Project name: SCAF4 and SCAF8, Transcriptional Anti-Terminator Proteins (mRNA-Seq)

Description: Accurate regulation of RNA Polymerase II (RNAPII) transcriptional termination is a prerequisite for correct gene expression. Here we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative polyadenylation sites (PASs). The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) modified on both Ser2 and Ser5, and are detected at early, alternative PASs. Concomitant knockout of human SCAF4 and SCAF8 results in altered PAS selection, leading to expression of truncated mRNAs and proteins lacking functional domains, and is cell-lethal. While SCAF4 and SCAF8 thus work redundantly to suppress early termination, they also have independent, non-essential functions. SCAF8 is a positive RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, directing PAS selection to ensure correct RNAPII transcriptional termination in human cells.Overall design: To study the function of SCAF4 and SCAF8 in human cells, we used CRISPR-Cas9 nickase technology to generate single and double knockouts (KOs) in HEK293 Flp-In TREX cells, which also contained a single copy of a doxycycline (Dox)-inducible SCAF4 or SCAF8 rescue construct, with the encoded GFP-tagged protein expressed at near-normal levels. All cell lines were maintained in the presence of Dox to ensure expression of the CRISPR-resistant rescue gene during and after KO cell line generation. Expression of the rescue protein reaches undetectable levels after 5 days growth in Dox-free media, resulting in effective single of double KOs. Due to the manner in which these cell lines were generated, a total of 12 cell types (6 different cell lines grown with or without Dox) were often analysed together. For example, SCAF4 SCAF8 double KOs were generated either by first knocking out SCAF4 and then SCAF8, or vice versa, and these different cell lines were in turn also derived from cell lines containing either a Dox-inducible SCAF4- or a SCAF8 rescue gene, giving rise to a total of 4 genotypically identical SCAF4 SCAF8 dKO cell lines. Moreover, a SCAF4 SCAF8 double KO that expresses a rescue gene (i.e. grown in the presence of doxycycline) is effectively a single KO; for example, a SCAF4 SCAF8 double KO expressing the SCAF4 rescue gene is genotypically and phenotypically a single SCAF8 KO cell line. For PAR-CLIP experiments, HEK293 Flp-In TREX cells stably expressing FLAG-tagged SCAF4 or SCAF8 were used for FLAG immunoprecipitations from RNA-protein crosslinked extracts.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Medical

Last updated: 2018-10-25