Singh, R. K. | Search bioRxiv

bioRxiv2018

Transient kinetic analysis of SWR1C-catalyzed H2A.Z deposition unravels the impact of nucleosome dynamics and the asymmetry of stepwise histone exchange

The SWR1C chromatin remodeling enzyme catalyzes an ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes, such as transcription and DNA repair. Here we investigate the transient kinetic mechanism of the histone exchange reaction employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATP-dependent replacement of nucleosomal dimers, promoted by localized DNA translocation. Taken together, our transient kinetic studies identify new intermediates and provide crucial insights into the SWR1C-catalyzed dimer exchange reaction, as well as shedding light on how the mechanics of H2A.Z deposition might contribute to transcriptional regulation in vivo.

biochemistry↗

bioRxiv2018

Rbfox splicing factors maintain skeletal muscle mass by regulating calpain3 and proteostasis

Alternative splicing promotes proteomic diversity important for cellular differentiation and cell fate determination. Here, we show that deletion of the highly conserved Rbfox1 and Rbfox2 alternative splicing regulators in adult mouse skeletal muscle causes rapid, severe loss of muscle mass. Homeostasis of skeletal muscle tissue requires a dynamic balance between protein synthesis and degradation (proteostasis) but the mechanisms that regulate this balance are not well understood. Rbfox deletion did not cause reduced global protein synthesis, but resulted in reduced autophagy flux and altered splicing of hundreds of transcripts including Capn3, which produced an active form of calpain3 protease. The results indicate Rbfox proteins regulate proteostasis in skeletal muscle tissue by control of calpain and autophagy-lysosome pathways.\n\nHighlightsO_LIProteostasis in adult skeletal muscle is post-transcriptionally regulated, in part by alternative splicing via Rbfox1/2\nC_LIO_LIRbfox1/2 regulate hundreds of targets in skeletal muscle, including Calpn3, to maintain muscle mass in adult mice\nC_LIO_LIAutophagy flux is markedly decreased in muscle lacking Rbfox1/2\nC_LIO_LIAs for neurons, altered proteostasis is detrimental to adult muscle\nC_LI

molecular biology↗