Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Essential role of a trypanosome U4-specific Sm core protein in small nuclear ribonucleoprotein assembly and splicing.

ABSTRACT: Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.

SUBMITTER: Jae N

PROVIDER: S-EPMC2837986 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Special Sm core complex functions in assembly of the U2 small nuclear ribonucleoprotein of Trypanosoma brucei.

Project description:The processing of polycistronic pre-mRNAs in trypanosomes requires the spliceosomal small ribonucleoprotein complexes (snRNPs) U1, U2, U4/U6, U5, and SL, each of which contains a core of seven Sm proteins. Recently we reported the first evidence for a core variation in spliceosomal snRNPs; specifically, in the trypanosome U2 snRNP, two of the canonical Sm proteins, SmB and SmD3, are replaced by two U2-specific Sm proteins, Sm15K and Sm16.5K. Here we identify the U2-specific, nuclear-localized U2B'' protein from Trypanosoma brucei. U2B'' interacts with a second U2 snRNP protein, U2-40K (U2A'), which in turn contacts the U2-specific Sm16.5K/15K subcomplex. Together they form a high-affinity, U2-specific binding complex. This trypanosome-specific assembly differs from the mammalian system and provides a functional role for the Sm core variation found in the trypanosomal U2 snRNP.

| S-EPMC2725569 | biostudies-literature

Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila.

Project description:RNAi is an evolutionarily conserved gene regulatory process that operates in a wide variety of organisms. During RNAi, long double-stranded RNA precursors are processed by Dicer proteins into ?21-nt siRNAs. Subsequently, siRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute-family proteins and guide RISC to target RNAs via complementary base pairing, leading to posttranscriptional gene silencing. Select pre-mRNA splicing factors have been implicated in RNAi in fission yeast, worms, and flies, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle implicated in splicing, is required for RNAi and antiviral immunity in cultured cells and in vivo. SmD1 interacts with both Dicer-2 and dsRNA precursors and is indispensable for optimal siRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the small interfering RISC without significantly affecting the expression of major canonical siRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the small interfering RISC, including Argonaute 2, both in flies and in humans. Notably, RNAi defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the RNAi and splicing machineries are physically and functionally distinct entities. Our results suggest that Drosophila SmD1 plays a direct role in RNAi-mediated gene silencing independently of its pre-mRNA splicing activity and indicate that the dual roles of splicing factors in posttranscriptional gene regulation may be evolutionarily widespread.

| S-EPMC3799365 | biostudies-literature

Identification of a 35S U4/U6.U5 tri-small nuclear ribonucleoprotein (tri-snRNP) complex intermediate in spliceosome assembly.

Project description:The de novo assembly and post-splicing reassembly of the U4/U6.U5 tri-snRNP remain to be investigated. We report here that ZIP, a protein containing a CCCH-type zinc finger and a G-patch domain, as characterized by us previously, regulates pre-mRNA splicing independent of RNA binding. We found that ZIP physically associates with the U4/U6.U5 tri-small nuclear ribonucleoprotein (tri-snRNP). Remarkably, the ZIP-containing tri-snRNP, which has a sedimentation coefficient of ∼35S, is a tri-snRNP that has not been described previously. We also found that the 35S tri-snRNP contains hPrp24, indicative of a state in which the U4/U6 di-snRNP is integrating with the U5 snRNP. We found that the 35S tri-snRNP is enriched in the Cajal body, indicating that it is an assembly intermediate during 25S tri-snRNP maturation. We showed that the 35S tri-snRNP also contains hPrp43, in which ATPase/RNA helicase activities are stimulated by ZIP. Our study identified, for the first time, a tri-snRNP intermediate, shedding new light on the de novo assembly and recycling of the U4/U6.U5 tri-snRNP.

| S-EPMC5672036 | biostudies-literature

Sm core variation in spliceosomal small nuclear ribonucleoproteins from Trypanosoma brucei.

Project description:Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP-specific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification-tagging and in vitro protein-protein interaction assays. Using in vitro reconstitutions with canonical and U2-specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA-specific Sm sites.

| S-EPMC1589986 | biostudies-literature

Convergent transcripts of the yeast PRP38-SMD1 locus encode two essential splicing factors, including the D1 core polypeptide of small nuclear ribonucleoprotein particles.

Project description:The PRP38 gene of Saccharomyces cerevisiae is necessary for the excision of intron sequences from pre-mRNA and required for the maintenance of maximal levels of U6 small nuclear RNA (snRNA). This report describes the identification of a gene of related function, SMD1, located immediately 3' to PRP38. The PRP38 and SMD1 transcription units are configured in an unusual "tail-to-tail" arrangement with their respective open reading frames terminating on opposite strands of a common 6-bp region. The predicted SMD1 polypeptide, Smd1p, is 40% identical to the D1 protein of human small nuclear ribonucleoprotein particles. Experimentally induced depletion of Smd1p blocks the first step of splicing and results in growth arrest. In addition, the levels of the trimethylguanosine-capped spliceosomal snRNAs, U1, U2, U4, and U5, but not the Prp38p-sensitive U6 snRNA, decrease in response to Smd1p depletion. The cap structures of snRNAs persisting in the absence of SMD1 expression appear to be peculiar, as they are poorly recognized by an anti-trimethylguanosine antibody. These data establish Smd1p as a required component of the cellular splicing apparatus and a factor in snRNA maturation and stability.

| S-EPMC45767 | biostudies-other

Structure and conformational plasticity of the U6 small nuclear ribonucleoprotein core.

Project description:U6 small nuclear RNA (snRNA) is a key component of the active site of the spliceosome, a large ribonucleoprotein complex that catalyzes the splicing of precursor messenger RNA. Prior to its incorporation into the spliceosome, U6 is bound by the protein Prp24, which facilitates unwinding of the U6 internal stem-loop (ISL) so that it can pair with U4 snRNA. A previously reported crystal structure of the `core' of the U6 small nuclear ribonucleoprotein (snRNP) contained an ISL-stabilized A62G mutant of U6 bound to all four RNA-recognition motif (RRM) domains of Prp24 [Montemayor et al. (2014), Nature Struct. Mol. Biol. 21, 544-551]. The structure revealed a novel topology containing interlocked rings of protein and RNA that was not predicted by prior biochemical and genetic data. Here, the crystal structure of the U6 snRNP core with a wild-type ISL is reported. This complex crystallized in a new space group, apparently owing in part to the presence of an intramolecular cross-link in RRM1 that was not observed in the previously reported U6-A62G structure. The structure exhibits the same protein-RNA interface and maintains the unique interlocked topology. However, the orientation of the wild-type ISL is altered relative to the A62G mutant structure, suggesting inherent structural dynamics that may facilitate its pairing with U4. Consistent with their similar architectures in the crystalline state, the wild-type and A62G variants of U6 exhibit similar Prp24-binding affinities and electrophoretic mobilities when analyzed by gel-shift assay.

| S-EPMC5331471 | biostudies-literature

U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

Project description:To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.

| S-EPMC362380 | biostudies-other

Targeting of U4/U6 small nuclear RNP assembly factor SART3/p110 to Cajal bodies.

Project description:The spliceosomal small nuclear RNAs (snRNAs) are distributed throughout the nucleoplasm and concentrated in nuclear inclusions termed Cajal bodies (CBs). A role for CBs in the metabolism of snRNPs has been proposed but is not well understood. The SART3/p110 protein interacts transiently with the U6 and U4/U6 snRNPs and promotes the reassembly of U4/U6 snRNPs after splicing in vitro. Here we report that SART3/p110 is enriched in CBs but not in gems or residual CBs lacking coilin. The U6 snRNP Sm-like (LSm) proteins, also involved in U4/U6 snRNP assembly, were localized to CBs as well. The levels of SART3/p110 and LSm proteins in CBs were reduced upon treatment with the transcription inhibitor alpha-amanitin, suggesting that CB localization reflects active processes dependent on transcription/splicing. The NH2-terminal HAT domain of SART3/p110 was necessary and sufficient for specific protein targeting to CBs. Overexpression of truncation mutants containing the HAT domain had dominant negative effects on U6 snRNP localization to CBs, indicating that endogenous SART3/p110 plays a role in targeting the U6 snRNP to CBs. We propose that U4 and U6 snRNPs accumulate in CBs for the purpose of assembly into U4/U6 snRNPs by SART3/p110.

| S-EPMC2173746 | biostudies-literature

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.

Project description:Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of ?-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.

| S-EPMC3799305 | biostudies-literature

Core structure of the U6 small nuclear ribonucleoprotein at 1.7-A resolution.

Project description:The spliceosome is a dynamic assembly of five small nuclear ribonucleoproteins (snRNPs) that removes introns from eukaryotic pre-mRNA. U6, the most conserved of the spliceosomal small nuclear RNAs (snRNAs), participates directly in catalysis. Here, we report the crystal structure of the Saccharomyces cerevisiae U6 snRNP core containing most of the U6 snRNA and all four RRM domains of the Prp24 protein. It reveals a unique interlocked RNP architecture that sequesters the 5' splice site-binding bases of U6 snRNA. RRMs 1, 2 and 4 of Prp24 form an electropositive groove that binds double-stranded RNA and may nucleate annealing of U4 and U6 snRNAs. Substitutions in Prp24 that suppress a mutation in U6 localize to direct RNA-protein contacts. Our results provide the most comprehensive view to date of a multi-RRM protein bound to RNA and reveal striking coevolution of protein and RNA structure.

| S-EPMC4141773 | 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