RESUMEN
MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicerâ¢-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.
Asunto(s)
MicroARNs , Ribonucleasa III , Ratones , Animales , Ribonucleasa III/metabolismo , Interferencia de ARN , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Portadoras/metabolismo , Mamíferos/metabolismoRESUMEN
Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.
Asunto(s)
Microscopía por Crioelectrón , Virus de la Influenza A , ARN Polimerasa II , ARN Polimerasa Dependiente del ARN , Proteínas Virales , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/genética , Virus de la Influenza A/enzimología , Gripe Humana/virología , Modelos Moleculares , Fosforilación , Unión Proteica , Dominios Proteicos , ARN Polimerasa II/metabolismo , ARN Polimerasa II/química , ARN Viral/metabolismo , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , Transcripción Genética , Proteínas Virales/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Replicación ViralRESUMEN
RNase III Dicer produces small RNAs guiding sequence-specific regulations, with important biological roles in eukaryotes. Major Dicer-dependent mechanisms are RNA interference (RNAi) and microRNA (miRNA) pathways, which employ distinct types of small RNAs. Small interfering RNAs (siRNAs) for RNAi are produced by Dicer from long double-stranded RNA (dsRNA) as a pool of different small RNAs. In contrast, miRNAs have specific sequences because they are precisely cleaved out from small hairpin precursors. Some Dicer homologs efficiently generate both, siRNAs and miRNAs, while others are adapted for biogenesis of one small RNA type. Here, we review the wealth of recent structural analyses of animal and plant Dicers, which have revealed how different domains and their adaptations contribute to substrate recognition and cleavage in different organisms and pathways. These data imply that siRNA generation was Dicer's ancestral role and that miRNA biogenesis relies on derived features. While the key element of functional divergence is a RIG-I-like helicase domain, Dicer-mediated small RNA biogenesis also documents the impressive functional versatility of the dsRNA-binding domain.
Asunto(s)
MicroARNs , Ribonucleasa III , Animales , Ribonucleasa III/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Bicatenario/genética , Interferencia de ARNRESUMEN
Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.
Asunto(s)
Adenosina Desaminasa/química , ARN Bicatenario/química , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Precursores del ARN/metabolismo , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN , Ratas , Receptores AMPA/genética , Alineación de SecuenciaRESUMEN
Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.
Asunto(s)
ADN Helicasas , ARN Helicasas , Terminación de la Transcripción Genética , Humanos , ADN Helicasas/genética , ADN Helicasas/metabolismo , Regulación de la Expresión Génica , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , Enfermedades Neurodegenerativas , Estructuras R-Loop , ARN Helicasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/metabolismo , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Polimerasa II/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Regulación Fúngica de la Expresión Génica , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , ARN de Hongos/metabolismo , ARN no Traducido/metabolismo , Saccharomyces cerevisiae/genética , Terminación de la Transcripción GenéticaRESUMEN
Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6's function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.
Asunto(s)
Nucleosomas , Proteínas de Saccharomyces cerevisiae , Microscopía por Crioelectrón , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ARN Polimerasa II/metabolismo , ARN de Hongos/metabolismo , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Terminación de la Transcripción Genética , Sitios de Unión , ADN Polimerasa Dirigida por ADN/química , Exosomas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Poliadenilación , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismoRESUMEN
RNA polymerase II contains a long C-terminal domain (CTD) that regulates interactions at the site of transcription. The CTD architecture remains poorly understood due to its low sequence complexity, dynamic phosphorylation patterns, and structural variability. We used integrative structural biology to visualize the architecture of the CTD in complex with Rtt103, a 3'-end RNA-processing and transcription termination factor. Rtt103 forms homodimers via its long coiled-coil domain and associates densely on the repetitive sequence of the phosphorylated CTD via its N-terminal CTD-interacting domain. The CTD-Rtt103 association opens the compact random coil structure of the CTD, leading to a beads-on-a-string topology in which the long rod-shaped Rtt103 dimers define the topological and mobility restraints of the entire assembly. These findings underpin the importance of the structural plasticity of the CTD, which is templated by a particular set of CTD-binding proteins.
Asunto(s)
ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/químicaRESUMEN
Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.
Asunto(s)
Prolina/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Serina/metabolismo , Supervivencia Celular , Modelos Moleculares , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.
Asunto(s)
ARN Polimerasa II/química , Proteínas de Saccharomyces cerevisiae/química , Treonina/química , Factores de Transcripción/química , Fosforilación , Unión Proteica , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteolisis , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Treonina/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Tirosina/metabolismoRESUMEN
The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for oligo(U) RNA-binding factors. Here, we demonstrate that NEXT displays preference for U-rich pyrimidine sequences and this RNA binding is mediated by the RNA recognition motif (RRM) of the RBM7 subunit. We solved the structure of RBM7 RRM and identified two phenylalanine residues that are critical for interaction with RNA. Furthermore, we showed that these residues are required for the NEXT interaction with snRNAs in vivo. Finally, we show that depletion of components of the NEXT complex alone or together with exosome nucleases resulted in the accumulation of mature as well as extended forms of snRNAs. Thus, our data suggest a new scenario in which the NEXT complex is involved in the surveillance of snRNAs and/or biogenesis of snRNPs.
Asunto(s)
ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Bases , Células HEK293 , Células HeLa , Humanos , Oligorribonucleótidos/metabolismo , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN Nuclear Pequeño/química , Proteínas de Unión al ARN/análisis , Nucleótidos de Uracilo/metabolismoRESUMEN
The Nrd1-Nab3-Sen1 (NNS) complex pathway is responsible for transcription termination of cryptic unstable transcripts and sn/snoRNAs. The NNS complex recognizes short motifs on the nascent RNA, but the presence of these sequences alone is not sufficient to define a functional terminator. We generated a homogeneous set of several hundreds of artificial, NNS-dependent terminators with an in vivo selection approach. Analysis of these terminators revealed novel and extended sequence determinants for transcription termination and NNS complex binding as well as supermotifs that are critical for termination. Biochemical and structural data revealed that affinity and specificity of RNA recognition by Nab3p relies on induced fit recognition implicating an α-helical extension of the RNA recognition motif. Interestingly, the same motifs can be recognized by the NNS or the mRNA termination complex depending on their position relative to the start of transcription, suggesting that they function as general transcriptional insulators to prevent interference between the non-coding and the coding yeast transcriptomes.
Asunto(s)
ADN Helicasas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Nucleares/metabolismo , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Terminación de la Transcripción Genética , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , ADN Helicasas/química , Datos de Secuencia Molecular , Proteínas Nucleares/química , Unión Proteica , ARN Helicasas/química , Proteínas de Unión al ARN/química , Técnica SELEX de Producción de Aptámeros , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
In Saccharomyces cerevisiae, the Nrd1-dependent termination and processing pathways play an important role in surveillance and processing of non-coding ribonucleic acids (RNAs). The termination and subsequent processing is dependent on the Nrd1 complex consisting of two RNA-binding proteins Nrd1 and Nab3 and Sen1 helicase. It is established that Nrd1 and Nab3 cooperatively recognize specific termination elements within nascent RNA, GUA[A/G] and UCUU[G], respectively. Interestingly, some transcripts do not require GUA[A/G] motif for transcription termination in vivo and binding in vitro, suggesting the existence of alternative Nrd1-binding motifs. Here we studied the structure and RNA-binding properties of Nrd1 using nuclear magnetic resonance (NMR), fluorescence anisotropy and phenotypic analyses in vivo. We determined the solution structure of a two-domain RNA-binding fragment of Nrd1, formed by an RNA-recognition motif and helix-loop bundle. NMR and fluorescence data show that not only GUA[A/G] but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. The broad substrate specificity is achieved by adaptable interaction surfaces of the RNA-recognition motif and helix-loop bundle domains that sandwich the RNA substrates. Our findings have implication for the role of Nrd1 in termination and processing of many non-coding RNAs arising from bidirectional pervasive transcription.
Asunto(s)
Proteínas de Unión al ARN/química , ARN/química , Proteínas de Saccharomyces cerevisiae/química , Dimerización , Modelos Moleculares , Mutación , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Asymmetric dimethylarginine (aDMA) marks are placed on histones and the C-terminal domain (CTD) of RNA Polymerase II (RNAP II) and serve as a signal for recruitment of appropriate transcription and processing factors in coordination with transcription cycle. In contrast to other Tudor domain-containing proteins, Tudor domain-containing protein 3 (TDRD3) associates selectively with the aDMA marks but not with other methylarginine motifs. Here, we report the solution structure of the Tudor domain of TDRD3 bound to the asymmetrically dimethylated CTD. The structure and mutational analysis provide a molecular basis for how TDRD3 recognizes the aDMA mark. The unique aromatic cavity of the TDRD3 Tudor domain with a tyrosine in position 566 creates a selectivity filter for the aDMA residue. Our work contributes to the understanding of substrate selectivity rules of the Tudor aromatic cavity, which is an important structural motif for reading of methylation marks.
Asunto(s)
Arginina/análogos & derivados , Proteínas/química , Secuencia de Aminoácidos , Aminoácidos/química , Arginina/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Proteínas/genética , Alineación de SecuenciaRESUMEN
Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , ARN Helicasas DEAD-box/química , ADN Polimerasa Dirigida por ADN/química , Proteínas de Unión al ARN/química , Ribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , ARN Helicasas DEAD-box/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid-like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response.
Asunto(s)
Proteínas de Unión al ADN , ARN Polimerasa II , ARN Polimerasa II/metabolismo , Proteínas de Unión al ADN/metabolismo , Separación de Fases , Reparación del ADN , Daño del ADNRESUMEN
Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the ß-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.
Asunto(s)
Proteínas Nucleares/química , Oligonucleótidos/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , Sitios de Unión , Espectroscopía de Resonancia Magnética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleótidos/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SolucionesRESUMEN
Although the abundant sterile alpha motif (SAM) domain was originally classified as a protein-protein interaction domain, it has recently been shown that certain SAM domains have the ability to bind RNA, defining a new type of post-transcriptional gene regulator. To further understand the function of SAM-RNA recognition, we determined the solution structures of the SAM domain of the Saccharomyces cerevisiae Vts1p (Vts1p-SAM) and the Smaug response element (SRE) stem-loop RNA as a complex and in isolation. The structures show that Vts1p-SAM recognizes predominantly the shape of the SRE rather than its sequence, with the exception of a G located at the tip of the pentaloop. Using microarray gene profiling, we identified several genes in S. cerevisiae that seem to be regulated by Vts1p and contain one or more copies of the SRE.
Asunto(s)
Conformación de Ácido Nucleico , ARN de Hongos/química , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Eliminación de Gen , Genoma Fúngico/genética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Elementos de Respuesta/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.