Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural studies of the spliceosome: zooming into the heart of the machine.

ABSTRACT: Spliceosomes are large, dynamic ribonucleoprotein complexes that catalyse the removal of introns from messenger RNA precursors via a two-step splicing reaction. The recent crystal structure of Prp8 has revealed Reverse Transcriptase-like, Linker and Endonuclease-like domains. The intron branch-point cross-link with the Linker domain of Prp8 in active spliceosomes and together with suppressors of 5' and 3' splice site mutations this unambiguously locates the active site cavity. Structural and mechanistic similarities with group II self-splicing introns have encouraged the notion that the spliceosome is at heart a ribozyme, and recently the ligands for two catalytic magnesium ions were identified within U6 snRNA. They position catalytic divalent metal ions in the same way as Domain V of group II intron RNA, suggesting that the spliceosome and group II intron use the same catalytic mechanisms.

SUBMITTER: Galej WP

PROVIDER: S-EPMC4045393 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Structural studies of the spliceosome: zooming into the heart of the machine.

Galej Wojciech P WP Nguyen Thi Hoang Duong TH Newman Andrew J AJ Nagai Kiyoshi K

Current opinion in structural biology 20140128

Spliceosomes are large, dynamic ribonucleoprotein complexes that catalyse the removal of introns from messenger RNA precursors via a two-step splicing reaction. The recent crystal structure of Prp8 has revealed Reverse Transcriptase-like, Linker and Endonuclease-like domains. The intron branch-point cross-link with the Linker domain of Prp8 in active spliceosomes and together with suppressors of 5' and 3' splice site mutations this unambiguously locates the active site cavity. Structural and mec ...[more]

PMID: 24480332

Similar Datasets

Challenging the spliceosome machine.

Project description:BackgroundUsing cDNA copies of transcripts and corresponding genomic sequences from the Berkeley Drosophila Genome Project, a set of 24,753 donor and acceptor splice sites were computed with a scanning algorithm that tested for single nucleotide insertion, deletion and substitution polymorphisms. Using this dataset, we developed a progressive partitioning approach to examining the effects of challenging the spliceosome system.ResultsOur analysis shows that information content increases near splice sites flanking progressively longer introns and exons, suggesting that longer splice elements require stronger binding of spliceosome components. Information also increases at splice sites near very short introns and exons, suggesting that short splice elements have crowding problems. We observe that the information found at individual splice sites depends upon a balance of splice element lengths in the vicinity, including both flanking and non-adjacent introns and exons.ConclusionThese results suggest an interdependence of multiple splicing events along the pre-mRNA, which may have implications for how the macromolecular spliceosome machine processes sets of neighboring splice sites.

| S-EPMC1431713 | biostudies-literature

Cryo-electron microscopy snapshots of the spliceosome: structural insights into a dynamic ribonucleoprotein machine.

Project description:The spliceosome excises introns from pre-messenger RNAs using an RNA-based active site that is cradled by a dynamic protein scaffold. A recent revolution in cryo-electron microscopy (cryo-EM) has led to near-atomic-resolution structures of key spliceosome complexes that provide insight into the mechanism of activation, splice site positioning, catalysis, protein rearrangements and ATPase-mediated dynamics of the active site. The cryo-EM structures rationalize decades of observations from genetic and biochemical studies and provide a molecular framework for future functional studies.

| S-EPMC6386135 | biostudies-literature

Zooming in on Cadherin-23: Structural Diversity and Potential Mechanisms of Inherited Deafness.

Project description:Cadherin-23 (CDH23) is an essential component of hair-cell tip links, fine filaments that mediate inner-ear mechanotransduction. The extracellular domain of CDH23 forms about three-fourths of the tip link with 27 extracellular cadherin (EC) repeats that are structurally similar but not identical to each other. Calcium (Ca2+) coordination at the EC linker regions is key for tip-link elasticity and function. There are ?116 sites in CDH23 affected by deafness-causing mutations, many of which alter conserved Ca2+-binding residues. Here we present crystal structures showing 18 CDH23 EC repeats, including the most and least conserved, a fragment carrying disease mutations, and EC repeats with non-canonical Ca2+-binding motif sequences and unusual secondary structure. Complementary experiments show deafness mutations' effects on stability and affinity for Ca2+. Additionally, a model of nine contiguous CDH23 EC repeats reveals helicity and potential parallel dimerization faces. Overall, our studies provide detailed structural insight into CDH23 function in mechanotransduction.

| S-EPMC6375707 | biostudies-literature

Malleable ribonucleoprotein machine: protein intrinsic disorder in the Saccharomyces cerevisiae spliceosome.

Project description:Recent studies revealed that a significant fraction of any given proteome is presented by proteins that do not have unique 3D structures as a whole or in significant parts. These intrinsically disordered proteins possess dramatic structural and functional variability, being especially enriched in signaling and regulatory functions since their lack of fixed structure defines their ability to be involved in interaction with several proteins and allows them to be re-used in multiple pathways. Among recognized disorder-based protein functions are interactions with nucleic acids and multi-target binding; i.e., the functions ascribed to many spliceosomal proteins. Therefore, the spliceosome, a multimegadalton ribonucleoprotein machine catalyzing the excision of introns from eukaryotic pre-mRNAs, represents an attractive target for the focused analysis of the abundance and functionality of intrinsic disorder in its proteinaceous components. In yeast cells, spliceosome consists of five small nuclear RNAs (U1, U2, U4, U5, and U6) and a range of associated proteins. Some of these proteins constitute cores of the corresponding snRNA-protein complexes known as small nuclear ribonucleoproteins (snRNPs). Other spliceosomal proteins have various auxiliary functions. To gain better understanding of the functional roles of intrinsic disorder, we have studied the prevalence of intrinsically disordered proteins in the yeast spliceosome using a wide array of bioinformatics methods. Our study revealed that similar to the proteins associated with human spliceosomes (Korneta & Bujnicki, 2012), proteins found in the yeast spliceosome are enriched in intrinsic disorder.

| S-EPMC3628832 | biostudies-literature

Structural basis for conformational equilibrium of the catalytic spliceosome.

Project description:The ATPase Prp16 governs equilibrium between the branching (B∗/C) and exon ligation (C∗/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. The exon-ligation factors Prp18 and Slu7 bind to Ci before ATP hydrolysis by Prp16 can destabilize the branching conformation. Biochemical assays suggest that these pre-bound factors prime the C complex for conversion to C∗ by Prp16. A complete model of the Prp19 complex (NTC) reveals how the branching factors Yju2 and Isy1 are recruited by the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain tethered to the NTC in the C∗ complex to promote exon ligation. Our results explain how Prp16 action modulates the dynamic binding of step-specific factors to alternatively stabilize the C or C∗ conformation and establish equilibrium of the catalytic spliceosome.

| S-EPMC8022279 | biostudies-literature

Structural basis of branch site recognition by the human spliceosome.

Project description:Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human 17S U2 snRNP and reconstituted in vitro its adenosine 5´-triphosphate (ATP)–dependent remodeling and binding to the pre–messenger RNA substrate. We determined a series of high-resolution (2.0 to 2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2 snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism based on branch helix stability.

| S-EPMC7614990 | biostudies-literature

Zooming in on Transcription Preinitiation.

Project description:Class II gene transcription commences with the assembly of the Preinitiation Complex (PIC) from a plethora of proteins and protein assemblies in the nucleus, including the General Transcription Factors (GTFs), RNA polymerase II (RNA pol II), co-activators, co-repressors, and more. TFIID, a megadalton-sized multiprotein complex comprising 20 subunits, is among the first GTFs to bind the core promoter. TFIID assists in nucleating PIC formation, completed by binding of further factors in a highly regulated stepwise fashion. Recent results indicate that TFIID itself is built from distinct preformed submodules, which reside in the nucleus but also in the cytosol of cells. Here, we highlight recent insights in transcription factor assembly and the regulation of transcription preinitiation.

| S-EPMC4906157 | biostudies-literature

Structural characterization of the fission yeast U5.U2/U6 spliceosome complex.

Project description:The spliceosome is a dynamic macromolecular machine that catalyzes the excision of introns from pre-mRNA. The megadalton-sized spliceosome is composed of four small nuclear RNPs and additional pre-mRNA splicing factors. The formation of an active spliceosome involves a series of regulated steps that requires the assembly and disassembly of large multiprotein/RNA complexes. The dynamic nature of the pre-mRNA splicing reaction has hampered progress in analyzing the structure of spliceosomal complexes. We have used cryo-electron microscopy to produce a 29-A density map of a stable 37S spliceosomal complex from the genetically tractable fission yeast, Schizosaccharomyces pombe. Containing the U2, U5, and U6 snRNAs, pre-mRNA splicing intermediates, U2 and U5 snRNP proteins, the Nineteen Complex (NTC), and second-step splicing factors, this complex closely resembles in vitro purified mammalian C complex. The density map reveals an asymmetric particle, approximately 30 x 20 x 18 nm in size, which is composed of distinct domains that contact each other at the center of the complex.

| S-EPMC1805518 | biostudies-literature

Structural requirement of Ntc77 for spliceosome activation and first catalytic step.

Project description:The Prp19-associated complex is required for spliceosome activation by stabilizing the binding of U5 and U6 on the spliceosome after the release of U4. The complex comprises at least eight proteins, among which Ntc90 and Ntc77 contain multiple tetratricopeptide repeat (TPR) elements. We have previously shown that Ntc90 is not involved in spliceosome activation, but is required for the recruitment of essential first-step factor Yju2 to the spliceosome. We demonstrate here that Ntc77 has dual functions in both spliceosome activation and the first catalytic step in recruiting Yju2. We have identified an amino-terminal region of Ntc77, which encompasses the N-terminal domain and the first three TPR motifs, dispensable for spliceosome activation but required for stable interaction of Yju2 with the spliceosome. Deletion of this region had no severe effect on the integrity of the NTC, binding of NTC to the spliceosome or spliceosome activation, but impaired splicing and exhibited a dominant-negative growth phenotype. Our data reveal functional roles of Ntc77 in both spliceosome activation and the first catalytic step, and distinct structural domains of Ntc77 required for these two steps.

| S-EPMC4231770 | biostudies-literature

Structural insights into the cross-exon to cross-intron spliceosome switch.

Project description:Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2-5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5' splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10-16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5' splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.

| S-EPMC11208138 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data