This project focused on unravelling the precise molecular mechanism of spliceosome activation.
The information necessary to synthesize proteins, which are the building blocks of any living organism, is contained in the genes in DNA. During gene expression DNA is first transcribed into messenger RNA, a labile copy of the precious genetic information. These mRNAs are then used by the translational machinery that decodes the building instructions to form proteins.
Eukaryotic messenger RNAs are initially produced as precursor molecules that contain non-coding sequences, the introns. RNA splicing consists of intron removal by a large molecular machine called the spliceosome. This central step in gene expression is subjected to extensive regulation since RNA can be spliced alternatively to form different proteins, which is thought to be one of the main determinants of the diversity among eukaryotes. The splicing process must be remarkably precise since production of erroneous mRNAs can have deleterious consequences for cells. Indeed, it has been suggested that numerous disease-causing mutations alter functional splicing signals within the pre-mRNAs making the study of splicing mechanisms particularly relevant to human health. Hence, the spliceosome has a great therapeutic potential but is largely unexploited due the poor understanding we have of this complex machinery.
A particularly obscure aspect of the spliceosome is the fact it is initially assembled as a pre-catalytic particle, which has no active site and needs to undergo a substantial activation process to become catalytically competent. Spliceosome activation is a pivotal transition in the splicing cycle since it is when the active site is formed and the upstream boundary of the intron (named 5' splice site) is introduced in this newly formed active site. These two molecular events must be perfectly executed and coordinated to ensure (1) that the active site is competent to perform catalysis and (2) that the upstream boundary of the intron is properly defined. Nevertheless, due to its complexity, molecular basis for spliceosome activation is poorly characterised.
In order to unravel the precise molecular mechanism of spliceosome activation, I have used precise biochemical strategies to capture the spliceosome during its activation. Isolated complexes were then characterised precisely by solving their three-dimensional structures, which constitute an important source of biological information.
Overall, this project was very successful as we reported the first high-resolution structure of a fully-assembled human spliceosome, captured at the initial stage of its activation as well as that of its precursor U4/U6.U5 tri-snRNP. This structural data explained numerous functional observations gathered over three decades. Firstly, it revealed the mode of action of a central DEAD-box RNA helicase in initiating spliceosome activation, a long-standing question in the field. It also provided the first mechanism for such an enzyme in the context of a large ribonucleoprotein. Secondly, our data unveiled how an intricate network of protein/RNA interactions ensures that RNA acceptor sequences present within pre-catalytic spliceosomes are primed to receive the pre-mRNA. This also revealed the role of metazoan-specific factors, that had previously been overlooked, in maintaining these sequences in their correct positions. Thirdly, this work uncovered how premature formation of the active site is prevented by inhibition of a key Ski2-like helicase.
These structural insights led us to propose a plausible model for how the recognition of the 5' splice site induces the formation of the active site via an allosteric coupling between RNA helicases.
