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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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RRM1 and RRM2 of HuC are important for binding to the NF1 and HuD pre-mRNAs. (A) RNA gel mobility shift assay. Radioactively labelled RNA oligonucleotides (1 ng) corresponding to wild-type or mutant Hu regulatory sequences upstream of NF1 exon 23a (23) were incubated with 200 ng of GST or increasing amounts (10, 25 or 200 ng) of GST–HuC mutants and run on a non-denaturing polyacrylamide gel. At the bottom are sequences of the wild-type and mutant NF1 oligonucleotides with mutated nucleotides underlined. (B) RNA gel mobility shift assay. Radioactively labelled RNA oligonucleotides (1 ng) corresponding to wild-type Hu regulatory sequences upstream of HuD exon 6 (26) were incubated with 50 ng of GST or increasing amounts (2, 10 or 50 ng) of GST–HuC mutants and run on a non-denaturing polyacrylamide gel. Mutant RNA oligonucleotides were incubated with 50 ng of each GST protein. At the bottom are sequences of the wild-type and mutant HuD oligonucleotides with mutated nucleotides underlined.
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gkt166-F3: RRM1 and RRM2 of HuC are important for binding to the NF1 and HuD pre-mRNAs. (A) RNA gel mobility shift assay. Radioactively labelled RNA oligonucleotides (1 ng) corresponding to wild-type or mutant Hu regulatory sequences upstream of NF1 exon 23a (23) were incubated with 200 ng of GST or increasing amounts (10, 25 or 200 ng) of GST–HuC mutants and run on a non-denaturing polyacrylamide gel. At the bottom are sequences of the wild-type and mutant NF1 oligonucleotides with mutated nucleotides underlined. (B) RNA gel mobility shift assay. Radioactively labelled RNA oligonucleotides (1 ng) corresponding to wild-type Hu regulatory sequences upstream of HuD exon 6 (26) were incubated with 50 ng of GST or increasing amounts (2, 10 or 50 ng) of GST–HuC mutants and run on a non-denaturing polyacrylamide gel. Mutant RNA oligonucleotides were incubated with 50 ng of each GST protein. At the bottom are sequences of the wild-type and mutant HuD oligonucleotides with mutated nucleotides underlined.

Mentions: Recombinant GST proteins were prepared from bacteria expressing HuC proteins from the pGEX-2TK vector using the B-PER GST spin purification kit (Thermo Scientific) (Figure 7B). Gel mobility shift assays were performed as described previously (26). Briefly, for the NF1 gel mobility shift assay, 1 ng (80 fmol) of radiolabelled WT or mutant RNA oligonucleotides (Figure 3A) (Thermo Fisher Scientific Inc.) were incubated with 200 ng of GST or 10, 25 or 200 ng of GST–HuC fusion proteins in 25 µl of reaction buffer for 30 min at 30°C and run on a non-denaturing polyacrylamide gel. For the HuD gel mobility shift assay, 1 ng (80 fmol) of radiolabelled WT RNA oligonucleotide (Figure 3B) (Thermo Fisher Scientific Inc.) was incubated with 50 ng of GST or 2, 10 or 50 ng of GST-tagged recombinant HuC proteins for 30 min at 30°C in 25 µl of reaction buffer and run on a non-denaturing polyacrylamide gel. Mutant HuD RNA oligonucleotides were incubated with 50 ng of each GST protein.Figure 3.


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)

RRM1 and RRM2 of HuC are important for binding to the NF1 and HuD pre-mRNAs. (A) RNA gel mobility shift assay. Radioactively labelled RNA oligonucleotides (1 ng) corresponding to wild-type or mutant Hu regulatory sequences upstream of NF1 exon 23a (23) were incubated with 200 ng of GST or increasing amounts (10, 25 or 200 ng) of GST–HuC mutants and run on a non-denaturing polyacrylamide gel. At the bottom are sequences of the wild-type and mutant NF1 oligonucleotides with mutated nucleotides underlined. (B) RNA gel mobility shift assay. Radioactively labelled RNA oligonucleotides (1 ng) corresponding to wild-type Hu regulatory sequences upstream of HuD exon 6 (26) were incubated with 50 ng of GST or increasing amounts (2, 10 or 50 ng) of GST–HuC mutants and run on a non-denaturing polyacrylamide gel. Mutant RNA oligonucleotides were incubated with 50 ng of each GST protein. At the bottom are sequences of the wild-type and mutant HuD oligonucleotides with mutated nucleotides underlined.
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getmorefigures.php?uid=PMC3643579&req=5

gkt166-F3: RRM1 and RRM2 of HuC are important for binding to the NF1 and HuD pre-mRNAs. (A) RNA gel mobility shift assay. Radioactively labelled RNA oligonucleotides (1 ng) corresponding to wild-type or mutant Hu regulatory sequences upstream of NF1 exon 23a (23) were incubated with 200 ng of GST or increasing amounts (10, 25 or 200 ng) of GST–HuC mutants and run on a non-denaturing polyacrylamide gel. At the bottom are sequences of the wild-type and mutant NF1 oligonucleotides with mutated nucleotides underlined. (B) RNA gel mobility shift assay. Radioactively labelled RNA oligonucleotides (1 ng) corresponding to wild-type Hu regulatory sequences upstream of HuD exon 6 (26) were incubated with 50 ng of GST or increasing amounts (2, 10 or 50 ng) of GST–HuC mutants and run on a non-denaturing polyacrylamide gel. Mutant RNA oligonucleotides were incubated with 50 ng of each GST protein. At the bottom are sequences of the wild-type and mutant HuD oligonucleotides with mutated nucleotides underlined.
Mentions: Recombinant GST proteins were prepared from bacteria expressing HuC proteins from the pGEX-2TK vector using the B-PER GST spin purification kit (Thermo Scientific) (Figure 7B). Gel mobility shift assays were performed as described previously (26). Briefly, for the NF1 gel mobility shift assay, 1 ng (80 fmol) of radiolabelled WT or mutant RNA oligonucleotides (Figure 3A) (Thermo Fisher Scientific Inc.) were incubated with 200 ng of GST or 10, 25 or 200 ng of GST–HuC fusion proteins in 25 µl of reaction buffer for 30 min at 30°C and run on a non-denaturing polyacrylamide gel. For the HuD gel mobility shift assay, 1 ng (80 fmol) of radiolabelled WT RNA oligonucleotide (Figure 3B) (Thermo Fisher Scientific Inc.) was incubated with 50 ng of GST or 2, 10 or 50 ng of GST-tagged recombinant HuC proteins for 30 min at 30°C in 25 µl of reaction buffer and run on a non-denaturing polyacrylamide gel. Mutant HuD RNA oligonucleotides were incubated with 50 ng of each GST protein.Figure 3.

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