RESUMO
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.
Assuntos
Neoplasias Hematológicas , Mutação , Fosfoproteínas , Fatores de Processamento de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Humanos , Animais , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/metabolismo , Camundongos , Splicing de RNA , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Hematopoese/genética , Células HEK293 , Íntrons , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Sequência de Bases , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleolares Pequenas , Técnica de Seleção de Aptâmeros , TranscriptomaRESUMO
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.