23 research outputs found
Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.
Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression
CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3
Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF65 and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain
The RNA Response to DNA Damage
Multicellular organisms must ensure genome integrity to prevent accumulation of mutations, cell death, and cancer. The DNA damage response (DDR) is a complex network that senses, signals, and executes multiple programs including DNA repair, cell cycle arrest, senescence, and apoptosis. This entails regulation of a variety of cellular processes: DNA replication and transcription, RNA processing, mRNA translation and turnover, and post-translational modification, degradation, and relocalization of proteins. Accumulated evidence over the past decades has shown that RNAs and RNA metabolism are both regulators and regulated actors of the DDR. This review aims to present a comprehensive overview of the current knowledge on the many interactions between the DNA damage and RNA fields.Fil: Giono, Luciana Eugenia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Nieto Moreno, NicolĂĄs. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Cambindo Botto, Adrian Edgardo. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Dujardin, Gwendal. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Muñoz, Manuel Javier. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; Argentin
LâĂ©pissage des ARN prĂ©-messagers : quand le splicĂ©osome perd pied
LâĂ©pissage des ARN prĂ©-messagers est une Ă©tape obligatoire pour lâassemblage de la trĂšs grande majoritĂ© des ARN messagers chez les organismes eucaryotes. Chez lâhomme, chacun des gĂšnes donne naissance Ă au moins deux transcrits, avec une moyenne de 6 Ă 8 transcrits par gĂšne. Câest lâĂ©tablissement dâune continuitĂ© entre les exons consĂ©cutifs qui permet la lecture de la sĂ©quence servant de matrice Ă la machinerie de synthĂšse protĂ©ique. NĂ©anmoins, lâĂ©pissage nâest pas univoque. Il admet des variantes, ou Ă©pissages alternatifs (EA), qui concourent Ă accroĂźtre la variabilitĂ© protĂ©ique, comblant ainsi en partie lâĂ©cart existant entre le nombre de gĂšnes â 20 000 Ă 25 000 chez lâhomme â et le nombre minimal de protĂ©ines synthĂ©tisĂ©es â au moins 100 000. En outre, bien que les EA relĂšvent souvent de besoins cellulaires spĂ©cifiques, un ensemble dâĂ©vĂ©nements aberrants peut survenir et participer au dĂ©veloppement de maladies gĂ©nĂ©tiques ou de pathologies acquises comme les maladies neuro-dĂ©gĂ©nĂ©ratives ou les cancers. Dans ces situations, les Ă©vĂ©nements dâEA peuvent servir de marqueurs de ces Ă©tats, voire de cibles potentielles pour des approches thĂ©rapeutiques visant Ă corriger les dĂ©fauts. Dans cette SynthĂšse, nous allons rappeler les principaux mĂ©canismes de rĂ©gulation de lâĂ©pissage et de lâEA. Nous prĂ©senterons notamment le splicĂ©osome, un macro-complexe ribonuclĂ©oprotĂ©ique constituant le cĆur de la machinerie dâĂ©pissage, rĂ©cemment identifiĂ© comme une cible privilĂ©giĂ©e de mutations dans plusieurs pathologies. Nous discuterons de certains des dĂ©fauts du splicĂ©osome associĂ©s Ă des altĂ©rations survenant en cis (sur lâADN, et donc sur lâARN prĂ©-messager) et en trans (dans des protĂ©ines interagissant avec lâARN prĂ©-messager), telles quâelles ont Ă©tĂ© rapportĂ©es dans des maladies gĂ©nĂ©tiques ou acquises
How Slow RNA Polymerase II Elongation Favors Alternative Exon Skipping
Splicing is functionally coupled to transcription, linking the rate of RNA polymerase II (Pol II) elongation and the ability of splicing factors to recognize splice sites (ss) of various strengths. In most cases, slow Pol II elongation allows weak splice sites to be recognized, leading to higher inclusion of alternative exons. Using CFTR alternative exon 9 (E9) as a model, we show here that slowing down elongation can also cause exon skipping by promoting the recruitment of the negative factor ETR-3 onto the UG-repeat at E9 3âČ splice site, which displaces the constitutive splicing factor U2AF65 from the overlapping polypyrimidine tract. Weakening of E9 5âČ ss increases ETR-3 binding at the 3âČ ss and subsequent E9 skipping, whereas strengthening of the 5âČ ss usage has the opposite effect. This indicates that a delay in the cotranscriptional emergence of the 5âČ ss promotes ETR-3 recruitment and subsequent inhibition of E9 inclusion.Fil: Dujardin, Gwendal. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Lafaille, Celina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: de la Mata, Manuel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Marasco, Luciano Edmundo. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Muñoz, Manuel Javier. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; ArgentinaFil: Le Jossic Corcos, Catherine. Inserm; FranciaFil: Corcos, Laurent. Inserm; FranciaFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; Argentin
Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy
Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function