Construction of a Protein Assembly Timeline for Spliceosomes by Relative MS-based Quantification

摘要

Eukaryotic pre-mRNAs consist of coding (exons) and non-coding (introns) sequences. One of the major steps during gene expression is therefore the excision of introns and the ligation of adjacent exons to yield mature mRNA (pre-mRNA splicing). The spliceosome is a multi-megadalton machinery that catalyzes pre-mRNA splicing. It assembles from different uridine-rich snRNPs (small nuclear ribonucleoprotein), snRNP specific proteins and numerous non-snRNP specific proteins. During pre-mRNA splicing, passes through sequential assembly states that differ in their RNA and protein composition (Figure 1; for review see [1]). In vitro assembled complexes can be purified from nuclear extract and were recently characterized in detail [2,3,4]. However, to date the assembly kinetics of spliceosomal proteins have not been investigated. We analyzed the protein assembly on splicing-active and splicing-inactive pre-mRNAs and compared the protein compositions at different time points during the pre-mRNA splicing process by stable isotope labeling with amino acids in cell culture (SILAC).