Identification of binding sites for hnRNP-K within the MBP mRNA. (A) Localization within the MBP mRNA of 12 RNA probes, synthesized and used for binding analysis. All probes are located in sequence stretches containing the UC_3–4(U/A) consensus motif (Thisted et al., 2001). In addition, a probe containing the hnRNP-A2-binding site, A2RE (p761–780), was used as a negative control. Probe sequences are specified in supplementary material Fig. S2A. (B) EMSA performed with 3′-end-biotinylated RNA probes (0.125 µM). The identity of the probes is indicated above the lanes. The absence (−) or presence (+) of purified recombinant hnRNP-K (1.2 µM) is indicated below the gel images. (C) Selected probes were analyzed as in B but with increasing concentration (150 nM–1.0 µM) of purified recombinant hnRNP-K. The EMSA results shown in B and C are representative of at least three independent experiments.

hnRNP-K and hnRNP-A2 are not involved in translational inhibition. (A) Western blotting for hnRNP-A2, hnRNP-K and MBP expression in oligodendrocytes generated from oligodendrocyte precursors following mock transfection (C1), or following transfection with non-targeting siRNA (C2) or siRNA targeting hnRNP-A2, hnRNP-K or both hnRNP-A2 and hnRNP-K, as indicated. Actin was used as a loading control. Note that knockdown of hnRNP-A2 and hnRNP-K, either individually or in combination, caused reduced MBP expression. (B) Immunocytochemistry for MBP (red) in oligodendrocytes generated from oligodendrocyte precursors that were transfected with non-targeting siRNA (control) or transfected with siRNA targeting either hnRNP-A2 or hnRNP-K, or both hnRNP-A2 and hnRNP-K, as indicated. Cells are also stained for MAG (green), a marker of mature oligodendrocytes. (C) The percentage of mature (MAG-positive) cells. Note that knockdown of either hnRNP-A2 or hnRNP-K, or the combined knockdown, did not have any effect on the percentage of mature cells. (D) The percentage of mature cells, which are positive for MBP. Note that knockdown of either hnRNP-A2 or hnRNP-K, or the combined knockdown, reduced the percentage of MBP-positive cells. (E) The morphology of the mature cells was scored as ‘simple’, ‘complex’ or ‘membrane’. The percentage of cells in each category is shown. Note that knockdown of neither hnRNP-A2 or hnRNP-K, nor the combined knockdown, had any effect on the morphology of the cells. (F) Using the EGFP reporter assay (Fig. 1), the effect on translational inhibition of a deletion (-RTS) or a disrupting point mutation (A8G) in the hnRNP-A2 binding site of the 3′-UTR of the MBP mRNA was analyzed. Note that neither the removal of the entire RTS sequence nor a mutation in the hnRNP-A2-binding site had any effect on the translational inhibition exerted by the 3′-UTR. (C–F) The cells were analyzed after 2.5 days in culture, and the data represent the mean±s.d. of at least three independent experiments. (C–E) For each independent experiment, ten randomly selected pictures from each of two coverslips were analyzed (∼200 cells per coverslip). *P<0.05; ****P<0.0001; ns, non-significant (one-way ANOVA followed by Tukey's multiple comparison test).

MBP expression is regulated by hnRNP-E1. (A) Immunocytochemistry of oligodendrocytes generated from oligodendrocyte precursors that were transfected with EGFP (green) or hnRNP-E1-FLAG (green), and were stained 2 days after their transfer to differentiation medium. The cells were also stained for MBP (red) and O4 (blue), a marker of maturing oligodendrocytes. The percentage of O4-positive cells that were also positive for MBP at day 2 (B) and day 3 (C), and the percentage of oligodendrocytes showing complex branching morphology at day 2 (D) and day 3 (E), are shown. The data represent the mean±s.d. of three independent experiments, with at least 130 transfected cells analyzed in each independent experiment. *P<0.05; **P<0.01; ns, non-significant (Student's t-test). (F–L) Oligodendrocyte precursors were mock transfected (C1), or transfected with non-targeting siRNA (C2) or siRNA targeting hnRNP-E1. (F) At 1.5 days after transfer to differentiation medium, the cells were stained for MBP (red) or MAG (green), a marker of mature oligodendrocytes. (G) The percentage of MBP-positive mature oligodendrocytes is shown. (H) The distribution of the mature cells between ‘simple’, ‘complex’ or ‘membrane’ morphology. A similar experiment was carried out in which the cells were stained and analyzed at 2.5 days after transfer to differentiation medium (I,J,L). In this experiment, the expression of hnRNP-E1 and MBP was assessed by western blotting using actin as a loading control (K). The percentage of MAG-positive cells at day 1.5 and 2.5 were ∼25% and 50%, respectively. The data show the mean±s.d. of three independent experiments. For each experiment, 10 randomly selected pictures from each of two coverslips were analyzed (∼200 cells per coverslip). *P<0.05; **P<0.01; ***P<0.001; ns, non-significant (one-way ANOVA followed by Tukey's multiple comparison test).

hnRNP-E1 binds directly to MBP mRNA and competes with hnRNP-K for binding. (A) RNA-immunoprecipitation from lysates of oligodendrocyte precursors that were differentiated for 4 days, using antibodies against hnRNP-E1 or hnRNP-K, or control IgG. The amount of co-precipitated MBP, PLP or G6PDH mRNA was quantified by using qRT-PCR, and the enrichment was calculated relative to the amount of mRNA obtained by immunoprecipitation with control IgG antibody. Data show the mean±s.d. of three independent experiments. Statistical significance was analyzed by one-way ANOVA followed by Tukey's multiple comparison test. (B) The localization of seven RNA probes within the MBP mRNA that were used for binding analysis. All probes are located in sequence stretches containing the [(A/U)C_3–5(A/U)-n_x-(A/U)C_3–5(A/U)] motif. A probe containing the hnRNP-A2-binding site, A2RE (p761–780), was used as a negative control. Probe sequences are specified in supplementary material Fig. S2B. (C) EMSA carried out with 3′-end-biotinylated RNA probes (0.125 µM). Probe identities are shown above the images, and the absence (−) or presence (+) of purified recombinant hnRNP-E1 (1.2 µM) is indicated below the gel images. (D) Selected probes were analyzed as in B but with increasing concentration (150 nM–1.0 µM) of purified recombinant hnRNP-E1. The images shown in C and D are representative of at least three independent experiments. (E) Schematic representation of the identified binding sites of hnRNP-E1 and hnRNP-K within the MBP mRNA. (F) In vitro transcribed MBP mRNA was preincubated with recombinant purified hnRNP-E1, followed by competition by hnRNP-K that was present at twofold or fivefold molar excess, as indicated. The mRNA that was associated with hnRNP-E1 was immunoprecipitated using antibodies against hnRNP-E1. The amount of co-precipitated MBP mRNA was quantified by using qRT-PCR, and the relative level of mRNA, compared with that obtained by using control IgG antibody incubated with mRNA without hnRNP-E1, was calculated. Data show the mean±s.e.m. of four independent experiments. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. *P<0.05; ***P<0.001; ns, non-significant.

hnRNP-K and hnRNP-E1 show different binding to hnRNP-A2 and distinct localization within differentiating oligodendrocytes. (A) Immunoprecipitation from lysates of mature oligodendrocytes using control IgG, or antibodies against hnRNP-K or hnRNP-E1, followed by incubation with or without RNase. The precipitates were analyzed by western blotting for hnRNP-E1, hnRNP-K and hnRNP-A2, as indicated. Note that in the absence of RNase treatment, hnRNP-A2 co-precipitated with both hnRNP-K and hnRNP-E1. No co-precipitation of hnRNP-K with hnRNP-E1 was observed. Also note that RNase treatment abolished the co-precipitation of hnRNP-A2 with hnRNP-K, whereas co-precipitation of hnRNP-E1 and hnRNP-A2 was still evident in the RNase-treated samples. (B) Western blots of lysates of oligodendrocyte precursor cells cultured for 1–4 days were probed for hnRNP-E1, hnRNP-K and MBP. Actin was used as a loading control. Note that differentiation affects the expression level of MBP but not that of hnRNP-E1 or hnRNP-K. (C) Oligodendrocyte precursors differentiated for 3 days in culture were stained with antibodies against hnRNP-K (red), hnRNP-E1 (green) and MBP (blue), as indicated. A representative MBP-negative cell (upper panels) and MBP-positive cell (lower panels) is shown. Note that hnRNP-K is localized mainly in the nucleus of the MBP-negative cell, but is also present in granules of the developing myelin sheets in the MBP-positive cell. By contrast, hnRNP-E1 is present mainly in the cell body and primary processes of both MBP-negative and MBP-positive cells. The enlargement of the boxed region in the middle lower panel shows that hnRNP-K and hnRNP-E1 are not colocalized outside the nucleus. Of more than 5000 hnRNP-K-positive granules in 15 cells, less than 3% were found to also contain hnRNP-E1.

hnRNP-K induces expression of MBP. (A) MBP mRNA (20 ng/µl), including the 3′-UTR, was in vitro translated using a rabbit reticulocyte lysate (RRL) in the absence or presence of hnRNP-K (0.15 µM), hnRNP-E1 (0.3 µM) or a combination thereof, as indicated. The amount of MBP synthesized was assessed by western blotting using MBP antibodies, and was quantified by densitometry. The data show the mean±s.d. of at least three independent experiments. Statistical significance was analyzed by one-way ANOVA followed by Dunnett's multiple comparision test. (B) A control experiment similar to A was performed using luciferase mRNA (20 ng/µl). (C) Immunocytochemistry of oligodendrocyte precursors that were transfected with EGFP (green) or hnRNP-K-FLAG (green), and stained 2 days after transfer to differentiation medium. The cells were also stained for MBP (red) and O4 (blue), a marker of maturing oligodendrocytes. The percentage of O4-positive cells that were positive for MBP at day 2 (D) and day 3 (E), and the percentage of oligodendrocytes showing complex branching morphology at day 2 (F) and day 3 (G) are shown. Data show the mean±s.d. of three independent experiments. Statistical significance was analyzed by Student's t-test. **P<0.01; ***P<0.001; ****P<0.0001; ns, non-significant.