SEARCH · PubMed Health

Results for “Spliceosomes”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources

PubMed1994

The yeast MUD2 protein: an interaction with PRP11 defines a bridge between commitment complexes and U2 snRNP addition.

In characterizing a series of yeast (Saccharomyces cerevisiae) mutants synthetic lethal with U1 RNA, we have identified a yeast gene (MUD2) with sequence similarity to the well-studied metazoan splicing factor U2AF65. The biochemical characterization indicates that the MUD2 gene product (MUD2P) contacts pre-mRNA directly and is a component of the pre-mRNA-U1 snRNP complex (commitment complex) that forms during early spliceosome assembly in yeast extracts. Unlike U1 snRNP itself, the association of MUD2P with pre-mRNA is dependent on a proper yeast branchpoint sequence. Genetic experiments indicate that MUD2P affects U2 snRNP addition. Moreover, experiments in the two-hybrid system show that PRP11P, a recently identified component of U2 snRNP, can interact directly with MUD2P. The experiments identify a specific inter-snRNP protein-protein contact that occurs during spliceosome assembly and more generally support substantial functional similarity between U2AF65 and MUD2P.

Amino Acid Sequence↗

PubMed1996

A U1/U4/U5 snRNP complex induced by a 2'-O-methyl-oligoribonucleotide complementary to U5 snRNA.

Nuclear messenger RNA splicing involves multiple interactions between the five spliceosomal small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5, and U6 and numerous spliceosomal proteins. Here it is shown that binding of a 2'-O-methyl-oligoribonucleotide complementary to U5 small nuclear RNA (snRNA) nucleotides 68 to 88 (BU5Ae) disrupts the initial U4/U5/U6 tri-snRNP complex, enhances the U2/U6 interaction, and induces a Ul/U4/U5 snRNP complex. The Ul/U4/U5 snRNP complex interacts specifically with an RNA oligonucleotide containing the 5' splice site sequence and may therefore represent a transitional stage in the displacement of U1 from the 5' splice site by U5 snRNP.

Base Sequence↗

PubMed1994

Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP.

Individual small nuclear ribonucleoproteins (snRNPs) U1, U2, and U4/U6 were removed from nuclear extracts of HeLa cells by antisense affinity depletion. Addition of a highly purified preparation of SR proteins fully restored splicing activity in reactions depleted of U1 snRNP but did not reconstitute splicing in reactions depleted of the other snRNPs. Affinity selection experiments revealed that spliceosomes lacking U1 snRNA formed in the U1 snRNP-depleted reactions reconstituted with SR proteins. Thus, high concentrations of SR proteins facilitate the assembly of precursor messenger RNA (pre-mRNA) into a spliceosome in the absence of interactions with U1 snRNP.

Base Sequence↗

PubMed2006

Yeast ntr1/spp382 mediates prp43 function in postspliceosomes.

The Ntr1 and Ntr2 proteins of Saccharomyces cerevisiae have been reported to interact with proteins involved in pre-mRNA splicing, but their roles in the splicing process are unknown. We show here that they associate with a postsplicing complex containing the excised intron and the spliceosomal U2, U5, and U6 snRNAs, supporting a link with a late stage in the pre-mRNA splicing process. Extract from cells that had been metabolically depleted of Ntr1 has low splicing activity and accumulates the excised intron. Also, the level of U4/U6 di-snRNP is increased but those of the free U5 and U6 snRNPs are decreased in Ntr1-depleted extract, and increased levels of U2 and decreased levels of U4 are found associated with the U5 snRNP protein Prp8. These results suggest a requirement for Ntr1 for turnover of the excised intron complex and recycling of snRNPs. Ntr1 interacts directly or indirectly with the intron release factor Prp43 and is required for its association with the excised intron. We propose that Ntr1 promotes release of excised introns from splicing complexes by acting as a spliceosome receptor or RNA-targeting factor for Prp43, possibly assisted by the Ntr2 protein.

DEAD-box RNA Helicases↗

PubMed1994

Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22.

In the budding yeast Saccharomyces cerevisiae, a number of PRP genes known to be involved in pre-mRNA processing have been genetically identified and cloned. Three PRP genes (PRP2, PRP16, and PRP22) were shown to encode putative RNA helicases of the family of proteins with DEAH boxes. However, any such splicing factor containing the helicase motifs in vertebrates has not been identified. To identify human homologs of this family, we designed PCR primers corresponding to the highly conserved region of the DEAH box protein family and successfully amplified five cDNA fragments, using HeLa poly(A)+ RNA as a substrate. One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which was previously shown to be involved in the release of spliced mRNAs from the spliceosomes. Expression of HRH1 in a S. cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype. These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein. Interestingly, HRH1 but not Prp22 contains an arginine- and serine-rich domain (RS domain) which is characteristic of some splicing factors, such as members of the SR protein family. We could show that HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain. We speculate that HRH1 might be targeted to the spliceosome through this interaction.

Amino Acid Sequence↗

PubMed1995

Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.

Amino Acid Sequence↗

PubMed1998

Detection of a novel ATP-dependent cross-linked protein at the 5' splice site-U1 small nuclear RNA duplex by methylene blue-mediated photo-cross-linking.

Assembly of spliceosomes involves a number of sequential steps in which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with the duplexes formed between pre-mRNA and small nuclear RNA (snRNA). We have detected a novel cross-link between a 65-kDa protein (p65) and the 5' splice site. A range of data suggest that p65 cross-links to the transient duplex formed by U1 snRNA and the 5' splice site. Moreover, although p65 cross-linking requires only a 5' splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splicing.

Adenosine Triphosphate↗

PubMed1998

Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells.

Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B' and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.

Amino Acid Sequence↗

PubMed2000

Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing.

Interaction between transcription and pre-mRNA processing via binding of polymerase II (Pol II) to factors involved in capping, splicing, and polyadenylation has recently been demonstrated. The C-terminal domain (CTD), a highly phosphorylated repeat sequence of the largest subunit of Pol II, has been implicated in this interaction because deletion of this domain affects downstream RNA processing events and because it is the binding site for numerous processing factors. Here we show that recombinant CTD, free of other components of Pol II, activated in vitro splicing and assembly of the spliceosome in nuclear extracts if, and only if, the assayed precursor RNA was recognized via exon definition, i.e., if the substrates contained complete exons with both 3' and 5' splice sites. Furthermore, depletion of intact Pol II inactivated splicing of this set of precursor RNAs and addition of recombinant CTD restored activity. The added recombinant CTD was quickly hyper- and hypophosphorylated in extract, became associated with the precursor RNA, and stimulated the association of U1 snRNPs but not ASF/SF2 with substrate RNA. These observations suggest that the mode of interaction between the CTD and splicing factors is integrally tied to exon definition and the mechanism whereby distal exons can be recognized and brought into juxtaposition during assembly of the spliceosome.

Animals↗

PubMed2001

Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m(7) guanosine cap.

In eukaryotes the majority of mRNAs have an m(7)G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3' end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure.

3T3 Cells↗

PubMed2004

Human U4/U6 snRNP recycling factor p110: mutational analysis reveals the function of the tetratricopeptide repeat domain in recycling.

After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

Animals↗

PubMed2004

snRNAs contain specific SMN-binding domains that are essential for snRNP assembly.

To serve in its function as an assembly machine for spliceosomal small nuclear ribonucleoprotein particles (snRNPs), the survival of motor neurons (SMN) protein complex binds directly to the Sm proteins and the U snRNAs. A specific domain unique to U1 snRNA, stem-loop 1 (SL1), is required for SMN complex binding and U1 snRNP Sm core assembly. Here, we show that each of the major spliceosomal U snRNAs (U2, U4, and U5), as well as the minor splicing pathway U11 snRNA, contains a domain to which the SMN complex binds directly and with remarkable affinity (low nanomolar concentration). The SMN-binding domains of the U snRNAs do not have any significant nucleotide sequence similarity yet they compete for binding to the SMN complex in a manner that suggests the presence of at least two binding sites. Furthermore, the SMN complex-binding domain and the Sm site are both necessary and sufficient for Sm core assembly and their relative positions are critical for snRNP assembly. These findings indicate that the SMN complex stringently scrutinizes RNAs for specific structural features that are not obvious from the sequence of the RNAs but are required for their identification as bona fide snRNAs. It is likely that this surveillance capacity of the SMN complex ensures assembly of Sm cores on the correct RNAs only and prevents illicit, potentially deleterious, assembly of Sm cores on random RNAs.

Animals↗

PubMed2005

ZPR1 is essential for survival and is required for localization of the survival motor neurons (SMN) protein to Cajal bodies.

Mutation of the survival motor neurons 1 (SMN1) gene causes motor neuron apoptosis and represents the major cause of spinal muscular atrophy in humans. Biochemical studies have established that the SMN protein plays an important role in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and that the SMN complex can interact with the zinc finger protein ZPR1. Here we report that targeted ablation of the Zpr1 gene in mice disrupts the subcellular localization of both SMN and spliceosomal snRNPs. Specifically, SMN localization to Cajal bodies and gems was not observed in cells derived from Zpr1-/- embryos and the amount of cytoplasmic snRNP detected in Zpr1-/- embryos was reduced compared with that in wild-type embryos. We found that Zpr1-/- mice die during early embryonic development, with reduced proliferation and increased apoptosis. These effects of Zpr1 gene disruption were confirmed and extended in studies of cultured motor neuron-like cells using small interfering RNA-mediated Zpr1 gene suppression; ZPR1 deficiency caused growth cone retraction, axonal defects, and apoptosis. Together, these data indicate that ZPR1 contributes to the regulation of SMN complexes and that it is essential for cell survival.

Animals↗

PubMed2026

Translational regulation of Sf1 integrates alternative splicing and hematopoietic stem cell fate.

The transition of hematopoietic stem cells (HSCs) from quiescence to lineage commitment requires precise posttranscriptional control, yet the contribution of mRNA isoform regulation remains poorly defined. Here, we identify a translationally controlled splicing program that contributes to HSC fate decisions. Using activity-based signatures of 305 splicing regulators, we uncover widespread posttranscriptional modulation of the spliceosome in stem and progenitor cells. The branch point recognition factor Sf1 emerges as a key node, regulated by a conserved structured 5' untranslated region (UTR) that cooperates with the RNA-binding protein Igf2bp2 to control its translation. Disrupting this cis-trans module reduces Sf1 protein synthesis and skews differentiation toward stem and erythroid programs. Mechanistically, Sf1-dependent alternative splicing remodels 5' UTRs of hematopoietic and DNA damage response genes, altering their translation and modulating DNA damage resolution. Together, these findings reveal an unrecognized translational layer controlling spliceosome activity and link RNA regulons, alternative splicing, and HSC fate determination.

Alternative Splicing↗

PubMed2005

Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements.

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.

Computer Simulation↗

PubMed2005

New maximum likelihood estimators for eukaryotic intron evolution.

The evolution of spliceosomal introns remains poorly understood. Although many approaches have been used to infer intron evolution from the patterns of intron position conservation, the results to date have been contradictory. In this paper, we address the problem using a novel maximum likelihood method, which allows estimation of the frequency of intron insertion target sites, together with the rates of intron gain and loss. We analyzed the pattern of 10,044 introns (7,221 intron positions) in the conserved regions of 684 sets of orthologs from seven eukaryotes. We determined that there is an average of one target site per 11.86 base pairs (bp) (95% confidence interval, 9.27 to 14.39 bp). In addition, our results showed that: (i) overall intron gains are approximately 25% greater than intron losses, although specific patterns vary with time and lineage; (ii) parallel gains account for approximately 18.5% of shared intron positions; and (iii) reacquisition following loss accounts for approximately 0.5% of all intron positions. Our results should assist in resolving the long-standing problem of inferring the evolution of spliceosomal introns.

Animals↗

PubMed2007

Intron dynamics in ribosomal protein genes.

The role of spliceosomal introns in eukaryotic genomes remains obscure. A large scale analysis of intron presence/absence patterns in many gene families and species is a necessary step to clarify the role of these introns. In this analysis, we used a maximum likelihood method to reconstruct the evolution of 2,961 introns in a dataset of 76 ribosomal protein genes from 22 eukaryotes and validated the results by a maximum parsimony method. Our results show that the trends of intron gain and loss differed across species in a given kingdom but appeared to be consistent within subphyla. Most subphyla in the dataset diverged around 1 billion years ago, when the "Big Bang" radiation occurred. We speculate that spliceosomal introns may play a role in the explosion of many eukaryotes at the Big Bang radiation.

Animals↗

PubMed1999

Dual posttranscriptional targets of retinoic acid-induced gene expression.

Retinoic acid-induced differentiation of the pre-osteoblastic cell line, UMR 201, is associated with a marked increase in the proficiency of posttranscriptional nuclear processing of alkaline phosphatase mRNA. In this study we attempted to correlate the posttranscriptional actions of retinoic acid with changes in phosphorylation, or abundance of spliceosome components, or both. Treatment with retinoic acid for periods of < or = 4 h resulted in dephosphorylation of nuclear U1 70K protein without affecting its abundance. Peptide mapping showed that U1 70K dephosphorylation was related to the disappearance of one specific phosphopeptide out of four major U1 70K phosphopeptides. A twofold decrease in mRNA expression of an isoform of alternative splicing factor that inhibits splicing was also observed over the same period. Tumor necrosis factor-alpha, which enhances the posttranscriptional action of retinoic acid, reduced U1 70K mRNA expression, while an inhibition of retinoic acid action by transforming growth factor-beta was associated with a marked increase in U1 70K mRNA levels. Our results draw attention to the complex interactions between short- and long-term alterations in the abundance and functional status of U1 70K, as well as SR proteins by growth and/or differentiation factors in the regulation of spliceosome formation and function.

Alkaline Phosphatase↗