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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 C-termini of UNC-75, ASD-1, and FOX-1 are the evolutionarily conserved nuclear localization signals.(A) Amino acid sequence alignment of the C-terminal ends of the CELF family proteins and the putative PY-NLSs from the related proteins C. elegans EXC-7, HRPF-1, human hnRNP F, hnRNP H1 and hnRNP H2 (left), human hnRNP A1, hnRNP D and TAP and the C-terminal ends of the RBFOX family proteins human RBFOX1, RBFOX2, mouse RBFOX3, Drosophila A2bp1, C. elegans ASD-1 and FOX-1 (right). The consensus sequences of the PY-NLS are indicated. The amino acid residues that match the consensus are shaded in yellow or orange. φ, hydrophobic residues. The underlines in the UNC-75 sequence indicate residues substituted with alanine in UNC-75(AAA) used in (C). The underlines in the ASD-1 and FOX-1 sequences indicate residues deleted in the truncated constructs used in (E) and (G), respectively. (B, C) Fluorescence images of the HeLa cells transfected with full-length UNC-75 (B) and UNC-75(AAA) (C) stained with anti-UNC-75 (left panels) and Hoechst 33258 (right panels). (D–G) Confocal images of the HeLa cells transfected with HA-tagged full-length ASD-1 (D), ASD-1(1–388) (E), full-length FOX-1 (F) and FOX-1(1–408) (G) stained with anti-ASD-1 (D and E, left panels) or anti-FOX-1 (F and G, left panels) and TO-PRO3 (right panels). Scale bar, 50 µm.
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pgen-1003337-g004: The C-termini of UNC-75, ASD-1, and FOX-1 are the evolutionarily conserved nuclear localization signals.(A) Amino acid sequence alignment of the C-terminal ends of the CELF family proteins and the putative PY-NLSs from the related proteins C. elegans EXC-7, HRPF-1, human hnRNP F, hnRNP H1 and hnRNP H2 (left), human hnRNP A1, hnRNP D and TAP and the C-terminal ends of the RBFOX family proteins human RBFOX1, RBFOX2, mouse RBFOX3, Drosophila A2bp1, C. elegans ASD-1 and FOX-1 (right). The consensus sequences of the PY-NLS are indicated. The amino acid residues that match the consensus are shaded in yellow or orange. φ, hydrophobic residues. The underlines in the UNC-75 sequence indicate residues substituted with alanine in UNC-75(AAA) used in (C). The underlines in the ASD-1 and FOX-1 sequences indicate residues deleted in the truncated constructs used in (E) and (G), respectively. (B, C) Fluorescence images of the HeLa cells transfected with full-length UNC-75 (B) and UNC-75(AAA) (C) stained with anti-UNC-75 (left panels) and Hoechst 33258 (right panels). (D–G) Confocal images of the HeLa cells transfected with HA-tagged full-length ASD-1 (D), ASD-1(1–388) (E), full-length FOX-1 (F) and FOX-1(1–408) (G) stained with anti-ASD-1 (D and E, left panels) or anti-FOX-1 (F and G, left panels) and TO-PRO3 (right panels). Scale bar, 50 µm.

Mentions: We noticed that the C-termini of the CELF family proteins as well as the RBFOX family proteins are evolutionarily conserved and match the consensus of the hydrophobic PY nuclear localization signal (PY-NLS) [30] (Figure 4A). To test this idea, we analyzed the effect of substitution or deletion of the C-terminal motifs upon subcellular localization of the proteins (Figure 4B–4G). The substitution of the three residues in the PY element of UNC-75 (Figure 4A) disrupted the nuclear localization of UNC-75 (Figure 4B, 4C), confirming that the C-terminal motif of UNC-75 functions as the PY-NLS. In the same way, the deletion of the 7 and 16 residues from the C-termini of ASD-1 and FOX-1, respectively (Figure 4A), disrupted the nuclear localization of the proteins (Figure 4D–4G), indicating that the C-terminal portions of ASD-1 and FOX-1 are the sole NLSs.


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 C-termini of UNC-75, ASD-1, and FOX-1 are the evolutionarily conserved nuclear localization signals.(A) Amino acid sequence alignment of the C-terminal ends of the CELF family proteins and the putative PY-NLSs from the related proteins C. elegans EXC-7, HRPF-1, human hnRNP F, hnRNP H1 and hnRNP H2 (left), human hnRNP A1, hnRNP D and TAP and the C-terminal ends of the RBFOX family proteins human RBFOX1, RBFOX2, mouse RBFOX3, Drosophila A2bp1, C. elegans ASD-1 and FOX-1 (right). The consensus sequences of the PY-NLS are indicated. The amino acid residues that match the consensus are shaded in yellow or orange. φ, hydrophobic residues. The underlines in the UNC-75 sequence indicate residues substituted with alanine in UNC-75(AAA) used in (C). The underlines in the ASD-1 and FOX-1 sequences indicate residues deleted in the truncated constructs used in (E) and (G), respectively. (B, C) Fluorescence images of the HeLa cells transfected with full-length UNC-75 (B) and UNC-75(AAA) (C) stained with anti-UNC-75 (left panels) and Hoechst 33258 (right panels). (D–G) Confocal images of the HeLa cells transfected with HA-tagged full-length ASD-1 (D), ASD-1(1–388) (E), full-length FOX-1 (F) and FOX-1(1–408) (G) stained with anti-ASD-1 (D and E, left panels) or anti-FOX-1 (F and G, left panels) and TO-PRO3 (right panels). Scale bar, 50 µm.
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Related In: Results  -  Collection

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

pgen-1003337-g004: The C-termini of UNC-75, ASD-1, and FOX-1 are the evolutionarily conserved nuclear localization signals.(A) Amino acid sequence alignment of the C-terminal ends of the CELF family proteins and the putative PY-NLSs from the related proteins C. elegans EXC-7, HRPF-1, human hnRNP F, hnRNP H1 and hnRNP H2 (left), human hnRNP A1, hnRNP D and TAP and the C-terminal ends of the RBFOX family proteins human RBFOX1, RBFOX2, mouse RBFOX3, Drosophila A2bp1, C. elegans ASD-1 and FOX-1 (right). The consensus sequences of the PY-NLS are indicated. The amino acid residues that match the consensus are shaded in yellow or orange. φ, hydrophobic residues. The underlines in the UNC-75 sequence indicate residues substituted with alanine in UNC-75(AAA) used in (C). The underlines in the ASD-1 and FOX-1 sequences indicate residues deleted in the truncated constructs used in (E) and (G), respectively. (B, C) Fluorescence images of the HeLa cells transfected with full-length UNC-75 (B) and UNC-75(AAA) (C) stained with anti-UNC-75 (left panels) and Hoechst 33258 (right panels). (D–G) Confocal images of the HeLa cells transfected with HA-tagged full-length ASD-1 (D), ASD-1(1–388) (E), full-length FOX-1 (F) and FOX-1(1–408) (G) stained with anti-ASD-1 (D and E, left panels) or anti-FOX-1 (F and G, left panels) and TO-PRO3 (right panels). Scale bar, 50 µm.
Mentions: We noticed that the C-termini of the CELF family proteins as well as the RBFOX family proteins are evolutionarily conserved and match the consensus of the hydrophobic PY nuclear localization signal (PY-NLS) [30] (Figure 4A). To test this idea, we analyzed the effect of substitution or deletion of the C-terminal motifs upon subcellular localization of the proteins (Figure 4B–4G). The substitution of the three residues in the PY element of UNC-75 (Figure 4A) disrupted the nuclear localization of UNC-75 (Figure 4B, 4C), confirming that the C-terminal motif of UNC-75 functions as the PY-NLS. In the same way, the deletion of the 7 and 16 residues from the C-termini of ASD-1 and FOX-1, respectively (Figure 4A), disrupted the nuclear localization of the proteins (Figure 4D–4G), indicating that the C-terminal portions of ASD-1 and FOX-1 are the sole NLSs.

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