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The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

Larocque D, Fragoso G, Huang J, Mushynski WE, Loignon M, Richard S, Almazan G - PLoS ONE (2009)

Bottom Line: In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation.Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital, Department of Oncology and Medicine, McGill University, Montréal, Québec, Canada.

ABSTRACT

Background: The quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v) mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.

Methodology/principal findings: To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.

Conclusions/significance: Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

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QKI-6 and QKI-7 deficient Schwann cells have reduced levels of MBP, Krox-20 and p27KIP1 mRNAs.(A) C6 rat glioma cells were transfected with QKI-specific siGENOME SMARTpool or luciferase specific siRNAs control (siCtrl), lysed and proteins were immunoblotted for QKI-6 and QKI-7 isoforms; Sam68 was used to ensure equal loading. The quantification by densitometric scanning is indicated below. (B) Co-cultures of Schwann cells/neurons were transfected with siCtrl or QKI-specific siGENOME SMARTpools in combination with siGlo, a transfection indicator. Transfection efficiency was assessed by the percentage of DAPI stained Schwann cells (blue) that incorporated siGlo (red) in the perinuclear region, and siGlo signal was absent in non-transfected co-cultures. (C) The mRNA levels of QKI, MBP, p27KIP1 and Krox-20 in siRNA-treated was quantified by real-time RT-PCR. The bars depict the mean plus the standard error of the mean (error bars) from three separate experiments.
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pone-0005867-g005: QKI-6 and QKI-7 deficient Schwann cells have reduced levels of MBP, Krox-20 and p27KIP1 mRNAs.(A) C6 rat glioma cells were transfected with QKI-specific siGENOME SMARTpool or luciferase specific siRNAs control (siCtrl), lysed and proteins were immunoblotted for QKI-6 and QKI-7 isoforms; Sam68 was used to ensure equal loading. The quantification by densitometric scanning is indicated below. (B) Co-cultures of Schwann cells/neurons were transfected with siCtrl or QKI-specific siGENOME SMARTpools in combination with siGlo, a transfection indicator. Transfection efficiency was assessed by the percentage of DAPI stained Schwann cells (blue) that incorporated siGlo (red) in the perinuclear region, and siGlo signal was absent in non-transfected co-cultures. (C) The mRNA levels of QKI, MBP, p27KIP1 and Krox-20 in siRNA-treated was quantified by real-time RT-PCR. The bars depict the mean plus the standard error of the mean (error bars) from three separate experiments.

Mentions: To confirm the positive regulatory role of ectopically expressed QKI-6 and QKI-7 in Schwann cell maturation, we used a knockdown approach using RNA interference to reduce the expression of the endogenous QKI-6 and QKI-7 isoforms. We first verified the efficiency of siGENOME SMARTpool siRNA targeting the rat QKI isoforms in C6 rat glioma cells. C6 glioma cells were transfected with QKI siRNAa for 72 hrs and the prepared cell lysates were immunoblotted with anti-QKI-6 or anti-QKI-7 antibodies and anti-Sam68 antibodies were used as a loading control. We observed that in comparison to control cultures (siCtrl), both QKI-6 and QKI-7 isoforms were down-regulated by >80% by QKI-specific siRNA (siQKI, Figure 5A). We next reduced the levels of QKI expression in Schwann/DRGN cultures using the same strategy except we used a more sensitive assay, quantitative RT-PCR, to monitor knockdown in rat Schwann cells [39]. To monitor the efficiency of siRNA delivery in the Schwann/DRGN cultures, we also included the siGlo transfection indicator. We observed a >70% transfection efficiency (Figure. 5B) which correlated with an ∼50% reduction in QKI isoform expression, as assessed by qRT-PCR (Figure 5C). Interestingly, a significant reduction in MBP, p27KIP1, and Krox-20 mRNA levels normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was observed (Figure 5C). Together these data further suggest that the QKI isoforms are essential regulators of Schwann cell myelination.


The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

Larocque D, Fragoso G, Huang J, Mushynski WE, Loignon M, Richard S, Almazan G - PLoS ONE (2009)

QKI-6 and QKI-7 deficient Schwann cells have reduced levels of MBP, Krox-20 and p27KIP1 mRNAs.(A) C6 rat glioma cells were transfected with QKI-specific siGENOME SMARTpool or luciferase specific siRNAs control (siCtrl), lysed and proteins were immunoblotted for QKI-6 and QKI-7 isoforms; Sam68 was used to ensure equal loading. The quantification by densitometric scanning is indicated below. (B) Co-cultures of Schwann cells/neurons were transfected with siCtrl or QKI-specific siGENOME SMARTpools in combination with siGlo, a transfection indicator. Transfection efficiency was assessed by the percentage of DAPI stained Schwann cells (blue) that incorporated siGlo (red) in the perinuclear region, and siGlo signal was absent in non-transfected co-cultures. (C) The mRNA levels of QKI, MBP, p27KIP1 and Krox-20 in siRNA-treated was quantified by real-time RT-PCR. The bars depict the mean plus the standard error of the mean (error bars) from three separate experiments.
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pone-0005867-g005: QKI-6 and QKI-7 deficient Schwann cells have reduced levels of MBP, Krox-20 and p27KIP1 mRNAs.(A) C6 rat glioma cells were transfected with QKI-specific siGENOME SMARTpool or luciferase specific siRNAs control (siCtrl), lysed and proteins were immunoblotted for QKI-6 and QKI-7 isoforms; Sam68 was used to ensure equal loading. The quantification by densitometric scanning is indicated below. (B) Co-cultures of Schwann cells/neurons were transfected with siCtrl or QKI-specific siGENOME SMARTpools in combination with siGlo, a transfection indicator. Transfection efficiency was assessed by the percentage of DAPI stained Schwann cells (blue) that incorporated siGlo (red) in the perinuclear region, and siGlo signal was absent in non-transfected co-cultures. (C) The mRNA levels of QKI, MBP, p27KIP1 and Krox-20 in siRNA-treated was quantified by real-time RT-PCR. The bars depict the mean plus the standard error of the mean (error bars) from three separate experiments.
Mentions: To confirm the positive regulatory role of ectopically expressed QKI-6 and QKI-7 in Schwann cell maturation, we used a knockdown approach using RNA interference to reduce the expression of the endogenous QKI-6 and QKI-7 isoforms. We first verified the efficiency of siGENOME SMARTpool siRNA targeting the rat QKI isoforms in C6 rat glioma cells. C6 glioma cells were transfected with QKI siRNAa for 72 hrs and the prepared cell lysates were immunoblotted with anti-QKI-6 or anti-QKI-7 antibodies and anti-Sam68 antibodies were used as a loading control. We observed that in comparison to control cultures (siCtrl), both QKI-6 and QKI-7 isoforms were down-regulated by >80% by QKI-specific siRNA (siQKI, Figure 5A). We next reduced the levels of QKI expression in Schwann/DRGN cultures using the same strategy except we used a more sensitive assay, quantitative RT-PCR, to monitor knockdown in rat Schwann cells [39]. To monitor the efficiency of siRNA delivery in the Schwann/DRGN cultures, we also included the siGlo transfection indicator. We observed a >70% transfection efficiency (Figure. 5B) which correlated with an ∼50% reduction in QKI isoform expression, as assessed by qRT-PCR (Figure 5C). Interestingly, a significant reduction in MBP, p27KIP1, and Krox-20 mRNA levels normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was observed (Figure 5C). Together these data further suggest that the QKI isoforms are essential regulators of Schwann cell myelination.

Bottom Line: In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation.Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital, Department of Oncology and Medicine, McGill University, Montréal, Québec, Canada.

ABSTRACT

Background: The quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v) mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.

Methodology/principal findings: To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.

Conclusions/significance: Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

Show MeSH
Related in: MedlinePlus