Separate transcription and splicing gene networks are linked and coordinated by the pRb-E2F pathway

E2F is a family of master transcription regulators involved in mediating diverse cell fates, and loss of control of the pathway is regarded as a hallmark of cancer. Recent studies have highlighted the pRb-E2F axis as a regulator of a large cellular network, which in addition to its classical target genes includes RNA splicing and non-coding genes. Here, we have addressed the generality of these effects by studying the role of the other key components of the pathway, including pRb, DP and PRMT5. Like E2F1, both pRb and DP1 influence transcription and alternative RNA splicing (AS) which is additionally impacted by PRMT5 activity. A detailed proteomics analysis of the E2F1 interactome revealed SRSF2 and HNRNPC as partner proteins that assist E2F1 in mediating the effects on RNA splicing. The ability of E2F1 to influence AS activity was apparent as cells progress through the cell cycle and during the DNA damage response. Our results highlight the role of the pRb-E2F pathway in mediating cell cycle regulation of gene expression mediated by transcription and RNA splicing, and provide a mechanistic understanding for how this is likely to occur. Overall design: To investigate the role played by PRMT5 and pRb-E2F pathway components in gene transcription and alternative splicing, we generated CRISPR cell lines in HCT116 and MCF7 cell backgrounds, in which E2F1, DP1, or pRb was knocked out. These cells were treated with DMSO or a PRMT5 inhibitor as required. Alternatively, HCT116 WT and E2F1Cr cells were synchronised at the G1/S boundary using a double thymidine block, and then released for 6 hours to allow them to progress into G2/M phase. We then performed gene expression and splicing analysis on RNA-seq data obtained from these various treatments and cell lines.