RNA Polymerase II interactome in presence or absence of SCAF1 in U2OS-SCAF1-AID cells

ABSTRACT: Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used an siRNA-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1, using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery, is synthetically lethal.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Pranjali Bhandare  

LAB HEAD: Elmar Wolf

PROVIDER: PXD054675 | Pride | 2024-12-20

REPOSITORIES: Pride