Abstract Body (Do not enter title and authors here): Background: hnRNPA1 is an important member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family, serves multiple functions. PPP1R1B-lncRNA is a functionally conserved long noncoding RNA that plays essential roles in promoting myogenic differentiation in cardiac and skeletal myocytes. We identified hnRNPA1 as the top proteins that dynamically interacts with PPP1R1B-lncRNA during myogenic differentiation.
Hypothesis: PPP1R1B-lncRNA function in Myogenic differentiation requires the interaction with hnRNPA1.
Goals: We aimed to investigate the role of hnRNPA1 in PPP1R1B-lncRNA-mediated regulation of myogenesis and identify the underlying molecular mechanisms.
Methods: Ppp1r1b-lncRNA-protein complex was isolated by RNA pulldown assay. The corresponding proteins were identified by mass spectrometry. The binding sites of hnRNPA1 and EZH2 (the enzymatic core unit of PRC2) on PPP1R1B-lncRNA were further evaluated by RNA pulldown assay and western blotting. After down-regulation of hnRNPA1, expression of myogenesis master regulators was measured by RT-PCR and western Blot. Furthermore, EZH2 RNA immunoprecipitation (RIP), H3K27me3 chromatin immunoprecipitation (CHIP); and Ppp1r1b-lncRNA chromatin isolation by RNA purification (CHIRP) were applied.
Results: hnRNPA1 and EZH2 both binds to PPP1R1B-lncRNA, located at different positions. After downregulation of hnRNPA1 in hiPSC-derived cardiomyocytes, EZH2 RIP and H3K27me3 CHIP show decreased EZH2 binding with PPP1R1B-lncRNA and corresponding increased H3K27me3 enrichment on promotors of myogenic differentiation factor genes TBX5, and GATA4. In tandem, the expression of TBX5 and GATA4 was significantly suppressed in the hnRNPA1-depleted cells at the mRNA and protein levels. Furthermore, CHIRP-PCR analysis indicated increased interaction of PPP1R1B-lncRNA with TBX5 and GATA4 promoters.
Conclusions: hnRNPA1 facilitate the integrity of PPP1R1B-lncRNA/PRC2 complex, which preserves correct regulation of myogenic differentiation factors expression. After hnRNPA1 suppression the elevated promoter occupancy of H3K27me3 corresponds with transcriptional repression. The hnRNPA1/PPP1R1B-lncRNA/PRC2 complex is a novel regulatory axis of myogenic differentiation that can be targeted for translational application in cardiac development and regeneration.