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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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Inclusion of MAG Exon 12 is reduced upon deletion of intronic G-runs. (A) Schematic of the MAG minigene reporter constructs illustrating deletion of individual or both intronic G-runs (mouse sequence). (B) qRT-PCR detection of mRNA isoforms derived from AS of the MAG minigene reporter that carries wild-type intronic G-runs (WT), deletion of the 5′-G-run (ΔSite#1), 3′-G-run (ΔSite#2) or both G-runs (ΔSite#1+2) expressed in the neuronal cell line CAD. Minigene-isoform-specific primers (11) were used for qPCR, and the ΔΔCt of inclusion versus exclusion was calculated. The % change of Exon 12 inclusion in each mutant minigene was calculated and statistically compared with the WT minigene. (C and D) Semi-quantitative RT-PCR detects reduced Exon 12 inclusion of the MAG minigene when both G-runs are deleted (ΔSite#1+2) in the OL cell line CG4 (C) and the neuronal cell line CAD (D). The same primer set used in Figure 4 was employed for (C) and (D). The % inclusion of the alternative exon in each sample is calculated and results are statistically compared between the WT and mutant minigene.
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Figure 5: Inclusion of MAG Exon 12 is reduced upon deletion of intronic G-runs. (A) Schematic of the MAG minigene reporter constructs illustrating deletion of individual or both intronic G-runs (mouse sequence). (B) qRT-PCR detection of mRNA isoforms derived from AS of the MAG minigene reporter that carries wild-type intronic G-runs (WT), deletion of the 5′-G-run (ΔSite#1), 3′-G-run (ΔSite#2) or both G-runs (ΔSite#1+2) expressed in the neuronal cell line CAD. Minigene-isoform-specific primers (11) were used for qPCR, and the ΔΔCt of inclusion versus exclusion was calculated. The % change of Exon 12 inclusion in each mutant minigene was calculated and statistically compared with the WT minigene. (C and D) Semi-quantitative RT-PCR detects reduced Exon 12 inclusion of the MAG minigene when both G-runs are deleted (ΔSite#1+2) in the OL cell line CG4 (C) and the neuronal cell line CAD (D). The same primer set used in Figure 4 was employed for (C) and (D). The % inclusion of the alternative exon in each sample is calculated and results are statistically compared between the WT and mutant minigene.

Mentions: To directly test whether hnRNP F/H target the G-run elements to control inclusion of MAG Exon 12, we utilized a MAG minigene in which the genomic sequence for regulating AS of Exon 12 was fused in-frame downstream of the EGFP coding sequence (Figure 4A). Upon knockdown of hnRNP F/H, Exon 12 inclusion from the MAG minigene was significantly reduced in transfected CG4 cells (Figure 4B). Interestingly, hnRNP F/H knockdown in the neuronal cell line CAD that does not naturally express MAG also reduced Exon 12 inclusion from the minigene (Figure 4C). This result suggests that hnRNP F/H can regulate inclusion of MAG Exon 12 independent of OL-specific factors. Importantly, deletion of either G-run alone from the minigene, as illustrated in Figure 5A, reduced Exon 12 inclusion by 30–40% based on qRT-PCR (Figure 5B). Moreover, deletion of both G-runs led to an additive effect, resulting in greater than 60% reduction of Exon 12 inclusion (Figure 5B), which largely recapitulates the effects caused by hnRNP F/H knockdown (Figure 4B). Such effects were observed in CG4 and CAD cells (Figure 5C and D. These results identify both G-runs as equivalent functional targets of hnRNP F/H for regulating MAG Exon 12 inclusion. Furthermore, although hnRNP A1 also regulates AS of MAG (11), the G-run deletion still causes comparable reduction of Exon 12 inclusion when hnRNP A1 is knocked down (Supplementary Figure S4). This result suggests that hnRNP F/H can regulate inclusion of MAG Exon 12 through the G-runs independent of hnRNP A1.


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)

Inclusion of MAG Exon 12 is reduced upon deletion of intronic G-runs. (A) Schematic of the MAG minigene reporter constructs illustrating deletion of individual or both intronic G-runs (mouse sequence). (B) qRT-PCR detection of mRNA isoforms derived from AS of the MAG minigene reporter that carries wild-type intronic G-runs (WT), deletion of the 5′-G-run (ΔSite#1), 3′-G-run (ΔSite#2) or both G-runs (ΔSite#1+2) expressed in the neuronal cell line CAD. Minigene-isoform-specific primers (11) were used for qPCR, and the ΔΔCt of inclusion versus exclusion was calculated. The % change of Exon 12 inclusion in each mutant minigene was calculated and statistically compared with the WT minigene. (C and D) Semi-quantitative RT-PCR detects reduced Exon 12 inclusion of the MAG minigene when both G-runs are deleted (ΔSite#1+2) in the OL cell line CG4 (C) and the neuronal cell line CAD (D). The same primer set used in Figure 4 was employed for (C) and (D). The % inclusion of the alternative exon in each sample is calculated and results are statistically compared between the WT and mutant minigene.
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Related In: Results  -  Collection

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Figure 5: Inclusion of MAG Exon 12 is reduced upon deletion of intronic G-runs. (A) Schematic of the MAG minigene reporter constructs illustrating deletion of individual or both intronic G-runs (mouse sequence). (B) qRT-PCR detection of mRNA isoforms derived from AS of the MAG minigene reporter that carries wild-type intronic G-runs (WT), deletion of the 5′-G-run (ΔSite#1), 3′-G-run (ΔSite#2) or both G-runs (ΔSite#1+2) expressed in the neuronal cell line CAD. Minigene-isoform-specific primers (11) were used for qPCR, and the ΔΔCt of inclusion versus exclusion was calculated. The % change of Exon 12 inclusion in each mutant minigene was calculated and statistically compared with the WT minigene. (C and D) Semi-quantitative RT-PCR detects reduced Exon 12 inclusion of the MAG minigene when both G-runs are deleted (ΔSite#1+2) in the OL cell line CG4 (C) and the neuronal cell line CAD (D). The same primer set used in Figure 4 was employed for (C) and (D). The % inclusion of the alternative exon in each sample is calculated and results are statistically compared between the WT and mutant minigene.
Mentions: To directly test whether hnRNP F/H target the G-run elements to control inclusion of MAG Exon 12, we utilized a MAG minigene in which the genomic sequence for regulating AS of Exon 12 was fused in-frame downstream of the EGFP coding sequence (Figure 4A). Upon knockdown of hnRNP F/H, Exon 12 inclusion from the MAG minigene was significantly reduced in transfected CG4 cells (Figure 4B). Interestingly, hnRNP F/H knockdown in the neuronal cell line CAD that does not naturally express MAG also reduced Exon 12 inclusion from the minigene (Figure 4C). This result suggests that hnRNP F/H can regulate inclusion of MAG Exon 12 independent of OL-specific factors. Importantly, deletion of either G-run alone from the minigene, as illustrated in Figure 5A, reduced Exon 12 inclusion by 30–40% based on qRT-PCR (Figure 5B). Moreover, deletion of both G-runs led to an additive effect, resulting in greater than 60% reduction of Exon 12 inclusion (Figure 5B), which largely recapitulates the effects caused by hnRNP F/H knockdown (Figure 4B). Such effects were observed in CG4 and CAD cells (Figure 5C and D. These results identify both G-runs as equivalent functional targets of hnRNP F/H for regulating MAG Exon 12 inclusion. Furthermore, although hnRNP A1 also regulates AS of MAG (11), the G-run deletion still causes comparable reduction of Exon 12 inclusion when hnRNP A1 is knocked down (Supplementary Figure S4). This result suggests that hnRNP F/H can regulate inclusion of MAG Exon 12 through the G-runs independent of hnRNP A1.

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