RESUMEN
Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. Multiomics analysis reveals that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery.
Asunto(s)
ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalmosomas/enzimología , Elongación de la Transcripción Genética , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Células HeLa , Humanos , Células K562 , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/genética , Factores de TiempoRESUMEN
Cell migration and mechanics are tightly regulated by the integrated activities of the various cytoskeletal networks. In cancer cells, cytoskeletal modulations have been implicated in the loss of tissue integrity and acquisition of an invasive phenotype. In epithelial cancers, for example, increased expression of the cytoskeletal filament protein vimentin correlates with metastatic potential. Nonetheless, the exact mechanism whereby vimentin affects cell motility remains poorly understood. In this study, we measured the effects of vimentin expression on the mechano-elastic and migratory properties of the highly invasive breast carcinoma cell line MDA231. We demonstrate here that vimentin stiffens cells and enhances cell migration in dense cultures, but exerts little or no effect on the migration of sparsely plated cells. These results suggest that cell-cell interactions play a key role in regulating cell migration, and coordinating cell movement in dense cultures. Our findings pave the way toward understanding the relationship between cell migration and mechanics in a biologically relevant context.
Asunto(s)
Neoplasias de la Mama/patología , Movimiento Celular , Invasividad Neoplásica/patología , Vimentina/metabolismo , Fenómenos Biomecánicos , Neoplasias de la Mama/metabolismo , Comunicación Celular , Línea Celular Tumoral , Elasticidad , Femenino , Humanos , Células MCF-7 , Vimentina/análisisRESUMEN
Enhancer activity drives cell differentiation and cell fate determination, but it remains unclear how enhancers cooperate during these processes. Here we investigate enhancer cooperation during transdifferentiation of human leukemia B-cells to macrophages. Putative enhancers are established by binding of the pioneer factor C/EBPα followed by chromatin opening and enhancer RNA (eRNA) synthesis from H3K4-monomethylated regions. Using eRNA synthesis as a proxy for enhancer activity, we find that most putative enhancers cooperate in an additive way to regulate transcription of assigned target genes. However, transcription from 136 target genes depends exponentially on the summed activity of its putative paired enhancers, indicating that these enhancers cooperate synergistically. The target genes are cell type-specific, suggesting that enhancer synergy can contribute to cell fate determination. Enhancer synergy appears to depend on cell type-specific transcription factors, and such interacting enhancers are not predicted from occupancy or accessibility data that are used to detect superenhancers.