Yra1-bound RNA-DNA hybrids cause orientation-independent transcription-replication collisions and telomere instability.

R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA-DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA-DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo. Importantly, an excess of Yra1 increases R-loop-mediated genome instability caused by transcription-replication collisions regardless of whether they are codirectional or head-on. It also induces telomere shortening in telomerase-negative cells and accelerates senescence, consistent with a defect in telomere replication. Our results indicate that RNA-DNA hybrids form transiently in cells regardless of replication and, after stabilization by excess Yra1, compromise genome integrity, in agreement with a two-step model of R-loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.

The Great Escape: mRNA Export through the Nuclear Pore Complex.

Nuclear export of messenger RNA (mRNA) through the nuclear pore complex (NPC) is an indispensable step to ensure protein translation in the cytoplasm of eukaryotic cells. mRNA is not translocated on its own, but it forms ribonuclear particles (mRNPs) in association with proteins that are crucial for its metabolism, some of which; like Mex67/MTR2-NXF1/NXT1; are key players for its translocation to the cytoplasm. In this review, I will summarize our current body of knowledge on the basic characteristics of mRNA export through the NPC. To be granted passage, the mRNP cargo needs to bind transport receptors, which facilitate the nuclear export. During NPC transport, mRNPs undergo compositional and conformational changes. The interactions between mRNP and the central channel of NPC are described; together with the multiple quality control steps that mRNPs undergo at the different rings of the NPC to ensure only proper export of mature transcripts to the cytoplasm. I conclude by mentioning new opportunities that arise from bottom up approaches for a mechanistic understanding of nuclear export.

The functional complexity of the RNA-binding protein Yra1: mRNA biogenesis, genome stability and DSB repair.

The mRNA export adaptor Yra1 is essential in S. cerevisiae, and conserved from yeast to human (ALY/REF). It is well characterized for its function during transcription elongation, 3' processing and mRNA export. Recently, different studies linked Yra1 to genome stability showing that Yra1 overexpression causes DNA Double Strand Breaks through DNA:RNA hybrids stabilization, and that Yra1 depletion affects DSB repair. However, the mechanisms through which Yra1 contributes to genome stability maintenance are not fully understood. Interestingly, our results showed that the Yra1 C-box domain is required for Yra1 recruitment to an HO-induced irreparable DSB following extensive resection, and that it is essential to repair an HO-induced reparable DSB. Furthermore, we defined that the C-box domain of Yra1 plays a crucial role in DSB repair through homologous recombination but not through non-homologous end joining. Future studies aim at deciphering the mechanism by which Yra1 contributes to DSB repair by searching for Yra1 partners important for this process. This review focuses on the functional complexity of the Yra1 protein, not only summarizing its role in mRNA biogenesis but also emphasizing its auto-regulation and implication in genome integrity either through DNA:RNA hybrids stabilization or DNA double strand break repair in S. cerevisiae.

A new regulatory pathway of mRNA export by an F-box protein, Mdm30.

Mdm30, an F-box protein in yeast, has been recently shown to promote mRNA export. However, it remains unknown how Mdm30 facilitates mRNA export. Here, we show that Mdm30 targets the Sub2 component of the TREX (Transcription/Export) complex for ubiquitylation and subsequent proteasomal degradation. Such a targeted degradation of Sub2 enhances the recruitment of the mRNA export adaptor, Yra1, to the active genes to promote mRNA export. Together, these results elucidate that Mdm30 promotes mRNA export by lowering Sub2's stability and consequently enhancing Yra1 recruitment, thus illuminating new regulatory mechanisms of mRNA export by Mdm30.

Single-molecule quantitation of RNA-binding protein occupancy and stoichiometry defines a role for Yra1 (Aly/REF) in nuclear mRNP organization.

RNA-binding proteins (RBPs) interact with mRNA to form supramolecular complexes called messenger ribonucleoprotein (mRNP) particles. These dynamic assemblies direct and regulate individual steps of gene expression; however, their composition and functional importance remain largely unknown. Here, we develop a total internal reflection fluorescence-based single-molecule imaging assay to investigate stoichiometry and co-occupancy of 15 RBPs within mRNPs from Saccharomyces cerevisiae. We show compositional heterogeneity of single mRNPs and plasticity across different growth conditions, with major co-occupants of mRNPs containing the nuclear cap-binding complex identified as Yra1 (1-10 copies), Nab2 (1-6 copies), and Npl3 (1-6 copies). Multicopy Yra1-bound mRNPs are specifically co-occupied by the THO complex and assembled on mRNAs biased by transcript length and RNA secondary structure. Yra1 depletion results in decreased compaction of nuclear mRNPs demonstrating a packaging function. Together, we provide a quantitative framework for gene- and condition-dependent RBP occupancy and stoichiometry in individual nuclear mRNPs.

What causes an RNA-DNA hybrid to compromise genome integrity?

Transcription is a source of genome instability that stimulates mutation and recombination. Part of the damage produced by transcription is mediated by R-loops, non-B DNA structures that normally form by the re-annealing of the nascent RNA with the template DNA outside the catalytic center of the RNA polymerase, displacing the non-template strand. Recent discoveries have revealed that R-loops might not be harmful by themselves. Instead, chromatin compaction triggered by these structures seems necessary, as deduced from the histone modifications frequently found associated with harmful R-loops. Remarkably, hybrids may also become harmful if stabilized by specific RNA binding proteins, one example of which is the yeast Yra1. We discuss here the possible mechanisms by which cells may stabilize R-loops and the consequences on transcription-replication conflicts and telomere homeostasis.