Abstract
SummaryAlthough correlations between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established, the mechanisms underlying these connections remain poorly understood. Here, we used a mutant version of the transcription elongation factor TFIIS (TFIISmut), aiming to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. Indeed, TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII; it affects mRNA splicing and termination as well. Remarkably, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed light on the relationship between transcription stress and R-loops and suggest that different classes of R-loops may exist, potentially with distinct consequences for genome stability.
Highlights
In contrast to ATP-driven molecular machines such as helicases, RNA polymerase (RNAP) moves by Brownian motion and may oscillate between productive and backtracked states at various positions on DNA
TFIISmut as a Tool to Study the Effects of Transcription Stress Yeast TFIISmut is incapable of supporting transcript cleavage by RNA polymerase II (RNAPII) in vitro, and its expression in cells gives rise to transcription stress and a dominant-negative effect on growth (Sigurdsson et al, 2010)
To investigate if similar effects are observed in human cells, the point mutations that characterize yeast TFIISmut were made in human TCEA1, generating TCEA1mut
Summary
In contrast to ATP-driven molecular machines such as helicases, RNA polymerase (RNAP) moves by Brownian motion and may oscillate between productive and backtracked states at various positions on DNA. Deep sequencing of the 30 ends of nascent RNA isolated with RNAPII elongation complexes (NET-seq) suggested the existence of >2 3 105 detectable pause sites in the compact yeast genome, of which more than 75% were associated with a backtracked polymerase (Churchman and Weissman, 2011). Backtracked RNAPII is recognized by transcription factor TFIIS (encoded by DST1 in the yeast Saccharomyces cerevisiae and TCEA1-3 in humans; the functional analogs are GreA and B in bacteria) (Nudler, 2012), which stimulates transcript cleavage by the polymerase active site, allowing RNAPII to regain control of the RNA end and resume transcript elongation (Izban and Luse, 1992; Kettenberger et al, 2003; Reines, 1992). Backtracking and transcript cleavage are an integral part of the elongation process, the likelihood of it occurring is greatly increased by any obstacle to forward translocation, such as nucleotide mis-incorporation, DNA sequences that are difficult to transcribe, nucleosomes, or other DNA-associated factors in the path of RNAPII, including other polymerases (see, for example, Kireeva et al, 2005; Saeki and Svejstrup, 2009; Sigurdsson et al, 2010)
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