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Results for “Spliceosomes”

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PubMed2002

Pre-mRNA splicing in Schizosaccharomyces pombe: regulatory role of a kinase conserved from fission yeast to mammals.

Most primary messenger RNA transcripts (pre-mRNAs) in eukaryotes contain intervening sequences that must be precisely removed to generate a functional mRNA. The excision of the intervening sequences, the introns, from a pre-mRNA and the concomitant joining of the flanking sequences, the exons, is called pre-mRNA splicing. Pre-mRNA splicing takes place in large ribonucleoprotein machinery, the spliceosome. Although the function and components of this machinery appear to be highly conserved between organisms, many distinct differences between budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe, have been found, emphasizing their evolutionary distance. Most interestingly, fission yeast appears to reflect the more conservative evolutionary development regarding pre-mRNA splicing. Many spliceosomal components, including the five small nuclear RNAs, which most likely form the catalytic core of the spliceosome, show a higher degree of similarity with the components of the splicing machinery found in mammals. In addition, several regulatory components of the spliceosome detected in mammals are absent in Sac. cerevisiae, but present in Sch. pombe. Here, we review recent progress made in our understanding of the control of pre-mRNA splicing in Sch. pombe. The focus is on Prp4p kinase, first discovered in fission yeast and also present in mammals, but absent in Sac. cerevisiae. Results from both mammals and Sch. pombe suggest that Prp4p plays a key role in regulating pre-mRNA splicing and in connecting this process with the cell cycle.

Animals↗

PubMed2002

Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18.

Prp18 functions in the second step of pre-mRNA splicing, joining the spliceosome just prior to the transesterification reaction that creates the mature mRNA. Prp18 interacts with Slu7, and the functions of the two proteins are intertwined. Using the X-ray structure of Prp18, we have designed mutants in Prp18 that imply that Prp18 has two distinct roles in splicing. Deletion mutations were used to delineate the surface of Prp18 that interacts with Slu7, and point mutations in Prp18 were used to define amino acids that contact Slu7. Experiments in which Slu7 and mutant Prp18 proteins were expressed at different levels support a model in which interaction between the proteins is needed for stable binding of both proteins to the spliceosome. Mutations in an evolutionarily conserved region show that it is critical for Prp18 function but is not involved in binding Slu7. Alleles with mutations in the conserved region are dominant negative, suggesting that the resulting mutant prp18 proteins make proper contacts with the spliceosome, but fail to carry out a Prp18-specific function. Prp18 thus appears to have two separable roles in splicing, one in stabilizing interaction of Slu7 with the spliceosome, and a second that requires the conserved loop.

Amino Acid Sequence↗

PubMed1991

Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA.

Splicing of pre-messenger RNA in eukaryotic cells occurs in a multicomponent complex termed the spliceosome, which contains small nuclear ribonucleoprotein particles (snRNPs), protein factors and substrate pre-mRNA. Assembly of the spliceosome involves the stepwise binding of snRNPs and protein factors to the pre-mRNA through a poorly understood mechanism which probably involves specific RNA-RNA, RNA-protein and protein-protein interactions. Of particular interest are the interactions between snRNPs, which are likely to be important not only for assembly of the spliceosome but also for catalysis. U1 snRNP interacts with the 5' splice site and U2 snRNP with the branch site of the pre-mRNA; both of these interactions involve Watson-Crick base pairing. But very little is known about how other factors such as the U4/U6 and U5 snRNPs reach the spliceosome and function in splicing. Here we report evidence that U6 snRNA interacts directly with U2 snRNA by a mechanism involving base-pairing, and that this interaction can be necessary for splicing of a mammalian pre-mRNA in vivo.

Base Composition↗

PubMed1996

In vitro studies of the Prp9.Prp11.Prp21 complex indicate a pathway for U2 small nuclear ribonucleoprotein activation.

Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9.Prp11.Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9.Prp11.Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome.

Electrophoresis, Polyacrylamide Gel↗

PubMed1999

A candidate U1 small nuclear RNA for trypanosomatid protozoa.

In trypanosomatid protozoa, all mRNAs obtain identical 5'-ends by trans-splicing of the 5'-terminal 39 nucleotides of a small spliced leader RNA to appropriate acceptor sites in pre-mRNA. Although this process involves spliceosomal small nuclear (sn) RNAs, it is thought that trypanosomatids do not contain a homolog of the cis-spliceosomal U1 snRNA. We show here that a trypanosomatid protozoon, Crithidia fasciculata, contains a novel small RNA that displays several features characteristic of a U1 snRNA, including (i) a methylguanosine cap and additional 5'-terminal modifications, (ii) a potential binding site for common core proteins that are present in other trans-spliceosomal ribonucleoproteins, (iii) a U1-like 5'-terminal sequence, and (iv) a U1-like stem/loop I structure. Because trypanosomatid pre-mRNAs do not appear to contain cis-spliced introns, we argue that this previously unrecognized RNA species is a good candidate to be a trans-spliceosomal U1 snRNA.

Animals↗

PubMed2001

In situ transcription and splicing in the Balbiani ring 3 gene.

The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of them are co-transcriptionally excised. We have determined the in situ structure of the active BR3 gene by electron tomography. Each of the 20-25 nascent transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here called the NTS complex. The results indicate that extensive changes in overall shape, substructure and molecular mass take place repeatedly within an NTS complex as it moves along the gene. The volume and calculated mass of the NTS complexes show that, maximally, one complete spliceosome is assembled on the multi-intron transcript at any given time point. The structural data show that the spliceosome is not a structurally well-defined unit in situ and that the C-terminal domain of the elongating RNA polymerase II cannot carry spliceosomal components for all introns in the BR3 transcript. Our data indicate that spliceosomal factors are continuously added to and released from the NTS complexes during transcription elongation.

Animals↗

PubMed2025

A CRISPR/Cas9 mutant resource for OsSm RNA-binding genes in rice.

Pre-mRNA, produced by eukaryotic DNA transcription, undergoes splicing by the spliceosome, which removes introns and joins exons to form mRNA. The spliceosome is a large and highly dynamic molecular machine. Its core components include five small nuclear ribonucleoproteins (snRNPs) and the various spliceosome-related proteins. The conserved Smith (Sm) complex and the Sm-like proteins (LSm) serve as primary components of the snRNPs. Sm proteins are involved in processes such as pre-mRNA splicing and mRNA degradation, which can regulate gene expression, thereby influencing plant growth, development, and stress responses. While 25 Sm proteins have been identified in rice, their specific roles in regulating rice growth and development remain unclear. In this study, we employed the CRISPR/Cas9 system to edit 15 OsSm genes, and 13 mutants were obtained, with mutation rates ranging from 20.83 to 83.87%. In comparison to the wild type (WT), the mutants exhibited dwarfism, reduced tiller numbers, lower seed-setting rates or sterility, and increased susceptibility to diseases. One Sm mutant, ossmf-2, exhibited dwarfism, delayed flowering, and small grains. Through transcriptome analysis, three target genes, OsMRG702, OsRGG2, and OsLA1, were identified. Mutations of the OsSmF protein may lead to the abnormal splicing of these genes and finally lead to the inhibition of growth and development. Our study first edited the OsSm genes and generated a mutant library in rice. Most of the mutants exhibited abnormal growth and development, underscoring the essential roles of OsSm proteins in rice physiology. Furthermore, this work addresses a critical gap in the functional characterization of Sm proteins in rice. The resulting mutant collection offers valuable germplasm resources and lays a theoretical foundation for elucidating the molecular regulatory networks involving spliceosomal components and their target genes in the control of crop growth, development, and reproduction.

Oryza↗

PubMed1996

Autoantibodies to the A/B proteins of the heterogeneous nuclear ribonucleoprotein complex: novel tools for the diagnosis of rheumatic diseases.

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are major constituents of the spliceosome. They are composed of approximately 30 different proteins which can bind to nascent pre-mRNA. Among these, the hnRNP-A/B proteins form a subgroup of highly related proteins: their N-terminal halves consist of two adjacent RNA-binding domains, whereas the C-terminal halves contain almost 50% glycine residues. These proteins represent a group of novel autoantigens which are targeted by autoantibodies from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD): thus, anti-A2/RA33 autoantibodies target the hnRNP proteins A2, B1, B2 (the 'RA33 complex'), and anti-A1 autoantibodies are directed to the hnRNP proteins A1 and A1b. In SLE, anti-hnRNP-A/B antibodies frequently occur together with antibodies to two other spliceosome-associated antigens, U1 small nuclear RNP (U1-snRNP) and Sm. Epitope-mapping studies have revealed that the major antibody binding sites are located in the RNA-binding regions. Furthermore, there is some indication of disease-specific epitope recognition. Studies in animal models have demonstrated the presence of anti-hnRNP-A/B antibodies in several lupus-prone mouse strains. Thus, autoantibodies to the spliceosomal hnRNP-A/B proteins are a common feature of RA, SLE, and MCTD. However, these diseases differ in their reactivities to other spliceosomal components, such as U1-snRNP and Sm antigens. Therefore, anti-hnRNP-A/B autoantibodies are not only valuable diagnostic markers but may also allow additional insights into the pathogenetic mechanisms of rheumatic autoimmune diseases.

Animals↗

PubMed1995

The role of PRP8 protein in nuclear pre-mRNA splicing in yeast.

The removal of introns from precursor messenger RNAs occurs in a large complex, the spliceosome, that contains many proteins and five small nuclear RNAs (snRNAs). The snRNAs interact with the intron-containing substrate RNA and with each other to form a dynamic network of RNA interactions that define the intron and promote splicing. There is evidence that protein splicing factors play important roles in regulating RNA interactions in the spliceosome. PRP8 is a highly conserved protein that is associated in particles with the U5 snRNA and directly binds the substrate RNA in spliceosomes. UV crosslinking has been used to map the binding sites, and shows extensive interaction between PRP8 protein and the 5' exon prior to the first step of splicing and with the 3' splice site region subsequently. It is proposed that PRP8 protein may stabilize fragile interactions between the U5 snRNA and exon sequences at the splice sites, to anchor and align them in the catalytic centre of the spliceosome.

Exons↗

PubMed2006

The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP.

The human 25S U4/U6.U5 tri-snRNP is a major building block of the U2-type spliceosome and contains, in addition to the U4, U6, and U5 snRNAs, at least 30 distinct proteins. To learn more about the molecular architecture of the tri-snRNP, we have investigated interactions between tri-snRNP proteins using the yeast two-hybrid assay and in vitro binding assays, and, in addition, have identified distinct protein domains that are critical for the connectivity of this protein network in the human tri-snRNP. These studies revealed multiple interactions between distinct domains of the U5 proteins hPrp8, hBrr2 (a DExH/D-box helicase), and hSnu114 (a putative GTPase), which are key players in the catalytic activation of the spliceosome, during which the U4/U6 base-pairing interaction is disrupted and U4 is released from the spliceosome. Both the U5-specific, TPR/HAT-repeat-containing hPrp6 protein and the tri-snRNP-specific hSnu66 protein interact with several U5- and U4/U6-associated proteins, including hBrr2 and hPrp3, which contacts the U6 snRNA. Thus, both proteins are located at the interface between U5 and U4/U6 in the tri-snRNP complex, and likely play an important role in transmitting the activity of hBrr2 and hSnu114 in the U5 snRNP to the U4/U6 duplex during spliceosome activation. A more detailed analysis of these protein interactions revealed that different HAT repeats mediate interactions with specific hPrp6 partners. Taken together, data presented here provide a detailed picture of the network of protein interactions within the human tri-snRNP.

Base Sequence↗

PubMed1992

Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing.

We have investigated the function of ATP hydrolysis in mammalian pre-mRNA in vitro splicing using adenosine phosphorothioates (ATP alpha S and ATP tau S) known to affect the activity of a number of ATP-requiring enzymes. Spliceosome assembly, but neither one of the two transesterification reactions involved in splicing, occurs with ATP alpha S suggesting that at least two types of ATP-requiring factors are brought into play. ATP alpha S has no effect in the presence of normal ATP and, therefore, spliceosomes assembled in the presence of ATP alpha S remain competent for splicing when supplied with normal ATP. ATP tau S noticeably and irreversibly inhibits the second transesterification reaction, i.e. at a time when most of the analog has been hydrolyzed and regenerated to normal ATP by creatine phosphate. This indicates that the inhibition results from an earlier event, most likely the thiophosphorylation of spliceosomal proteins. Under this assumption, the inhibition could be due to the failure of the thiophosphorylated proteins to be dephosphorylated. Indeed, okadaic acid, a potent inhibitor of protein phosphatases, inhibits the second step of a reaction in the presence of normal ATP. We propose that some splicing factors undergo phosphorylation-dephosphorylation cycles during spliceosome assembly and splicing, while others that could be the mammalian equivalents of the RNA helicase-like proteins recently discovered in yeast most likely bind and hydrolyze ATP.

Adenosine Triphosphatases↗

PubMed1998

Lupus humoral autoimmunity induced in a primate model by short peptide immunization.

BACKGROUND: Systemic lupus erythematosus (SLE) is characterized by humoral autoimmunity against the spliceosomal proteins, including Sm B/B'. In SLE patients with anti-Sm B/B' antibodies the proline rich sequence, PPPGMRPP, is the predominant Sm B/B' autoimmune epitope and appears to be an early target in the development of the anti-Sm B/B' response. METHODS: Two female baboons were immunized with the PPPGMRPP peptide from the Sm B/B' spliceosomal protein constructed on a MAP backbone in Freund's adjuvant. One female control baboon was immunized with Freund's adjuvant alone. Baboon sera were collected and assessed for antibody binding to the spliceosomal proteins and compared to SLE patient and control sera. RESULTS: Peptide immunized baboons developed antibodies to multiple regions of the Sm B/B' protein, as well as reactivity against other spliceosomal proteins. Consistent with serologic manifestations found in SLE, experimental baboons also acquired anti-nuclear antibodies, anti-nuclear ribonucleoprotein (nRNP) antibodies and, in one animal, anti-double stranded DNA antibodies. The control animal had none of these immunologic findings. CONCLUSIONS: Immunization with PPPGMRPP is capable of initiating a humoral autoimmune response in primates against the Sm, nRNP complex from which the peptide was derived. The additional autoantibody specificities generated in experimental animals are similar to those found in human SLE sera. This study is the first evidence of peptide induction of SLE humoral autoimmunity in a primate model.

Amino Acid Sequence↗

PubMed1994

Functional association of essential splicing factor(s) with PRP19 in a protein complex.

We have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs. It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly. We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing. Taking advantage of the epitope tagging technique, we have isolated the PRP19-associated complex by affinity chromatography. The isolated complex is functional for complementation for the heat-inactivated prp19 mutant extract, and consists of at least seven polypeptides in addition to PRP19. At least three of these can interact directly with the PRP19 protein. We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins.

Base Sequence↗

PubMed1995

SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing.

Understanding the mechanism of pre-mRNA splicing requires the characterization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes containing lariat intermediate and exon 1 from free proteins present in the whole-cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second-step splicing factors. We show that SLU7 protein and a novel activity that we named SSF1 (second-step factor 1) were required in concert with PRP16 to promote progression through the second catalytic step of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.

Actins↗

PubMed1999

Identification of RNA-protein contacts within functional ribonucleoprotein complexes by RNA site-specific labeling and UV crosslinking.

A variety of cellular processes are carried out by highly complex ribonucleoprotein (RNP) particles in which multiple RNA-RNA, RNA-protein, and protein-protein interactions occur. The spliceosome, which executes the nuclear pre-mRNA splicing reaction, is a particularly striking example of a complex RNP, containing a minimum of 50 distinct protein components as well as five small nuclear RNAs. In order to identify which among the numerous proteins may play critical roles in the splicing reaction, we have assembled spliceosomal complexes on pre-mRNA containing a single 32P-labeled nucleotide, isolated the complexes by gel filtration, and then carried out UV crosslinking. The combination of these three methods has allowed the identification of proteins that crosslink to critical sequence elements during each stage in spliceosome assembly. These methods should be generally applicable to the analysis of RNP complexes assembled in vitro.

Chromatography, Affinity↗

PubMed1995

Unexpected diversity in U6 snRNA sequences from trypanosomatids.

We have isolated and sequenced the genes for the trans-spliceosomal U6 small nuclear RNAs (snRNAs) from the trypanosomatid species Leishmania mexicana (Lm) and Phytomonas sp. (Ps). Compared with the Trypanosoma brucei (Tb) U6 snRNA, the Lm U6 snRNA contains only a single additional G-C bp in the 5' terminal stem-loop. In contrast, the Ps U6 snRNA sequence contains a G-->C change at the last nucleotide of the highly conserved and functionally important ACAGAG hexanucleotide and three additional changes in conserved positions. Our results indicate that trans-spliceosomal U6 snRNAs from trypanosomatid species do not always conform to the consensus sequence of cis-spliceosomal U6 snRNAs.

Animals↗

PubMed1993

Splicing of pre-mRNA: mechanism, regulation and role in development.

Over the past year, significant progress has been made in the understanding of how RNA-binding factors may facilitate splice-site selection and spliceosome assembly, and confer fidelity to the pre-mRNA splicing reaction. In addition, a number of studies have revealed a complex network of RNA-RNA interactions in the spliceosome, strengthening the structural and functional parallels between nuclear pre-mRNA splicing and the self-splicing group I and group II introns. These new data further support the idea that pre-mRNA splicing occurs by RNA-mediated catalysis and illustrate quite dramatically the dynamic nature of conformational changes in the spliceosome cycle. With respect to tissue-specific pre-mRNA splicing, a number of studies have begun to illuminate mechanisms underlying control of splice-site selection and how so-called 'general' RNA-binding proteins, such as heterogeneous nuclear ribonucleoproteins, may be involved in determining different splicing patterns. Finally, an emerging theme involving the role of splicing in development is that differential transcriptional programs can be triggered in different cell types by alternative splicing patterns that generate transcription factor isoforms with different activities or DNA-binding specificities.

Animals↗

PubMed1997

Protein functions in pre-mRNA splicing.

Proteins have been implicated in an expanding variety of functions during pre-mRNA splicing. Molecular cloning has identified genes encoding spliceosomal proteins that potentially act as novel RNA helicases, GTPases, or protein isomerases. Novel protein-protein and protein-RNA interactions that are required for functional spliceosome formation have also been described. Finally, growing evidence suggests that proteins may contribute directly to the spliceosome's active sites.

Fungal Proteins↗