 µm in (A), (D) and (E). (F) Top, radiolabelled wild-type (WT) and mutant (Mut) intron 7b probes. The substituted bases are underlined. Lowercase indicates the sequence derived from the T7 promoter. Bottom left, Neutral PAGE and CBB staining of the recombinant FLAG-tagged ASD-1 and FOX-1 proteins. Bottom right, EMSA using the WT and Mut probes without (−) or with 4-fold dilution series of FLAG-ASD-1 and -FOX-1.
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Related In: Results  -  Collection

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pgen-1003337-g002: The UGCAUG stretch and the RBFOX family proteins are involved in the neuron-specific selection of exon 7a from the unc-32 exon 7 reporter.(A) Fluorescence images of the exon 7 reporter worms ybIs1622 [rgef-1::unc-32E7a-EGFP rgef-1::unc-32E7b-mRFP] with dual-bandpass (Dual), green (GFP2) and red (DSR) filters and a bright field image (BF). Note that individual neurons differentially express E7a-EGFP and E7b-mRFP in a cell-type-specific pattern. (B) Nucleotide sequence alignment of intron 7b from C. briggsae, C. elegans and C. remanei. Residues shaded in black and gray are conserved among three and two species, respectively. Conserved stretches are boxed in red and the sequences of the M1 and M2 mutant pairs of the reporter minigenes are indicated. (C) Schematic illustrations of the mutated pairs of the exon 7 reporter minigenes. Red arrows indicate the positions of the modification. (D) A fluorescence image of the M1 mutant reporter worms with a dual-bandpass filter. (E) Fluorescence images of the ybIs1622 worms in the asd-1 (yb978) (top), fox-1 (e2643) (middle) and asd-1; fox-1 (bottom) backgrounds with a dual-bandpass filter. Scale bars, 200 µm in (A), (D) and (E). (F) Top, radiolabelled wild-type (WT) and mutant (Mut) intron 7b probes. The substituted bases are underlined. Lowercase indicates the sequence derived from the T7 promoter. Bottom left, Neutral PAGE and CBB staining of the recombinant FLAG-tagged ASD-1 and FOX-1 proteins. Bottom right, EMSA using the WT and Mut probes without (−) or with 4-fold dilution series of FLAG-ASD-1 and -FOX-1.
Mentions: To focus on the neuron-specific selection of exon 7a, we utilized the rgef-1 (also known as F25B3.3) promoter to drive pan-neuronal expression of the exon 7 reporter. As expected, transgenic worms with an integrated reporter allele ybIs1622 [rgef-1::unc-32E7a-EGFP rgef-1::unc-32E7b-mRFP] predominantly expressed E7a-EGFP in the nervous system and appeared Green with a dual-bandpass filter (Figure 2A). We therefore used the rgef-1 promoter for further analyses described below.

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