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HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform.

Nasrin F, Rahman MA, Masuda A, Ohe K, Takeda J, Ohno K - Sci Rep (2014)

Bottom Line: Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L.Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping.Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.

ABSTRACT
Muscle specific receptor tyrosine kinase (MuSK) is an essential postsynaptic transmembrane molecule that mediates clustering of acetylcholine receptors (AChR). MUSK exon 10 is alternatively skipped in human, but not in mouse. Skipping of this exon disrupts a cysteine-rich region (Fz-CRD), which is essential for Wnt-mediated AChR clustering. To investigate the underlying mechanisms of alternative splicing, we exploited block-scanning mutagenesis with human minigene and identified a 20-nucleotide block that contained exonic splicing silencers. Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L. Antibody-mediated in vitro protein depletion and scanning mutagenesis additionally revealed that binding of hnRNP C to RNA subsequently promotes binding of YB-1 and hnRNP L to the immediate downstream sites and enhances exon skipping. Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping. Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1.

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Binding of hnRNP C, YB-1, and hnRNP L to specific motifs enhances coordinated skipping of MUSK exon 10.(a, b) Scanning mutagenesis to map the binding motifs of hnRNP C, YB-1, and hnRNP L in ESS5 (H-B5). RNA probe sequences are shown in the upper panels, where discordant nucleotides between human and mouse are underlined in H-B5 (ESS5). Artificial mutations are shown in red. RNA affinity-purified products are detected by immunoblotting in the lower panels. The results are indicated on the right side by “+” and “−” for positive and negative binding to each probe, respectively. (c) The resulting binding site of each factor from panels (a) and (b) is schematically shown. Essential binding nucleotides are indicated by large green letters and are underlined. (d) Schematic of a reporter minigene (pSPL3-human-MUSK-MS2-PP7). MS2 coat protein-binding hairpin RNA (blue) is substituted for the first half of ESS5 (binding site of hnRNP C). Similarly, PP7 coat protein-binding hairpin RNA (orange) is substituted for the second half of ESS5 (binding sites of hnRNP L and YB-1). (e) RT-PCR of pSPL3-human-MUSK-MS2-PP7 minigene in HeLa cells that are co-transfected with the indicated effectors. Blue and orange letters match to those in (d). The mean and SD (n = 3) of the ratio of exon skipping in each treatment is shown below the gel image.
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f5: Binding of hnRNP C, YB-1, and hnRNP L to specific motifs enhances coordinated skipping of MUSK exon 10.(a, b) Scanning mutagenesis to map the binding motifs of hnRNP C, YB-1, and hnRNP L in ESS5 (H-B5). RNA probe sequences are shown in the upper panels, where discordant nucleotides between human and mouse are underlined in H-B5 (ESS5). Artificial mutations are shown in red. RNA affinity-purified products are detected by immunoblotting in the lower panels. The results are indicated on the right side by “+” and “−” for positive and negative binding to each probe, respectively. (c) The resulting binding site of each factor from panels (a) and (b) is schematically shown. Essential binding nucleotides are indicated by large green letters and are underlined. (d) Schematic of a reporter minigene (pSPL3-human-MUSK-MS2-PP7). MS2 coat protein-binding hairpin RNA (blue) is substituted for the first half of ESS5 (binding site of hnRNP C). Similarly, PP7 coat protein-binding hairpin RNA (orange) is substituted for the second half of ESS5 (binding sites of hnRNP L and YB-1). (e) RT-PCR of pSPL3-human-MUSK-MS2-PP7 minigene in HeLa cells that are co-transfected with the indicated effectors. Blue and orange letters match to those in (d). The mean and SD (n = 3) of the ratio of exon skipping in each treatment is shown below the gel image.

Mentions: Having identified the critical cis-element and their cognate-binding partners, we next analyzed the molecular basis of specific binding of each trans-factor to ESS5. HnRNP C prefers to bind to poly-T stretch motifs2444 and both hnRNP L and YB-1 prefer to bind to C/A-rich motifs293245. ESS5 carries a stretch of five T's in the first half of the block and a C/A-rich sequence in the second half of the block (H-B5 in Fig. 5a and b). In vitro SELEX studies of hnRNP L demonstrated that CACA and ACAC sequences confer high-affinity binding motifs and that CAAC and CACC confer low-affinity binding motifs for hnRNP L32, where motifs present in ESS5 are underlined. On the contrary, in vitro SELEX studies of YB-1 revealed that CATC and CACC sequences confer high-affinity binding motifs for YB-129, where a motif present in ESS5 is underlined. Therefore, the second half of ESS5 harbors overlapping binding motifs of both YB-1 and hnRNP L. To characterize the precise binding sites of the associated factors, we introduced a series of artificial point mutations into the human ESS5 RNA probe and checked the binding of each factor by RNA-affinity purification followed by Western blotting (Fig. 5a and b). We observed that poly-T stretch-disrupting mutations in the first half indeed abolished the binding of hnRNP C (Fig. 5a, lanes 3, 4, and 5), and four or more consecutive T-nucleotides are necessary for hnRNP C binding. To our surprise, we noticed that binding of YB-1 and hnRNP L was also compromised along with disruption of the hnRNP C binding (Fig. 5a, lanes 3, 4 and 5). This suggested that binding of YB-1 and hnRNP L was dependent on poly T-stretch. On the other hand, introduction of mutations in the second half (C/A- rich sequences) compromised binding of YB-1 and hnRNP L, but not of hnRNP C (Fig. 5b). In addition, characterization of essential nucleotides for binding of hnRNP L (CAACA) and YB-1 (ACACCT) revealed that binding motifs of hnRNP L and YB-1 indeed overlap (CAACACCT) in the second half of ESS5, where the overlapping nucleotides are underlined. Considering the overall findings (Fig. 5c), we predicted that binding of hnRNP C to the poly-T stretch facilitates the binding of YB-1 and hnRNP L to the adjacent downstream site. To test this hypothesis, we depleted hnRNP C from a HeLa nuclear extract using a specific antibody (Fig. S4a) and performed RNA affinity purification assays. As we had expected, depletion of hnRNP C ified the binding of YB-1 and hnRNP L (Fig. S4b).


HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform.

Nasrin F, Rahman MA, Masuda A, Ohe K, Takeda J, Ohno K - Sci Rep (2014)

Binding of hnRNP C, YB-1, and hnRNP L to specific motifs enhances coordinated skipping of MUSK exon 10.(a, b) Scanning mutagenesis to map the binding motifs of hnRNP C, YB-1, and hnRNP L in ESS5 (H-B5). RNA probe sequences are shown in the upper panels, where discordant nucleotides between human and mouse are underlined in H-B5 (ESS5). Artificial mutations are shown in red. RNA affinity-purified products are detected by immunoblotting in the lower panels. The results are indicated on the right side by “+” and “−” for positive and negative binding to each probe, respectively. (c) The resulting binding site of each factor from panels (a) and (b) is schematically shown. Essential binding nucleotides are indicated by large green letters and are underlined. (d) Schematic of a reporter minigene (pSPL3-human-MUSK-MS2-PP7). MS2 coat protein-binding hairpin RNA (blue) is substituted for the first half of ESS5 (binding site of hnRNP C). Similarly, PP7 coat protein-binding hairpin RNA (orange) is substituted for the second half of ESS5 (binding sites of hnRNP L and YB-1). (e) RT-PCR of pSPL3-human-MUSK-MS2-PP7 minigene in HeLa cells that are co-transfected with the indicated effectors. Blue and orange letters match to those in (d). The mean and SD (n = 3) of the ratio of exon skipping in each treatment is shown below the gel image.
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f5: Binding of hnRNP C, YB-1, and hnRNP L to specific motifs enhances coordinated skipping of MUSK exon 10.(a, b) Scanning mutagenesis to map the binding motifs of hnRNP C, YB-1, and hnRNP L in ESS5 (H-B5). RNA probe sequences are shown in the upper panels, where discordant nucleotides between human and mouse are underlined in H-B5 (ESS5). Artificial mutations are shown in red. RNA affinity-purified products are detected by immunoblotting in the lower panels. The results are indicated on the right side by “+” and “−” for positive and negative binding to each probe, respectively. (c) The resulting binding site of each factor from panels (a) and (b) is schematically shown. Essential binding nucleotides are indicated by large green letters and are underlined. (d) Schematic of a reporter minigene (pSPL3-human-MUSK-MS2-PP7). MS2 coat protein-binding hairpin RNA (blue) is substituted for the first half of ESS5 (binding site of hnRNP C). Similarly, PP7 coat protein-binding hairpin RNA (orange) is substituted for the second half of ESS5 (binding sites of hnRNP L and YB-1). (e) RT-PCR of pSPL3-human-MUSK-MS2-PP7 minigene in HeLa cells that are co-transfected with the indicated effectors. Blue and orange letters match to those in (d). The mean and SD (n = 3) of the ratio of exon skipping in each treatment is shown below the gel image.
Mentions: Having identified the critical cis-element and their cognate-binding partners, we next analyzed the molecular basis of specific binding of each trans-factor to ESS5. HnRNP C prefers to bind to poly-T stretch motifs2444 and both hnRNP L and YB-1 prefer to bind to C/A-rich motifs293245. ESS5 carries a stretch of five T's in the first half of the block and a C/A-rich sequence in the second half of the block (H-B5 in Fig. 5a and b). In vitro SELEX studies of hnRNP L demonstrated that CACA and ACAC sequences confer high-affinity binding motifs and that CAAC and CACC confer low-affinity binding motifs for hnRNP L32, where motifs present in ESS5 are underlined. On the contrary, in vitro SELEX studies of YB-1 revealed that CATC and CACC sequences confer high-affinity binding motifs for YB-129, where a motif present in ESS5 is underlined. Therefore, the second half of ESS5 harbors overlapping binding motifs of both YB-1 and hnRNP L. To characterize the precise binding sites of the associated factors, we introduced a series of artificial point mutations into the human ESS5 RNA probe and checked the binding of each factor by RNA-affinity purification followed by Western blotting (Fig. 5a and b). We observed that poly-T stretch-disrupting mutations in the first half indeed abolished the binding of hnRNP C (Fig. 5a, lanes 3, 4, and 5), and four or more consecutive T-nucleotides are necessary for hnRNP C binding. To our surprise, we noticed that binding of YB-1 and hnRNP L was also compromised along with disruption of the hnRNP C binding (Fig. 5a, lanes 3, 4 and 5). This suggested that binding of YB-1 and hnRNP L was dependent on poly T-stretch. On the other hand, introduction of mutations in the second half (C/A- rich sequences) compromised binding of YB-1 and hnRNP L, but not of hnRNP C (Fig. 5b). In addition, characterization of essential nucleotides for binding of hnRNP L (CAACA) and YB-1 (ACACCT) revealed that binding motifs of hnRNP L and YB-1 indeed overlap (CAACACCT) in the second half of ESS5, where the overlapping nucleotides are underlined. Considering the overall findings (Fig. 5c), we predicted that binding of hnRNP C to the poly-T stretch facilitates the binding of YB-1 and hnRNP L to the adjacent downstream site. To test this hypothesis, we depleted hnRNP C from a HeLa nuclear extract using a specific antibody (Fig. S4a) and performed RNA affinity purification assays. As we had expected, depletion of hnRNP C ified the binding of YB-1 and hnRNP L (Fig. S4b).

Bottom Line: Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L.Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping.Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.

ABSTRACT
Muscle specific receptor tyrosine kinase (MuSK) is an essential postsynaptic transmembrane molecule that mediates clustering of acetylcholine receptors (AChR). MUSK exon 10 is alternatively skipped in human, but not in mouse. Skipping of this exon disrupts a cysteine-rich region (Fz-CRD), which is essential for Wnt-mediated AChR clustering. To investigate the underlying mechanisms of alternative splicing, we exploited block-scanning mutagenesis with human minigene and identified a 20-nucleotide block that contained exonic splicing silencers. Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L. Antibody-mediated in vitro protein depletion and scanning mutagenesis additionally revealed that binding of hnRNP C to RNA subsequently promotes binding of YB-1 and hnRNP L to the immediate downstream sites and enhances exon skipping. Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping. Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1.

Show MeSH