Descrizione del progetto
La caratterizzazione dei percorsi dello spliceosoma minore nelle cellule umane
Gli introni sono segmenti non codificanti di geni eucariotici eliminati dai precursori dell’mRNA dallo spliceosoma. Nelle cellule umane, la maggior parte degli introni è elaborata dallo spliceosoma dipendente dalle molecole U2. Ciononostante, circa lo 0,5 % degli introni umani è elaborato dallo spliceosoma minore, che dipende dal piccolo RNA nucleare U12. Questi introni si trovano spesso in geni caratterizzati da funzioni cellulari di importanza critica e le mutazioni nei componenti dello spliceosoma minore portano a disturbi di carattere genetico. Il progetto MinorSplice, finanziato dall’UE, lavorerà per realizzare una completa caratterizzazione strutturale e funzionale del percorso di splicing dipendente dalle molecole U12. Una combinazione di metodi che prevedono saggi cellulari, proteomica e sequenziamento di prossima generazione fornirà informazioni dettagliate sulla composizione dello spliceosoma minore e sui meccanismi regolatori associati.
Obiettivo
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.
Campo scientifico
Programma(i)
Argomento(i)
Meccanismo di finanziamento
ERC-STG - Starting GrantIstituzione ospitante
69117 Heidelberg
Germania