Periodic Reporting for period 1 - SpliceosomeStructure (Structural role of protein splicing factors in promoting an active configuration of the spliceosome's RNA catalytic core)
Reporting period: 2016-03-01 to 2018-02-28
To synthesize the building blocks of living organisms – proteins – cells use the information contained in their genetic material (DNA) as a blueprint. During gene expression cells make copies of their DNA in the form of pre-messenger RNAs (pre-mRNA). When first synthesized, the information coding for proteins is interrupted in pre-mRNAs by non-coding sequences, called introns. Accurate expression of the genetic material, and thus accurate protein synthesis, relies on the correct removal of introns from pre-mRNAs to produce mature messenger RNAs (mRNAs). Intron removal is called splicing and is performed by the spliceosome, a very dynamic cellular machine composed of both proteins and RNA. Errors in splicing result in distorted mRNAs and can lead to abnormal proteins that interfere with the normal functions of cells and often cause disease.
Over the past 40 years much research was being carried out to understand how the spliceosome recognizes correct splicing sequences in pre-mRNAs and how specific mutations of such sequences can lead to aberrant splicing. However, such detailed understanding of splicing had been severely impaired by the lack of a proper understanding of the three-dimensional arrangement at the atomic level of specific RNA and protein components of the spliceosome. The goal of this work was to utilize recent advances in the three-dimensional study of macromolecules in combination with novel methods to purify spliceosomes from cells in order to obtain such a detailed picture of the spatial arrangement of the spliceosome with high resolution. Specifically, the work aimed to understand how each component of the spliceosome interacts with other components to form this complex cellular machine and promote its catalytic activity.
Through this work we have elucidated the high-resolution structure of the spliceosome in several states and have shed light not only on the arrangement of its protein and RNA components at the molecular level but also on the dynamic rearrangements of these components during the splicing cycle. The structures of the catalytic spliceosome resulting from this work have further revealed the physical basis for how the spliceosome assembles properly at the correct pre-mRNA sequences and promotes proper splicing, thus rationalizing decades of previous biochemical and genetic research.
Over the past 40 years much research was being carried out to understand how the spliceosome recognizes correct splicing sequences in pre-mRNAs and how specific mutations of such sequences can lead to aberrant splicing. However, such detailed understanding of splicing had been severely impaired by the lack of a proper understanding of the three-dimensional arrangement at the atomic level of specific RNA and protein components of the spliceosome. The goal of this work was to utilize recent advances in the three-dimensional study of macromolecules in combination with novel methods to purify spliceosomes from cells in order to obtain such a detailed picture of the spatial arrangement of the spliceosome with high resolution. Specifically, the work aimed to understand how each component of the spliceosome interacts with other components to form this complex cellular machine and promote its catalytic activity.
Through this work we have elucidated the high-resolution structure of the spliceosome in several states and have shed light not only on the arrangement of its protein and RNA components at the molecular level but also on the dynamic rearrangements of these components during the splicing cycle. The structures of the catalytic spliceosome resulting from this work have further revealed the physical basis for how the spliceosome assembles properly at the correct pre-mRNA sequences and promotes proper splicing, thus rationalizing decades of previous biochemical and genetic research.
To produce mRNA, the spliceosome excises introns from pre-mRNAs in two sequential reactions – branching and exon ligation. During this process of splicing, the spliceosome recognizes the 5’ and 3’ ends of the intron as well as a conserved adenosine nucleotide (termed branch site) and undergoes a dynamic rearrangement between these two reactions. This rearrangement is mediated by Prp16, as protein that uses energy from breaking down ATP to remodel the spliceosome after branching.
In this work we used budding yeast as a model organism to purify native spliceosomes that were trapped right after branching by using a mutation in Prp16 that blocks remodeling after the first catalytic step. We then prepared a frozen sample of these spliceosomes and utilized an electron microscope operated under cryogenic conditions (cryo-EM) to visualize individual spliceosome particles and obtain a 3D image of the complex that allowed us to build a near-atomic model of all of the spliceosome’s RNA and protein components. The resulting structure of the spliceosome in the branching conformation confirmed previous biochemical studies and showed that the active site is composed of RNA, whereas proteins promote formation for he active site and docking of the pre-mRNA substrate in the proper configuration necessary for catalysis. Importantly, the structure elucidated the molecular basis for recognition of the 5' end of the intron and of the branch adenosine.
To elucidate the structural consequences of Prp16 action, we then assembled spliceosomes on a pre-mRNA substrate containing a mutation that allows the Prp16 remodelling step but prevents mRNA formation. We then used cryo-EM to obtain a 3D image and build a near-atomic model of the spliceosome right before exon ligation. We found that the main consequence of Prp16 activity is to undock the RNA helix containing the branch site from the catalytic core in order to allow docking of the 3’ end of the intron. When undocked from the active site, the branch helix in the second step conformation would clash with first step proteins, thus explaining why dissociation of first step proteins is required for the second step of splicing. Moreover, our structure revealed how second step proteins stabilize the undocked conformation of the branch helix and suggests a plausible model for how the 3’ end of the intron binds in the active site.
Our two spliceosome structures in the branching and exon ligation states reveal the two active conformations of the spliceosome and elucidate the molecular basis for dynamics of the catalytic spliceosome.
In this work we used budding yeast as a model organism to purify native spliceosomes that were trapped right after branching by using a mutation in Prp16 that blocks remodeling after the first catalytic step. We then prepared a frozen sample of these spliceosomes and utilized an electron microscope operated under cryogenic conditions (cryo-EM) to visualize individual spliceosome particles and obtain a 3D image of the complex that allowed us to build a near-atomic model of all of the spliceosome’s RNA and protein components. The resulting structure of the spliceosome in the branching conformation confirmed previous biochemical studies and showed that the active site is composed of RNA, whereas proteins promote formation for he active site and docking of the pre-mRNA substrate in the proper configuration necessary for catalysis. Importantly, the structure elucidated the molecular basis for recognition of the 5' end of the intron and of the branch adenosine.
To elucidate the structural consequences of Prp16 action, we then assembled spliceosomes on a pre-mRNA substrate containing a mutation that allows the Prp16 remodelling step but prevents mRNA formation. We then used cryo-EM to obtain a 3D image and build a near-atomic model of the spliceosome right before exon ligation. We found that the main consequence of Prp16 activity is to undock the RNA helix containing the branch site from the catalytic core in order to allow docking of the 3’ end of the intron. When undocked from the active site, the branch helix in the second step conformation would clash with first step proteins, thus explaining why dissociation of first step proteins is required for the second step of splicing. Moreover, our structure revealed how second step proteins stabilize the undocked conformation of the branch helix and suggests a plausible model for how the 3’ end of the intron binds in the active site.
Our two spliceosome structures in the branching and exon ligation states reveal the two active conformations of the spliceosome and elucidate the molecular basis for dynamics of the catalytic spliceosome.
Our two spliceosome structures in the branching and exon ligation states reveal the two active conformations of the spliceosome and elucidate the molecular basis for dynamics of the catalytic spliceosome. They further provide a starting point for future finer, temporal studies of the conformational transitions that occur during the catalytic stage. Indeed these three-dimensional structures of the spliceosome will revolutionize the way research is carried out in the splicing field, by allowing more targeted design of experiments, and could enable the design of small molecules to modulate the activity of the spliceosome and potentially serve as therapeutic drugs.