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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 mutually exclusive exons of the unc-32 gene are regulated in tissue-specific manners.(A) Schematic structure of the unc-32 gene. Numbered boxes indicate exons. The position of the 3′-splice site mutation in the unc-32 (e189) allele is indicated. Black triangles indicate positions and directions of the exonic primers used in the RT-PCR analyses shown in (B). (B) RT-PCR analyses of unc-32 exon 4 (left) and exon 7 (right) at the L1 and L4 larval stages. (C, D) Schematic illustration of the symmetric trio of the exon 4 reporter minigenes (C) and the symmetric pair of the exon 7 reporter minigenes (D) and the mRNA isoforms derived from them. The cDNA cassettes and the predicted ORFs for Venus/EGFP, mRFP and ECFP are colored in green, magenta and cyan, respectively. Green circles and red diamonds indicate the artificially-introduced initiation and termination codons, respectively. (E) Fluorescence images of an L4 larva of the exon 4 reporter worm ybIs1891 [eft-3::unc-32E4a-Venus eft-3::unc-32E4b-mRFP eft-3::unc-32E4c-ECFP]. E4a-Venus, E4b-mRFP and E4c-ECFP images in black-white and a merged image with pseudo colors. (F, G) Confocal images of the exon 7 reporter worms ybEx1440 [eft-3::unc-32E7-EGFP eft-3::unc-32E7b-mRFP]. (H) A fluorescence image of the unc-32 transcriptional fusion reporter worm ybEx1896 [unc-32::EGFP] containing 1.7-kb of the unc-32 promoter. int, intestine; N, neurons in head ganglia; phx, pharynx. Scale bars in (E), (F) and (H), 100 µm; in (G), 20 µm.
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pgen-1003337-g001: The mutually exclusive exons of the unc-32 gene are regulated in tissue-specific manners.(A) Schematic structure of the unc-32 gene. Numbered boxes indicate exons. The position of the 3′-splice site mutation in the unc-32 (e189) allele is indicated. Black triangles indicate positions and directions of the exonic primers used in the RT-PCR analyses shown in (B). (B) RT-PCR analyses of unc-32 exon 4 (left) and exon 7 (right) at the L1 and L4 larval stages. (C, D) Schematic illustration of the symmetric trio of the exon 4 reporter minigenes (C) and the symmetric pair of the exon 7 reporter minigenes (D) and the mRNA isoforms derived from them. The cDNA cassettes and the predicted ORFs for Venus/EGFP, mRFP and ECFP are colored in green, magenta and cyan, respectively. Green circles and red diamonds indicate the artificially-introduced initiation and termination codons, respectively. (E) Fluorescence images of an L4 larva of the exon 4 reporter worm ybIs1891 [eft-3::unc-32E4a-Venus eft-3::unc-32E4b-mRFP eft-3::unc-32E4c-ECFP]. E4a-Venus, E4b-mRFP and E4c-ECFP images in black-white and a merged image with pseudo colors. (F, G) Confocal images of the exon 7 reporter worms ybEx1440 [eft-3::unc-32E7-EGFP eft-3::unc-32E7b-mRFP]. (H) A fluorescence image of the unc-32 transcriptional fusion reporter worm ybEx1896 [unc-32::EGFP] containing 1.7-kb of the unc-32 promoter. int, intestine; N, neurons in head ganglia; phx, pharynx. Scale bars in (E), (F) and (H), 100 µm; in (G), 20 µm.

Mentions: The unc-32 gene of C. elegans, analyzed in this study, encodes the a subunit of V0 complex of vacuolar-type H+-ATPases considered to be proton pumps that acidify intracellular organelles [23], [24]. The unique property of the unc-32 gene as a model for studying alternative splicing regulation is that it has two sets of mutually exclusive exons (Figure 1A). Only one exon at a time is selected from three exons 4a, 4b and 4c; only one exon is selected at a time from two exons 7a and 7b. Of the six possible combinations of exons 4 and 7, the three isoforms UNC-32A (4a/7b), UNC-32B (4b/7a) and UNC-32C (4c/7b) were predominantly detected [25] and appear to be developmentally regulated [18], raising questions about the exact selection patterns and the regulation mechanisms in vivo. In the present study, we demonstrate that unc-32 exon 4 and exon 7 are selected in tissue-specific manners and that a neuron-specific RNA-binding protein UNC-75 regulates the neuron-specific selection of exons 4b and 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)

The mutually exclusive exons of the unc-32 gene are regulated in tissue-specific manners.(A) Schematic structure of the unc-32 gene. Numbered boxes indicate exons. The position of the 3′-splice site mutation in the unc-32 (e189) allele is indicated. Black triangles indicate positions and directions of the exonic primers used in the RT-PCR analyses shown in (B). (B) RT-PCR analyses of unc-32 exon 4 (left) and exon 7 (right) at the L1 and L4 larval stages. (C, D) Schematic illustration of the symmetric trio of the exon 4 reporter minigenes (C) and the symmetric pair of the exon 7 reporter minigenes (D) and the mRNA isoforms derived from them. The cDNA cassettes and the predicted ORFs for Venus/EGFP, mRFP and ECFP are colored in green, magenta and cyan, respectively. Green circles and red diamonds indicate the artificially-introduced initiation and termination codons, respectively. (E) Fluorescence images of an L4 larva of the exon 4 reporter worm ybIs1891 [eft-3::unc-32E4a-Venus eft-3::unc-32E4b-mRFP eft-3::unc-32E4c-ECFP]. E4a-Venus, E4b-mRFP and E4c-ECFP images in black-white and a merged image with pseudo colors. (F, G) Confocal images of the exon 7 reporter worms ybEx1440 [eft-3::unc-32E7-EGFP eft-3::unc-32E7b-mRFP]. (H) A fluorescence image of the unc-32 transcriptional fusion reporter worm ybEx1896 [unc-32::EGFP] containing 1.7-kb of the unc-32 promoter. int, intestine; N, neurons in head ganglia; phx, pharynx. Scale bars in (E), (F) and (H), 100 µm; in (G), 20 µm.
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Related In: Results  -  Collection

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pgen-1003337-g001: The mutually exclusive exons of the unc-32 gene are regulated in tissue-specific manners.(A) Schematic structure of the unc-32 gene. Numbered boxes indicate exons. The position of the 3′-splice site mutation in the unc-32 (e189) allele is indicated. Black triangles indicate positions and directions of the exonic primers used in the RT-PCR analyses shown in (B). (B) RT-PCR analyses of unc-32 exon 4 (left) and exon 7 (right) at the L1 and L4 larval stages. (C, D) Schematic illustration of the symmetric trio of the exon 4 reporter minigenes (C) and the symmetric pair of the exon 7 reporter minigenes (D) and the mRNA isoforms derived from them. The cDNA cassettes and the predicted ORFs for Venus/EGFP, mRFP and ECFP are colored in green, magenta and cyan, respectively. Green circles and red diamonds indicate the artificially-introduced initiation and termination codons, respectively. (E) Fluorescence images of an L4 larva of the exon 4 reporter worm ybIs1891 [eft-3::unc-32E4a-Venus eft-3::unc-32E4b-mRFP eft-3::unc-32E4c-ECFP]. E4a-Venus, E4b-mRFP and E4c-ECFP images in black-white and a merged image with pseudo colors. (F, G) Confocal images of the exon 7 reporter worms ybEx1440 [eft-3::unc-32E7-EGFP eft-3::unc-32E7b-mRFP]. (H) A fluorescence image of the unc-32 transcriptional fusion reporter worm ybEx1896 [unc-32::EGFP] containing 1.7-kb of the unc-32 promoter. int, intestine; N, neurons in head ganglia; phx, pharynx. Scale bars in (E), (F) and (H), 100 µm; in (G), 20 µm.
Mentions: The unc-32 gene of C. elegans, analyzed in this study, encodes the a subunit of V0 complex of vacuolar-type H+-ATPases considered to be proton pumps that acidify intracellular organelles [23], [24]. The unique property of the unc-32 gene as a model for studying alternative splicing regulation is that it has two sets of mutually exclusive exons (Figure 1A). Only one exon at a time is selected from three exons 4a, 4b and 4c; only one exon is selected at a time from two exons 7a and 7b. Of the six possible combinations of exons 4 and 7, the three isoforms UNC-32A (4a/7b), UNC-32B (4b/7a) and UNC-32C (4c/7b) were predominantly detected [25] and appear to be developmentally regulated [18], raising questions about the exact selection patterns and the regulation mechanisms in vivo. In the present study, we demonstrate that unc-32 exon 4 and exon 7 are selected in tissue-specific manners and that a neuron-specific RNA-binding protein UNC-75 regulates the neuron-specific selection of exons 4b and 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