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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 three HuC RNA recognition motifs and the hinge region are all important for splicing regulation in HeLa cells. (A) Schematic of HuC domain deletion mutants (35). Dotted lines indicate the deleted portion of the protein. (B–D) Reporter NF1 exon 23a (23) (B), endogenous Fas exon 6 (C) and reporter HuD exon 6 (26) (D) inclusion in HeLa cells after expression of HuC proteins as measured by RT–PCR. Western blot analysis using anti-Myc antibody to detect Myc-tagged HuC domain deletion constructs. U1 70K and γ-tubulin are loading controls. Arrows indicate locations of RT–PCR primers. Error bars represent 1 SD. N > 3.
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gkt166-F1: The three HuC RNA recognition motifs and the hinge region are all important for splicing regulation in HeLa cells. (A) Schematic of HuC domain deletion mutants (35). Dotted lines indicate the deleted portion of the protein. (B–D) Reporter NF1 exon 23a (23) (B), endogenous Fas exon 6 (C) and reporter HuD exon 6 (26) (D) inclusion in HeLa cells after expression of HuC proteins as measured by RT–PCR. Western blot analysis using anti-Myc antibody to detect Myc-tagged HuC domain deletion constructs. U1 70K and γ-tubulin are loading controls. Arrows indicate locations of RT–PCR primers. Error bars represent 1 SD. N > 3.

Mentions: An unanswered question is how the domain structure of Hu proteins influences their splicing regulatory functions. The four mammalian Hu proteins are highly conserved among each other and share a similar domain structure (Figure 1A) (36). Each has three RNA recognition motifs (RRMs), referred to as RRM1, RRM2 and RRM3, that share >90% sequence identity among the Hu family members. In between RRM2 and RRM3 is a basic hinge region, and each Hu protein contains a unique short N-terminal region. Although Hu proteins contain three RRMs, it is RRM1 and RRM2 that cooperate to recognize and bind to U-rich sequences (37–41). RRM1 and RRM2 are required for binding to and regulating numerous mRNA targets, although this has not yet been shown for pre-mRNA splicing targets (38,42–46).Figure 1.


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 three HuC RNA recognition motifs and the hinge region are all important for splicing regulation in HeLa cells. (A) Schematic of HuC domain deletion mutants (35). Dotted lines indicate the deleted portion of the protein. (B–D) Reporter NF1 exon 23a (23) (B), endogenous Fas exon 6 (C) and reporter HuD exon 6 (26) (D) inclusion in HeLa cells after expression of HuC proteins as measured by RT–PCR. Western blot analysis using anti-Myc antibody to detect Myc-tagged HuC domain deletion constructs. U1 70K and γ-tubulin are loading controls. Arrows indicate locations of RT–PCR primers. Error bars represent 1 SD. N > 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt166-F1: The three HuC RNA recognition motifs and the hinge region are all important for splicing regulation in HeLa cells. (A) Schematic of HuC domain deletion mutants (35). Dotted lines indicate the deleted portion of the protein. (B–D) Reporter NF1 exon 23a (23) (B), endogenous Fas exon 6 (C) and reporter HuD exon 6 (26) (D) inclusion in HeLa cells after expression of HuC proteins as measured by RT–PCR. Western blot analysis using anti-Myc antibody to detect Myc-tagged HuC domain deletion constructs. U1 70K and γ-tubulin are loading controls. Arrows indicate locations of RT–PCR primers. Error bars represent 1 SD. N > 3.
Mentions: An unanswered question is how the domain structure of Hu proteins influences their splicing regulatory functions. The four mammalian Hu proteins are highly conserved among each other and share a similar domain structure (Figure 1A) (36). Each has three RNA recognition motifs (RRMs), referred to as RRM1, RRM2 and RRM3, that share >90% sequence identity among the Hu family members. In between RRM2 and RRM3 is a basic hinge region, and each Hu protein contains a unique short N-terminal region. Although Hu proteins contain three RRMs, it is RRM1 and RRM2 that cooperate to recognize and bind to U-rich sequences (37–41). RRM1 and RRM2 are required for binding to and regulating numerous mRNA targets, although this has not yet been shown for pre-mRNA splicing targets (38,42–46).Figure 1.

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