Fig. 7

Loss of RBM22 impairs the association of the elongation factor SPT5 with RNAPII. a Recombinant proteins from pull-down assays visualized by immunoblotting. Input (purified 6HIS-GFP, 6HIS-GFP-RBM22 before and after the cleavage of HRV 3C protease and SPT5) and eluted proteins from immunoprecipitations (GFP or GFP-RBM22 incubated with SPT5) were visualized with the GFP and SPT5 antibodies (* indicates non-specific signal). b Barplot showing the number of SPT5 target genes within each category of RBM22 ChIP-seq binding, pause release, elongation slower, and readthrough genes. c Metagene analysis showing the change in SPT5 ChIP-seq signal at all protein-coding genes in control or RBM22 knockdown HepG2 cells. d Metagene profiles of RNAPII-normalized SPT5 occupancy levels at the promoter, gene body, and termination zone in control and RBM22 knockdown HepG2 cells. The 9076 genes with RNAPII binding were used. e Co-IP assay examining the interaction between endogenous SPT5 and Ser2P RNAPII (POLR2A-Ser2P) in mAID-RBM22 cells with DMSO or 5-Ph-IAA treatment. f TT-qPCR quantification of transcriptional level and transcriptional readthrough at three representative protein-coding genes in control, SPT5 knockdown (siSPT5) or HEXIM1 knockdown (siHEXIM1) HepG2 cells. Graphs show the ratios of relative readthrough, normalized to control. The p values are based on a two-tailed unpaired t test; *P < 0.05, **P < 0.01. g During early elongation by RNAPII at many protein-coding genes, RBM22 may initially be recruited by promoter-paused RNAPII, whose CTD is modified by Ser5 phosphorylation. This recruitment may stabilize promoter-associated inhibitory 7SK-P-TEFb complex to globally restrict P-TEFb PGT and subsequent RNAPII pause release in the sense and antisense direction. Moreover, RBM22 interacts with SPT5 and sustains the association between SPT5 and RNAPII, thus promoting RNAPII pausing at promoters. During productive elongation and termination at most genes, RBM22 may enhance the association of SPT5 to maintain the speed of elongating RNAPII and ensure efficient transcription termination. These roles of RBM22 facilitate the maintenance of RNAPII transcription homeostasis