RESUMEN
The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the Saccharomyces cerevisiae catalytic center, we characterize factors that modulate the function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-41AACAAU46 region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 alleles. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second-step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.
Asunto(s)
Factores de Empalme de ARN , Empalme del ARN , ARN Nuclear Pequeño , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Empalmosomas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , ARN Nuclear Pequeño/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Empalmosomas/genética , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/química , Intrones/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Microscopía por Crioelectrón , Mutación , Unión Proteica , Dominio Catalítico , Alelos , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/química , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U5 , ARN HelicasasRESUMEN
PRPF40A plays an important role in the regulation of pre-mRNA splicing by mediating protein-protein interactions in the early steps of spliceosome assembly. By binding to proteins at the 5´ and 3´ splice sites, PRPF40A promotes spliceosome assembly by bridging the recognition of the splices. The PRPF40A WW domains are expected to recognize proline-rich sequences in SF1 and SF3A1 in the early spliceosome complexes E and A, respectively. Here, we combine NMR, SAXS and ITC to determine the structure of the PRPF40A tandem WW domains in solution and characterize the binding specificity and mechanism for proline-rich motifs recognition. Our structure of the PRPF40A WW tandem in complex with a high-affinity SF1 peptide reveals contributions of both WW domains, which also enables tryptophan sandwiching by two proline residues in the ligand. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A mediates intramolecular interactions with the WW tandem. Using NMR, ITC, mutational analysis in vitro, and immunoprecipitation experiments in cells, we show that the intramolecular interaction acts as an autoinhibitory filter for proof-reading of high-affinity proline-rich motifs in bona fide PRPF40A binding partners. We propose that similar autoinhibitory mechanisms are present in most WW tandem-containing proteins to enhance binding selectivity and regulation of WW/proline-rich peptide interaction networks.
Asunto(s)
Prolina , Unión Proteica , Dominios WW , Humanos , Secuencias de Aminoácidos , Modelos Moleculares , Prolina/metabolismo , Prolina/química , Empalme del ARN , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/química , Factores de Empalme de ARN/genética , Dispersión del Ángulo Pequeño , Empalmosomas/metabolismo , Difracción de Rayos XRESUMEN
N6-Adenosine methylation (m6A) is a prevalent post-transcriptional modification of mRNA, with YTHDC1 being the reader protein responsible for recognizing this modification in the cell nucleus. Here, we present a protein structure-based medicinal chemistry campaign that resulted in the YTHDC1 inhibitor 40, which shows an equilibrium dissociation constant (Kd) of 49 nM. The crystal structure of the complex (1.6 Å resolution) validated the design. Compound 40 is selective against the cytoplasmic m6A-RNA readers YTHDF1-3 and YTHDC2 and shows antiproliferative activity against the acute myeloid leukemia (AML) cell lines THP-1, MOLM-13, and NOMO-1. For the series of compounds that culminated into ligand 40, the good correlation between the affinity in the biochemical assay and antiproliferative activity in the THP-1 cell line provides evidence of YTHDC1 target engagement in the cell. The binding to YTHDC1 in the cell is further supported by the cellular thermal shift assay. Thus, ligand 40 is a tool compound for studying the role of YTHDC1 in AML.
Asunto(s)
Diseño de Fármacos , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Proteínas del Tejido Nervioso , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/química , Relación Estructura-Actividad , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/farmacologíaRESUMEN
Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2-BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.
Asunto(s)
Modelos Moleculares , Factores de Empalme de ARN , Empalmosomas , Humanos , Empalmosomas/metabolismo , Empalmosomas/química , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/química , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/genética , Microscopía por Crioelectrón , Empalme del ARN , Precursores del ARN/metabolismo , Conformación de Ácido Nucleico , ARN Nuclear Pequeño/metabolismo , ARN Nuclear Pequeño/química , FosfoproteínasRESUMEN
The spliceosomal gene SF3B1 is frequently mutated in cancer. While it is known that SF3B1 hotspot mutations lead to loss of splicing factor SUGP1 from spliceosomes, the cancer-relevant SF3B1-SUGP1 interaction has not been characterized. To address this issue, we show by structural modeling that two regions flanking the SUGP1 G-patch make numerous contacts with the region of SF3B1 harboring hotspot mutations. Experiments confirmed that all the cancer-associated mutations in these regions, as well as mutations affecting other residues in the SF3B1-SUGP1 interface, not only weaken or disrupt the interaction but also alter splicing similarly to SF3B1 cancer mutations. Finally, structural modeling of a trimeric protein complex reveals that the SF3B1-SUGP1 interaction "loops out" the G-patch for interaction with the helicase DHX15. Our study thus provides an unprecedented molecular view of a protein complex essential for accurate splicing and also reveals that numerous cancer-associated mutations disrupt the critical SF3B1-SUGP1 interaction.
Asunto(s)
Neoplasias , Empalmosomas , Humanos , ARN Mensajero/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo , Factores de Empalme de ARN/química , Empalme del ARN/genética , Neoplasias/genética , Neoplasias/metabolismo , Mutación , Fosfoproteínas/metabolismoRESUMEN
During spliceosome assembly, the 3' splice site is recognized by sequential U2AF2 complexes, first with Splicing Factor 1 (SF1) and second by the SF3B1 subunit of the U2 small nuclear ribonuclear protein particle. The U2AF2-SF1 interface is well characterized, comprising a U2AF homology motif (UHM) of U2AF2 bound to a U2AF ligand motif (ULM) of SF1. However, the structure of the U2AF2-SF3B1 interface and its importance for pre-mRNA splicing are unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8-Å resolution. We discovered a distinctive trajectory of the SF3B1 ULM across the U2AF2 UHM surface, which differs from prior UHM/ULM structures and is expected to modulate the orientations of the full-length proteins. We established that the binding affinity of the U2AF2 UHM for the cocrystallized SF3B1 ULM rivals that of a nearly full-length U2AF2 protein for an N-terminal SF3B1 region. An additional SF3B6 subunit had no detectable effect on the U2AF2-SF3B1 binding affinities. We further showed that key residues at the U2AF2 UHM-SF3B1 ULM interface contribute to coimmunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2-SF3B1 interface changed splicing of representative human transcripts. From analysis of genome-wide data, we found that many of the splice sites coregulated by U2AF2 and SF3B1 differ from those coregulated by U2AF2 and SF1. Taken together, these findings support distinct structural and functional roles for the U2AF2-SF1 and U2AF2-SF3B1 complexes during the pre-mRNA splicing process.
Asunto(s)
Precursores del ARN , Factores de Empalme de ARN/química , Empalme del ARN , Factor de Empalme U2AF/química , Humanos , Ligandos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , Factor de Empalme U2AF/metabolismoRESUMEN
In mammals, the structural basis for the interaction between U1 and U2 small nuclear ribonucleoproteins (snRNPs) during the early steps of splicing is still elusive. The binding of the ubiquitin-like (UBL) domain of SF3A1 to the stem-loop 4 of U1 snRNP (U1-SL4) contributes to this interaction. Here, we determined the 3D structure of the complex between the UBL of SF3A1 and U1-SL4 RNA. Our crystallography, NMR spectroscopy, and cross-linking mass spectrometry data show that SF3A1-UBL recognizes, sequence specifically, the GCG/CGC RNA stem and the apical UUCG tetraloop of U1-SL4. In vitro and in vivo mutational analyses support the observed intermolecular contacts and demonstrate that the carboxyl-terminal arginine-glycine-glycine-arginine (RGGR) motif of SF3A1-UBL binds sequence specifically by inserting into the RNA major groove. Thus, the characterization of the SF3A1-UBL/U1-SL4 complex expands the repertoire of RNA binding domains and reveals the capacity of RGG/RG motifs to bind RNA in a sequence-specific manner.
Asunto(s)
Factores de Empalme de ARN/química , Ribonucleoproteína Nuclear Pequeña U1/química , Ribonucleoproteína Nuclear Pequeña U2/química , Cristalografía por Rayos X , Humanos , Resonancia Magnética Nuclear Biomolecular , Motivos de Nucleótidos , Factores de Empalme de ARN/genética , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U2/genéticaRESUMEN
Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human 17S U2 snRNP and reconstituted in vitro its adenosine 5´-triphosphate (ATP)dependent remodeling and binding to the premessenger RNA substrate. We determined a series of high-resolution (2.0 to 2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2 snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism based on branch helix stability.
Asunto(s)
Intrones , Precursores del ARN/química , Ribonucleoproteína Nuclear Pequeña U2/química , Empalmosomas/química , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Precursores del ARN/metabolismo , Empalme del ARN , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalmosomas/metabolismo , Transactivadores/química , Transactivadores/metabolismoRESUMEN
The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre-mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B-8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B-8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome-mutant cancer cells remains elusive. Here, microsecond-long all-atom simulations addressed the binding/dissociation mechanism of H3B-8800 to wild type and K700E SF3B1-containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B-8800 binding. This supports the hypothesis that the selectivity of H3B-8800 towards mutant cancer cells is unrelated to its preferential targeting of K700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B-8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug-discovery efforts to specifically target mutant SF3b.
Asunto(s)
Mutación , Neoplasias/patología , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Piperazinas/metabolismo , Piridinas/metabolismo , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Empalme del ARN , Humanos , Simulación de Dinámica Molecular , Neoplasias/genética , Neoplasias/metabolismo , Fenotipo , Fosfoproteínas/genética , Piperazinas/química , Conformación Proteica , Piridinas/química , Factores de Empalme de ARN/genéticaRESUMEN
The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We report the high-resolution cryoelectron microscopy structures of maturing human small subunit (SSU) processomes at resolutions of 2.7 to 3.9 angstroms. These structures reveal the molecular mechanisms that enable crucial progressions during SSU processome maturation. RNA folding states within these particles are communicated to and coordinated with key enzymes that drive irreversible steps such as targeted exosome-mediated RNA degradation, protein-guided site-specific endonucleolytic RNA cleavage, and tightly controlled RNA unwinding. These conserved mechanisms highlight the SSU processome's impressive structural plasticity, which endows this 4.5-megadalton nucleolar assembly with the distinctive ability to mature the small ribosomal subunit from within.
Asunto(s)
Nucléolo Celular/ultraestructura , Pliegue del ARN , Estabilidad del ARN , ARN Nucleolar Pequeño/química , Nucléolo Celular/metabolismo , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/química , Humanos , División del ARN , Factores de Empalme de ARN/químicaRESUMEN
Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.
Asunto(s)
Genes del Desarrollo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Poliadenilación , Toxoplasma/metabolismo , Transcriptoma , Arabidopsis/genética , Sitios de Unión , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Regulación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/química , Metiltransferasas/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Factores de Empalme de ARN/química , ARN Mensajero/metabolismo , Lectura , Análisis de Secuencia de ARN , Dedos de ZincRESUMEN
RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.
Asunto(s)
Fosfoproteínas/antagonistas & inhibidores , Piranos/farmacología , Factores de Empalme de ARN/antagonistas & inhibidores , ARN Largo no Codificante/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Femenino , Células HeLa , Humanos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Poliadenilación/efectos de los fármacos , Piranos/química , Empalme del ARN/efectos de los fármacos , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/química , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Compuestos de Espiro/química , Células Tumorales CultivadasRESUMEN
SF3B1, an essential component of the U2 snRNP, is frequently mutated in cancers. Cancer-associated SF3B1 mutation causes aberrant RNA splicing, mostly at 3' splice sites (3'ss). RNA splicing of DVL2, a regulator of Notch signaling, is affected by SF3B1 mutation. Here, we report that the mutated SF3B1 use an alternative branchpoint sequence (BPS) for the aberrant splicing of DVL2, which has a higher affinity to U2 snRNA than the BPS for the canonical splicing of DVL2. Swapping the position of the alternative BPS with the position of the canonical BPS decreased the aberrant splicing of DVL2, suggesting that the mutated SF3B1 prefers to use BPS with high affinity to U2 snRNA for splicing. Additionally, swapping the positions of two BPSs associated with the canonical splicing of DVL2 demonstrated that both the affinity to the U2 snRNA and the distance to the 3'ss are important to the selection of BPS. Importantly, the aberrant splicing of DVL2 does not require the canonical 3'ss and the canonical polypyrimidine tract, which reveals a novel type of aberrant splicing induced by SF3B1 mutation. These findings provide a more comprehensive understanding of the mechanisms underlying aberrant splicing induced by SF3B1 mutation in cancer.
Asunto(s)
Empalme Alternativo , Proteínas Dishevelled/genética , Mutación , Neoplasias/genética , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Secuencia de Bases , Proteínas Dishevelled/química , Humanos , Fosfoproteínas/química , Sitios de Empalme de ARN/genética , Factores de Empalme de ARN/química , ARN Nuclear Pequeño/genéticaRESUMEN
N6-Methyladenosine (m6A) is the most frequent modification in eukaryotic messenger RNA (mRNA) and its cellular processing and functions are regulated by the reader proteins YTHDCs and YTHDFs. However, the mechanism of m6A recognition by the reader proteins is still elusive. Here, we investigate this recognition process by combining atomistic simulations, site-directed mutagenesis, and biophysical experiments using YTHDC1 as a model. We find that the N6 methyl group of m6A contributes to the binding through its specific interactions with an aromatic cage (formed by Trp377 and Trp428) and also by favoring the association-prone conformation of m6A-containing RNA in solution. The m6A binding site dynamically equilibrates between multiple metastable conformations with four residues being involved in the regulation of m6A binding (Trp428, Met438, Ser378, and Thr379). Trp428 switches between two conformational states to build and dismantle the aromatic cage. Interestingly, mutating Met438 and Ser378 to alanine does not alter m6A binding to the protein but significantly redistributes the binding enthalpy and entropy terms, i.e., enthalpy-entropy compensation. Such compensation is reasoned by different entropy-enthalpy transduction associated with both conformational changes of the wild-type and mutant proteins and the redistribution of water molecules. In contrast, the point mutant Thr379Val significantly changes the thermal stability and binding capability of YTHDC1 to its natural ligand. Additionally, thermodynamic analysis and free energy calculations shed light on the role of a structural water molecule that synergistically binds to YTHDC1 with m6A and acts as the hub of a hydrogen-bond network. Taken together, the experimental data and simulation results may accelerate the discovery of chemical probes, m6A-editing tools, and drug candidates against reader proteins.
Asunto(s)
Adenosina/análogos & derivados , Proteínas del Tejido Nervioso/química , Factores de Empalme de ARN/química , Termodinámica , Adenosina/química , Calorimetría/métodos , Cristalografía por Rayos X , Metilación , Conformación Molecular , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Dominios Proteicos , Agua/químicaRESUMEN
The AAA-ATPase NVL2 associates with an RNA helicase MTR4 and the nuclear RNA exosome in the course of ribosome biogenesis. In our proteomic screen, we had identified a ribosome biogenesis factor WDR74 as a MTR4-interacting partner, whose dissociation is stimulated by the ATP hydrolysis of NVL2. In this study, we report the identification of splicing factor 30 (SPF30), another MTR4-interacting protein with a similar regulatory mechanism. SPF30 is a pre-mRNA splicing factor harboring a Tudor domain in its central region, which regulates various cellular events by binding to dimethylarginine-modified proteins. The interaction between SPF30 and the exosome core is mediated by MTR4 and RRP6, a catalytic component of the nuclear exosome. The N- and C-terminal regions, but not the Tudor domain, of SPF30 are involved in the association with MTR4 and the exosome. The knockdown of SPF30 caused subtle delay in the 12S pre-rRNA processing to mature 5.8S rRNA, even though no obvious effect was observed on the ribosome subunit profile in the cells. Shotgun proteomic analysis to search for SPF30-interacting proteins indicated its role in ribosome biogenesis, pre-mRNA splicing, and box C/D snoRNA biogenesis. These results suggest that SPF30 collaborates with the MTR4-exosome machinery to play a functional role in multiple RNA metabolic pathways, some of which may be regulated by the ATP hydrolysis of NVL2.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Exosomas/genética , ARN Helicasas/metabolismo , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Estabilidad del ARN , Proteínas del Complejo SMN/química , Proteínas del Complejo SMN/metabolismo , Humanos , Unión Proteica , Dominios ProteicosRESUMEN
The minor spliceosome mediates splicing of the rare but essential U12-type precursor messenger RNA. Here, we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-angstrom resolution. The 5' splice site and branch point sequence of the U12-type intron are recognized by the U6atac and U12 small nuclear RNAs (snRNAs), respectively. Five newly identified proteins stabilize the conformation of the catalytic center: The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome, the RBM48-ARMC7 complex binds the γ-monomethyl phosphate cap at the 5' end of U6atac snRNA, the U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 small nuclear ribonucleoprotein (snRNP), and CRIPT stabilizes U12 snRNP. Our study provides a framework for the mechanistic understanding of the function of the human minor spliceosome.
Asunto(s)
Empalmosomas/química , Empalmosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/metabolismo , Microscopía por Crioelectrón , Ciclofilinas/química , Ciclofilinas/metabolismo , Humanos , Intrones , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Dominios Proteicos , Precursores del ARN/química , Precursores del ARN/metabolismo , Empalme del ARN , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , ARN Nuclear Pequeño/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/química , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
Defects in the functions of RNA binding proteins (RBPs) are at the origin of many diseases; however, targeting RBPs with conventional drugs has proven difficult. PROTACs are a new class of drugs that mediate selective degradation of a target protein through a cell's ubiquitination machinery. PROTACs comprise a moiety that binds the selected protein, conjugated to a ligand of an E3 ligase. Herein, we introduce RNA-PROTACs as a new concept in the targeting of RBPs. These chimeric structures employ small RNA mimics as targeting groups that dock the RNA-binding site of the RBP, whereupon a conjugated E3-recruiting peptide derived from the HIF-1α protein directs the RBP for proteasomal degradation. We performed a proof-of-concept demonstration with the degradation of two RBPs-a stem cell factor LIN28 and a splicing factor RBFOX1-and showed their use in cancer cell lines. The RNA-PROTAC approach opens the way to rapid, selective targeting of RBPs in a rational and general fashion.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Humanos , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Proteolisis , ARN/química , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/química , Ubiquitina-Proteína Ligasas/químicaRESUMEN
The 55-residue OCRE domains of the splicing factors RBM5 and RBM10 contain 15 tyrosines in compact, globular folds. At 25 °C, all 15 tyrosines show symmetric 1 H NMR spectra, with averaged signals for the pairs of δ- and ϵ-ring hydrogens. At 4 °C, two tyrosines were identified as showing 1 H NMR line-broadening due to lowered frequency of the ring-flipping. For the other 13 tyrosine rings, it was not evident, from the 1 H NMR data alone, whether they were either all flipping at high frequencies, or whether slowed flipping went undetected due to small chemical-shift differences between pairs of exchanging ring hydrogen atoms. Here, we integrate 1 H NMR spectroscopy and molecular dynamics (MD) simulations to determine the tyrosine ring-flip frequencies. In the RBM10-OCRE domain, we found that, for 11 of the 15 tyrosines, these frequencies are in the range 2.0×106 to 1.3×108 â s-1 , and we established an upper limit of <1.0×106 â s-1 for the remaining four residues. The experimental data and the MD simulation are mutually supportive, and their combined use extends the analysis of aromatic ring-flip events beyond the limitations of routine 1 H NMR line-shape analysis into the nanosecond frequency range.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , Simulación de Dinámica Molecular , Factores de Empalme de ARN/química , Proteínas de Unión al ARN/química , Proteínas Supresoras de Tumor/química , Tirosina/química , Secuencias de Aminoácidos , Humanos , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase.
Asunto(s)
Proteínas de Ciclo Celular/genética , Enfermedades Cerebelosas/genética , Microcefalia/genética , Mutación/genética , Isomerasa de Peptidilprolil/genética , Factores de Empalme de ARN/genética , Empalmosomas/genética , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Enfermedades Cerebelosas/complicaciones , Enfermedades Cerebelosas/diagnóstico por imagen , Estudios de Cohortes , Femenino , Técnicas de Inactivación de Genes/métodos , Células HEK293 , Trastornos Heredodegenerativos del Sistema Nervioso/complicaciones , Trastornos Heredodegenerativos del Sistema Nervioso/diagnóstico por imagen , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microcefalia/complicaciones , Microcefalia/diagnóstico por imagen , Linaje , Isomerasa de Peptidilprolil/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Factores de Empalme de ARN/químicaRESUMEN
To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.