MYRF Is a Membrane-Associated Transcription Factor That Autoproteolytically Cleaves to Directly Activate Myelin Genes
Figure 2
Autoproteolytic processing via the ICD and a NLS are required for nuclear localization of MYRF.
(A) ClustalW2 alignment of the peptide sequence of MYRF and the ICD domain of the bacteriophage GA-1 Neck appendage protein. The serine/lysine dyad residues subjected to mutagenesis are highlighted. (B) Western blot analysis of un-mutated or ICD mutant (K592R, K592H, K592M) Myc-tagged MYRF constructs in 293T cells. (C) Immunofluorescence for the N-terminal Myc tagged MYRF and the K587H mutant in CG-4 cells. Prevention of the cleavage is associated with a loss of nuclear localization of the N-terminus. (D) Primary rat OPC cultures co-transfected with GFP and either empty vector (pcDNA3) or pcDNA3 containing MYRF or the S587A and K592H mutant constructs, stained for MOG. (E) Quantification of the percentage of transfected (GFP+) cells expressing MOG 48 h posttransfection in each condition. **p<0.01 by t test. (F) Predicted NLS within the proline-rich region of MYRF showing the KKR to AAA mutation in the Myc-MYRFΔNLS construct. (G) Western analysis of the Myc-MYRF and Myc-MYRFΔNLS construct; mutation of the NLS has no effects on the cleavage of MYRF, though it routinely led to an increase in protein levels. (H) Representative images of immunostaining for the N-terminal Myc tag showing shift from nuclear to extranuclear staining in the Myc-MYRFΔNLS construct. (I) Quantification of the proportion of predominantly nuclear, mixed, or predominantly extranuclear staining seen with each construct (100 cells assessed/condition). Scale bars, (C and H) 10 µm and (D) 50 µm.
