RESUMEN
The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited 'closed' state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active 'open' state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants (i.e. poorly processive, slow, and mechanochemically uncoupled) can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower RNA binding kinetics and enhanced ATP-stimulated RNA dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2 and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
Asunto(s)
Adenosina Trifosfatasas , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas , ARN Mensajero , Humanos , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Cinética , Mutación , Unión Proteica , ARN Helicasas/metabolismo , ARN Helicasas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Transactivadores/metabolismo , Transactivadores/genéticaRESUMEN
The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited "closed" state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active "open" state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
RESUMEN
Proteins containing DZF (domain associated with zinc fingers) modules play important roles throughout gene expression, from transcription to translation. Derived from nucleotidyltransferases but lacking catalytic residues, DZF domains serve as heterodimerization surfaces between DZF protein pairs. Three DZF proteins are widely expressed in mammalian tissues, ILF2, ILF3 and ZFR, which form mutually exclusive ILF2-ILF3 and ILF2-ZFR heterodimers. Using eCLIP-Seq, we find that ZFR binds across broad intronic regions to regulate the alternative splicing of cassette and mutually exclusive exons. ZFR preferentially binds dsRNA in vitro and is enriched on introns containing conserved dsRNA elements in cells. Many splicing events are similarly altered upon depletion of any of the three DZF proteins; however, we also identify independent and opposing roles for ZFR and ILF3 in alternative splicing regulation. Along with widespread involvement in cassette exon splicing, the DZF proteins control the fidelity and regulation of over a dozen highly validated mutually exclusive splicing events. Our findings indicate that the DZF proteins form a complex regulatory network that leverages dsRNA binding by ILF3 and ZFR to modulate splicing regulation and fidelity.
Asunto(s)
Empalme Alternativo , Empalme del ARN , Animales , Intrones/genética , Exones/genética , Nucleotidiltransferasas , MamíferosRESUMEN
The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work; K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.
Asunto(s)
Adenosina Trifosfatasas , Degradación de ARNm Mediada por Codón sin Sentido , Humanos , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Transactivadores/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ADN Helicasas/genética , ARN/metabolismoRESUMEN
Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Although initially beneficial, TMZ resistance is universal and second-line interventions are an unmet clinical need. Here, we took advantage of the known mechanism of action of TMZ to target guanines (G) and investigated G-rich G-quadruplex (G4) and splice site changes that occur upon TMZ resistance. We report that TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4-stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2-like kinase. Last, we show that the G4 and RNA binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker.
Asunto(s)
Antineoplásicos , Neoplasias Encefálicas , Glioblastoma , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , ADN/farmacología , Resistencia a Antineoplásicos/genética , Glioblastoma/metabolismo , Guanina/farmacología , Humanos , Mutación , ARN , Temozolomida/farmacología , Temozolomida/uso terapéuticoRESUMEN
The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination in order to degrade transcripts with premature stop codons and regulate thousands of human genes. Here, we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL , enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to UPF1SL , the major UPF1 isoform. Using biochemical and transcriptome-wide approaches, we find that UPF1LL can circumvent the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate transcripts with long 3'UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, offering the possibility to rapidly rewire NMD specificity in response to cellular stress.
Asunto(s)
Codón sin Sentido , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas , Transactivadores , Regiones no Traducidas 3' , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Proteína de Unión al Tracto de Polipirimidina/genética , Isoformas de Proteínas/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Alternative polyadenylation (APA) produces transcript 3' untranslated regions (3'UTRs) with distinct sequences, lengths, stabilities and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3'UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3'UTR products of APA, leading to their systematic downregulation. Counteracting this mechanism, the multifunctional RNA-binding protein PTBP1 regulates the balance of short and long 3'UTR isoforms by inhibiting NMD, in addition to its previously described modulation of co-transcriptional polyadenylation (polyA) site choice. Further, we find that many transcripts with altered APA isoform abundance across multiple tumor types are controlled by NMD. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , Poliadenilación , Regiones no Traducidas 3' , Células HEK293 , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Proteína de Unión al Tracto de Polipirimidina/metabolismo , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Transactivadores/metabolismo , TranscriptomaRESUMEN
The sequence-specific RNA-binding proteins PTBP1 (polypyrimidine tract-binding protein 1) and HNRNP L (heterogeneous nuclear ribonucleoprotein L) protect mRNAs from nonsense-mediated decay (NMD) by preventing the UPF1 RNA helicase from associating with potential decay targets. Here, by analyzing in vitro helicase activity, dissociation of UPF1 from purified mRNPs, and transcriptome-wide UPF1 RNA binding, we present the mechanistic basis for inhibition of NMD by PTBP1. Unlike mechanisms of RNA stabilization that depend on direct competition for binding sites among protective RNA-binding proteins and decay factors, PTBP1 promotes displacement of UPF1 already bound to potential substrates. Our results show that PTBP1 directly exploits the tendency of UPF1 to release RNA upon ATP binding and hydrolysis. We further find that UPF1 sensitivity to PTBP1 is coordinated by a regulatory loop in domain 1B of UPF1. We propose that the UPF1 regulatory loop and protective proteins control kinetic proofreading of potential NMD substrates, presenting a new model for RNA helicase regulation and target selection in the NMD pathway.
Asunto(s)
Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Proteína de Unión al Tracto de Polipirimidina/metabolismo , ARN Helicasas/metabolismo , Transactivadores/metabolismo , Adenosina Trifosfato/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/química , Humanos , Modelos Moleculares , Proteína de Unión al Tracto de Polipirimidina/química , Dominios Proteicos , ARN Helicasas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/química , Transcripción GenéticaRESUMEN
Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS-FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS-FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS-FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS-FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS-FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS-FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.
Asunto(s)
Carcinogénesis/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/genética , Factores de Transcripción/genética , Empalme Alternativo/genética , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Nucleares/química , Proteínas de Fusión Oncogénica/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estabilidad Proteica , Proteína Proto-Oncogénica c-fli-1/química , Proteína EWS de Unión a ARN/química , Sarcoma de Ewing/patología , Factores de Transcripción/químicaRESUMEN
The nonsense-mediated mRNA decay pathway selects and degrades its targets using a dense network of RNA-protein and protein-protein interactions. Together, these interactions allow the pathway to collect copious information about the translating mRNA, including translation termination status, splice junction positions, mRNP composition, and 3'UTR length and structure. The core NMD machinery, centered on the RNA helicase UPF1, integrates this information to determine the efficiency of decay. A picture of NMD is emerging in which many factors contribute to the dynamics of decay complex assembly and disassembly, thereby influencing the probability of decay. The ability of the NMD pathway to recognize mRNP features of diverse potential substrates allows it to simultaneously perform quality control and regulatory functions. In vertebrates, increased transcriptome complexity requires balance between these two functions since high NMD efficiency is desirable for maintenance of quality control fidelity but may impair expression of normal mRNAs. NMD has adapted to this challenge by employing mechanisms to enhance identification of certain potential substrates, while using sequence-specific RNA-binding proteins to shield others from detection. These elaborations on the conserved NMD mechanism permit more sensitive post-transcriptional gene regulation but can have severe deleterious consequences, including the failure to degrade pathogenic aberrant mRNAs in many B cell lymphomas. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Mensajero/genética , Transcriptoma , Animales , Humanos , ARN Mensajero/metabolismoRESUMEN
The human nonsense-mediated mRNA decay pathway (NMD) performs quality control and regulatory functions within complex post-transcriptional regulatory networks. In addition to degradation-promoting factors, efficient and accurate detection of NMD substrates involves proteins that safeguard normal mRNAs. Here, we identify hnRNP L as a factor that protects mRNAs with NMD-inducing features including long 3'UTRs. Using biochemical and transcriptome-wide approaches, we provide evidence that the susceptibility of a given transcript to NMD can be modulated by its 3'UTR length and ability to recruit hnRNP L. Integrating these findings with the previously defined role of polypyrimidine tract binding protein 1 in NMD evasion enables enhanced prediction of transcript susceptibility to NMD. Unexpectedly, this system is subverted in B cell lymphomas harboring translocations that produce BCL2:IGH fusion mRNAs. CRISPR/Cas9 deletion of hnRNP L binding sites near the BCL2 stop codon reduces expression of the fusion mRNAs and induces apoptosis. Together, our data indicate that protection by hnRNP L overrides the presence of multiple 3'UTR introns, allowing these aberrant mRNAs to evade NMD and promoting BCL2 overexpression and neoplasia.
Asunto(s)
Regiones no Traducidas 3'/genética , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo L/metabolismo , Linfoma de Células B/patología , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo L/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Linfoma de Células B/genética , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , TranscriptomaRESUMEN
Cell-free systems are widely used to study mechanisms and regulation of translation, but the use of in vitro transcribed (IVT) mRNAs as translation substrates limits their efficiency and utility. Here, we present an approach for in vitro translation of messenger ribonucleoprotein (mRNP) complexes affinity purified in association with tagged mRNAs expressed in mammalian cells. We show that in vitro translation of purified mRNPs is much more efficient than that achieved using standard IVT mRNA substrates and is compatible with physiological ionic conditions. The high efficiency of affinity-purified mRNP in vitro translation is attributable to both copurified protein components and proper mRNA processing and modification. Further, we use translation inhibitors to show that translation of purified mRNPs consists of separable phases of run-off elongation by copurified ribosomes and de novo initiation by ribosomes present in the translation extracts. We expect that this in vitro system will enhance mechanistic studies of eukaryotic translation and translation-associated processes by allowing the use of endogenous mRNPs as translation substrates under physiological buffer conditions.
Asunto(s)
Biosíntesis de Proteínas , Ribonucleoproteínas/metabolismo , Sistema Libre de Células , Células HEK293 , Humanos , Magnesio/fisiología , Iniciación de la Cadena Peptídica Traduccional , Potasio/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/aislamiento & purificación , Ribosomas/metabolismoRESUMEN
The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5-10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC.
Asunto(s)
Proteínas Portadoras/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , Biosíntesis de Proteínas/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Codón sin Sentido/genética , Exones/genética , Regulación de la Expresión Génica , Humanos , Dominios Proteicos/genética , Interferencia de ARN , Proteínas Ribosómicas/genéticaRESUMEN
Control of type I interferon production is crucial to combat infection while preventing deleterious inflammatory responses, but the extent of the contribution of post-transcriptional mechanisms to innate immune regulation is unclear. Here, we show that human zinc finger RNA-binding protein (ZFR) represses the interferon response by regulating alternative pre-mRNA splicing. ZFR expression is tightly controlled during macrophage development; monocytes express truncated ZFR isoforms, while macrophages induce full-length ZFR to modulate macrophage-specific alternative splicing. Interferon-stimulated genes are constitutively activated by ZFR depletion, and immunostimulation results in hyper-induction of interferon ß (IFNß/IFNB1). Through whole-genome analyses, we show that ZFR controls interferon signaling by preventing aberrant splicing and nonsense-mediated decay of histone variant macroH2A1/H2AFY mRNAs. Together, our data suggest that regulation of ZFR in macrophage differentiation guards against aberrant interferon responses and reveal a network of mRNA processing and decay that shapes the transcriptional response to infection.
Asunto(s)
Inmunidad Innata , ARN Mensajero/genética , Proteínas de Unión al ARN/inmunología , Empalme Alternativo , Diferenciación Celular , Línea Celular , Histonas/genética , Histonas/metabolismo , Humanos , Interferón beta/genética , Interferón beta/inmunología , Macrófagos/citología , Macrófagos/inmunología , Degradación de ARNm Mediada por Codón sin Sentido , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero/inmunología , Proteínas de Unión al ARN/genéticaRESUMEN
Cellular mRNA levels are determined by the competing forces of transcription and decay. A wide array of cellular mRNA decay pathways carry out RNA turnover either on a constitutive basis or in response to changing cellular conditions. Here, we outline a method to investigate mRNA decay that employs RNAi knockdown of known or putative decay factors in commercially available Tet-off cell systems. Reporter mRNAs of interest are expressed under the control of a tetracycline-regulated promoter, allowing pulse-chase mRNA decay assays to be conducted. Levels of reporter and constitutively expressed control RNAs throughout the decay assay time course are detected by traditional northern blot analysis and used to calculate mRNA half-lives. We describe the utility of this approach to study nonsense-mediated mRNA decay substrates and factors, but it can be readily adapted to investigate key mechanistic features that dictate the specificity and functions of any mRNA decay pathway.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Interferencia de ARN , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Regulación de la Expresión Génica , Genes Reporteros/genética , Semivida , Células HeLa , Humanos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/genética , Tetraciclina/farmacología , Transactivadores/genética , Transgenes/genéticaRESUMEN
The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing premature termination codons, limiting the expression of potentially deleterious truncated proteins. This activity positions the pathway as a regulator of the severity of genetic diseases caused by nonsense mutations. Because many genetic diseases result from nonsense alleles, therapeutics inducing readthrough of premature termination codons and/or inhibition of NMD have been of great interest. Several means of enhancing translational readthrough have been reported to concomitantly inhibit NMD efficiency, but tools for systematic analysis of mammalian NMD inhibition by translational readthrough are lacking. Here, we introduce a system that allows concurrent analysis of translational readthrough and mRNA decay. We use this system to show that diverse readthrough-promoting RNA elements have similar capacities to inhibit NMD. Further, we provide evidence that the level of translational readthrough required for protection from NMD depends on the distance of the suppressed termination codon from the end of the mRNA.
Asunto(s)
Codón sin Sentido/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , Biosíntesis de Proteínas/genética , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Virus de la Fiebre por Garrapatas del Colorado/genética , Epidermólisis Ampollosa/genética , Enfermedades Genéticas Congénitas/genética , Células HEK293 , Células HeLa , Humanos , Secuencias Invertidas Repetidas/genética , Virus de la Leucemia Murina de Moloney/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
The RNA field has been revolutionized by methods that allow genome-scale identification of RNA-protein interaction sites. Two reports now introduce more efficient approaches, opening the technology to wider adoption (Van Nostrand et al., 2016; Zarnegar et al., 2016).
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunoprecipitación/métodos , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Motivos de Nucleótidos , Unión Proteica , ARN/química , ARN/genética , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Retroviral reverse transcriptase (RT) of Moloney murine leukemia virus (MoMLV) is expressed in the form of a large Gag-Pol precursor protein by suppression of translational termination in which the maximal efficiency of stop codon read-through depends on the interaction between MoMLV RT and peptidyl release factor 1 (eRF1). Here, we report the crystal structure of MoMLV RT in complex with eRF1. The MoMLV RT interacts with the C-terminal domain of eRF1 via its RNase H domain to sterically occlude the binding of peptidyl release factor 3 (eRF3) to eRF1. Promotion of read-through by MoMLV RNase H prevents nonsense-mediated mRNA decay (NMD) of mRNAs. Comparison of our structure with that of HIV RT explains why HIV RT cannot interact with eRF1. Our results provide a mechanistic view of how MoMLV manipulates the host translation termination machinery for the synthesis of its own proteins.
Asunto(s)
Virus de la Leucemia Murina de Moloney/metabolismo , Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Animales , Calorimetría , Codón de Terminación , Proteínas de Fusión gag-pol/metabolismo , Células HEK293 , Transcriptasa Inversa del VIH/metabolismo , Células HeLa , Humanos , Ratones , Mutación , Degradación de ARNm Mediada por Codón sin Sentido , Unión Proteica , Dominios Proteicos , ARN Mensajero/metabolismo , Ribonucleasa H/química , Ribonucleasa H/metabolismoRESUMEN
Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance.
Asunto(s)
Interacciones Huésped-Patógeno , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Virus/patogenicidad , Regulación Viral de la Expresión Génica , Humanos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genéticaRESUMEN
The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD.