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A cytoplasmic quaking I isoform regulates the hnRNP F/H-dependent alternative splicing pathway in myelinating glia.

Mandler MD, Ku L, Feng Y - Nucleic Acids Res. (2014)

Bottom Line: We found that the cytoplasmic isoform QKI-6 regulates AS of polyguanine (G-run)-containing transcripts in OLs and rescues aberrant AS in the qk(v) mutant by repressing expression of two canonical splicing factors, heterologous nuclear ribonucleoproteins (hnRNPs) F and H.Interestingly, AS targets of the QKI-6-hnRNP F/H pathway in OLs are differentially affected in SWCs, suggesting that additional cell-type-specific factors modulate AS during CNS and PNS myelination.Together, our studies provide the first evidence that cytoplasmic QKI-6 acts upstream of hnRNP F/H, which forms a novel pathway to control AS in myelinating glia.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University, Atlanta, GA 30329, USA.

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hnRNP F/H promotes inclusion of Exon 12 in MAG pre-mRNA. (A) Schematic of inclusion or exclusion of the alternative Exon 12 (marked black) in MAG pre-mRNA. Exons are boxed/numbered, introns are displayed as lines, and intronic G-run (rat) sequences are indicated. Primers that simultaneously detect AS isoforms of MAG mRNAs are depicted by half-arrows. (B) Immunoblot detects siRNA knockdown of hnRNP F/H (siF/H) as compared to negative control siRNA (siNC) in CG4 cells (top panel). Signal density of hnRNP F/H was normalized to β-actin and graphically displayed (bottom panel). (C and D) Representative image of semi-quantitative RT-PCR products (top panels) of alternatively spliced endogenous MAG pre-mRNA (C, S-MAG and L-MAG) and PLP pre-mRNA (D, PLP/DM20). The alternative exon is marked black and inclusion is depicted on the side for the corresponding band. The % inclusion of the alternative exon in each sample is calculated and results are statistically compared between siF/H and siNC-treated cells (bottom panels).
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Figure 3: hnRNP F/H promotes inclusion of Exon 12 in MAG pre-mRNA. (A) Schematic of inclusion or exclusion of the alternative Exon 12 (marked black) in MAG pre-mRNA. Exons are boxed/numbered, introns are displayed as lines, and intronic G-run (rat) sequences are indicated. Primers that simultaneously detect AS isoforms of MAG mRNAs are depicted by half-arrows. (B) Immunoblot detects siRNA knockdown of hnRNP F/H (siF/H) as compared to negative control siRNA (siNC) in CG4 cells (top panel). Signal density of hnRNP F/H was normalized to β-actin and graphically displayed (bottom panel). (C and D) Representative image of semi-quantitative RT-PCR products (top panels) of alternatively spliced endogenous MAG pre-mRNA (C, S-MAG and L-MAG) and PLP pre-mRNA (D, PLP/DM20). The alternative exon is marked black and inclusion is depicted on the side for the corresponding band. The % inclusion of the alternative exon in each sample is calculated and results are statistically compared between siF/H and siNC-treated cells (bottom panels).

Mentions: We next asked whether hnRNP F/H might control AS of pre-mRNAs that are dysregulated in the qkv/qkv OLs. Inclusion of MAG Exon 12 is aberrantly increased in qkv/qkv OLs (11), which is rescued by the FLAG-QKI-6 transgene (11,23). Noticeably, long stretches of G-runs, analogous to the consensus sequence targeted by hnRNP F/H in numerous pre-mRNAs (25,34–35), are found in the introns within 250 nt of both the 3′ and 5′ splice sites that define Exon 12 in the mouse, rat and human MAG gene (Supplementary Figure S3), all within optimal distance known for regulation by hnRNP F/H (26). To test whether hnRNP F/H may regulate AS of endogenous MAG pre-mRNA that harbor G-runs flanking Exon 12 (Figure 3A), we knocked down hnRNP F and hnRNP H simultaneously in the rat OL cell line CG4 (Figure 3B) using a previously validated siRNA (25). As a result, inclusion of Exon 12 was significantly reduced based on semi-quantitative RT-PCR that detects both MAG mRNA isoforms with a single primer set (Figure 3C). Knockdown hnRNP F/H in CG4 cells in a parallel experiment also significantly affected AS of the PLP pre-mRNA (Figure 3D), which is a well-characterized target of hnRNP F/H (25,28,34).


A cytoplasmic quaking I isoform regulates the hnRNP F/H-dependent alternative splicing pathway in myelinating glia.

Mandler MD, Ku L, Feng Y - Nucleic Acids Res. (2014)

hnRNP F/H promotes inclusion of Exon 12 in MAG pre-mRNA. (A) Schematic of inclusion or exclusion of the alternative Exon 12 (marked black) in MAG pre-mRNA. Exons are boxed/numbered, introns are displayed as lines, and intronic G-run (rat) sequences are indicated. Primers that simultaneously detect AS isoforms of MAG mRNAs are depicted by half-arrows. (B) Immunoblot detects siRNA knockdown of hnRNP F/H (siF/H) as compared to negative control siRNA (siNC) in CG4 cells (top panel). Signal density of hnRNP F/H was normalized to β-actin and graphically displayed (bottom panel). (C and D) Representative image of semi-quantitative RT-PCR products (top panels) of alternatively spliced endogenous MAG pre-mRNA (C, S-MAG and L-MAG) and PLP pre-mRNA (D, PLP/DM20). The alternative exon is marked black and inclusion is depicted on the side for the corresponding band. The % inclusion of the alternative exon in each sample is calculated and results are statistically compared between siF/H and siNC-treated cells (bottom panels).
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Figure 3: hnRNP F/H promotes inclusion of Exon 12 in MAG pre-mRNA. (A) Schematic of inclusion or exclusion of the alternative Exon 12 (marked black) in MAG pre-mRNA. Exons are boxed/numbered, introns are displayed as lines, and intronic G-run (rat) sequences are indicated. Primers that simultaneously detect AS isoforms of MAG mRNAs are depicted by half-arrows. (B) Immunoblot detects siRNA knockdown of hnRNP F/H (siF/H) as compared to negative control siRNA (siNC) in CG4 cells (top panel). Signal density of hnRNP F/H was normalized to β-actin and graphically displayed (bottom panel). (C and D) Representative image of semi-quantitative RT-PCR products (top panels) of alternatively spliced endogenous MAG pre-mRNA (C, S-MAG and L-MAG) and PLP pre-mRNA (D, PLP/DM20). The alternative exon is marked black and inclusion is depicted on the side for the corresponding band. The % inclusion of the alternative exon in each sample is calculated and results are statistically compared between siF/H and siNC-treated cells (bottom panels).
Mentions: We next asked whether hnRNP F/H might control AS of pre-mRNAs that are dysregulated in the qkv/qkv OLs. Inclusion of MAG Exon 12 is aberrantly increased in qkv/qkv OLs (11), which is rescued by the FLAG-QKI-6 transgene (11,23). Noticeably, long stretches of G-runs, analogous to the consensus sequence targeted by hnRNP F/H in numerous pre-mRNAs (25,34–35), are found in the introns within 250 nt of both the 3′ and 5′ splice sites that define Exon 12 in the mouse, rat and human MAG gene (Supplementary Figure S3), all within optimal distance known for regulation by hnRNP F/H (26). To test whether hnRNP F/H may regulate AS of endogenous MAG pre-mRNA that harbor G-runs flanking Exon 12 (Figure 3A), we knocked down hnRNP F and hnRNP H simultaneously in the rat OL cell line CG4 (Figure 3B) using a previously validated siRNA (25). As a result, inclusion of Exon 12 was significantly reduced based on semi-quantitative RT-PCR that detects both MAG mRNA isoforms with a single primer set (Figure 3C). Knockdown hnRNP F/H in CG4 cells in a parallel experiment also significantly affected AS of the PLP pre-mRNA (Figure 3D), which is a well-characterized target of hnRNP F/H (25,28,34).

Bottom Line: We found that the cytoplasmic isoform QKI-6 regulates AS of polyguanine (G-run)-containing transcripts in OLs and rescues aberrant AS in the qk(v) mutant by repressing expression of two canonical splicing factors, heterologous nuclear ribonucleoproteins (hnRNPs) F and H.Interestingly, AS targets of the QKI-6-hnRNP F/H pathway in OLs are differentially affected in SWCs, suggesting that additional cell-type-specific factors modulate AS during CNS and PNS myelination.Together, our studies provide the first evidence that cytoplasmic QKI-6 acts upstream of hnRNP F/H, which forms a novel pathway to control AS in myelinating glia.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University, Atlanta, GA 30329, USA.

Show MeSH