A SUMO-dependent pathway controls elongating RNA Polymerase II upon UV-induced damage

Abstract

RnA polymerase ii (RnApii) is the workhorse of eukaryotic transcription and produces messenger RnAs and small nuclear RnAs. Stalling of RnApii caused by transcription obstacles such as DnA damage threatens functional gene expression and is linked to transcription-coupled DnA repair. to restore transcription, persistently stalled RnApii can be disassembled and removed from chromatin. this process involves several ubiquitin ligases that have been implicated in RnApii ubiquitylation and proteasomal degradation. transcription by RnApii is heavily controlled by phosphorylation of the C-terminal domain of its largest subunit Rpb1. Here, we show that the elongating form of Rpb1, marked by S2 phosphorylation, is specifically controlled upon UV-induced DNA damage. Regulation of S2phosphorylated Rpb1 is mediated by SUMOylation, the SUMO-targeted ubiquitin ligase Slx5-Slx8, the Cdc48 segregase as well as the proteasome. Our data suggest an RNAPII control pathway with striking parallels to known disassembly mechanisms acting on defective RnA polymerase iii. DNA is the macromolecule that harbors all information required for life. In eukaryotes three nuclear RNA polymerases (RNAPI-III) are necessary to read out this information. While RNAPI transcribes ribosomal RNA (rRNA) 1 , RNAPIII mediates transcription of rRNA, transfer RNAs (tRNA) and other small RNAs 2. RNAPII is important for transcription of protein-coding genes, as well as for synthesis of non-coding RNAs 3-5. All three RNA polymerases are under control of several posttranslational modifications. However, specifically transcription by RNAPII is regulated by phosphorylation that chiefly targets Rpb1's carboxyl-terminal domain (CTD) heptapeptide repeats (consensus sequence YSPTSPS) 6 , which serve as platform for factors involved in transcription, chromatin modification, mRNA splicing and export 7,8. Phosphorylation of serine-5 (S5P) of the CTD repeats appears to be largely specific for Rpb1 molecules localized to promoters 9,10. Serine-2 phosphorylation (S2P) in contrast is a hallmark for elongating polymerases and ChIP signals using S2P-specific antibodies steadily rise downstream of transcription start sites and culminate at the 3'-ends of genes 9,11-13. Highly problematic for all RNA Polymerases are bulky DNA lesions on the coding strand as they may behave as "roadblocks" leading to persistent stalling of the progressing protein complexes. A key mechanism to remove bulky DNA adducts and to avoid cell cycle arrest and cell death is nucleotide excision repair (NER) 14,15. Yet to conduct DNA repair the machinery first needs to gain access to the lesion that is masked by a stalled polymerase and here, several possibilities exist. First, the RNA polymerase can simply be released from the DNA to allow access. This has been shown to occur for RNAPI and RNAPII, but the underlying mechanism is poorly understood 16-18. Second, RNAPII backtracking from the lesion can facilitate repair without dissociation. This is made possible by the proofreading activity of RNAPII, which al...

DNA–protein crosslink proteases in genome stability

“…Its degradation might be a consequence of transcription stalling due to roadblocks ahead of RNA polymerase II. In fact, under these circumstances Rpb1 degradation was described to rely on the ubiquitin-proteasome system, the ATPase Cdc48/p97 and SUMO 91 , 92 . At any rate, a stalled, non-crosslinked Rpb1 would be equally “eligible” for degradation by DPC proteases, since other “non-DPC” substrates exist and will be discussed later in this review.…”

Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II

“…Although these data strongly link RNAPIII sumoylation with a positive effect on its transcriptional activity, using genetic analysis, another study found that impairing sumoylation could at least partly rescue the growth defects associated with mutant forms of RNAPIII subunits, suggesting that in some conditions, sumoylation can repress RNAPIII activity [25]. Also in yeast, the elongation-associated form of RNAPII was found to become sumoylated on its large subunit, Rpb1, in response to UV irradiation or impairment of transcription, implicating the modification in the resolution of transcriptionally stalled RNAPII complexes at DNA lesions [26,27]. In human cells, both TBP and multiple TAF subunits of TFIID are known SUMO targets, and in in vitro assays, sumoylation of hsTAF5 was found to inhibit binding of this GTF to an immobilized DNA template, suggesting that TFIID sumoylation might be involved in regulating the assembly of the complex with promoters [6,9,28,29].…”

Repair of DNA double-strand breaks leaves heritable impairment to genome function

“…Prominent among those is CTCF, a canonical TAD insulator, whose known accumulation at DSB-flanking chromatin microdomains ( 68 , 69 ) is consistent with transcriptional deregulation described in our study. Firstly, CTCF anchor engagement per se seems enhanced under conditions of RNAPII removal ( 70 ), such as the one that can occur at damaged chromatin ( 71–73 ). Additionally, several non-coding RNAs have been implicated with CTCF eviction to regulate local chromatin folding ( 74–76 ), consistent with the possibility that reduced retention of RNA species at post-repair chromatin as shown in our study could foster ectopic CTCF accumulation.…”