Project description
Characterisation of the minor spliceosome pathway in human cells
Introns are non-coding segments of eukaryotic genes removed from mRNA precursors by the spliceosome. In human cells, most of the introns are processed by the U2-dependent spliceosome. However, around 0.5 % of human introns are processed by the minor spliceosome, which depends on the U12 small nuclear RNA. These introns are often located in genes with critical cellular functions, and mutations in minor spliceosome components lead to genetic disorders. The EU-funded MinorSplice project will work towards the comprehensive structural and functional characterisation of the U12-dependent splicing pathway. A combination of cell-based assays, proteomics and next-generation sequencing methods will provide detailed information about minor spliceosome composition and related regulatory mechanisms.
Objective
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.
Fields of science
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Funding Scheme
ERC-STG - Starting GrantHost institution
69117 Heidelberg
Germany