Periodic Reporting for period 1 - StructuRNP (Cryo-EM structural analysis of small nuclear ribonucleoprotein particles (snRNP) biogenesis)
Periodo di rendicontazione: 2021-10-01 al 2023-09-30
Pre-mRNA splicing is carried out by the dynamic and multi-megadalton spliceosome, which assembles anew on each intron from pre-formed small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and non-snRNP proteins. The U2-U6 snRNPs undergo major compositional and conformational changes throughout spliceosome assembly, activation, catalysis, and disassembly. Among these, the U5 snRNP undergoes particularly dramatic changes, while serving as the ~1 megadalton “heart” of the spliceosome around which pre-mRNA, U1, U2, U4, and U6 snRNPs, and non snRNP proteins organize for splicing. After spliceosome disassembly, the post splicing U5 snRNP is recycled into a ‘20S U5 snRNP’, which subsequently scaffolds the formation of the ~2 megadalton U4/U6.U5 tri-snRNP for the next round of splicing. While the molecular basis of pre-mRNA splicing has been extensively studied the structural mechanism of U5 snRNP biogenesis and recycling remained unknown.
Impaired recycling and biogenesis of the spliceosomal components has been linked to a plethora of diseases, such as Lobular Neoplasia, Cardiofaciocutaneous Syndrome or Amyotrophic Lateral Sclerosis. Hence, our objective was to study the cryo-EM structure of native human U5 snRNP in its recycling state, to shed light on the functional aspects of the spliceosome recycling process that may also have implications to understand these diseases.
Impaired recycling and biogenesis of the spliceosomal components has been linked to a plethora of diseases, such as Lobular Neoplasia, Cardiofaciocutaneous Syndrome or Amyotrophic Lateral Sclerosis. Hence, our objective was to study the cryo-EM structure of native human U5 snRNP in its recycling state, to shed light on the functional aspects of the spliceosome recycling process that may also have implications to understand these diseases.
The goal of this project was to perform structural and functional studies of the biogenesis steps of the spliceosomal snRNPs, which in early steps involves the survival of motor neuron (SMN)-complex. To that end, we aimed to reconstitute in vitro and uncover the cryo-EM structures of three key states of snRNP biogenesis: (1) the complete SMN complex comprising SMN and Gemin2-8 proteins, (2) the SMN complex with a loaded Sm ring, and (3) the complete SMN-Sm-ring complex captured during the loading onto an snRNA.
While we made substantial progress towards the reconstitution of the complete and various SMN subcomplexes, EM studies proved challenging due to remaining complex heterogeneity and poor behavior. In addition, we purified endogenous SMN-complex assemblies bound to the U1 snRNP via its protein U1-70K. We were successful in purifying a stoichiometric sample containing all known U1 proteins and SMN complex components, but EM studies of this sample also proved challenging due to inherent sample heterogeneity.
Since later steps in snRNP biogenesis and recycling are thought to converge to similar complexes, we then chose to investigate late biogenesis steps and recycling of the human U5 snRNP. To gain structural insights into U5 snRNP biogenesis and recycling, we overexpressed in human K562 cells the GFP-tagged CD2BP2 protein, a known recycling factor of the U5, and purified the endogenous 20S U5 snRNP via the GFP-tag from nuclear extract. The purified complexes contained the U5 snRNP core, including the known recycling chaperones CD2BP2 and TSSC4. Purification of GFP-CD2BP2 complexes from cytoplasmic extract yielded U5 snRNPs of the same composition suggesting that late steps of U5 snRNP biogenesis and recycling may converge at CD2BP2 and TSSC4 chaperones.
Using this endogenous sample, we determined a consensus cryo-EM density of the human nuclear 20S U5 snRNP at an overall resolution of 2.6 Å with the help of which we could describe four sequential recycling states of the U5 snRNP. This revealing cryo-EM structures of the recycling 20S U5 snRNP, based on which we could assign, unknown until now, roles to the CD2BP2 and TSSC4 chaperones.
We discovered that CD2BP2 is responsible for stabilizing the core protein of the U5- PRP8 and for tethering a later tri-snRNP factor DIM1, while TSSC4 is tethering the essential BRR2 helicase before its docking to the fully assembled tri-snRNP. Additionally, we found that the docking, the function and the dissociation of these chaperones is uniquely ATP-independent and rely on protein-protein interactions: in the end of their function TSSC4 and CD2BP2 are replaced by the DDX23 helicase and DIM1 respectively, within the complex of the assembled and functional tri-snRNP.
In the duration of this fellowship, the U5 snRNP project was presented in talks at the FISEB 2023 conference in Israel, at the RNA microsymposium 2023 in Austria, at the Polish RNA meeting 2023 in Poland, and as a poster at the Cold Spring Harbor RNA processing meeting 2023. Internally, this work was presented to the RNA community at the Vienna BioCenter in the “Biochemistry Coffee Club” and as part of VBC Monday seminar lecture series. Additionally, this project turned into a manuscript that is currently undergoing revisions in Nature Structural Molecular Biology journal. During the manuscript submission process, the structures and the cryo-EM densities determined were deposited to the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB).
While we made substantial progress towards the reconstitution of the complete and various SMN subcomplexes, EM studies proved challenging due to remaining complex heterogeneity and poor behavior. In addition, we purified endogenous SMN-complex assemblies bound to the U1 snRNP via its protein U1-70K. We were successful in purifying a stoichiometric sample containing all known U1 proteins and SMN complex components, but EM studies of this sample also proved challenging due to inherent sample heterogeneity.
Since later steps in snRNP biogenesis and recycling are thought to converge to similar complexes, we then chose to investigate late biogenesis steps and recycling of the human U5 snRNP. To gain structural insights into U5 snRNP biogenesis and recycling, we overexpressed in human K562 cells the GFP-tagged CD2BP2 protein, a known recycling factor of the U5, and purified the endogenous 20S U5 snRNP via the GFP-tag from nuclear extract. The purified complexes contained the U5 snRNP core, including the known recycling chaperones CD2BP2 and TSSC4. Purification of GFP-CD2BP2 complexes from cytoplasmic extract yielded U5 snRNPs of the same composition suggesting that late steps of U5 snRNP biogenesis and recycling may converge at CD2BP2 and TSSC4 chaperones.
Using this endogenous sample, we determined a consensus cryo-EM density of the human nuclear 20S U5 snRNP at an overall resolution of 2.6 Å with the help of which we could describe four sequential recycling states of the U5 snRNP. This revealing cryo-EM structures of the recycling 20S U5 snRNP, based on which we could assign, unknown until now, roles to the CD2BP2 and TSSC4 chaperones.
We discovered that CD2BP2 is responsible for stabilizing the core protein of the U5- PRP8 and for tethering a later tri-snRNP factor DIM1, while TSSC4 is tethering the essential BRR2 helicase before its docking to the fully assembled tri-snRNP. Additionally, we found that the docking, the function and the dissociation of these chaperones is uniquely ATP-independent and rely on protein-protein interactions: in the end of their function TSSC4 and CD2BP2 are replaced by the DDX23 helicase and DIM1 respectively, within the complex of the assembled and functional tri-snRNP.
In the duration of this fellowship, the U5 snRNP project was presented in talks at the FISEB 2023 conference in Israel, at the RNA microsymposium 2023 in Austria, at the Polish RNA meeting 2023 in Poland, and as a poster at the Cold Spring Harbor RNA processing meeting 2023. Internally, this work was presented to the RNA community at the Vienna BioCenter in the “Biochemistry Coffee Club” and as part of VBC Monday seminar lecture series. Additionally, this project turned into a manuscript that is currently undergoing revisions in Nature Structural Molecular Biology journal. During the manuscript submission process, the structures and the cryo-EM densities determined were deposited to the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB).
A central step in the maturation of eukaryotic mRNA is the removal of non-coding introns from precursor mRNA, in a process termed splicing. This activity is mediated by the spliceosome and much is known about its assembly and activity. However, despite its significance as the rate limiting step of splicing, the recycling of spliceosomal snRNPs remains poorly understood.
Our results reveal how the U5 snRNP-specific chaperones CD2BP2 and TSSC4 facilitate the ATP-independent biogenesis and recycling of the human U5 snRNP and prime the assembly of the U4/U6.U5 tri-snRNP for a new round of splicing. The modes of CD2BP2 and TSSC4 chaperones action suggest a framework to understand how snRNP-specific chaperones use protein-protein interactions to position key, mobile snRNP domains for the efficient binding of snRNP-specific subunits or snRNAs.
Our results reveal how the U5 snRNP-specific chaperones CD2BP2 and TSSC4 facilitate the ATP-independent biogenesis and recycling of the human U5 snRNP and prime the assembly of the U4/U6.U5 tri-snRNP for a new round of splicing. The modes of CD2BP2 and TSSC4 chaperones action suggest a framework to understand how snRNP-specific chaperones use protein-protein interactions to position key, mobile snRNP domains for the efficient binding of snRNP-specific subunits or snRNAs.