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TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed DNA-/RNA-binding protein that has been linked to numerous aspects of the mRNA life cycle. Similar to many RNA-binding proteins, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Cell function and survival depend on the strict control of TDP-43 protein levels. TDP-43 has been identified as the major constituent of ubiquitin-positive inclusions in patients with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several observations argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulation of the cycle of TDP-43 production might therefore provide a new therapeutic strategy. Using a Drosophila model mimicking key features of the TDP-43 autoregulatory feedback loop, we identified CG42724 as a genetic modulator of TDP-43 production in vivo. We found that CG42724 protein influences qualitatively and quantitatively the TDP-43 mRNA transcript pattern. CG42724 overexpression promotes the production of transcripts that can be efficiently released into the cytoplasm for protein translation. Importantly, we showed that TCERG1, the human homolog of the Drosophila CG42724 protein, also caused an increase of TDP-43 protein steady-state levels in mammalian cells. Therefore, our data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies. ; This work was supported by a grant from the European Union and the Région Normandy. Europe gets involved in Normandy through the European Regional Development Fund (ERDF). MP is a PhD fellow of the French Ministry of Higher Education and Research. ; Peer reviewed

Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas. We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-xL, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis. ; This work was supported by grants from the Spanish Ministry of Economy and Competitiveness [grant numbers BFU2011-24577 and BFU2014-54660-R] and the Andalusian Government [Excellence Projects CVI-4626/2009 and BIO-2515/2012] to CS; the Spanish Ministry of Economy and Competitiveness [grant number BFU2013-44660-R] and the Andalusian Government [Excellence Project CTS-6587] to CHM; the Spanish Ministry of Economy and Competitiveness [grant numbers SAF2013-44677-R and FIS PI12/00506], the SPANISH AIDS Research Network RD12/0017/0015 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011, Instituto de Salud Carlos III, European Region Development Fund, ERDF (FEDER) to MC and JA. MM was supported by a fellowship from the Spanish Ministry of Education (FPU program). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Sí

Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas.We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-xL, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis. ; This work was supported by grants from the Spanish Ministry of Economy and Competitiveness [grant numbers BFU2011-24577 and BFU2014-54660-R] and the Andalusian Government [Excellence Projects CVI-4626/2009 and BIO-2515/2012] to CS; the Spanish Ministry of Economy and Competitiveness [grant number BFU2013-44660-R] and the Andalusian Government [Excellence Project CTS-6587] to CHM; the Spanish Ministry of Economy and Competitiveness [grant numbers SAF2013-44677-R and FIS PI12/00506], the SPANISH AIDS Research Network RD12/0017/ 0015 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011, Instituto de Salud Carlos III, European Region Development Fund, ERDF (FEDER) to MC and JA. MM was supported by a fellowship from the Spanish Ministry of Education (FPU program) ; Peer reviewed

BACKGROUND: Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo. RESULTS: We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication. CONCLUSIONS: Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication. ; This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2011-24577), the Foundation for Research and Prevention of AIDS in Spain (FIPSE-36768/08), and the Andalusian Government (Excellence Project CVI-4626/2009) to C.S.; by the Spanish Ministry of Science and Innovation (BFU2009-08796), and the Andalusian Government (Excellence Project CTS-6587) to C.H.M; and by FIPSE (360924/10), the Spanish Ministry of Economy and Competitiveness (SAF2010-18388; FIS PI0120506), the Spanish Ministry of Health (EC11-285, -278), Instituto de Salud Carlos III, AIDS Network ISCIII-RETIC (RD12/0017/0015), and the Health Programme 2009 on Combined Highly Active Anti-Retroviral Microbicides (CHAARM) to M.C. and J.A. Support from the European Region Development Fund, ERDF (FEDER) is also acknowledged. M.M. was supported by a fellowship from the Spanish Ministry of Education (FPU program). M.R.L.H was supported by a fellowship from the European Union (CHAARM). C.L.S. was funded by a fellowship from the Foundation for Medical Research (F.R.M., France) and by funds from NIH RO1 GM071037 (USA) to M.A.G-B. ; Sí

Abstract Background Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo. Results We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication. Conclusions Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication ; This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2011-24577), the Foundation for Research and Prevention of AIDS in Spain (FIPSE-36768/08), and the Andalusian Government (Excellence Project CVI-4626/2009) to C.S.; by the Spanish Ministry of Science and Innovation (BFU2009-08796), and the Andalusian Government (Excellence Project CTS-6587) to C.H.M; and by FIPSE (360924/10), the Spanish Ministry of Economy and Competitiveness (SAF2010-18388; FIS PI0120506), the Spanish Ministry of Health (EC11-285, -278), Instituto de Salud Carlos III, AIDS Network ISCIII-RETIC (RD12/0017/0015), and the Health Programme 2009 on Combined Highly Active Anti-Retroviral Microbicides (CHAARM) to M.C. and J.A. Support from the European Region Development Fund, ERDF (FEDER) is also acknowledged. M.M. was supported by a fellowship from the Spanish Ministry of Education (FPU program). M.R.L.H was supported by a fellowship from the European Union (CHAARM). C.L.S. was funded by a fellowship from the Foundation for Medical Research (F.R.M., France) and by funds from NIH RO1 GM071037 (USA) to M.A.G-B. ; Peer Reviewed

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing. ; Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación [BFU2016-77728-C3-1-P to S.C. and BFU2017-85420-R to J.C.R.] co-financed with European Union funds (FEDER); Andalusian Government [P12-BIO1938MO, BIO271, US-1256285 to S.C., BIO321 to J.C.R.]; Junta de Andalucía (to L.P.-B.). Funding for open access charge: Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación [BFU2016-77728-C3-1-P].