Li and Dewey, 2011) with the –v2 argument. NMD also vary during differentiation. Hematopoietic deletion of prevented fetal liver cells from undergoing terminal differentiation (Thoren et al., 2010). Finally, NMD has been directly implicated in repressing neural differentiation. Overexpression or knockdown of in skeletal musclewhich causes facioscapulohumeral muscular dystrophyresults in UPF1 protein degradation and severe inhibition of NMD (Feng et al., 2015). Determining the normal role of NMD during myogenesis is usually therefore of direct biomedical relevance. In this study, we describe a direct and mechanistic role for the central NMD factor UPF1 in repressing human muscle mass differentiation. UPF1 promotes proteasome-mediated degradation of MYOD protein, a grasp regulator of the myogenic process. UPF1 promotes MYOD proteolysis by acting as an E3 ubiquitin ligase via its RING domain while leaving MYOD mRNA stability unaffected. Mutating UPF1s RING domain name alleviates UPF1-dependent MYOD proteolysis and reverses UPF1s repressive role in myogenesis. RESULTS knockdown accelerates human myogenesis To determine whether the reported decrease in UPF1 levels and NMD efficiency that occurs during myogenesis (Gong et al., 2009) plays a regulatory and causative role, rather than being a by-product of the myogenic process, we tested whether forced reductions in UPF1 levels altered the efficiency of myoblast differentiation. We transfected two genetically unique myoblast cell lines generated from healthy human Gestrinone muscle mass (54-1 and MB135 cells) with two different siRNAs against UPF1, reducing UPF1 protein to 24.7% and 63.0% of levels in cells transfected with two different non-targeting siRNAs (Determine 1A-B, S1A-C). We induced differentiation two days post-transfection (day 0) by switching the confluent myoblasts from high-serum growth media to low-serum differentiation media, which promotes cell cycle stalling and the expression Gestrinone of myogenic markers. Open in a separate window Physique 1 knockdown accelerates myoblast differentiation(A) Schematic of a time course of human myoblast differentiation following knockdown (KD). Two genetically unique human myoblast cell lines (54-1 and MB135 cells) were transfected with two different siRNAs against UPF1, as well as two different non-targeting siRNAs as controls (Day -2). When the transfected cells reached full confluency (Day 0), differentiation was induced by switching from high-serum growth media to low-serum differentiation media (DM). 54-1 and MB135 cells respectively differentiate less and more rapidly, and so were differentiated for seven and four days, respectively. (B) Immunoblot for UPF1 protein from 54-1 cells following control or KD immediately prior to (Day 0) or two days after BA554C12.1 (Day 2) induction of differentiation. Equivalent amounts of protein were loaded based on the BCA assay. -tubulin, loading control. Bar plot, quantification of UPF1 protein levels relative to the loading control. (C) Immunofluorescence labeling of 54-1 cells with an antibody against BrdU (reddish) at the indicated time points, prior to significant cell fusion. At each time point, cells were fixed after one hour of BrdU labeling. Gestrinone Box plot, percentage of BrdU+ nuclei measured over 10 fields. Whiskers, maximum and min over the fields. */**/***, 0.05/0.01/0.001. (D-E) Immunofluorescence labeling of 54-1 or Gestrinone MB135 cells with antibodies against Myogenin (MYOG, reddish) and Myosin Heavy Chain (MHC, green) at the indicated time points. MB135 differentiate more rapidly than do 54-1 cells and so a shorter time course was used. Box plot, percentage of MYOG+ nuclei measured over 10 fields. Whiskers, maximum and min over the fields. */**/***, 0.05/0.01/0.001. See also Figure S1. knockdown (KD) resulted in accelerated differentiation throughout the myogenic process in both 54-1 and MB135 myoblasts. We first analyzed cells.
Li and Dewey, 2011) with the –v2 argument
