Periodic Reporting for period 1 - SPLICEOSACT (Biochemical and CryoEM studies of spliceosome activation.)
Reporting period: 2019-03-01 to 2021-02-28
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.
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.
"In this work, we used human nuclear extracts to capture the spliceosome during activation. Specifically, we targeted a crucial step that initiates activation: the so-called 5' splice site (5'SS) transfer during which the upstream boundary of an intron is docked in the future active site. 5'SS transfer is catalysed by the central RNA helicase Prp28 and triggers a cascade of molecular events that lead to active site formation.
We started by designing an experimental strategy to obtain the human pre-catalytic spliceosome stalled just before 5’SS transfer, known as ""pre-B complex"". To that end, we used a yeast expression system to produce recombinantly a mutant version of human Prp28 that is unable to catalyse the 5'SS transfer. We then optimised the conditions of assembly of the pre-B complex by monitoring the effect of this mutant protein on the splicing activity using a well-established splicing assay. With these defined conditions, we assembled spliceosomes from important quantities of human nuclear extracts onto a pre-mRNA harbouring specific sequences (MS2-loops) that can be used for affinity purification. The assembled spliceosomes were subsequently isolated using a double affinity purification strategy targeting first, the MS2 loops-containing substrate pre-mRNA and, second, the tagged mutant Prp28 protein.
Once the spliceosomes isolated, we used a combination of various biophysical and biochemistry methods, to characterize precisely the purified complex. In particular, we used electron cryo-microscopy (cryo-EM) to study the 3D structure of the complex. We first optimised the conditions for cryo-EM grids preparation and collected several datasets on high end 300kV electron microscopes. This cryo-EM data was then processed to calculate very precise density maps for the different parts of the complex. These maps were finally combined and used as a guide to build atomic models describing the 3D structure of the whole complexe. Thanks to that approach, we could solve the structures of both human pre-B complex (65 proteins and 6 RNA molecules) but also of its precursor U4/U6.U5 tri-snRNP (35 proteins, 3 RNA molecules) arising from dissociation of pre-B during grid preparation.
Our structures reveals the organization of the human spliceosome as it initially assembles on an intron and gives insights into how proper recognition of the pre-mRNA triggers active site formation."
We started by designing an experimental strategy to obtain the human pre-catalytic spliceosome stalled just before 5’SS transfer, known as ""pre-B complex"". To that end, we used a yeast expression system to produce recombinantly a mutant version of human Prp28 that is unable to catalyse the 5'SS transfer. We then optimised the conditions of assembly of the pre-B complex by monitoring the effect of this mutant protein on the splicing activity using a well-established splicing assay. With these defined conditions, we assembled spliceosomes from important quantities of human nuclear extracts onto a pre-mRNA harbouring specific sequences (MS2-loops) that can be used for affinity purification. The assembled spliceosomes were subsequently isolated using a double affinity purification strategy targeting first, the MS2 loops-containing substrate pre-mRNA and, second, the tagged mutant Prp28 protein.
Once the spliceosomes isolated, we used a combination of various biophysical and biochemistry methods, to characterize precisely the purified complex. In particular, we used electron cryo-microscopy (cryo-EM) to study the 3D structure of the complex. We first optimised the conditions for cryo-EM grids preparation and collected several datasets on high end 300kV electron microscopes. This cryo-EM data was then processed to calculate very precise density maps for the different parts of the complex. These maps were finally combined and used as a guide to build atomic models describing the 3D structure of the whole complexe. Thanks to that approach, we could solve the structures of both human pre-B complex (65 proteins and 6 RNA molecules) but also of its precursor U4/U6.U5 tri-snRNP (35 proteins, 3 RNA molecules) arising from dissociation of pre-B during grid preparation.
Our structures reveals the organization of the human spliceosome as it initially assembles on an intron and gives insights into how proper recognition of the pre-mRNA triggers active site formation."
The conceptual advances made on the spliceosome activation greatly improve our understanding of this previously poorly characterised process.
The structures we reported offer a view of the human splicing machinery in a pre-catalytic state at an unprecedented level of detail. They will serve as a starting point for functional studies focusing on the human splicing machinery (generation of mutant, selective disruptions of interfaces…). The structures could further be used for rational drug design aiming at blocking or modulating spliceosome activity, that could be used to exploit the therapeutic potential of the spliceosome.
In this structural study, we reported a formidable example of a dynamic and flexible biological macromolecule. This achievement was permitted by recent developments in the method of electron cryo-microscopy (cryo-EM). By depositing both the raw and generated data onto public repository we made it publicly available and thereby contributed to the advance of this method. Indeed, this data has been and will be used by cryo-EM software developers to improve the current processing programs that aim at handling flexibility and heterogeneity in cryo-EM datasets.
The structures we reported offer a view of the human splicing machinery in a pre-catalytic state at an unprecedented level of detail. They will serve as a starting point for functional studies focusing on the human splicing machinery (generation of mutant, selective disruptions of interfaces…). The structures could further be used for rational drug design aiming at blocking or modulating spliceosome activity, that could be used to exploit the therapeutic potential of the spliceosome.
In this structural study, we reported a formidable example of a dynamic and flexible biological macromolecule. This achievement was permitted by recent developments in the method of electron cryo-microscopy (cryo-EM). By depositing both the raw and generated data onto public repository we made it publicly available and thereby contributed to the advance of this method. Indeed, this data has been and will be used by cryo-EM software developers to improve the current processing programs that aim at handling flexibility and heterogeneity in cryo-EM datasets.