RNA biogenesis and RNA metabolism factors as R-loop suppressors: a hidden role in genome integrity.

Genome integrity relies on the accuracy of DNA metabolism, but as appreciated for more than four decades, transcription enhances mutation and recombination frequencies. More recent research provided evidence for a previously unforeseen link between RNA and DNA metabolism, which is often related to the accumulation of DNA-RNA hybrids and R-loops. In addition to physiological roles, R-loops interfere with DNA replication and repair, providing a molecular scenario for the origin of genome instability. Here, we review current knowledge on the multiple RNA factors that prevent or resolve R-loops and consequent transcription-replication conflicts and thus act as modulators of genome dynamics.

Rat1 promotes premature transcription termination at R-loops.

Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast. Importantly, we demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such structures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, providing an entry site for Rat1. Thus, we have uncovered an unanticipated function of Rat1 as a transcription restoring factor opening up the possibility that it may also promote transcription through other genomic DNA structures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relieved by Rat1, it will cause genomic instability, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.

Expression of human RECQL5 in Saccharomyces cerevisiae causes transcription defects and transcription-associated genome instability.

RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast Saccharomyces cerevisiae, which does not have a RECQL5 ortholog. We found that RECQL5 expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in S. cerevisiae.

Fast spin echo MRI of reservoir core plugs with a variable field magnet.

Fast Spin Echo MRI is now widely employed in biomedicine for proton density and T2 contrast imaging. Fast Spin Echo methods provide rapid data acquisition by employing multiple echoes to determine multiple k-space lines with single excitations. Due to the multi-exponential behavior of T2 in typical porous media, and the strong dependence of T2 on the details of the experiment, acquiring a proton density image with Fast Spin Echo methods requires favorable sample and acquisition parameters. In recent years, we have shown the value of pure phase encode Free Induction Decay based methods such as SPRITE. However, in a reservoir rock, a typical T2* is hundreds of µs, whereas a typical T2 is hundreds of ms. Hence, there is merit in considering spin echo-based MRI measurements such as the Fast Spin Echo for rock core plug studies. A variable field superconducting magnet was employed in this study. This is a new class of magnet for MR/MRI. These magnets have the flexibility of operation in the field range of 0.01 Tesla to 3 Tesla. This is advantageous when working with rock core plugs, as it allows one to maximize sample magnetization, by increasing the static field while controlling magnetic susceptibility mismatch effects, and thereby T2 and T2*, through reducing the static field. The magnetic fields employed in the study were 0.79, 1.5, and 3 Tesla. Measurements were undertaken on five brine-saturated reservoir rock core plugs (Bentheimer, Berea, Buff Berea, Nugget, and Wallace). The results show that Fast Spin Echo measurements are more sensitive than SPRITE methods in amenable samples and usually feature higher resolution. Quantification of saturation with Fast Spin Echo methods requires correction for T2 attenuation. The results also show that 3 Tesla is too high a static field in general for rock core MRI studies with either method. While the current study is focused on five representative reservoir rock cores, the conclusions which result are general for MRI of fluids in porous media.

SIN3A histone deacetylase action counteracts MUS81 to promote stalled fork stability.

During genome duplication, replication forks (RFs) can be stalled by different obstacles or by depletion of replication factors or nucleotides. A limited number of histone post-translational modifications at stalled RFs are involved in RF protection and restart. Provided the recent observation that the SIN3A histone deacetylase complex reduces transcription-replication conflicts, we explore the role of the SIN3A complex in protecting RFs under stressed conditions. We observe that Sin3A protein is enriched at replicating DNA in the presence of hydroxyurea. In this situation, Sin3A-depleted cells show increased RF stalling, H3 acetylation, and DNA breaks at stalled RFs. Under Sin3A depletion, RF recovery is impaired, and DNA damage accumulates. Importantly, these effects are partially dependent on the MUS81 endonuclease, which promotes DNA breaks and MRE11-dependent DNA degradation of such breaks. We propose that chromatin deacetylation triggered by the SIN3A complex limits MUS81 cleavage of stalled RFs, promoting genome stability when DNA replication is challenged.

Human DNA topoisomerase I poisoning causes R loop-mediated genome instability attenuated by transcription factor IIS.

DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.

Topological regulation of the estrogen transcriptional response by ZATT-mediated inhibition of TOP2B activity.

Human type-II topoisomerases, TOP2A and TOP2B, remove transcription associated DNA supercoiling, thereby affecting gene-expression programs, and have recently been associated with 3D genome architecture. Here, we study the regulatory roles of TOP2 paralogs in response to estrogen, which triggers an acute transcriptional induction that involves rewiring of genome organization. We find that, whereas TOP2A facilitates transcription, as expected for a topoisomerase, TOP2B limits the estrogen response. Consistent with this, TOP2B activity is locally downregulated upon estrogen treatment to favor the establishment and stabilization of regulatory chromatin contacts, likely through an accumulation of DNA supercoiling. We show that estrogen-mediated inhibition of TOP2B requires estrogen receptor α (ERα), a non-catalytic function of TOP2A, and the action of the atypical SUMO-ligase ZATT. This mechanism of topological transcriptional-control, which may be shared by additional gene-expression circuits, highlights the relevance of DNA topoisomerases as central actors of genome dynamics.

Contextualización de las ecuaciones para la estimación de la estatura a partir de la hemibraza en una población santiaguera / Equations for the height estimate from the hemimainbrace in Santiago de Cuba population

Se realizó un estudio descriptivo y transversal de 265 adultos (101 hombres y 164 féminas) de la Facultad de Medicina No. 1 de Santiago de Cuba, desde septiembre hasta octubre del 2014, con vistas a determinar las ecuaciones para la estimación de la estatura y verificar si las que fueron validadas en adultos de otras nacionalidades tienen aplicabilidad en esta población. Se encontró una elevada correlación entre la estatura y la hemibraza (p<0,001), r=0,889; 0,803 y 0,765 para hombres y mujeres, respectivamente. El error entre los modelos y la estatura medida fue menor (0,27 para ellos y 0,36 para ellas) en el propuesto que en los de Bassey (8,83; 9,89) y Shahar (11,72; 12,11). La correlación entre la estatura y la hemibraza posibilitó la obtención de ecuaciones contextualizadas en este medio, lo cual reafirmó la necesidad de poseer dichas ecuaciones para cada población.

A descriptive and cross-sectional study of 265 adults (101 men and 164 women) of the Medicine Faculty No. 1 in Santiago de Cuba, was carried out from September to October, 2014, aimed at determining the equations for the height estimate and verifying if those that were validated in adults of other nationalities have applicability in this population. A high correlation between the height and the hemimainbrace (p<0.001), r=0.889; 0.803 and 0.765 for men and women, respectively was found. The error between the models and the measured height was lower (0.27 for men and 0.36 for women) in the proposed model than in Bassey (8.83; 9.89) and Shahar models (11.72; 12.11). The correlation between height and hemimainbrace facilitated the obtaining of contextualized equations in this mean, which confirmed the necessity to possess these equations for each population.