FIGURE 4.

MBP synthesis is influenced by hnRNP F. A, knockdown of hnRNP F specifically affects MBP levels. Primary mouse oligodendrocytes were treated with hnRNP F or control siRNA and allowed to differentiate for 3 days. Protein levels were quantified by densitometric analysis of Western blots, and the expression of MBP, CNP, and MOG was normalized to GAPDH. B, knockdown of hnRNP F has no impact on levels of MBP mRNA. Primary mouse oligodendrocytes were treated as in A, but here mRNA levels were analyzed by qRT-PCR and normalized to β-actin mRNA. C, altered hnRNP F levels impair the translation of MBP luciferase reporters. hnRNP F levels in Oli-neu cells were reduced by transfection of hnRNP F-directed siRNA or increased by transfection of hnRNP F expression vectors. Subsequently, luciferase-based translational reporters containing parts of the 3′UTR of MBP as regulatory elements were used to measure translational activity in DualGlo assays (see “Experimental Procedures” for details). Relative luciferase activity is reduced in cells treated with hnRNP F siRNA as well as in hnRNP F-overexpressing cells that were related to control siRNA- or GFP-transfected cells, respectively. D, effect of hnRNP F levels on the translation of MBP reporters, including or lacking the A2RE. Cells were treated as in C, but here a luciferase reporter construct lacking the A2RE was included. E, reduced MBP levels in response to hnRNP F knockdown are independent of proteasomal activity in primary oligodendrocytes. The experiment was performed according to A. Before lysis, the siRNA-treated cells were incubated with the proteasomal inhibitor ALLN or as control with DMSO. F, statistical evaluation of four independent experiments as shown in E. Note the persistence in MBP reduction despite inhibition of the proteasome. *, p < 0.05 (Student's t test (D and F) or Wilcoxon signed-rank test (A and C)).