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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 is required for the selection exon 7a from the unc-32 exon 7 reporter in the nervous system.(A) Fluorescence images of the ybIs1622 worms with the unc-75 mutant alleles yb1700, yb1725, yb1714 and yb1701 with a dual-bandpass filter. Scale bar, 200 µm. (B) Schematic structure of the unc-75 gene and the positions and consequence of the mutations. The ORF is colored in yellow and the regions corresponding to the three RRMs are in orange. The allele names are in the colors representing the color phenotypes. SA, splice acceptor; SD, splice donor. (C) Amino acid sequence alignments of the three RRMs from the CELF3–6 subfamily members C. elegans UNC-75, human CELF3, CELF4, CELF5 and CELF6 and the CELF1–2 subfamily members C. elegans ETR-1, human CELF1 and CELF2. Conserved residues are shaded in black. Residues with similar properties to the consensus are shaded in gray. The secondary structure elements determined for human CELF1 [51], [52] are depicted with the blue rectangles (β-sheets) and the red ellipses (α-helices) below the alignments. The highly conserved RNP1 and RNP2 motifs in each RRM are boxed in magenta. The positions of the missense mutations and the short deletion are indicated with the allele names. Note that yb1725 generated a cryptic splice donor site almost exclusively used in the mutant, which resulted in the in-frame deletion of the 4-amino acid stretch indicated. (D) RT-PCR analysis of the UNC-75 mRNAs from the synchronized L1 larvae of N2 (wt), asd-1 (yb978); fox-1 (e2643) and unc-75 (yb1701).
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pgen-1003337-g003: UNC-75 is required for the selection exon 7a from the unc-32 exon 7 reporter in the nervous system.(A) Fluorescence images of the ybIs1622 worms with the unc-75 mutant alleles yb1700, yb1725, yb1714 and yb1701 with a dual-bandpass filter. Scale bar, 200 µm. (B) Schematic structure of the unc-75 gene and the positions and consequence of the mutations. The ORF is colored in yellow and the regions corresponding to the three RRMs are in orange. The allele names are in the colors representing the color phenotypes. SA, splice acceptor; SD, splice donor. (C) Amino acid sequence alignments of the three RRMs from the CELF3–6 subfamily members C. elegans UNC-75, human CELF3, CELF4, CELF5 and CELF6 and the CELF1–2 subfamily members C. elegans ETR-1, human CELF1 and CELF2. Conserved residues are shaded in black. Residues with similar properties to the consensus are shaded in gray. The secondary structure elements determined for human CELF1 [51], [52] are depicted with the blue rectangles (β-sheets) and the red ellipses (α-helices) below the alignments. The highly conserved RNP1 and RNP2 motifs in each RRM are boxed in magenta. The positions of the missense mutations and the short deletion are indicated with the allele names. Note that yb1725 generated a cryptic splice donor site almost exclusively used in the mutant, which resulted in the in-frame deletion of the 4-amino acid stretch indicated. (D) RT-PCR analysis of the UNC-75 mRNAs from the synchronized L1 larvae of N2 (wt), asd-1 (yb978); fox-1 (e2643) and unc-75 (yb1701).

Mentions: To identify other regulator(s) that confer the neuron-specificity to the exon 7 reporter, we mutagenized the ybIs1622 strain to screen for mutants exhibiting altered colors. We successfully isolated many homozygous viable strains with Yellow, Orange or Red phenotype (Figure 3A, Figure S2). In some other strains, most neurons turned red while some remained green (Red/Green) (Figure 3A, Figure S2). The color phenotypes were completely penetrated within the strains. Notably, all the Red and Red/Green strains also showed an uncoordinated (Unc) phenotype while the Orange or Yellow strains did not.


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 is required for the selection exon 7a from the unc-32 exon 7 reporter in the nervous system.(A) Fluorescence images of the ybIs1622 worms with the unc-75 mutant alleles yb1700, yb1725, yb1714 and yb1701 with a dual-bandpass filter. Scale bar, 200 µm. (B) Schematic structure of the unc-75 gene and the positions and consequence of the mutations. The ORF is colored in yellow and the regions corresponding to the three RRMs are in orange. The allele names are in the colors representing the color phenotypes. SA, splice acceptor; SD, splice donor. (C) Amino acid sequence alignments of the three RRMs from the CELF3–6 subfamily members C. elegans UNC-75, human CELF3, CELF4, CELF5 and CELF6 and the CELF1–2 subfamily members C. elegans ETR-1, human CELF1 and CELF2. Conserved residues are shaded in black. Residues with similar properties to the consensus are shaded in gray. The secondary structure elements determined for human CELF1 [51], [52] are depicted with the blue rectangles (β-sheets) and the red ellipses (α-helices) below the alignments. The highly conserved RNP1 and RNP2 motifs in each RRM are boxed in magenta. The positions of the missense mutations and the short deletion are indicated with the allele names. Note that yb1725 generated a cryptic splice donor site almost exclusively used in the mutant, which resulted in the in-frame deletion of the 4-amino acid stretch indicated. (D) RT-PCR analysis of the UNC-75 mRNAs from the synchronized L1 larvae of N2 (wt), asd-1 (yb978); fox-1 (e2643) and unc-75 (yb1701).
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Related In: Results  -  Collection

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

pgen-1003337-g003: UNC-75 is required for the selection exon 7a from the unc-32 exon 7 reporter in the nervous system.(A) Fluorescence images of the ybIs1622 worms with the unc-75 mutant alleles yb1700, yb1725, yb1714 and yb1701 with a dual-bandpass filter. Scale bar, 200 µm. (B) Schematic structure of the unc-75 gene and the positions and consequence of the mutations. The ORF is colored in yellow and the regions corresponding to the three RRMs are in orange. The allele names are in the colors representing the color phenotypes. SA, splice acceptor; SD, splice donor. (C) Amino acid sequence alignments of the three RRMs from the CELF3–6 subfamily members C. elegans UNC-75, human CELF3, CELF4, CELF5 and CELF6 and the CELF1–2 subfamily members C. elegans ETR-1, human CELF1 and CELF2. Conserved residues are shaded in black. Residues with similar properties to the consensus are shaded in gray. The secondary structure elements determined for human CELF1 [51], [52] are depicted with the blue rectangles (β-sheets) and the red ellipses (α-helices) below the alignments. The highly conserved RNP1 and RNP2 motifs in each RRM are boxed in magenta. The positions of the missense mutations and the short deletion are indicated with the allele names. Note that yb1725 generated a cryptic splice donor site almost exclusively used in the mutant, which resulted in the in-frame deletion of the 4-amino acid stretch indicated. (D) RT-PCR analysis of the UNC-75 mRNAs from the synchronized L1 larvae of N2 (wt), asd-1 (yb978); fox-1 (e2643) and unc-75 (yb1701).
Mentions: To identify other regulator(s) that confer the neuron-specificity to the exon 7 reporter, we mutagenized the ybIs1622 strain to screen for mutants exhibiting altered colors. We successfully isolated many homozygous viable strains with Yellow, Orange or Red phenotype (Figure 3A, Figure S2). In some other strains, most neurons turned red while some remained green (Red/Green) (Figure 3A, Figure S2). The color phenotypes were completely penetrated within the strains. Notably, all the Red and Red/Green strains also showed an uncoordinated (Unc) phenotype while the Orange or Yellow strains did not.

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