Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1, and TIA1

C.V. was recipient of an FPI-Severo Ochoa Fellowship from the Spanish Ministry of Economy and Competitiveness. Work in J.V. laboratory is supported by the European Research Council (ERC AdvG 670146), AGAUR, Spanish Ministry of Economy and Competitiveness (BFU 2017 89308-P) and the Centre of Excellence Severo Ochoa. Work in T.G.'s laboratory was supported by the European Research Council FP7/2007-2013 (ERC Synergy Grant 4D-Genome) the Ministerio de Educación y Ciencia (SAF.2012-37167) and AGAUR. We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership and the CERCA Programme / Generalitat de Catalunya.UDTRIASBackground: Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results: We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. Conclusions: Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets

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Resumen basado en el de la revista en catalánEl entusiasmo, la curiosidad y la emoción demostrados por los niños de la escuela frente a cualquier estímulo han sido aprovechados por el equipo de maestros de ciclo inicial, no sólo para conseguir trabajar el tema escogido por los niños, sino también para iniciarles en un trabajo serio de investigación, en la búsqueda del conocimiento impulsando la autonomía, la organización personal del trabajo, la valoración del esfuerzo y sobre todo el compromiso para poder llegar al final de una meta con responsabilidad.CataluñaES

Coordinated post-transcriptional control of oncogene-induced senescence by UNR/CSDE1

Oncogene-induced senescence (OIS) is a form of stable cell-cycle arrest arising in response to oncogenic stimulation. OIS must be bypassed for transformation, but the mechanisms of OIS establishment and bypass remain poorly understood, especially at the post-transcriptional level. Here, we show that the RNA-binding protein UNR/CSDE1 enables OIS in primary mouse keratinocytes. Depletion of CSDE1 leads to senescence bypass, cell immortalization, and tumor formation, indicating that CSDE1 behaves as a tumor suppressor. Unbiased high-throughput analyses uncovered that CSDE1 promotes OIS by two independent molecular mechanisms: enhancement of the stability of senescence-associated secretory phenotype (SASP) factor mRNAs and repression of Ybx1 mRNA translation. Importantly, depletion of YBX1 from immortal keratinocytes rescues senescence and uncouples proliferation arrest from the SASP, revealing multilayered mechanisms exerted by CSDE1 to coordinate senescence. Our data highlight the relevance of post-transcriptional control in the regulation of senescence.We thank Bill Keyes and members of his lab Mekayla Storer, Birgit Ritschka, and Alba Mas for expert advice and help at the initial stages of this project. We also thank Jernej Ule for sharing reagents and expertise on iCLIP, Ola Larsson for guidance on anota2seq, and Bill Keyes and Juan Valcárcel for carefully reading the manuscript. We acknowledge Raquel García Olivas of the CRG Advanced Light Microscopy Unit (ALMU) for her support and assistance in this work. We thank the CRG Bioinformatics Unit, the CRG Genomics Facility, the CRG/UPF FACS Unit, and the IRB Histopathology Facility for high-throughput sequencing and analysis, cell sorting, and histopathology analysis, respectively. M.I.-F. and A.C. were supported by FPI fellowships from the Spanish Ministry of Science and Innovation (MICINN) . F.G. was supported by grants from MICINN ( PGC2018-099697-B-I00 and BFU2015-68741 ), “La Caixa” Foundation (ID 100010434 ) under the agreement LCF/PR/HR17/52150016, the Catalan Agency for Research and Universities ( 2017SGR534 ), and the Centre of Excellence Severo Ochoa

Co-option of the piRNA pathway for germline-specific alternative splicing of C. elegans TOR

Includes supplementary materials for the online appendix.Many eukaryotic genes contain embedded antisense transcripts and repetitive sequences of unknown function. We report that male germline-specific expression of an antisense transcript contained in an intron of C. elegans Target of Rapamycin (TOR, let-363) is associated with (1) accumulation of endo-small interfering RNAs (siRNAs) against an embedded Helitron transposon and (2) activation of an alternative 3′ splice site of TOR. The germline-specific Argonaute proteins PRG-1 and CSR-1, which participate in self/nonself RNA recognition, antagonistically regulate the generation of these endo-siRNAs, TOR mRNA levels, and 3′ splice-site selection. Supply of exogenous double-stranded RNA against the region of sense/antisense overlap reverses changes in TOR expression and splicing and suppresses the progressive multigenerational sterility phenotype of prg-1 mutants. We propose that recognition of a “nonself” intronic transposon by endo-siRNAs/the piRNA system provides physiological regulation of expression and alternative splicing of a host gene that, in turn, contributes to the maintenance of germline function across generations.We thank A. Fire, S. Ahmed, and E. Miska for sharing information and helpful discussions, members of our laboratories for critical feedback, the Caenorhabditis Genetics Center (CGC) for providing strains, Matthew Ragle for collection of sperm samples, Alan Zahler for assistance and critical feedback, and Montserrat Porta de la Riva for technical assistance. S.B.-S. was supported by an EMBO postdoctoral fellowship. A.T.L. is a Taub Fellow, supported by the Taub Foundation. J.C. is a Miguel Servet Junior Investigator. Work in J.V.’s lab is supported by Fundación Botín, Consolider RNAREG and Ministerio de Economía y Competitividad (MINECO). Work in B.L.’s lab is supported by the European Research Council (ERC), MINECO Plan Nacional grants BFU2008-00365 and BFU2011-26206, ERASysBio+ ERANET project EUI2009-04059 GRAPPLE, the EMBO Young Investigator Program, EU Framework 7 project 277899 4DCellFate, the AXA Research Fund, and the EMBL/CRG Systems Biology Program. Support from the CRG Genomics Facility is also acknowledged

Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1, and TIA1

Background: Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results: We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. Conclusions: Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets.C.V. was recipient of an FPI-Severo Ochoa Fellowship from the Spanish Ministry of Economy and Competitiveness. Work in J.V. laboratory is supported by the European Research Council (ERC AdvG 670146), AGAUR, Spanish Ministry of Economy and Competitiveness (BFU 2017 89308-P) and the Centre of Excellence Severo Ochoa. Work in T.G.’s laboratory was supported by the European Research Council FP7/2007-2013 (ERC Synergy Grant 4D-Genome) the Ministerio de Educación y Ciencia (SAF.2012-37167) and AGAUR. We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership and the CERCA Programme / Generalitat de Catalunya

Combination of Thrombolysis and Statins in Acute Stroke Is Safe: Results of the STARS Randomized Trial (Stroke Treatment With Acute Reperfusion and Simvastatin).

The STARS trial (Stroke Treatment With Acute Reperfusion and Simvastatin) was conducted to demonstrate the efficacy and safety of simvastatin treatment in acute stroke. STARS07 was a multicentre, phase IV, prospective, randomized, double-blind, placebo-controlled trial. Patients with Acute ischemic stroke recruited within 12 hours from symptom onset were randomized to oral simvastatin 40 mg or placebo, once daily for 90 days. Primary outcome was proportion of independent patients (modified Rankin Scale score of ≤2) at 90 days. Safety end points were hemorrhagic transformation, hemorrhagic events, death, infections, and serious adverse events. From April 2009 to March 2014, 104 patients were included. Fifty-five patients received intravenous tissue-type plasminogen activator. No differences were found between treatment arms regarding the primary outcome (adjusted odds ratio, 0.99 [0.35-2.78]; P=0.98). Concerning safety, no significant differences were found in the rate of hemorrhagic transformation of any type, nor symptomatic hemorrhagic transformation. There were no differences in other predefined safety outcomes. In post hoc analyses, for patients receiving tissue-type plasminogen activator, a favorable effect for simvastatin treatment was noted with higher proportion of patients experiencing major neurological recovery (adjusted odds ratio, 4.14 [1.18-14.4]; P=0.02). Simvastatin plus tissue-type plasminogen activator combination seems safe in acute stroke, with low rates of bleeding complications. Because of the low recruitment, the STARS trial was underpowered to detect differences in simvastatin efficacy. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01073007

Effect of general anaesthesia on functional outcome in patients with anterior circulation ischaemic stroke having endovascular thrombectomy versus standard care: a meta-analysis of individual patient data

Background: General anaesthesia (GA) during endovascular thrombectomy has been associated with worse patient outcomes in observational studies compared with patients treated without GA. We assessed functional outcome in ischaemic stroke patients with large vessel anterior circulation occlusion undergoing endovascular thrombectomy under GA, versus thrombectomy not under GA (with or without sedation) versus standard care (ie, no thrombectomy), stratified by the use of GA versus standard care. Methods: For this meta-analysis, patient-level data were pooled from all patients included in randomised trials in PuMed published between Jan 1, 2010, and May 31, 2017, that compared endovascular thrombectomy predominantly done with stent retrievers with standard care in anterior circulation ischaemic stroke patients (HERMES Collaboration). The primary outcome was functional outcome assessed by ordinal analysis of the modified Rankin scale (mRS) at 90 days in the GA and non-GA subgroups of patients treated with endovascular therapy versus those patients treated with standard care, adjusted for baseline prognostic variables. To account for between-trial variance we used mixed-effects modelling with a random effect for trials incorporated in all models. Bias was assessed using the Cochrane method. The meta-analysis was prospectively designed, but not registered. Findings: Seven trials were identified by our search; of 1764 patients included in these trials, 871 were allocated to endovascular thrombectomy and 893 were assigned standard care. After exclusion of 74 patients (72 did not undergo the procedure and two had missing data on anaesthetic strategy), 236 (30%) of 797 patients who had endovascular procedures were treated under GA. At baseline, patients receiving GA were younger and had a shorter delay between stroke onset and randomisation but they had similar pre-treatment clinical severity compared with patients who did not have GA. Endovascular thrombectomy improved functional outcome at 3 months both in patients who had GA (adjusted common odds ratio (cOR) 1·52, 95% CI 1·09–2·11, p=0·014) and in those who did not have GA (adjusted cOR 2·33, 95% CI 1·75–3·10, p<0·0001) versus standard care. However, outcomes were significantly better for patients who did not receive GA versus those who received GA (covariate-adjusted cOR 1·53, 95% CI 1·14–2·04, p=0·0044). The risk of bias and variability between studies was assessed to be low. Interpretation: Worse outcomes after endovascular thrombectomy were associated with GA, after adjustment for baseline prognostic variables. These data support avoidance of GA whenever possible. The procedure did, however, remain effective versus standard care in patients treated under GA, indicating that treatment should not be withheld in those who require anaesthesia for medical reasons