Periodic Reporting for period 3 - SPLICE3D (CryoEM studies of the spliceosome)
Periodo di rendicontazione: 2019-06-01 al 2020-09-30
The spliceosome is a molecular machine that plays an important role in gene expression in higher organisms. When the gene is copied into RNA the spliceosome cuts non-coding sequences (introns) out of messenger RNA (mRNA) precursors, and joins together the useful coding sequences (exons) to make continuous protein coding chain. The spliceosome performs this in two steps. First, the start of an intron is recognised, cut, and joined to a specific point in the middle of that intron, forming a lasso-like looped structure. In the second step, the end of the intron is recognised, cut, and the exons joined together. This process called splicing is made more complex in higher organism such as the humans. Different mature mRNAs are produced by joining multiple exons in different combinations. Many different forms of proteins can be produced from a single gene so that proteins can be sent to different parts of the cell or can have silightly different functions. This is called alternative splicing and is crucial in increasing the complexity of the organisms from the limited number of genes. Many human diseases are caused by a failure to remove introns correctly.
Understanding the molecular mechanism of pre-mRNA splicing is very important to understand the cause of many inheritable genetic diseases which is the first step to find cures for these diseases. This will benefit society enormously.
The first important step for the understanding of the mechanism of pre-mRNA is to learn what the spliceosome looks like and to see how it works.
The overall objectives of our project are to biochemically isolate the spliceosome at particular steps of splicing reaction. Making a movie of spliceosome during its action will tell us how the spliceosomes recognise the junction between the exon and intron (splice site) and how it cuts and joins them.
Understanding the molecular mechanism of pre-mRNA splicing is very important to understand the cause of many inheritable genetic diseases which is the first step to find cures for these diseases. This will benefit society enormously.
The first important step for the understanding of the mechanism of pre-mRNA is to learn what the spliceosome looks like and to see how it works.
The overall objectives of our project are to biochemically isolate the spliceosome at particular steps of splicing reaction. Making a movie of spliceosome during its action will tell us how the spliceosomes recognise the junction between the exon and intron (splice site) and how it cuts and joins them.
Our first important achievement was to solved the structure of yeast U4/U6.U5 tri-snRNP which will becomes a part of the active site of the spliceosome and contain proteins which cradle the active site. The is the first structure of the spliceomal complex which revealed how the protein and RNA components are put together to form the spliceosome. We then determined the structure of the spliceosomes at different steps of the splicing reaction. The first such structure was the spliceosome just completed the first reaction and we were able to visualise the substrates (first exon and a lariat intron-3'exon intermediate) bound to the active site. This revealed how the first reaction is catalysed and what the active looks like. In order to carry of the second reaction the spliceosome has to undergo a large conformational change to be able to accommodate the 3' splice site (juction between the intron and the 2nd exon). The cryoEM structure of the C* complex showed how the spliceosome changes its shape and make a room for the 3'SS. Despite this conformational change the 3'SS was not bound in the active site so we could not visualise how the 3'SS is identified by the spliceosome. We decided to freeze the spliceosome after completing the second reaction (exon ligation) and look at the structure of the spliceosome immediately after completing the exon-ligation reaction so that we can still learn how the 3'SS is recognised if the product still remain in the active site. The structure of the post-catalytic spliceosome (P complex) indeed revealed how the 3'SS is recognised. The nucleotides preceding the 3'SS site is recognised by nucleotide within the substrate (the first intron nucleotide and branch point adenosise. Also the structure explains why the GU nucleotide at the begnning of the intron and the AG di-nucleotide at the end of the intron are almost invariant. This solved the long standing question in molecular biology. We also determined the structure of the spliceosomal complex at the beginning of the assembly. Our work provided profound mechanistic insights into the molecular mechanism of pre-mRNA splicing.
We now have good structural understanding of pre-mRNA splicing in yeast. Human spliceosomes are much larger than the yeast couterpart and contain many more human specific proteins. We are going to determine the structures of human spliceosomes by cryoEM and try to shed light into the mechanism of much more complex splicing events in human. The structures of the human spliceosomes are determined but we now have evidence that spliceosomes are slightly different in different cell types and depending on which pre-mRNA is being spliced. We are going to look at the variability of the spliceosome structure (protein composition) by cryoEM until the end of the project.
By the end of this reporting period we have determined the cryo-EM structure of human P complex spliceosomes, providing new insight into how metazoan-specific splicing factors promote exon ligation. Of particular interest, we showed that the FAM32A protein (previously identified independently as the tumour suppressor OTAG-12) inserts its conserved C-terminus into the spliceosome's active site, acting as an exon ligation factor. We have optimised functional assays for the FAM32A and Prp18 exon ligation factors. One interesting possibility is that pre-mRNA splicing might be regulated by different exon ligation factors in humans, perhaps in a tissue-specific fashion.
We also determined the cryo-EM structure of human pre-B complex spliceosomes and human U4/U6.U5 tri-snRNP particles. These structures provide important new insights into the mechanism of 5'SS transfer from U1 snRNP to U6 snRNA during human spliceosome activation, a complex but crucial process that is still only partly understood. The structures suggest how 5'SS transfer to U6 is coupled to a remodelling step that relocates a crucial ATPase enzyme that plays a central role in spliceosome activation.
By the end of this reporting period we have determined the cryo-EM structure of human P complex spliceosomes, providing new insight into how metazoan-specific splicing factors promote exon ligation. Of particular interest, we showed that the FAM32A protein (previously identified independently as the tumour suppressor OTAG-12) inserts its conserved C-terminus into the spliceosome's active site, acting as an exon ligation factor. We have optimised functional assays for the FAM32A and Prp18 exon ligation factors. One interesting possibility is that pre-mRNA splicing might be regulated by different exon ligation factors in humans, perhaps in a tissue-specific fashion.
We also determined the cryo-EM structure of human pre-B complex spliceosomes and human U4/U6.U5 tri-snRNP particles. These structures provide important new insights into the mechanism of 5'SS transfer from U1 snRNP to U6 snRNA during human spliceosome activation, a complex but crucial process that is still only partly understood. The structures suggest how 5'SS transfer to U6 is coupled to a remodelling step that relocates a crucial ATPase enzyme that plays a central role in spliceosome activation.