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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 RBFOX family and UNC-75 differentially regulate the intron excision from the unc-32 pre-mRNA.(A) RT-PCR analysis of the mature mRNAs from the endogenous unc-32 gene (left) and the exon 7 reporter transgene ybIs1622 (right) in the wild-type (lane 1), asd-1 (yb978); fox-1 (e2643) double (lane 2) and unc-75 (yb1725) (lane 3) backgrounds. Note that the isoform with double inclusion or double skipping of exons 7a and 7b was not detected. (B) RT-PCR analyses of the partially spliced RNAs from ybIs1622 in the wild-type, asd-1; fox-1 and unc-75 backgrounds. The schematic structures of the RT-PCR products are indicated on the right. Black and blue arrowheads indicate the positions and directions of the exonic and the intronic primers, respectively. RTase, reverse transcriptase. (C) Averages of the relative amounts of the partially spliced RNAs to the pre-mRNA. Error bars indicate S.E.M. (n = 3). *p<0.005 and **p<0.001, Student's t-test. (D) Schematic models of the mutually exclusive selection of unc-32 exons 7a and 7b. See Discussion for detail.
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pgen-1003337-g007: The RBFOX family and UNC-75 differentially regulate the intron excision from the unc-32 pre-mRNA.(A) RT-PCR analysis of the mature mRNAs from the endogenous unc-32 gene (left) and the exon 7 reporter transgene ybIs1622 (right) in the wild-type (lane 1), asd-1 (yb978); fox-1 (e2643) double (lane 2) and unc-75 (yb1725) (lane 3) backgrounds. Note that the isoform with double inclusion or double skipping of exons 7a and 7b was not detected. (B) RT-PCR analyses of the partially spliced RNAs from ybIs1622 in the wild-type, asd-1; fox-1 and unc-75 backgrounds. The schematic structures of the RT-PCR products are indicated on the right. Black and blue arrowheads indicate the positions and directions of the exonic and the intronic primers, respectively. RTase, reverse transcriptase. (C) Averages of the relative amounts of the partially spliced RNAs to the pre-mRNA. Error bars indicate S.E.M. (n = 3). *p<0.005 and **p<0.001, Student's t-test. (D) Schematic models of the mutually exclusive selection of unc-32 exons 7a and 7b. See Discussion for detail.

Mentions: Next we analyzed the effects of the RBFOX family and UNC-75 on the endogenous unc-32 gene. In the wild-type L1 larvae, the exon 7a and exon 7b mRNA isoforms were almost equally detected (Figure 7A, left, lane 1). The relative amount of the exon 7a isoform was reduced in the asd-1; fox-1 double mutant (lane 2) and unc-75 mutant (lane 3) backgrounds. A double inclusion isoform or a double skipping isoform was not detected in either of the mutants. These results are consistent with their color phenotypes and the splicing patterns of the exon 7 reporter expressed in the nervous system (Figure 7A, right) and confirm that the RBFOX family and UNC-75 regulate the mutually exclusive splicing of exons 7a and 7b of the endogenous unc-32 gene.


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 RBFOX family and UNC-75 differentially regulate the intron excision from the unc-32 pre-mRNA.(A) RT-PCR analysis of the mature mRNAs from the endogenous unc-32 gene (left) and the exon 7 reporter transgene ybIs1622 (right) in the wild-type (lane 1), asd-1 (yb978); fox-1 (e2643) double (lane 2) and unc-75 (yb1725) (lane 3) backgrounds. Note that the isoform with double inclusion or double skipping of exons 7a and 7b was not detected. (B) RT-PCR analyses of the partially spliced RNAs from ybIs1622 in the wild-type, asd-1; fox-1 and unc-75 backgrounds. The schematic structures of the RT-PCR products are indicated on the right. Black and blue arrowheads indicate the positions and directions of the exonic and the intronic primers, respectively. RTase, reverse transcriptase. (C) Averages of the relative amounts of the partially spliced RNAs to the pre-mRNA. Error bars indicate S.E.M. (n = 3). *p<0.005 and **p<0.001, Student's t-test. (D) Schematic models of the mutually exclusive selection of unc-32 exons 7a and 7b. See Discussion for detail.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003337-g007: The RBFOX family and UNC-75 differentially regulate the intron excision from the unc-32 pre-mRNA.(A) RT-PCR analysis of the mature mRNAs from the endogenous unc-32 gene (left) and the exon 7 reporter transgene ybIs1622 (right) in the wild-type (lane 1), asd-1 (yb978); fox-1 (e2643) double (lane 2) and unc-75 (yb1725) (lane 3) backgrounds. Note that the isoform with double inclusion or double skipping of exons 7a and 7b was not detected. (B) RT-PCR analyses of the partially spliced RNAs from ybIs1622 in the wild-type, asd-1; fox-1 and unc-75 backgrounds. The schematic structures of the RT-PCR products are indicated on the right. Black and blue arrowheads indicate the positions and directions of the exonic and the intronic primers, respectively. RTase, reverse transcriptase. (C) Averages of the relative amounts of the partially spliced RNAs to the pre-mRNA. Error bars indicate S.E.M. (n = 3). *p<0.005 and **p<0.001, Student's t-test. (D) Schematic models of the mutually exclusive selection of unc-32 exons 7a and 7b. See Discussion for detail.
Mentions: Next we analyzed the effects of the RBFOX family and UNC-75 on the endogenous unc-32 gene. In the wild-type L1 larvae, the exon 7a and exon 7b mRNA isoforms were almost equally detected (Figure 7A, left, lane 1). The relative amount of the exon 7a isoform was reduced in the asd-1; fox-1 double mutant (lane 2) and unc-75 mutant (lane 3) backgrounds. A double inclusion isoform or a double skipping isoform was not detected in either of the mutants. These results are consistent with their color phenotypes and the splicing patterns of the exon 7 reporter expressed in the nervous system (Figure 7A, right) and confirm that the RBFOX family and UNC-75 regulate the mutually exclusive splicing of exons 7a and 7b of the endogenous unc-32 gene.

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