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Edited by Kevin Struhl, Harvard Medical School, Boston, MA, and approved August 29, 2017 (received for review May 11, 2017)
Significance
During transcription, the mRNA may hybridize back with its template DNA, forming a structure called R loop. These structures have been associated with genome instability and human disease. Using budding yeast as a model organism to screen for new genes preventing R loops, we identified MLP1 and subsequently showed that the nuclear basket protein Mlp1/2 has a role in preventing R loop formation and genome instability in yeast. Our work indicates that R loops are formed in the nucleoplasm and that proximity of transcribed chromatin to the nuclear pore constrains R loop formation. Our study opens additional perspectives to understand the role of RNA in the control of genome integrity as a function of nuclear location.
Abstract
During transcription, the mRNA may hybridize with DNA, forming an R loop, which can be physiological or pathological, constituting in this case a source of genomic instability. To understand the mechanism by which eukaryotic cells prevent harmful R loops, we used human activation-induced cytidine deaminase (AID) to identify genes preventing R loops. A screening of 400 Saccharomyces cerevisiae selected strains deleted in nuclear genes revealed that cells lacking the Mlp1/2 nuclear basket proteins show AID-dependent genomic instability and replication defects that were suppressed by RNase H1 overexpression. Importantly, DNA–RNA hybrids accumulated at transcribed genes in mlp1/2 mutants, indicating that Mlp1/2 prevents R loops. Consistent with the Mlp1/2 role in gene gating to nuclear pores, artificial tethering to the nuclear periphery of a transcribed locus suppressed R loops in mlp1∆ cells. The same occurred in THO-deficient hpr1∆ cells. We conclude that proximity of transcribed chromatin to the nuclear pore helps restrain pathological R loops.
Footnotes
- ↵1To whom correspondence may be addressed. Email: gaillard{at}us.es or aguilo{at}us.es.
Author contributions: F.G.-B., H.G., and A.A. designed research; F.G.-B. and H.G. performed research; F.G.-B., H.G., and A.A. analyzed data; and H.G. and A.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1707845114/-/DCSupplemental.
Nuclear pore proximity prevents R loop formation
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- Biological Sciences
- Genetics
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