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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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HuC RRM1, RRM2, RRM3 and hinge are all important for splicing regulation in a neuron-like cell type. (A and B) Splicing pattern for NF1 exon 23a (A) and HuD exon 6 (B) reporters in CA77 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 is a loading control. Arrows indicate locations of RT–PCR primers. Error bars represent 1 SD. N = 3.
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gkt166-F2: HuC RRM1, RRM2, RRM3 and hinge are all important for splicing regulation in a neuron-like cell type. (A and B) Splicing pattern for NF1 exon 23a (A) and HuD exon 6 (B) reporters in CA77 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 is a loading control. Arrows indicate locations of RT–PCR primers. Error bars represent 1 SD. N = 3.

Mentions: For reverse transcription (RT)–PCR experiments, 0.5–1 µg of splicing reporter plasmid (HMT-NF1 863/499 WT for NF1, E6 for HuD or no splicing reporter for Fas) and 2–4 µg of Myc-tagged HuC constructs (Figures 1, 2 and 4) or Xpress-tagged HuC splice variants (Figure 5) were co-transfected into HeLa cells or CA77 cells that were grown on 6-cm plates. RNA and protein were harvested 24–48 h post-transfection, and RT-PCR was performed as described previously (59). Primers 1 and 2 (Supplementary Table S2) and 17–20 cycles were used for HuD and NF1 splicing reporter RT–PCR. Primers 3 and 4 (Supplementary Table S2) and 22–25 cycles were used for endogenous Fas RT–PCR. Percentage exon inclusion {[exon included/(exon included + exon skipped)] × 100} was measured using a Typhoon Trio Variable Mode Imager (GE Healthcare), and results were averaged using at least three independent experiments. To measure protein expression, western blot analysis was performed using 20–50 µg of total protein and anti-Myc (1:10 000, Invitrogen) or anti-Xpress (1:3500, Invitrogen), as well as anti-U1 70K (1:250, a gift from Susan Berget, Baylor College of Medicine) or anti-γ-tubulin (1:10 000, Sigma) as primary antibodies and goat anti-mouse IgG (1:2000, Thermo Scientific) as the secondary antibody.Figure 2.


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)

HuC RRM1, RRM2, RRM3 and hinge are all important for splicing regulation in a neuron-like cell type. (A and B) Splicing pattern for NF1 exon 23a (A) and HuD exon 6 (B) reporters in CA77 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 is a loading control. 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-F2: HuC RRM1, RRM2, RRM3 and hinge are all important for splicing regulation in a neuron-like cell type. (A and B) Splicing pattern for NF1 exon 23a (A) and HuD exon 6 (B) reporters in CA77 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 is a loading control. Arrows indicate locations of RT–PCR primers. Error bars represent 1 SD. N = 3.
Mentions: For reverse transcription (RT)–PCR experiments, 0.5–1 µg of splicing reporter plasmid (HMT-NF1 863/499 WT for NF1, E6 for HuD or no splicing reporter for Fas) and 2–4 µg of Myc-tagged HuC constructs (Figures 1, 2 and 4) or Xpress-tagged HuC splice variants (Figure 5) were co-transfected into HeLa cells or CA77 cells that were grown on 6-cm plates. RNA and protein were harvested 24–48 h post-transfection, and RT-PCR was performed as described previously (59). Primers 1 and 2 (Supplementary Table S2) and 17–20 cycles were used for HuD and NF1 splicing reporter RT–PCR. Primers 3 and 4 (Supplementary Table S2) and 22–25 cycles were used for endogenous Fas RT–PCR. Percentage exon inclusion {[exon included/(exon included + exon skipped)] × 100} was measured using a Typhoon Trio Variable Mode Imager (GE Healthcare), and results were averaged using at least three independent experiments. To measure protein expression, western blot analysis was performed using 20–50 µg of total protein and anti-Myc (1:10 000, Invitrogen) or anti-Xpress (1:3500, Invitrogen), as well as anti-U1 70K (1:250, a gift from Susan Berget, Baylor College of Medicine) or anti-γ-tubulin (1:10 000, Sigma) as primary antibodies and goat anti-mouse IgG (1:2000, Thermo Scientific) as the secondary antibody.Figure 2.

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