Periodic Reporting for period 2 - MinorSplice (Functional and structural studies of the U12-dependent splicing in human cells)
Période du rapport: 2022-07-01 au 2023-12-31
In living organisms, genetic information is used to create proteins, which are vital for the functioning of all cells. This information is stored in DNA; however, it is not continuous and contains segments of coding information needed to build proteins, called exons, separated by non-coding segments known as introns. Before the genetic information can be translated into proteins, introns need to be removed in a process known as pre-mRNA splicing, which is carried out by a complex set of molecules assembled into the spliceosome. The spliceosome identifies specific positions in the messenger molecules of the genetic information (pre-mRNAs) and cuts them to remove introns. This process has a very simple chemistry but requires enormously complex molecular machinery that is needed to coordinate it in time and space in a crowded cellular environment. The fidelity of the pre-mRNA splicing is essential for ensuring that the resulting proteins are produced in their functional forms. Failure to do so would have fatal consequences for the host organism. Therefore, to ensure precise removal of the non-coding information, the spliceosome has to accurately determine exon-intron boundaries and it does so by using specialised components built from RNA and proteins, known as snRNPs. There are five snRNPs that build up the spliceosome (U1, U2, U4, U5 and U6). While these snRNPs can support the splicing of the vast majority of human introns, there is a small subset of introns (so-called U12-dependent introns), which cannot be processed by them and require a distinct molecular machinery. This machinery is known as the minor spliceosome (as opposed to the major spliceosome) and contains a set of unique snRNPs (U11, U12, U4atac, U5 and U6atac) and proteins, in addition to the components shared with the major spliceosome. Although U12-dependent introns are not very frequent, they are located in the genes with critical cellular functions and their removal is essential. Consequently, several diseases are caused by malfunctions of the minor spliceosome or mutations in the U12-dependent introns. Therefore, understanding how the minor spliceosome works on the molecular level is of critical importance for the treatment of these diseases and makes minor spliceosomes a medically relevant drug target.
The molecular mechanism of pre-mRNA splicing has been investigated for more than four decades using biochemistry, genetics and structural biology approaches. Some of the major breakthroughs were achieved recently through the determination of high-resolution structures of numerous major spliceosome assembly intermediates. Despite this progress, our understanding of the composition, structure and mechanism of the minor spliceosome remains limited. Therefore, in the framework of this project, we propose to investigate the molecular mechanism of the minor spliceosome. This will be achieved by studying specific proteins (so-called helicases), which may be potentially involved in controlling the splicing reaction. We are planning to develop a set of new tools to isolate minor splicing complexes and characterise their composition and structure. We will do it by using the latest technologies available to engineer mammalian cell lines to allow specific isolation of molecules of interest, which will be then imaged with electron microscopes operated at cryogenic temperature (cryo-EM). Such direct observation of the minor splicing machinery will allow us to gain insights into its potential mode of action.
Taken together, our project aims to address a fundamental question of the molecular mechanism of pre-mRNA splicing. It is estimated that up to 50% of genetic disorders are caused by malfunction of the splicing machinery, therefore the results produced during this project will have a direct impact on human health and benefit society.
The molecular mechanism of pre-mRNA splicing has been investigated for more than four decades using biochemistry, genetics and structural biology approaches. Some of the major breakthroughs were achieved recently through the determination of high-resolution structures of numerous major spliceosome assembly intermediates. Despite this progress, our understanding of the composition, structure and mechanism of the minor spliceosome remains limited. Therefore, in the framework of this project, we propose to investigate the molecular mechanism of the minor spliceosome. This will be achieved by studying specific proteins (so-called helicases), which may be potentially involved in controlling the splicing reaction. We are planning to develop a set of new tools to isolate minor splicing complexes and characterise their composition and structure. We will do it by using the latest technologies available to engineer mammalian cell lines to allow specific isolation of molecules of interest, which will be then imaged with electron microscopes operated at cryogenic temperature (cryo-EM). Such direct observation of the minor splicing machinery will allow us to gain insights into its potential mode of action.
Taken together, our project aims to address a fundamental question of the molecular mechanism of pre-mRNA splicing. It is estimated that up to 50% of genetic disorders are caused by malfunction of the splicing machinery, therefore the results produced during this project will have a direct impact on human health and benefit society.
During this reporting period, our main focus was on establishing methods and protocols that are needed for advancing two main objectives of the project: structural characterization of the minor splicing complexes and the involvement of RNA helicases in the U12-dependent splicing. So far, the key intermediate results that were obtained are the optimized conditions for sample purification and vitrification for the cryo-EM analysis and preliminary structural analysis of one of the analysed complexes. Additionally, we performed a number of biochemical experiments to uncover the composition of the complexes involved in U12-dependent splicing and the role of RNA helicases in this process. During this part of the project, we generated a number of cell lines and research tools that will used during subsequent stages of the project.
During the initial phases of the project, we focused our efforts on the structural characterization of the early splicing complexes purified from human cells. We established protocols to purify U2 snRNP and U11/U12 di-snRNP and perform structural characterization of several U2 snRNP-containing complexes (Tholen et al., Science 2022). Our work identified a new spliceosome assembly intermediate and provided several high-resolution snapshots into a complex process of the intron branch site recognition. The methods and experience obtained during this part of the project will be used in future studies of similar complexes in the minor splicing pathway.