The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination - PubMed (original) (raw)

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

Daniel Larocque et al. PLoS One. 2009.

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The QKI isoforms induce Schwann cell cycle arrest.

Primary Schwann cells/neuron co-cultures were infected for 48 hr with control GFP adenovirus (A and B) and adenoviruses encoding for QKI-6 (C and D), QKI-7 (E and F), and the combination of QKI-6/7 (G and H). The green fluorescence denotes infected cells (panels A, C, E and G). Incorporation of BrdU (16 hr pulse) was detected with an anti-BrdU antibody followed by a goat anti-mouse conjugated to Alexa 546 (red) (B, D, F and H). Arrowheads denote the GFP positive Schwann cells that are also BrdU positive in the controls infected with GFP-adenovirus. (I). The expression of each QKI isoform causes inhibition of Schwann cell proliferation. The graph shows the percentage of GFP positive cells that are also BrdU positive. The proliferation was quantified from _n_>500 cells from three different experiments (p<0.001, ANOVA). The error bars represent +/− standard deviation of the mean.

Figure 2

Figure 2. QKI-6 and QKI-7 induce the expression of Schwann cell differentiation markers and enhance their maturation.

Co-cultures of Schwann cells/neurons were transduced with control adenoviruses (Adv GFP, A and B), adenoviruses encoding QKI-6 (C and D), QKI-7 (E and F), or a combination of QKI-6/7 (G and H). Myelination was induced for 5 days with ascorbic acid and the culture stained with anti-MBP antibodies (red). (I) Parallel cultures were lysed and analyzed by immunoblotting for MBP, and β-actin was used to ensure equal loading. The relative band intensities quantified by densitometry are indicated below.

Figure 3

Figure 3. Electron microscopy of the myelin layers of the QKI-transduced Schwann cells/neurons co-cultures.

Co-cultures of Schwann cells/neurons were transduced with adenoviruses encoding GFP alone (A and B), QKI-6 (C and D), QKI-7 (E and F), combination of QKI-6 and QKI-7 (G to J). Myelin layers were analyzed using electron microscopy. Each picture is a representative image from 30 different axons for each treatment showing a detailed view of the myelin sheath.

Figure 4

Figure 4. QKI-6/7 transduced Schwann cells/neurons co-cultures display elevated nuclear p27KIP1 staining and directional MBP nerve fibers.

Co-cultures of Schwann cells/neurons were transduced with adenoviruses coding for GFP (A, B and C), QKI-6 (D, E and F), QKI-7 (G, H and I), or the combination of QKI-6 and QKI-7 (J, K and L). After infection, co-cultures were allowed to myelinate for 5 days with ascorbic acid. Cells were immunostained with anti-p27KIP1 antibodies (red), anti-MBP antibodies (purple) and visualized by fluorescence confocal microscopy. The green cells denote infected Schwann cells as well as infected axons. Ectopic expression of QKI-6 (E), QKI-7 (H) and QKI-6/7 combination (K) activate the expression of p27KIP1 in the nucleus of Schwann cells. Schwann cells are in the vicinity of MBP+ axons fibers (F, I and L). The inset (L) is an image showing a merge from p27KIP1-positive Schwann cells and MBP-positive axons (arrowheads). (M) This image shows the double staining for p27KIP1 (red) and MBP (purple) from a combined infection by QKI-6 and QKI-7 adenoviruses and demonstrate that p27KIP1 Schwann cells are associated with MBP-positive axonal internodes (arrows). (N) Denotes the green fluorescence of the GFP-positive cells of panel M. (O) Schwann/DRGN co-cultures infected with control or the indicated QKI adenoviruses were lysed and the proteins analyzed by immunoblotting for p27KIP1 and β-actin to ensure equal loading. The relative band intensities quantified by densitometry are indicated below.

Figure 5

Figure 5. 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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