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All three RNA recognition motifs and the hinge region of HuC play distinct roles in the regulation of alternative splicing.

Hinman MN, Zhou HL, Sharma A, Lou H - Nucleic Acids Res. (2013)

Bottom Line: Each of the Hu proteins contains a divergent N-terminus, three highly conserved RNA recognition motifs (RRM1, RRM2 and RRM3) and a hinge region separating RRM2 and RRM3.The roles of each domain in splicing regulation are not well understood.Finally, we find that the portions of RRM3 required for HuC-HuC interaction overlap with those required for splicing regulation of all three targets, suggesting a role of HuC-HuC interaction in splicing regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
The four Hu [embryonic lethal abnormal vision-like (ELAVL)] protein family members regulate alternative splicing by binding to U-rich sequences surrounding target exons and affecting the interaction of the splicing machinery and/or local chromatin modifications. Each of the Hu proteins contains a divergent N-terminus, three highly conserved RNA recognition motifs (RRM1, RRM2 and RRM3) and a hinge region separating RRM2 and RRM3. The roles of each domain in splicing regulation are not well understood. Here, we investigate how HuC, a relatively poorly characterized family member, regulates three target pre-mRNAs: neurofibromatosis type I, Fas and HuD. We find that the HuC N-terminus is dispensable for splicing regulation, and the three RRMs are required for splicing regulation of each target, whereas the hinge region contributes to regulation of only some targets. Interestingly, the regions of the hinge and RRM3 required for regulating different targets only partially overlap, implying substrate-specific mechanisms of HuC-mediated splicing regulation. We show that RRM1 and RRM2 are required for binding to target pre-mRNAs, whereas the hinge and RRM3 are required for HuC-HuC self-interaction. Finally, we find that the portions of RRM3 required for HuC-HuC interaction overlap with those required for splicing regulation of all three targets, suggesting a role of HuC-HuC interaction in splicing regulation.

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The hinge and RRM3 domains of HuC are important for HuC–HuC interaction in cell lysates. (A and B) Myc- and Xpress-tagged HuC domain deletion mutants (A), HuC partial domain deletion mutants (B) and HuC splice variants (B) were co-transfected into HeLa cells. Top of A and B: IP of cell lysates was carried out using either IgG or Myc beads, with or without RNase treatment, followed by western blot analysis with anti-Xpress antibody. To the right of the figure is a chart summarizing the results of the co-IP experiment, with + indicating an interaction between the two exogenously expressed proteins, and − indicating a lack of interaction. Bottom of A and B: western blot analysis using 10% input with anti-Myc antibody to confirm that Myc-tagged proteins were expressed in each of the experiments shown in the top portion of the figure. Transfection numbers refer to the same experiment for the top and bottom portions of the figure.
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gkt166-F6: The hinge and RRM3 domains of HuC are important for HuC–HuC interaction in cell lysates. (A and B) Myc- and Xpress-tagged HuC domain deletion mutants (A), HuC partial domain deletion mutants (B) and HuC splice variants (B) were co-transfected into HeLa cells. Top of A and B: IP of cell lysates was carried out using either IgG or Myc beads, with or without RNase treatment, followed by western blot analysis with anti-Xpress antibody. To the right of the figure is a chart summarizing the results of the co-IP experiment, with + indicating an interaction between the two exogenously expressed proteins, and − indicating a lack of interaction. Bottom of A and B: western blot analysis using 10% input with anti-Myc antibody to confirm that Myc-tagged proteins were expressed in each of the experiments shown in the top portion of the figure. Transfection numbers refer to the same experiment for the top and bottom portions of the figure.

Mentions: Hu proteins are known to interact with themselves (52–57). We sought to understand which portions of HuC are important for binding to itself, and whether these same portions of HuC are required for splicing regulation. IP experiments were performed in which Xpress- and Myc-tagged HuC mutants were both expressed in HeLa cells. Expression of Xpress-tagged HuC proteins was confirmed by western blot analysis (Figure 6A and B, top portion, input lanes), as was the expression of Myc-tagged proteins (Figure 6A and B, bottom portion). Myc-tagged HuC proteins were pulled down using beads coated with anti-Myc antibody, and western blot analysis was performed with anti-Xpress antibody to detect co-immunoprecipitated Xpress-tagged HuC proteins (Figure 6A and B, top portion, α-Myc lanes). Pull-downs using IgG-coated beads were performed as negative controls (Figure 6A and B, top portion, IgG lanes).Figure 6.


All three RNA recognition motifs and the hinge region of HuC play distinct roles in the regulation of alternative splicing.

Hinman MN, Zhou HL, Sharma A, Lou H - Nucleic Acids Res. (2013)

The hinge and RRM3 domains of HuC are important for HuC–HuC interaction in cell lysates. (A and B) Myc- and Xpress-tagged HuC domain deletion mutants (A), HuC partial domain deletion mutants (B) and HuC splice variants (B) were co-transfected into HeLa cells. Top of A and B: IP of cell lysates was carried out using either IgG or Myc beads, with or without RNase treatment, followed by western blot analysis with anti-Xpress antibody. To the right of the figure is a chart summarizing the results of the co-IP experiment, with + indicating an interaction between the two exogenously expressed proteins, and − indicating a lack of interaction. Bottom of A and B: western blot analysis using 10% input with anti-Myc antibody to confirm that Myc-tagged proteins were expressed in each of the experiments shown in the top portion of the figure. Transfection numbers refer to the same experiment for the top and bottom portions of the figure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643579&req=5

gkt166-F6: The hinge and RRM3 domains of HuC are important for HuC–HuC interaction in cell lysates. (A and B) Myc- and Xpress-tagged HuC domain deletion mutants (A), HuC partial domain deletion mutants (B) and HuC splice variants (B) were co-transfected into HeLa cells. Top of A and B: IP of cell lysates was carried out using either IgG or Myc beads, with or without RNase treatment, followed by western blot analysis with anti-Xpress antibody. To the right of the figure is a chart summarizing the results of the co-IP experiment, with + indicating an interaction between the two exogenously expressed proteins, and − indicating a lack of interaction. Bottom of A and B: western blot analysis using 10% input with anti-Myc antibody to confirm that Myc-tagged proteins were expressed in each of the experiments shown in the top portion of the figure. Transfection numbers refer to the same experiment for the top and bottom portions of the figure.
Mentions: Hu proteins are known to interact with themselves (52–57). We sought to understand which portions of HuC are important for binding to itself, and whether these same portions of HuC are required for splicing regulation. IP experiments were performed in which Xpress- and Myc-tagged HuC mutants were both expressed in HeLa cells. Expression of Xpress-tagged HuC proteins was confirmed by western blot analysis (Figure 6A and B, top portion, input lanes), as was the expression of Myc-tagged proteins (Figure 6A and B, bottom portion). Myc-tagged HuC proteins were pulled down using beads coated with anti-Myc antibody, and western blot analysis was performed with anti-Xpress antibody to detect co-immunoprecipitated Xpress-tagged HuC proteins (Figure 6A and B, top portion, α-Myc lanes). Pull-downs using IgG-coated beads were performed as negative controls (Figure 6A and B, top portion, IgG lanes).Figure 6.

Bottom Line: Each of the Hu proteins contains a divergent N-terminus, three highly conserved RNA recognition motifs (RRM1, RRM2 and RRM3) and a hinge region separating RRM2 and RRM3.The roles of each domain in splicing regulation are not well understood.Finally, we find that the portions of RRM3 required for HuC-HuC interaction overlap with those required for splicing regulation of all three targets, suggesting a role of HuC-HuC interaction in splicing regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
The four Hu [embryonic lethal abnormal vision-like (ELAVL)] protein family members regulate alternative splicing by binding to U-rich sequences surrounding target exons and affecting the interaction of the splicing machinery and/or local chromatin modifications. Each of the Hu proteins contains a divergent N-terminus, three highly conserved RNA recognition motifs (RRM1, RRM2 and RRM3) and a hinge region separating RRM2 and RRM3. The roles of each domain in splicing regulation are not well understood. Here, we investigate how HuC, a relatively poorly characterized family member, regulates three target pre-mRNAs: neurofibromatosis type I, Fas and HuD. We find that the HuC N-terminus is dispensable for splicing regulation, and the three RRMs are required for splicing regulation of each target, whereas the hinge region contributes to regulation of only some targets. Interestingly, the regions of the hinge and RRM3 required for regulating different targets only partially overlap, implying substrate-specific mechanisms of HuC-mediated splicing regulation. We show that RRM1 and RRM2 are required for binding to target pre-mRNAs, whereas the hinge and RRM3 are required for HuC-HuC self-interaction. Finally, we find that the portions of RRM3 required for HuC-HuC interaction overlap with those required for splicing regulation of all three targets, suggesting a role of HuC-HuC interaction in splicing regulation.

Show MeSH
Related in: MedlinePlus