Nuevos genes humanos asociados a la biogénesis de mRNPs necesarios para la integridad del genoma

La integridad del genoma es una condición necesaria para la transmisión fidedigna de la información genética. Numerosos procesos altamente regulados trabajan de forma coordinada para evitar o solucionar problemas que pueden comprometer la estabilidad del genoma. Su inestabilidad es una patología celular que se manifiesta generalmente en forma de mutaciones y reordenaciones cromosómicas y se encuentra asociada a la predisposición a cáncer y envejecimiento. El origen de la inestabilidad genética es variado y no sólo es consecuencia de la acción de agentes genotóxicos externos, sino resultado del propio metabolismo celular, estando ligada a procesos básicos como la replicación, transcripción y recombinación. En esta tesis nos hemos centrado en la transcripción y en el metabolismo del ARN mensajero (ARNm) como fuentes endógenas de inestabilidad genética. La transcripción puede suponer una amenaza para la integridad del genoma debido a que durante la misma se facilita la aparición de ADN de cadena sencilla, que es más susceptible a daños que la doble cadena. Paralelamente, la transcripción puede suponer un obstáculo para la replicación que puede derivar en un incremento de roturas en el ADN y recombinación, responsable de reordenaciones cromosómicas. Estos fenómenos se pueden agravar cuando además se forman unas estructuras denominadas bucles R (R loops), estructuras compuestas por un híbrido de ARN-ADN y la cadena sencilla de ADN desplazada. La formación de R loops se produce cuando el ARN naciente resultante de la transcripción, hibrida con la hebra molde de ADN, desplazando así a la hebra no transcrita que queda como cadena sencilla. Aunque los R loops pueden desempeñar papeles positivos, se ha demostrado que también pueden amenazar la integridad del genoma. Muchos factores implicados en las diferentes etapas del procesamiento del ARNm contribuyen a proteger el genoma de la formación de R loops. En esta tesis nos hemos centrado en factores con un papel en la biogénesis de las ribonucleoproteínas mensajeras (mRNPs), y en concreto en el complejo THO/TREX. Durante la transcripción, el ARNm necesita ser correctamente empaquetado en mRNPs, permitiendo así la elongación de la transcripción, la integridad y el procesamiento del ARNm y su transporte al citoplasma. Para ello, numerosas proteínas de unión al ARN (RBPs) se asocian con el ARN naciente, de forma que este queda empaquetado y protegido, reduciendo así la probabilidad de que el ARN hibride con el ADN molde y forme R loops. Uno de los factores claves en este proceso es THO/TREX, conservado de levaduras a humanos con un papel en el acoplamiento de la transcripción con la biogénesis y transporte de mRNPs. Los mutantes de este complejo acumulan R loops y muestran alta inestabilidad genética. Esto se explica en gran medida por el hecho de que en ausencia de este complejo la mRNP no se forma correctamente y por tanto el ARNm queda más desprotegido, facilitando así la formación de R loops e incrementando la inestabilidad genética. En esta tesis hemos querido profundizar sobre los mecanismos por los cuales la correcta biogénesis de mRNPs contribuye a la integridad del genoma. Para ello, hemos realizado un escrutinio para identificar nuevas proteínas que interaccionen con el complejo THO/TREX humano. Como resultado, hemos identificado dos nuevas interacciones. Hemos mostrado que la subunidad THOC1 del complejo THO/TREX humano, interacciona con el complejo histona desacetilasa Sin3A, y con MFAP1, un factor asociado al madurosoma (spliceosome). THOC1 interacciona físicamente con las subunidades SAP130 y SIN3 del complejo Sin3A. El silenciamiento de las subunidades del complejo SAP130, SIN3, SAP30 y SUDS3 causa inestabilidad genética, determinada por el incremento de roturas en el ADN. Esta inestabilidad, al igual que sucede en ausencia de THO, está mediada por la formación de R loops, puesto que el incremento de roturas en el ADN en ausencia de subunidades del complejo Sin3A tales como SAP130 y SIN3 se suprime mediante la sobreexpressión de RNasa H, la cual degrada los híbridos de ARN-ADN. Hemos demostrado que la inhibición de la desacetilación de histonas mediante compuestos químicos conduce a una acumulación de R loops, y aún más importante, que la inhibición de la acetilación de histonas mediante compuestos químicos suprime el daño en el ADN y la formación de R loops causados por el silenciamiento de THOC1. En general, la acetilación de las histonas da lugar a una cromatina más abierta y una activación de la transcripción, mientras que la desacetilación de histonas se asocia con una cromatina más cerrada o compactada y a una represión de la transcripción. Por tanto, estos resultados permiten proponer un modelo en el que la desacetilacion de histonas sería necesaria para prevenir la formación de R loops tras el paso de la ARN polimerasa. THO podría interaccionar con Sin3A para contribuir a la desacetilación de histonas, con objeto de cerrar la cromatina transitoriamente y así prevenir que el ARN naciente hibride con el ADN molde. El silenciamiento de MFAP1, en cambio, provoca roturas en el ADN que no dependen de la formación de R loops. Los análisis globales de expresión génica y maduración de intrones (splicing) en células silenciadas para MFAP1 sugieren que el impacto de este factor en la estabilidad del genoma puede deberse principalmente a su papel en splicing. El hecho de que el silenciamiento de MFAP1 afecte en gran medida al splicing de genes implicados en la reparación del ADN, el ciclo celular y la organización y modificación de la cromatina entre otros, sugiere que el papel de MFAP1 en la estabilidad del genoma es indirecto; es decir, mediado por los genes cuyo splicing regula. El splicing de algunos de estos genes también presenta cambios en ausencia de THOC1. Dado que THO sí desempeña un papel directo en el mantenimiento de la estabilidad del genoma, estos cambios se podrían explicar como una consecuencia indirecta de la inestabilidad genética dependiente de transcripción y R loops que causa la ausencia de THOC1. No obstante, no se puede descartar que THOC1 también regule la integridad del genoma a través de su interacción con MFAP1 y su efecto en el splicing de algunos genes. Estos datos son particularmente relevantes al sugerir que no todas las proteínas de unión a ARN tienen un papel directo en estabilidad del genoma, pudiendo ser su efecto en muchos casos indirecto, consecuencia de su efecto en la regulación de la expresión de otros genes

Human THO–Sin3A interaction reveals new mechanisms to prevent R-loops that cause genome instability

R-loops, formed by co-transcriptional DNA–RNA hybrids and a displaced DNA single strand (ssDNA), fulfill certain positive regulatory roles but are also a source of genomic instability. One key cellular mechanism to prevent R-loop accumulation centers on the conserved THO/TREX complex, an RNA-binding factor involved in transcription elongation and RNA export that contributes to messenger ribonucleoprotein (mRNP) assembly, but whose precise function is still unclear. To understand how THO restrains harmful R-loops, we searched for new THO-interacting factors. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress co-transcriptional R-loops, DNA damage, and replication impairment. Functional analyses show that histone hypo-acetylation prevents accumulation of harmful R-loops and RNA-mediated genomic instability. Diminished histone deacetylase activity in THO- and Sin3A-depleted cell lines correlates with increased R-loop formation, genomic instability, and replication fork stalling. Our study thus uncovers physical and functional crosstalk between RNA-binding factors and chromatin modifiers with a major role in preventing R-loop formation and RNA-mediated genome instability.European Research Council ERC2014 AdG669898 TARLOOPMinisterio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-PJunta de Andalucía BIO123

The THO Complex as a Paradigm for the Prevention of Cotranscriptional R-Loops

Different proteins associate with the nascent RNA and the RNA polymerase (RNAP) to catalyze the transcription cycle and RNA export. If these processes are not properly controlled, the nascent RNA can thread back and hybridize to the DNA template forming R-loops capable of stalling replication, leading to DNA breaks. Given the transcriptional promiscuity of the genome, which leads to large amounts of RNAs from mRNAs to different types of ncRNAs, these can become a major threat to genome integrity if they form R-loops. Consequently, cells have evolved nuclear factors to prevent this phenomenon that includes THO, a conserved eukaryotic complex acting in transcription elongation and RNA processing and export that upon inactivation causes genome instability linked to R-loop accumulation. We revise and discuss here the biological relevance of THO and a number of RNA helicases, including the THO partner UAP56/DDX39B, as a paradigm of the cellular mechanisms of cotranscriptional R-loop prevention

Depletion of the MFAP1/SPP381 Splicing Factor Causes R-Loop-Independent Genome Instability

THO/TREX is a conserved complex with a role in messenger ribonucleoprotein biogenesis that links gene expression and genome instability. Here, we show that human THO interacts with MFAP1 (microfibrillar-associated protein 1), a spliceosome-associated factor. Interestingly, MFAP1 depletion impairs cell proliferation and genome integrity, increasing γH2AX foci and DNA breaks. This phenotype is not dependent on either transcription or RNA-DNA hybrids. Mutations in the yeast orthologous gene SPP381 cause similar transcription-independent genome instability, supporting a conserved role. MFAP1 depletion has a wide effect on splicing and gene expression in human cells, determined by transcriptome analyses. MFAP1 depletion affects a number of DNA damage response (DDR) genes, which supports an indirect role of MFAP1 on genome integrity. Our work defines a functional interaction between THO and RNA processing and argues that splicing factors may contribute to genome integrity indirectly by regulating the expression of DDR genes rather than by a direct role.European ResearchCouncil (grant ERC2014 AdG669898 TARLOOP)Junta de Andalucía Spain (grant BIO1238)Spanish Ministry of Economy and Competitiveness (grant BFU2016-75058-P

New insights into the function of THO/TREX mRNP biogenesis and export factor in yeast and human cells

Póster presentado al 22nd IUBMB & 37th FEBS Congress: From Single Molecules to Systems Biology, celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012Coupling of transcription with mRNA processing and export has been shown to be relevant to efficient gene expression. A number of studies have determined that THO/TREX, a nuclear protein complex conserved from yeast to humans, plays an important role in mRNP biogenesis connecting transcription elongation, mRNA export and preventing genetic instability. Recent data indicates that THO could be relevant to different mRNA processing steps, including the 3'-end formation, transcript release and export. Novel connections of THO to proteins related to the splicing machinery, provide new views about possible functions of THO in mRNP biogenesis. In this review, we summarize the previous and new results concerning the impact of THO in transcription and its biological implications, with a special emphasis on the relationship with THSC/TREX-2 and other functionally related factors involved in mRNA biogenesis and export. The emerging picture presents THO as a dynamic complex interacting with the nascent RNA and with different factors connecting nuclear functions necessary for mRNP biogenesis with genome integrity, cellular homeostasis and development. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.Peer Reviewe

Role of chromatin and the DNA damage response in transcription-associated genome instability

Resumen del trabajo presentado a la Conferencia Dynamic DNA Structure in Biology, celebrada en Saxtons River, Vermont (US) del 10 al 15 de julio de 2016.Coordination of DNA replication with DNA-damage sensing, repair and cell cycle progression ensures with high probability genome integrity during cell divisions, thus preventing mutations and DNA rearrangements. Such events may be harmful for the cell and the organism, and are usually associated with pathological disorders. One important type of genome instability is that associated with transcription. R-loops are transcriptional by-products that can be formed naturally as key intermediates in specific cellular processes, but they are also a major source of genome instability. Specific RNA processing factors have been shown to play a role in preventing R-loop and transcription-associated genome instability. The first one identified with this role was the yeast THO complex, a conserved factor working at the interface between transcription and RNA export. However, other proteins directly or indirectly related with a function in RNA processing, such as human SRSF2 and AQR or yeast Trf4 or Npl3 among others, also prevent RNA-DNA hybrids. The working hypothesis suggests that in cells defective in such factors a suboptimal nascent mRNA-protein particle is formed, enhancing the probability that the nascent RNA interacts with the DNA. However, it is unclear how this mechanism occurs. To explore further the mechanism by which RNA processing factors control genome integrity, we have screened a human library for proteins that physically interact with components of the human THO complex using the yeast two-hybrid system. Further confirmation of this interaction via co-immunoprecipitation and Proximity Ligation Assay, has permitted us to identify a chromatin remodeling complex. Functional analyses of this interaction and of the effect of depleting cells from these factors has permitted us to propose a new model to explain how cells prevent co-transcriptional RNA-DNA hybrid formation and transcription-associated genome instability. Our work open news perspectives to understand the different mechanisms used by the cells to prevent the accumulation of DNA structures that compromises genome integrity.Peer Reviewe

Human THO–Sin3A interaction reveals new mechanisms to prevent R-loops that cause genome instability

R-loops, formed by co-transcriptional DNA–RNA hybrids and a displaced DNA single strand (ssDNA), fulfill certain positive regulatory roles but are also a source of genomic instability. One key cellular mechanism to prevent R-loop accumulation centers on the conserved THO/TREX complex, an RNA-binding factor involved in transcription elongation and RNA export that contributes to messenger ribonucleoprotein (mRNP) assembly, but whose precise function is still unclear. To understand how THO restrains harmful R-loops, we searched for new THO-interacting factors. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress co-transcriptional R-loops, DNA damage, and replication impairment. Functional analyses show that histone hypo-acetylation prevents accumulation of harmful R-loops and RNA-mediated genomic instability. Diminished histone deacetylase activity in THO- and Sin3A-depleted cell lines correlates with increased R-loop formation, genomic instability, and replication fork stalling. Our study thus uncovers physical and functional crosstalk between RNA-binding factors and chromatin modifiers with a major role in preventing R-loop formation and RNA-mediated genome instability.Research was funded by the European Research Council (ERC2014 AdG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2013-42918-P and BFU2016-75058-P), the Junta de Andalucía (BIO1238), and the European Union (FEDER). I.S.A. and C.P.C were recipients of FPU and FPI pre-doctoral training grants from the Spanish Ministries of Education, Culture and Sports and of Economy and Competitiveness, respectively.Peer Reviewe

Depletion of the MFAP1/SPP381 Splicing Factor Causes R-Loop-Independent Genome Instability

THO/TREX is a conserved complex with a role in messenger ribonucleoprotein biogenesis that links gene expression and genome instability. Here, we show that human THO interacts with MFAP1 (microfibrillar-associated protein 1), a spliceosome-associated factor. Interestingly, MFAP1 depletion impairs cell proliferation and genome integrity, increasing γH2AX foci and DNA breaks. This phenotype is not dependent on either transcription or RNA-DNA hybrids. Mutations in the yeast orthologous gene SPP381 cause similar transcription-independent genome instability, supporting a conserved role. MFAP1 depletion has a wide effect on splicing and gene expression in human cells, determined by transcriptome analyses. MFAP1 depletion affects a number of DNA damage response (DDR) genes, which supports an indirect role of MFAP1 on genome integrity. Our work defines a functional interaction between THO and RNA processing and argues that splicing factors may contribute to genome integrity indirectly by regulating the expression of DDR genes rather than by a direct role. THO, an mRNA biogenesis factor, interacts with MFAP1, a conserved spliceosome-associated protein. Salas-Armenteros et al. show that MFAP1/SPP381 depletion alters splicing and gene expression and increases genome instability in an RNA-DNA hybrid-independent manner. Therefore, RNA-DNA hybrid accumulation is not an intrinsic consequence of splicing defects.Research was funded by the European Research Council (grant ERC2014 AdG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (grant BFU2016-75058-P), the Junta de Andalucía Spain (grant BIO1238), and the European Union Regional Funds (FEDER). I.S.-A. was a recipient of a FPU pre-doctoral training grant from the Spanish Ministry of Education, Culture and Sports

Understanding R loop-mediated genome instability: a new role for histones and chromatin modifications

Resumen del trabajo presentado a la 12th International Conference & 5th Asian Congress on Environmental Mutagens with the 33rd Annual Meeting of KSOT/KEMS, celebradas en Songdo Convensia, Incheon (Korea) del 12 al 16 de noviembre de 2017.Coordination of DNA replication with DNA-damage sensing, repair and cell cycle progression ensures with high probability genome integrity during cell divisions. One important type of genome instability is that associated with transcription. R loops, structures formed by a DNA-RNA hybrid and the displaced single-stranded DNA (ssDNA) molecule, are transcriptional by-products that can be formed naturally as key intermediates in specific cellular processes. Nevertheless, they are also a major source of transcription-associated genome instability and compelling evidence supports that this is mainly caused by replication fork impairment. Consequently, the relevance of R loopmediated genome instability as a mechanism of environmental mutagenesis needs to be studied. Our analysis of R loop-mediated instability in human cells depleted of the THO complex involved in RNA biogenesis reveals a new role for chromatin modifications in R loop accumulation. In addition, using human activation-induced cytidine deaminase (AID), we have identified yeast histone mutants that facilitate R loop formation without leading to genome instability. These R loops are similar in size to those causing genome instability. However, these yeast histone mutants do not lead to the same chromatin alterations. Importantly, we are able to suppress R loop-mediated genome instability by specific histone mutations as well as by altering the pattern of co-transcriptional chromatin modifications in yeast and human cells. Our results imply a new role for chromatin on the sources or R loop formation as well as on the mechanisms of transcription-associated genome instability. The relevance of our conclusions in the context of environmental genotoxicity will be discussed.Peer Reviewe

Active DNA damage eviction by HLTF stimulates nucleotide excision repair

Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a “cut-and-patch”-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3′-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity