RESUMEN
Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.
Asunto(s)
Neoplasias Hematológicas , Mutación , Fosfoproteínas , Factores de Empalme de ARN , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Humanos , Animales , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/metabolismo , Ratones , Empalme del ARN , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Hematopoyesis/genética , Células HEK293 , Intrones , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
RNA binding proteins (RBPs) regulate nearly all post-transcriptional processes within cells. To fully understand RBP function, it is essential to identify their in vivo targets. Standard techniques for profiling RBP targets, such as crosslinking immunoprecipitation (CLIP) and its variants, are limited or suboptimal in some situations, e.g. when compatible antibodies are not available and when dealing with small cell populations such as neuronal subtypes and primary stem cells. This review summarizes and compares several genetic approaches recently designed to identify RBP targets in such circumstances. TRIBE (targets of RNA binding proteins identified by editing), RNA tagging, and STAMP (surveying targets by APOBEC-mediated profiling) are new genetic tools useful for the study of post-transcriptional regulation and RBP identification. We describe the underlying RNA base editing technology, recent applications, and therapeutic implications.
Asunto(s)
Técnicas Genéticas , Edición de ARN , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/genética , ARN/genética , Animales , Sitios de Unión , Humanos , Unión Proteica , ARN/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.
Asunto(s)
Regulación de la Expresión Génica , Análisis de la Célula Individual/métodos , Animales , Cromatina/química , Cromatina/genética , Citoplasma/genética , Perfilación de la Expresión Génica/métodos , Humanos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Madre/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging. Constitutive mRNAs (MDN1 and DOA1) and highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in Saccharomyces cerevisiae degrade normally. As a result, short-lived mRNAs were imaged throughout their complete life cycle. The MBSV6 reporter revealed that, in contrast to previous findings, coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur, and mRNA degradation was heterogeneously distributed in the cytoplasm.
Asunto(s)
Proteínas de la Cápside/metabolismo , Estabilidad del ARN , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Proteínas de la Cápside/genética , Citoplasma/metabolismo , Humanos , Hibridación Fluorescente in Situ , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análisis de la Célula Individual , Células Tumorales CultivadasRESUMEN
CYP106A2 (cytochrome P450meg) is a bacterial enzyme originally isolated from B. megaterium, and has been shown to hydroxylate a wide variety of substrates, including steroids. The regio- and stereochemistry of CYP106A2 hydroxylation has been shown to be dependent on a variety of factors, and hydroxylation often occurs at more than one site and/or with lack of stereospecificity for some substrates. Comprehensive backbone 15N, 1H and 13C resonance assignments based on multidimensional nuclear magnetic resonance (NMR) experiments performed with uniform and selective isotopically labeled CYP106A2 samples are reported herein, and broadening and splitting of resonances assigned to regions of the enzyme shown to be affected by substrate binding in other P450 enzymes indicate that substrate binding does not reduce structural heterogeneity as has been observed previously in P450 enzymes CYP101A1 and MycG. Paramagnetic relaxation enhancement (PRE) due to proximity between substrate protons and the heme iron were measured for three different substrates, and the relatively uniform nature of the PREs support the proposal that multiple substrate binding modes are occupied at saturating substrate concentrations.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Esteroides , Modelos Moleculares , Sistema Enzimático del Citocromo P-450/metabolismo , Conformación Molecular , Espectroscopía de Resonancia Magnética , Especificidad por Sustrato , Hidroxilación , Proteínas Bacterianas/químicaRESUMEN
Cell motility requires strict spatiotemporal control of protein expression. During cell migration, mRNA localization and local translation in subcellular areas like the leading edge and protrusions are particularly advantageous for regulating the reorganization of the cytoskeleton. Fidgetin-Like 2 (FL2), a microtubule severing enzyme (MSE) that restricts migration and outgrowth, localizes to the leading edge of protrusions where it severs dynamic microtubules. FL2 is primarily expressed during development but in adulthood, is spatially upregulated at the leading edge minutes after injury. Here, we show mRNA localization and local translation in protrusions of polarized cells are responsible for FL2 leading edge expression after injury. The data suggests that the RNA binding protein IMP1 is involved in the translational regulation and stabilization of FL2 mRNA, in competition with the miRNA let-7. These data exemplify the role of local translation in microtubule network reorganization during migration and elucidate an unexplored MSE protein localization mechanism.
RESUMEN
Targets of RNA-binding proteins discovered by editing (TRIBE) determines RNA-proteins interactions and nuclear organization with minimal false positives. We detail necessary steps for performing mammalian cell RBP-TRIBE to determine the targets of RNA-binding proteins and MS2-TRIBE to determine RNA-RNA interactions within the nucleus. Necessary steps for performing a TRIBE experiment are detailed, starting with plasmid/cell line generation, cellular transfection, and RNA sequencing library preparation and concluding with bioinformatics analysis of RNA editing sites and identification of target RNAs. For complete details on the use and execution of this protocol, please refer to Biswas et al. (2020).
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Animales , Línea Celular , Biología Computacional , Humanos , Plásmidos , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
Within the nucleus, messenger RNA is generated and processed in a highly organized and regulated manner. Messenger RNA processing begins during transcription initiation and continues until the RNA is translated and degraded. Processes such as 5' capping, alternative splicing, and 3' end processing have been studied extensively with biochemical methods and more recently with single-molecule imaging approaches. In this review, we highlight how imaging has helped understand the highly dynamic process of RNA processing. We conclude with open questions and new technological developments that may further our understanding of RNA processing.
Asunto(s)
Núcleo Celular , ARN , Empalme Alternativo , Núcleo Celular/metabolismo , ARN/metabolismo , ARN Mensajero/metabolismo , Imagen Individual de Molécula , Transcripción GenéticaRESUMEN
Both UV-cross-linking and immunoprecipitation (CLIP) and RNA editing (TRIBE) can identify the targets of RNA-binding proteins. To evaluate false-positives of CLIP and TRIBE, endogenous ß-actin mRNA was tagged with MS2 stem loops, making it the only bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE detected ß-actin, albeit with false-positives. False-positive CLIP signals were attributed to nonspecific antibody interactions. In contrast, putative false-positive TRIBE targets were genes spatially proximal to the ß-actin gene. MCP-ADAR edited nearby nascent transcripts consistent with interchromosomal contacts observed in Hi-C. The identification of nascent contacts implies RNA regulatory proteins (e.g., splicing factors) associated with multiple nascent transcripts, forming domains of post-transcriptional activity. Repeating these results with an integrated inducible MS2 reporter indicated that MS2-TRIBE can be applied to a broad array of cells and transcripts to study spatial organization and nuclear RNA regulation.
RESUMEN
Translation is the fundamental biological process that converts the genetic information in messenger RNAs (mRNAs) into functional proteins. Translation regulation allows cells to control when, where, and how many proteins are synthesized. Much of what we know about translation comes from ensemble approaches that measure the average of many cells. The cellular and molecular heterogeneity in the regulation of translation remains largely elusive. Fluorescence microscopy allows interrogation of biological problems with single-molecule, single-cell sensitivity. In recent years, improved design of reagents and microscopy tools has led to improved spatial and temporal resolution of translation imaging. It is now possible to track global translation in specific subcellular compartments and follow the translation dynamics of single transcripts. Highlighted here is the recent progress in translation imaging with emphasis on in vivo translation dynamics. These tools will be invaluable to the study of translation regulation.
Asunto(s)
Imagen Óptica/métodos , Biosíntesis de Proteínas , Análisis de la Célula Individual , CinéticaRESUMEN
The fate of an RNA, from its localization, translation, and ultimate decay, is dictated by interactions with RNA binding proteins (RBPs). ß-actin mRNA has functioned as the classic example of RNA localization in eukaryotic cells. Studies of ß-actin mRNA over the past three decades have allowed understanding of how RBPs, such as ZBP1 (IGF2BP1), can control both RNA localization and translational status. Here, we summarize studies of ß-actin mRNA and focus on how ZBP1 serves as a model for understanding interactions between RNA and their binding protein(s). Central to the study of RNA and RBPs were technological developments that occurred along the way. We conclude with a future outlook highlighting new technologies that may be used to address still unanswered questions about RBP-mediated regulation of mRNA during its life cycle, within the cell.
RESUMEN
The IGF2 mRNA-binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine systematic evolution of ligands by exponential enrichment (SELEX) and NMR spectroscopy to demonstrate that the major RNA-binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.