R-Loop Mediated Transcription-Associated Recombination in trf4D Mutants Reveals New Links between RNA Surveillance and Genome Integrity

To get further insight into the factors involved in the maintenance of genome integrity we performed a screening of Saccharomyces cerevisiae deletion strains inducing hyperrecombination. We have identified trf4 , a gene encoding a non- canonical polyA-polymerase involved in RNA surveillance, as a factor that prevents recombination between DNA repeats. We show that trf4 D confers a transcription-associated recombination phenotype that is mediated by the nascent mRNA. In addition, trf4 D also leads to an increase in the mutation frequency. Both genetic instability phenotypes can be suppressed by overexpression of RNase H and are exacerbated by overexpression of the human cytidine deaminase AID. These results suggest that in the absence of Trf4 R-loops accumulate co-transcriptionally increasing the recombination and mutation frequencies. Altogether our data indicate that Trf4 is necessary for both mRNA surveillance and maintenance of genome integrity, serving as a link between RNA and DNA metabolism in S. cerevisia

Excess of Yra1 RNA-Binding Factor Causes Transcription-Dependent Genome Instability, Replication Impairment and Telomere Shortening

Yra1 is an essential nuclear factor of the evolutionarily conserved family of hnRNP-like export factors that when overexpressed impairs mRNA export and cell growth. To investigate further the relevance of proper Yra1 stoichiometry in the cell, we overexpressed Yra1 by transforming yeast cells with YRA1 intron-less constructs and analyzed its effect on gene expression and genome integrity. We found that YRA1 overexpression induces DNA damage and leads to a transcription-associated hyperrecombination phenotype that is mediated by RNA:DNA hybrids. In addition, it confers a genome-wide replication retardation as seen by reduced BrdU incorporation and accumulation of the Rrm3 helicase. In addition, YRA1 overexpression causes a cell senescence-like phenotype and telomere shortening. ChIP-chip analysis shows that overexpressed Yra1 is loaded to transcribed chromatin along the genome and to Y’ telomeric regions, where Rrm3 is also accumulated, suggesting an impairment of telomere replication. Our work not only demonstrates that a proper stoichiometry of the Yra1 mRNA binding and export factor is required to maintain genome integrity and telomere homeostasis, but suggests that the cellular imbalance between transcribed RNA and specific RNA-binding factors may become a major cause of genome instability mediated by co-transcriptional replication impairment.España Ministerio de Ciencia e Innovación BFU2010-16372, BFU2013-42918Andalucía, Junta de Andalucía BIO102, CVI4567 and BIO123

REX-001, a BM-MNC Enriched Solution, Induces Revascularization of Ischemic Tissues in a Murine Model of Chronic Limb-Threatening Ischemia

Background: Bone Marrow Mononuclear Cells (BM-MNC) constitute a promising alternative for the treatment of Chronic Limb-Threatening ischemia (CLTI), a disease characterized by extensive blockade of peripheral arteries, clinically presenting as excruciating pain at rest and ischemic ulcers which may lead to gangrene and amputation. BM-MNC implantation has shown to be efficient in promoting angiogenesis and ameliorating ischemic symptoms in CLTI patients. However, the variability seen between clinical trials makes necessary a further understanding of the mechanisms of action of BM-MNC, and moreover, to improve trial characteristics such as endpoints, inclusion/exclusion criteria or drug product compositions, in order to implement their use as stem-cell therapy. Materials: Herein, the effect of REX-001, a human-BM derived cell suspension enriched for mononuclear cells, granulocytes and CD34+ cells, has been assessed in a murine model of CLTI. In addition, a REX-001 placebo solution containing BM-derived red blood cells (BM-RBCs) was also tested. Thus, 24 h after double ligation of the femoral artery, REX-001 and placebo were administrated intramuscularly to Balb-c nude mice (n:51) and follow-up of ischemic symptoms (blood flow perfusion, motility, ulceration and necrosis) was carried out for 21 days. The number of vessels and vascular diameter sizes were measured within the ischemic tissues to evaluate neovascularization and arteriogenesis. Finally, several cell-tracking assays were performed to evaluate potential biodistribution of these cells. Results: REX-001 induced a significant recovery of blood flow by increasing vascular density within the ischemic limbs, with no cell translocation to other organs. Moreover, cell tracking assays confirmed a decrease in the number of infused cells after 2 weeks post-injection despite on-going revascularization, suggesting a paracrine mechanism of action. Conclusion: Overall, our data supported the role of REX-001 product to improve revascularization and ischemic reperfusion in CLTI

Characterization of the CPAP-treated patient population in Catalonia

There are different phenotypes of obstructive sleep apnoea (OSA), many of which have not been characterised. Identification of these different phenotypes is important in defining prognosis and guiding the therapeutic strategy. The aim of this study was to characterise the entire population of continuous positive airway pressure (CPAP)-treated patients in Catalonia and identify specific patient profiles using cluster analysis. A total of 72,217 CPAP-treated patients who contacted the Catalan Health System (CatSalut) during the years 2012 and 2013 were included. Six clusters were identified, classified as “Neoplastic patients” (Cluster 1, 10.4%), “Metabolic syndrome patients” (Cluster 2, 27.7%), “Asthmatic patients” (Cluster 3, 5.8%), “Musculoskeletal and joint disorder patients” (Cluster 4, 10.3%), “Patients with few comorbidities” (Cluster 5, 35.6%) and “Oldest and cardiac disease patients” (Cluster 6, 10.2%). Healthcare facility use and mortality were highest in patients from Cluster 1 and 6. Conversely, patients in Clusters 2 and 4 had low morbidity, mortality and healthcare resource use. Our findings highlight the heterogeneity of CPAP-treated patients, and suggest that OSA is associated with a different prognosis in the clusters identified. These results suggest the need for a comprehensive and individualised approach to CPAP treatment of OSA.This study was supported by the Spanish Respiratory Society (SEPAR), Associació Lleidatana de Respiratori (ALLER) and ResMed, a company that provides diagnostic services and treatment for sleep apnoea

Genetic evidence for R-loop formation in <b><i>trf4</i></b>

<p>Δ <b>mutants.</b> Effect of RNaseH1 and AID over-expression on the mutation frequency in <i>trf4</i> mutants. Upper panel shows the analysis of recombination frequencies in W303-1A (WT) and TRF4D-C5 (<i>trf4Δ</i>) cells containing the recombination system LYΔNS, without RNaseH1 overexpression (-RNH1) or with over-expression of RNaseH1 (+RNH1). The latter was achivied with the multicopy plasmid pGAL-RNH1 carrying RNH1 under the GAL1 promoter. Lower panel shows recombination frequencies as in the upper one, but over-expressing AID from plasmid p413GALAID. The average median value and SD of 3–4 fluctuation tests are shown. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065541#pone-0065541-g002" target="_blank">Figure 2</a>. Asterisks indicate statistically significant differences, according to Student's t tests (***, Ρ<0.0005).</p

Nascent mRNA-dependency of the hyperrecombination phenotype of <b><i>trf4</i></b>

<p>Δ <b>mutants.</b> (<b>A</b>) Direct-repeat recombination systems GL-<i>Rib⁺</i> and GL-<i>rib^m</i> containing the <i>PHO5-Rib⁺</i> or <i>PHO5- rib^m</i> sequences flanked by two truncated copies of <i>LEU2</i> in direct orientation under the <i>GAL1</i> promoter. These systems contain respectively an active or inactive 52-bp ribozyme (<i>Rib</i>). The <i>rib^m</i> system (inactive ribozyme) yields a long transcript, whereas in the <i>Rib⁺</i> system (active ribozyme) self-cleavage of the <i>PHO5-Rib</i> transcript leads to a shorter mRNA (represented by arrows). (<b>B</b>) Recombination frequencies in W303-1A (WT) and TRF4D-C5 (<i>trf4Δ</i>) cells containing the recombination systems GL-<i>Rib⁺</i> and GL-<i>rib^m</i>. Experiments were performed in 2% galactose to allow expression of the direct repeats. The average median value and SD of 3–4 fluctuation tests are shown. Asterisk indicates statistically significant differences, according to Student's t-tests (*, Ρ<0.05).</p

Figure 3

<p><b>Transcription analysis of the </b><b><i>trf4Δ</i></b><b> strain.</b> (<b>A</b>) Analysis of the ability of W303-1A and TRF4D-C5 (<i>trf4Δ</i>) strains carrying the <i>Ptet::lacZ-URA3</i> (LAUR) fusion construct (plasmid pCM184-LAUR) to form colonies on SC-trp-ura medium and to form blue colonies on SC-Trp complemented with X-Gal. (<b>B</b>) Northern analysis of the expression of the <i>Ptet::lacZ-URA3</i>. RNA was isolated from two different mid-log phase cultures from each strain, grown in SC-trp. We used the 3-kb <i>Bam</i>HI <i>lacZ</i> fragment and an internal 589-bp 25S rDNA fragment obtained by PCR, as probes.</p

Effect of the level of transcription on the <b><i>trf4Δ</i></b><b> hyperrecombination phenotype.</b>

<p>Isogenic strains W303-1A (WT) and TRF4D-C5 (<i>trf4Δ</i>) were transformed with plasmids pSCH204 (L-<i>lacZ</i> recombination system) or pRS314GL-lacZ (GL-<i>lacZ)</i> in which transcription is under the control of <i>LEU2</i> and <i>GAL1-10</i> promoters, respectively. Gray boxes represent <i>LEU2</i> repeats that flank the <i>lacZ</i> sequence. Arrow indicates the transcript produced. P. Promoter. Recombination frequencies are plotted as a function of the transcription levels. Low transcription refers to the GL-<i>lacZ</i> systems in strains cultured in 2% glucose; medium refers to L-<i>lacZ</i> in 2% glucose, and high to GL-<i>lacZ</i> in 2% galactose. The average median value and SD of 3-4 fluctuation tests are shown. Asterisks indicate statistically significant differences between the strains indicated, according to Student's t-tests (*, Ρ<0.05; ***, Ρ<0.0005).</p

Table of Strains used in this work.

<p>Table of Strains used in this work.</p

Figure 7

<p><b>Spontaneous and AID-induced mutation frequencies in wild-type and </b><b><i>trf4</i></b>Δ <b>strains.</b> Mutation frequency of W303-1A (WT), TRF4D-C5 (<i>trf4</i>Δ) strains, using the LAUR fusion construct. Ura^- mutants are selected in SC+FOA. The human <i>AID</i> gene was overexpressed in 2% galactose medium using plasmid p413GALAID. The median values of mutation frequencies and SD of 3–4 different fluctuation tests are shown. Asterisks indicate statistically significant differences, according to Student's t tests (*, P<0.05; **, Ρ<0.005).</p