Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
Science ; 370(6523)2020 12 18.
Article in English | MEDLINE | ID: mdl-33243851

ABSTRACT

Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA.


Subject(s)
RNA Folding , RNA Splicing , RNA, Small Nuclear/chemistry , RNA-Binding Proteins/chemistry , Spliceosomes/chemistry , Catalytic Domain , Cryoelectron Microscopy , Humans , Protein Conformation , RNA, Catalytic/chemistry , RNA, Small Nuclear/genetics , Spliceosomes/genetics
2.
Mol Cell ; 80(1): 127-139.e6, 2020 10 01.
Article in English | MEDLINE | ID: mdl-33007253

ABSTRACT

Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.


Subject(s)
RNA Splicing Factors/chemistry , RNA Splicing Factors/metabolism , Spliceosomes/metabolism , Animals , Catalysis , HeLa Cells , Humans , Introns/genetics , Models, Molecular , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Protein Binding , Protein Stability , RNA/chemistry , RNA/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae/metabolism , Species Specificity , Time Factors
3.
Nature ; 583(7815): 310-313, 2020 07.
Article in English | MEDLINE | ID: mdl-32494006

ABSTRACT

The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52-7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.


Subject(s)
Cryoelectron Microscopy , Ribonucleoprotein, U2 Small Nuclear/chemistry , Ribonucleoprotein, U2 Small Nuclear/ultrastructure , Base Sequence , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , HeLa Cells , Humans , Models, Molecular , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Binding , Protein Conformation , RNA Splicing Factors/chemistry , RNA Splicing Factors/metabolism , Ribonucleoprotein, U2 Small Nuclear/genetics , Ribonucleoprotein, U2 Small Nuclear/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism
4.
Proc Natl Acad Sci U S A ; 115(23): E5334-E5343, 2018 06 05.
Article in English | MEDLINE | ID: mdl-29777089

ABSTRACT

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell-specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell-specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.


Subject(s)
Cell-Free Nucleic Acids/blood , Adult , Biomarkers/blood , Cell-Free Nucleic Acids/genetics , Cell-Free Nucleic Acids/isolation & purification , Female , Gene Library , High-Throughput Nucleotide Sequencing , Humans , Male , MicroRNAs/blood , MicroRNAs/genetics
5.
Cell ; 170(4): 701-713.e11, 2017 Aug 10.
Article in English | MEDLINE | ID: mdl-28781166

ABSTRACT

Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5' splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step. The molecular organization of several B-specific proteins suggests that they are involved in negatively regulating Brr2, positioning the U6/5'ss helix, and stabilizing the B complex structure. Our results indicate significant differences between the early activation phase of human and yeast spliceosomes.


Subject(s)
Spliceosomes/chemistry , Cell Nucleus/chemistry , Cryoelectron Microscopy , HeLa Cells , Humans , Models, Molecular , RNA-Binding Proteins/chemistry , Ribonucleoproteins, Small Nuclear/chemistry , Saccharomyces cerevisiae/chemistry , Spliceosomes/ultrastructure
6.
Nature ; 542(7641): 318-323, 2017 02 16.
Article in English | MEDLINE | ID: mdl-28076346

ABSTRACT

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP.


Subject(s)
Cryoelectron Microscopy , RNA Splicing , Spliceosomes/metabolism , Spliceosomes/ultrastructure , Adenosine/metabolism , Base Sequence , Biocatalysis , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/ultrastructure , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/ultrastructure , Exons/genetics , Humans , Introns/genetics , Models, Molecular , Movement , Protein Domains , RNA Splicing Factors/chemistry , RNA Splicing Factors/metabolism , RNA Splicing Factors/ultrastructure , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , Ribonuclease H/chemistry , Ribonuclease H/metabolism , Ribonucleoproteins, Small Nuclear/chemistry , Ribonucleoproteins, Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/ultrastructure , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology , Spliceosomes/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL
...