Descripción del proyecto
Caracterización de la ruta del empalmosoma menor en las células humanas
Los intrones son segmentos no codificantes de genes eucariotas que el empalmosoma elimina de precursores del ARNm. En las células humanas, la mayoría de los intrones son procesados por el empalmosoma dependiente de U2. Sin embargo, cerca del 0,5 % de los intrones humanos son procesados por el empalmosoma menor, que depende del ARN nuclear pequeño U12. Esos intrones suelen localizarse en genes con funciones celulares esenciales, y las mutaciones en componentes menores del empalmosoma conducen a trastornos genéticos. El proyecto financiado con fondos europeos MinorSplice trabajará en la caracterización funcional y estructural exhaustiva de la ruta de empalme dependiente de U12. Una combinación de ensayos celulares, proteómica y métodos de secuenciación de próxima generación proporcionará información detallada de la composición del empalmosoma menor y los mecanismos reguladores relacionados.
Objetivo
Introns are non-coding segment of eukaryotic genes, which are removed from precursors of messenger RNAs (pre-mRNAs) by a large and dynamic RNA-protein complex known as the spliceosome. In human cells, most of the introns are processed by the canonical U2-dependent, major spliceosome. Around 0.5% of human introns utilise an alternative splicing pathway, catalysed by the minor spliceosome, which depends on the U12 small nuclear RNA (snRNA). While U12-dependent introns are rare, often they are located in genes with critical cellular functions and mutations in the minor spliceosome components lead to several genetic disorders. MinorSplice project aims to perform a comprehensive structural and functional characterization of the U12-dependent splicing pathway. The functional studies will be focused on the proteomic characterization of the minor spliceosome assembly intermediates and the involvement of the conserved spliceosomal RNA helicases in the U12-dependent splicing pathway. By combining cell-based assays, proteomics and next generation sequencing methods we will create a detailed picture of the minor spliceosome composition and associated regulatory mechanisms. The core of the projects aims to determine a series of minor spliceosome’s structures using single particle electron cryo microscopy (cryo-EM). We expect that high-resolution structural information will answer some of the fundamental mechanistic questions about the minor spliceosome assembly, U12-dependent intron recognition and will shed a light on structural similarities and differences between the major and minor splicing pathways. The last part of the project will aim to visualise spliceosomes in the native cellular environment using electron cryo tomography (cryo-ET). By doing so, we anticipate to obtain functional insights into the coupling between different nuclear processes.
Ámbito científico
Programa(s)
Régimen de financiación
ERC-STG - Starting GrantInstitución de acogida
69117 Heidelberg
Alemania