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Results for “Spliceosomes”

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PubMed1996

The nuclear matrix protein p255 is a highly phosphorylated form of RNA polymerase II largest subunit which associates with spliceosomes.

The monoclonal antibody CC-3 recognizes a phosphodependent epitope on a 255 kDa nuclear matrix protein (p255) recently shown to associate with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle [Chabot et al. (1995) Nucleic Acids Res. 23, 3206-3213]. In mouse and Drosophila cultured cells the electrophoretic mobility of p255, faster in the latter species, was identical to that of the hyperphosphorylated form of RNA polymerase II largest subunit (IIo). The CC-3 immunoreactivity of p255 was abolished by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which is known to cause the dephosphorylation of the C-terminal domain of subunit IIo by inhibiting the TFIIH-associated kinase. The identity of p255 was confirmed by showing that CC-3-immunoprecipitated p255 was recognized by POL3/3 and 8WG16, two antibodies specific to RNA polymerase II largest subunit. Lastly, the recovery of RNA polymerase II largest subunit from HeLa splicing mixtures was compromised by EDTA, which prevents the interaction of p255 with splicing complexes and inhibits splicing. Our results indicate that p255 represents a highly phosphorylated form of RNA polymerase II largest subunit physically associated with spliceosomes and possibly involved in coupling transcription to RNA processing.

3T3 Cells↗

PubMed1998

At least three linear regions but not the zinc-finger domain of U1C protein are exposed at the surface of the protein in solution and on the human spliceosomal U1 snRNP particle.

No structural information on U1C protein either in its free state or bound to the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) particle is currently available. Using rabbit antibodies raised against a complete set of 15 U1C overlapping synthetic peptides (16-30 residues long) in different immunochemical tests, linear regions exposed at the surface of free and U1 snRNP-bound U1C were identified. Epitopes within at least three regions spanning residues 31-62, 85-103 and 116-159 were recognized on free and plastic-immobilized recombinant human U1C expressed in Escherichia coli, on in vitro translated U1C protein and on U1C bound to the U1 snRNP particle present in HeLa S100 extract. Using a zinc affinity labeling method, we further showed that the N-terminal U1C peptide containing a zinc-finger motif (peptide 5-34) effectively binds65Zn2+. The N-terminal region of U1C, which is functional in U1 snRNP assembly, is apparently not located at the surface of the U1 snRNP particle.

Amino Acid Sequence↗

PubMed2003

Two protein-protein interaction sites on the spliceosome-associated human cyclophilin CypH.

Cyclophilins are a family of proteins that share a common, highly conserved sequence motif. Cyclophilins bind transiently to other proteins and facilitate their folding. One member of the family, hCypH, is part of the human spliceosomal [U4/U6.U5] tri-snRNP complex; it associates specifically and stably with the U4/U6-specific protein 60K. Here, we demonstrate that recombinant hCypH exhibits peptidyl-prolyl isomerase (PPIase) activity, and describe mutagenesis studies demonstrating that it shares the catalytic pocket with other members of the cyclophilin family. However, neither the PPIase activity nor the catalytic pocket is required for binding of protein 60K. Rather, hCypH contains a small insertion in a loop of the otherwise conserved cyclophilin backbone, and this minor change creates a highly specific binding site that is responsible for the association of this cyclophilin, but not others, with protein 60K. hCypH is thus the first small cyclophilin shown to have a second protein-protein interaction site and the ability to bind stably to another protein. Since the catalytic pocket and the second binding site are located on opposite sides of the cyclophilin structure, this opens up the interesting possibility that hCypH may serve as a bridge mediating interactions between protein 60K of the U4/U6 snRNP and other as yet unknown factors.

Binding Sites↗

PubMed2006

U12DB: a database of orthologous U12-type spliceosomal introns.

U12-type introns are spliced by the U12-dependent spliceosome and are present in the genomes of many higher eukaryotic lineages including plants, chordates and some invertebrates. However, due to their relatively recent discovery and a systematic bias against recognition of non-canonical splice sites in general, the introns defined by U12-type splice sites are under-represented in genome annotations. Such under-representation compounds the already difficult problem of determining gene structures. It also impedes attempts to study these introns genome-wide or phylum-wide. The resource described here, the U12 Intron Database (U12DB), aims to catalog the U12-type introns of completely sequenced eukaryotic genomes in a framework that groups orthologous introns with each other. This will aid further investigations into the evolution and mechanism of U12-dependent splicing as well as assist ongoing genome annotation efforts. Public access to the U12DB is available at http://genome.imim.es/cgi-bin/u12db/u12db.cgi.

Animals↗

PubMed2005

Targeted 'knockdown' of spliceosome function in mammalian cells.

The existence of two sophisticated parallel splicing machineries in multicellular organisms has raised intriguing questions--ranging from their impact on proteome expansion to the evolution of splicing and of metazoan genomes. Exploring roles for the distinct splicing systems in vivo has, however, been restricted by the lack of techniques to selectively inhibit their function in cells. In this study, we show that morpholino oligomers complementary to the branch-site recognition elements of U2 or U12 small nuclear RNA specifically suppress the function of the two splicing systems in mammalian cells. The data provide the first evidence for a role of distinct spliceosomes in pre-mRNA splicing from endogenous mammalian genes and establish a tool to define roles for the different splicing machineries in vivo.

Animals↗

PubMed1996

Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A.

A critical step in the pre-mRNA splicing reaction is the stable binding of U2 snRNP to the branchpoint sequence (BPS) to form the A complex. The multimeric U2 snRNP protein complexes SF3a and SF3b are required for A complex assembly, but their specific roles in this process are not known. Saccharomyces cerevisiae homologs of all of the SF3a, but none of the SF3b, subunits have been identified. Here we report the isolation of a cDNA encoding the mammalian SF3b subunit SAP 145 and the identification of its probable yeast homolog (29% identity). This first indication that the homology between yeast and metazoan A complex proteins can be extended to SF3b adds strong new evidence that the mechanism of A complex assembly is highly conserved. To investigate this mechanism in the mammalian system we analyzed proteins that cross-link to 32P-site-specifically labeled pre-mRNA in the A complex. This analysis revealed that SAP 145, together with four other SF3a/SF3b subunits, UV cross-links to pre-mRNA in a 20-nucleotide region upstream of the BPS. Mutation of this region, which we have designated the anchoring site, has no apparent effect on U2 snRNP binding. In contrast, when a 2'O methyl oligonucleotide complementary to the anchoring site is added to the spliceosome assembly reaction, A complex assembly and cross-linking of the SF3a/SF3b subunits are blocked. These data indicate that sequence-independent binding of the highly conserved SF3a/SF3b subunits upstream of the branch site is essential for anchoring U2 snRNP to pre-mRNA.

Amino Acid Sequence↗

PubMed1999

Overproduction, purification, crystallization and preliminary x-ray diffraction studies of the human spliceosomal protein U5-15kD.

The gene coding for the human spliceosomal U5 snRNP-specific 15 kDa protein (U5-15kD) was overexpressed in Escherichia coli, its product purified to homogeneity and crystallized. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops and subsequent macroseeding. The crystals belong to the orthorhombic space group P21212 with a = 62.3, b = 65.7, c = 37.1 A. They diffract to at least 3.0 A and contain one molecule in the asymmetric unit. A selenomethionine derivative of the protein was prepared and crystallized for multiwavelength anomalous diffraction (MAD) data collection.

Cloning, Molecular↗

PubMed2005

Overproduction, purification, crystallization and preliminary X-ray diffraction studies of the human spliceosomal protein TXNL4B.

The human gene coding for the spliceosomal protein thioredoxin-like 4B (TXNL4B) was overexpressed in Escherichia coli and the encoded protein was purified and crystallized. Well diffracting single crystals were obtained by the vapor-diffusion method in hanging drops. The crystals belong to the primitive monoclinic space group P2, with unit-cell parameters a = 39.0, b = 63.6, c = 51.0 A, beta = 92.484 degrees,, and diffract to at least 1.50 A. A SeMet derivative of the protein was prepared and crystallized for MAD phasing.

Cell Cycle Proteins↗

PubMed2004

Variants of U1 small nuclear RNA assemble into spliceosomal complexes.

In this study, the existence of additional U1 snRNA variants in the posterior silk gland of the Bombyx mori Nistari strain from India was investigated. Three new U1 variants were detected. One of the new isoforms (U1 SG1) was found to be preferentially assembled into high molecular weight spliceosomal complexes in comparison with the total cellular lysate RNA control. Structural and nucleotide differences were examined in these new isoforms and compared with the previously reported U1 variants. Free energy (Delta G) values for the entire U1 snRNA secondary structures as well as the individual stem/loops (I, II, III and IV) domains of the isoforms were estimated to determine their structural stability.

Animals↗

PubMed1998

A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles.

The spliceosome removes introns from pre-messenger RNAs by a mechanism that entails extensive remodeling of RNA structure. The most conspicuous rearrangement involves disruption of 24 base pairs between U4 and U6 small nuclear RNAs (snRNAs). Here, the yeast RNA binding protein Prp24 is shown to reanneal these snRNAs. When Prp24 is absent, unpaired U4 and U6 small nuclear ribonucleoprotein particles (snRNPs) accumulate; with time, splicing becomes inhibited. Addition of purified Prp24 protein regenerates duplex U4/U6 snRNPs for new rounds of splicing. The reannealing reaction catalyzed by Prp24 proceeds more efficiently with snRNPs than with deproteinized snRNAs.

Adenosine Triphosphate↗

PubMed2001

Herpes simplex virus IE63 (ICP27) protein interacts with spliceosome-associated protein 145 and inhibits splicing prior to the first catalytic step.

The multifunctional herpes simplex virus type 1 (HSV-1) protein IE63 (ICP27) interacts with the essential pre-mRNA splicing factor, spliceosome-associated protein 145 (SAP145), and in infected cells IE63 and SAP145 colocalize. This interaction was reduced or abrogated completely using extracts from cells infected with IE63 viral mutants, with mutations in IE63 KH and Sm homology domains, which do not exhibit host shutoff or inhibit splicing. In the presence of IE63, splicing in vitro was inhibited prior to the first catalytic step and the B/C complex formed during splicing was shifted up in mobility and reduced in intensity. With the use of splicing extracts, IE63 and SAP145 both comigrated with the B/C complex, suggesting that they interact within this complex to inhibit B/C complex formation or conversion. The inhibition of splicing may facilitate the export of viral or cellular transcripts, possibly via other protein partners of IE63. These data provide important new insights into how IE63 influences pre-mRNA processing during HSV-1 infection.

Binding Sites↗

PubMed1993

Mammalian U6 small nuclear RNA undergoes 3' end modifications within the spliceosome.

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.

Base Composition↗

PubMed1995

Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations.

Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.

Base Sequence↗

PubMed1997

A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract.

The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes. We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex, here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate is a minimal RNA oligonucleotide, containing only a branch sequence and polypyrimidine tract. Interactions at the branch site adenosine and requirements for polypyrimidine tract-binding proteins for the Amin complex are the same as those of authentic prespliceosome complex A. Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snRNP per se, but rather are necessary for accessibility of components on longer RNA substrates. Furthermore, there is an ATP-dependent activity that releases or destabilizes U2 snRNP from branch sequences. The simplicity of the Amin complex will facilitate a detailed understanding of the assembly of prespliceosomes.

Adenosine Triphosphate↗

PubMed1997

Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae.

The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae. Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions. Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex. Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs. These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.

Fungal Proteins↗

PubMed2006

A high density of ancient spliceosomal introns in oxymonad excavates.

BACKGROUND: Certain eukaryotic genomes, such as those of the amitochondriate parasites Giardia and Trichomonas, have very low intron densities, so low that canonical spliceosomal introns have only recently been discovered through genome sequencing. These organisms were formerly thought to be ancient eukaryotes that diverged before introns originated, or at least became common. Now however, they are thought to be members of a supergroup known as excavates, whose members generally appear to have low densities of canonical introns. Here we have used environmental expressed sequence tag (EST) sequencing to identify 17 genes from the uncultivable oxymonad Streblomastix strix, to survey intron densities in this most poorly studied excavate group. RESULTS: We find that Streblomastix genes contain an unexpectedly high intron density of about 1.1 introns per gene. Moreover, over 50% of these are at positions shared between a broad spectrum of eukaryotes, suggesting they are very ancient introns, potentially present in the last common ancestor of eukaryotes. CONCLUSION: The Streblomastix data show that the genome of the ancestor of excavates likely contained many introns and the subsequent evolution of introns has proceeded very differently in different excavate lineages: in Streblomastix there has been much stasis while in Trichomonas and Giardia most introns have been lost.

Amino Acid Sequence↗

PubMed2007

Arginine methyltransferase Capsuleen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila.

Although arginine modification has been implicated in a number of cellular processes, the in vivo requirement of protein arginine methyltransferases (PRMTs) in specific biological processes remain to be clarified. In this study we characterize the Drosophila PRMT Capsuléen, homologous to human PRMT5. During Drosophila oogenesis, catalytic activity of Capsuléen is necessary for both the assembly of the nuage surrounding nurse cell nuclei and the formation of the pole plasm at the posterior end of the oocyte. In particular, we show that the nuage and pole plasm localization of Tudor, an essential component for germ cell formation, are abolished in csul mutant germ cells. We identify the spliceosomal Sm proteins as in vivo substrates of Capsuléen and demonstrate that Capsuléen, together with its associated protein Valois, is essential for the synthesis of symmetric di-methylated arginyl residues in Sm proteins. Finally, we show that Tudor can be targeted to the nuage in the absence of Sm methylation by Capsuléen, indicating that Tudor localization and Sm methylation are separate processes. Our results thus reveal the role of a PRMT in protein localization in germ cells.

Amino Acid Sequence↗

PubMed1995

The organization of spliceosomal components in the nuclei of higher plants.

To analyze the organization of spliceosomal snRNPs in plant nuclei, we have used both immunofluorescence labelling with the antibody 4G3, raised against the human snRNP-specific protein U2B", and in situ hybridization with anti-sense probes to conserved regions of U1, U2 and U6 snRNAs. The organization comprises a fibrous interchromatin network, which may include both interchromatin fibrils and granules, and very prominent nuclear and nucleolar-associated bodies. Double labelling with an anti-p80 coilin antibody shows that these are coiled bodies. Dynamic changes in the labelling pattern were observed through the cell cycle, and in response to and on recovery from heat shock. The similarity of this organization to that observed in mammalian nuclei is strong evidence that it is fundamental to the processing of pre-mRNA in eucaryotes in general.

Base Sequence↗