RESUMO
Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small-molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges the tandem U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Cells expressing a cancer-associated U2AF1 mutant are preferentially killed by treatment with the compound. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing assembly intermediates may offer a useful approach for small-molecule inhibition of macromolecular machines.
Assuntos
Precursores de RNA/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , RNA Neoplásico/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Processamento U2AF/antagonistas & inibidores , Feminino , Células HEK293 , Humanos , Células K562 , Simulação de Acoplamento Molecular , Estrutura Molecular , Precursores de RNA/genética , Splicing de RNA/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismoRESUMO
Intron retention (IR) is the least well-understood alternative splicing type in animals, and its prevalence and function in physiological and pathological processes have long been underestimated. Cellular senescence contributes to individual aging and age-related diseases and can also serve as an important cancer prevention mechanism. Dynamic IR events have been observed in senescence models and aged tissues; however, whether and how IR impacts senescence remain unclear. Through analyzing polyA+ RNA-seq data from human replicative senescence models, we found IR was prevalent and dynamically regulated during senescence and IR changes negatively correlated with expression alteration of corresponding genes. We discovered that knocking down (KD) splicing factor U2AF1, which showed higher binding density to retained introns and decreased expression during senescence, led to senescence-associated phenotypes and global IR changes. Intriguingly, U2AF1-KD-induced IR changes also negatively correlated with gene expression. Furthermore, we demonstrated that U2AF1-mediated IR of specific gene (CPNE1 as an example) contributed to cellular senescence. Decreased expression of U2AF1, higher IR of CPNE1, and reduced expression of CPNE1 were also discovered in dermal fibroblasts with age. We discovered prevalent IR could fine-tune gene expression and contribute to senescence-associated phenotypes, largely extending the biological significance of IR.
Assuntos
Processamento Alternativo/genética , Senescência Celular/genética , Íntrons , Fator de Processamento U2AF/genética , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Regulação para Baixo , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Fator de Processamento U2AF/antagonistas & inibidores , Fator de Processamento U2AF/metabolismoRESUMO
The essential splicing factor U2AF65 is known to help anchoring U2 snRNP at the branch site. Its C-terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF65 and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF65 drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF65 or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine-rich motifs. These results support a model in which liquid-like assemblies of U2AF65 and CAPERα on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF65 and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.
Assuntos
Processamento Alternativo , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Spliceossomos/genética , Fator de Processamento U2AF/genética , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , DNA Complementar/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Motivos de Nucleotídeos , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/metabolismo , Fator de Processamento U2AF/antagonistas & inibidores , Fator de Processamento U2AF/metabolismoRESUMO
Splicing factor gene mutations are the most frequent mutations found in patients with the myeloid malignancy myelodysplastic syndrome (MDS), suggesting that spliceosomal dysfunction plays a major role in disease pathogenesis. The aberrantly spliced target genes and deregulated cellular pathways associated with the commonly mutated splicing factor genes in MDS (SF3B1, SRSF2 and U2AF1) are being identified, illuminating the molecular mechanisms underlying MDS. Emerging data from mouse modeling studies indicate that the presence of splicing factor gene mutations can lead to bone marrow hematopoietic stem/myeloid progenitor cell expansion, impaired hematopoiesis and dysplastic differentiation that are hallmarks of MDS. Importantly, recent evidence suggests that spliceosome inhibitors and splicing modulators may have therapeutic value in the treatment of splicing factor mutant myeloid malignancies.
Assuntos
Antineoplásicos/farmacologia , Mutação , Síndromes Mielodisplásicas/genética , Fosfoproteínas/antagonistas & inibidores , Piranos/farmacologia , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina/antagonistas & inibidores , Compostos de Espiro/farmacologia , Fator de Processamento U2AF/antagonistas & inibidores , Animais , Proliferação de Células , Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Splicing de RNA/efeitos dos fármacos , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Spliceossomos/patologia , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismoRESUMO
U2AF homology motifs (UHMs) are atypical RNA recognition motif domains that mediate critical protein-protein interactions during the regulation of alternative pre-mRNA splicing and other processes. The recognition of UHM domains by UHM ligand motif (ULM) peptide sequences plays important roles during early steps of spliceosome assembly. Splicing factor 45 kDa (SPF45) is an alternative splicing factor implicated in breast and lung cancers, and splicing regulation of apoptosis-linked pre-mRNAs by SPF45 was shown to depend on interactions between its UHM domain and ULM motifs in constitutive splicing factors. We have developed cyclic peptide inhibitors that target UHM domains. By screening a focused library of linear and cyclic peptides and performing structure-activity relationship analysis, we designed cyclic peptides with 4-fold improved binding affinity for the SPF45 UHM domain compared to native ULM ligands and 270-fold selectivity to discriminate UHM domains from alternative and constitutive splicing factors. These inhibitors are useful tools to modulate and dissect mechanisms of alternative splicing regulation.