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PubMed1993

The spliceosome.

The spliceosome is a large RNA-protein complex that catalyses the removal of introns from nuclear pre-mRNA. A wide range of biochemical and genetical studies shows that the spliceosome comprises three major RNA-protein subunits, the U1, U2 and [U4/U6.U5] small nuclear ribonucleoprotein particles (snRNPs), and an additional group of non-snRNP protein splicing factors. Rapid progress is being made in unravelling the interactions which take place between these factors during the splicing reaction. The emerging picture of the spliceosome reveals a highly dynamic structure that assembles on pre-mRNA transcripts in a stepwise pathway and is organised, at least in part, by complex RNA base-pairing interactions between the small nuclear RNAs (snRNAs) and the intron substrate. Many of these interactions can be detected both in mammalian and yeast spliceosomes, suggesting that the basic splicing mechanism is an ancient one that has been highly conserved during evolution.

Base Composition↗

PubMed2001

Stoichiometry of the Sm proteins in yeast spliceosomal snRNPs supports the heptamer ring model of the core domain.

Seven Sm proteins (B/B', D1, D2, D3, E, F and G proteins) containing a common sequence motif form a globular core domain within the U1, U2, U5 and U4/U6 spliceosomal snRNPs. Based on the crystal structure of two Sm protein dimers we have previously proposed a model of the snRNP core domain consisting of a ring of seven Sm proteins. This model postulates that there is only a single copy of each Sm protein in the core domain. In order to test this model we have determined the stoichiometry of the Sm proteins in yeast spliceosomal snRNPs. We have constructed seven different yeast strains each of which produces one of the Sm proteins tagged with a calmodulin-binding peptide (CBP). Further, each of these strains was transformed with one of seven different plasmids coding for one of the seven Sm proteins tagged with protein A. When one Sm protein is expressed as a CBP-tagged protein from the chromosome and a second protein was produced with a protein A-tag from the plasmid, the protein A-tag was detected strongly in the fraction bound to calmodulin beads, demonstrating that two different tagged Sm proteins can be assembled into functional snRNPs. In contrast when the CBP and protein A-tagged forms of the same Sm protein were co-expressed, no protein A-tag was detectable in the fraction bound to calmodulin. These results indicate that there is only a single copy of each Sm protein in the spliceosomal snRNP core domain and therefore strongly support the heptamer ring model of the spliceosomal snRNP core domain.

Amino Acid Motifs↗

PubMedDate not supplied

Regular spliceosomal introns are invasive in Chlamydomonas reinhardtii: 15 introns in the recently relocated mitochondrial cox2 and cox3 genes.

In the unicellular green alga, Chlamydomonas reinhardtii, cytochrome oxidase subunit 2 (cox2) and 3 (cox3) genes are missing from the mitochondrial genome. We isolated and sequenced a BAC clone that carries the whole cox3 gene and its corresponding cDNA. Almost the entire cox2 gene and its cDNA were also determined. Comparison of the genomic and the corresponding cDNA sequences revealed that the cox3 gene contains as many as nine spliceosomal introns and that cox2 bears six introns. Putative mitochondria targeting signals were predicted at each N terminal of the cox genes. These spliceosomal introns were typical GT-AG-type introns, which are very common not only in Chlamydomonas nuclear genes but also in diverse eukaryotic taxa. We found no particular distinguishing features in the cox introns. Comparative analysis of these genes with the various mitochondrial genes showed that 8 of the 15 introns were interrupting the conserved mature protein coding segments, while the other 7 introns were located in the N-terminal target peptide regions. Phylogenetic analysis of the evolutionary position of C. reinhardtii in Chlorophyta was carried out and the existence of the cox2 and cox3 genes in the mitochondrial genome was superimposed in the tree. This analysis clearly shows that these cox genes were relocated during the evolution of Chlorophyceae. It is apparent that long before the estimated period of relocation of these mitochondrial genes, the cytosol had lost the splicing ability for group II introns. Therefore, at least eight introns located in the mature protein coding region cannot be the direct descendant of group II introns. Here, we conclude that the presence of these introns is due to the invasion of spliceosomal introns, which occurred during the evolution of Chlorophyceae. This finding provides concrete evidence supporting the "intron-late" model, which rests largely on the mobility of spliceosomal introns.

Amino Acid Sequence↗

PubMed2005

Native spliceosomes assemble with pre-mRNA to form supraspliceosomes.

Regulation of eukaryotic gene expression is achieved at different levels, which require accurate coordination. Macromolecular assemblies that exist as pre-formed entities can account for such coordination. Processing of pre-mRNA represents one step in this cascade of regulatory events but, moreover, provides explanation for protein versatility. The cellular machine where splicing of pre-mRNA, as well as additional processing events, take place in vivo is termed the supraspliceosome. Here, we show that the supraspliceosome is composed of four active spliceosomes, termed native spliceosomes, connected to each other by the pre-mRNA. Cleavage of pre-mRNA shows that its integrity is essential for the stability of the supraspliceosome. Furthermore, supraspliceosomes can be reconstituted in vitro, from purified native spliceosomes by addition of synthetic pre-mRNAs, providing further support to the supraspliceosome as a preassembled biological complex. The internal setting of the native spliceosomes within the supraspliceosome is most suitable to enable the communication between these structures, which is crucial in order to achieve regulated splicing.

HeLa Cells↗

PubMed2006

Organization of core spliceosomal components U5 snRNA loop I and U4/U6 Di-snRNP within U4/U6.U5 Tri-snRNP as revealed by electron cryomicroscopy.

In eukaryotes, pre-mRNA exons are interrupted by large noncoding introns. Alternative selection of exons and nucleotide-exact removal of introns are performed by the spliceosome, a highly dynamic macromolecular machine. U4/U6.U5 tri-snRNP is the largest and most conserved building block of the spliceosome. By 3D electron cryomicroscopy and labeling, the exon-aligning U5 snRNA loop I is localized at the center of the tetrahedrally shaped tri-snRNP reconstructed to approximately 2.1 nm resolution in vitrified ice. Independent 3D reconstructions of its subunits, U4/U6 and U5 snRNPs, show how U4/U6 and U5 combine to form tri-snRNP and, together with labeling experiments, indicate a close proximity of the spliceosomal core components U5 snRNA loop I and U4/U6 at the center of tri-snRNP. We suggest that this central tri-snRNP region may be the site to which the prespliceosomal U2 snRNA has to approach closely during formation of the catalytic core of the spliceosome.

Base Sequence↗

PubMed1997

Pre-mRNA splicing: the discovery of a new spliceosome doubles the challenge.

A rare class of pre-mRNA introns with non-canonical consensus sequences has been identified in metazoan genes. The novel, low-abundance spliceosome that excises these introns contains one small nuclear ribonucleoprotein (snRNP) in common with the major spliceosome (U5) and four snRNPs that are distinct from, but structurally and functionally analogous to U1, U2 and U4-U6. The architecture of RNA components at the presumptive core of the AT-AC splicesome supports current models of the spliceosomal active center and raises tantalizing questions about spliceosome evolution.

Animals↗

PubMed2002

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.

We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.

Adenosine Triphosphatases↗

PubMed2002

The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly.

In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing. We have further defined the roles of key features of yeast U6 snRNA in the splicing process. By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia. These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing. These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5' splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie [Cell, 1992, 71: 803-817]. In the 3' intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP. Base pairing of the U6 3' stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3' stem-loop structure.

Base Sequence↗

PubMed2006

An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition.

Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosome's dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4-U6-U5 tri-snRNP-containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway.

Adenosine Triphosphate↗

PubMed2002

Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly.

We have investigated the role played in precursor mRNA (pre-mRNA) splicing by the protein pair of molecular size 72/74 kDa, which are integral components of a discrete subset of heterogeneous nuclear (hn) ribonucleoproteins (RNPs) named large heterogeneous nuclear RNP (LH-nRNP). This 72/74 kDa pair of proteins has been shown to belong to the hnRNP M group, and are referred to as 72/74(M). By applying specific immunoprecipitation assays in a consecutive series of splicing reactions in vitro, the antigenic 72/74(M) protein species were found to associate with the pre-mRNA and not the intermediate or final products of splicing. Kinetic studies, combined with isolation of pre-spliceosomal and spliceosomal complexes from the splicing reaction, indicated a loose association of 72/74(M) with both the initially formed H assembly and the first splicing-committed E complex. Stable binding was seen at a later stage of the reaction, well in advance of the appearance of the first intermediate products of RNA splicing. Evidence is provided that supports the almost exclusive association of 72/74(M) with pre-mRNA within the pre-spliceosomal A complex. This dynamic binding appeared to involve pre-mRNA sites similar to those of spliceosomal U1 and U2 small nuclear RNP complexes. Moreover, a preferential binding to a truncated RNA containing the 5' exon-intron part, rather than the intron-3' exon part, of pre-mRNA was observed.

Electrophoresis, Polyacrylamide Gel↗

PubMed2002

A spliceosomal intron in Giardia lamblia.

Short introns occur in numerous protist lineages, but there are no reports of intervening sequences in the protists Giardia lamblia and Trichomonas vaginalis, which may represent the deepest known branches in the eukaryotic line of descent. We have discovered a 35-bp spliceosomal intron in a gene encoding a putative [2Fe-2S] ferredoxin of G. lamblia. The Giardia intron contains a canonical splice site at its 3' end (AG), a noncanonical splice site at its 5' end (CT), and a branch point sequence that fits the yeast consensus sequence of TACTAAC except for the first nucleotide (AACTAAC). We have also identified several G. lamblia genes with spliceosomal peptides, including homologues of eukaryote-specific spliceosomal peptides (Prp8 and Prp11), several DExH-box RNA-helicases that have homologues in eubacteria, but serve essential functions in the splicing of introns in eukaryotes, and 11 predicted archaebacteria-like Sm and like-Sm core peptides, which coat small nuclear RNAs. Phylogenetic analyses show the Giardia Sm core peptides are the products of multiple, ancestral gene duplications followed by divergence, but they retain strong similarity to Sm and like-Sm peptides of other eukaryotes. Although we have documented only a single intron in Giardia, it likely has other introns and fully functional, spliceosomal machinery. If introns were added during eukaryotic evolution (the introns-late hypothesis), then these results push back the date of this event before the branching of G. lamblia.

Amino Acid Sequence↗

PubMed1995

Purification of the spliceosome A-complex and its visualization by electron microscopy.

Pre-mRNA splicing occurs on spliceosomes, a family of ribonucleoprotein particles. Spliceosome assembly on exogenous adenovirus pre-mRNA was blocked at the A-complex (or pre-spliceosome) stage, either by destruction of the small nuclear ribonucleoproteins (snRNPs) that comprise the U4/U5/U6 tri-snRNP complex, or by interference in tri-snRNP assembly and interactions. The A-complex was isolated by size exclusion chromatography; homogeneity was shown by electrophoresis in nondenaturing polyacrylamide gels, gradient sedimentation, and electron microscopy. Northern hybridization showed U1 and U2 snRNAs to be present in the preparation, but not U4, U5, or U6. Antibodies specific for a component of the U1 snRNP or for a component that is common to all snRNPs (except U6) each precipitated an A-complex containing pre-mRNA, U1 and U2 snRNPs. Electron micrographs showed 230 x 270-A particles whose two components appear similar to individual U1 and U2 snRNPs. Electron micrographs of an A-complex-5'-biotinyl oligonucleotide-streptavidin-gold composite allowed identification of the U2 snRNP within the structure and the localization of the 5'-segment of U2 snRNA at a unique site in the A-complex. This region of U2 RNA is adjacent to the developing catalytic center of the spliceosome.

Base Sequence↗

PubMed1997

Dynamics of the U1 small nuclear ribonucleoprotein during yeast spliceosome assembly.

U1 small nuclear ribonucleoprotein (snRNP) may function during several steps of spliceosome assembly. Most spliceosome assembly assays, however, fail to detect the U1 snRNP. Here, I used a new native gel electrophoretic assay to find the yeast U1 snRNP in three pre-splicing complexes (delta, beta1, alpha2) formed in vitro. The order of complex formation is deduced to be delta --> beta1 --> alpha2 --> alpha1 --> beta2, the active spliceosome. The delta complex is formed when U1 snRNP binds to pre-mRNA in the absence of ATP. There are two forms of delta: a major one, deltaun, unstable to competitor RNA; and a minor one, deltacommit, committed to the splicing pathway. The other complexes are formed in the presence of ATP and contain the following snRNPs: beta1, the pre-spliceosome, has both U1 and U2; alpha2 has all five, however, U1 is reduced compared with the others; and alpha1 and beta2 have U2, U5, and U6. Prior work by others suggests that U1 is "handing off" the 5' splice site region to the U5 and U6 snRNPs before splicing begins. The reduced levels of U1 snRNP in the alpha2 complex suggests that the handoff occurs during formation of this complex.

Adenosine Triphosphate↗

PubMed2002

The protein phosphatase-1 regulator NIPP1 is also a splicing factor involved in a late step of spliceosome assembly.

NIPP1 is a ubiquitous regulator of protein phosphatase-1 (PP1) and is targeted to the splicing factor storage sites (speckles) in the nucleus by its forkhead-associated domain. We show here that NIPP1 is also a component of the spliceosomes in HeLa cell-splicing extracts and that the interaction with the spliceosomes requires a functional forkhead-associated domain. The in vitro splicing of beta-globin pre-mRNA was not affected by exogenous wild type NIPP1 but was blocked by mutants that lacked residues 225-329. The inhibition by these dominant negative mutants resulted from a block in a late phase of spliceosome assembly, i.e. at the transition between the B-complex and the C-complex. Site-directed mutagenesis furthermore showed that this spliceosomal function of NIPP1 was unrelated to its ability to bind PP1 or RNA. Our data suggest that NIPP1 can function independently as a splicing factor and a phosphatase regulator.

Alternative Splicing↗

PubMed1998

The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.

Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase. Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity. Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome.

Adenosine Triphosphatases↗

PubMed1994

Genetic interactions between the yeast RNA helicase homolog Prp16 and spliceosomal snRNAs identify candidate ligands for the Prp16 RNA-dependent ATPase.

Pre-mRNA splicing occurs in a large and dynamic ribonucleoprotein complex, the spliceosome. Several protein factors involved in splicing are homologous to a family of RNA-dependent ATPases, the so-called DEAD/DEAH proteins. A subset of these factors exhibit RNA helicase activity in vitro. The DEAD/DEAH proteins involved in splicing are thought to mediate RNA conformational rearrangements during spliceosome assembly. However, the RNA ligands for these factors are currently unknown. Here, we present genetic evidence in Saccharomyces cerevisiae for a functional interaction between the DEAH protein Prp16, and the U6 and U2 spliceosomal snRNAs. Using a library of mutagenized U6 snRNA genes, we have identified 14 strong suppressors of the cold-sensitive (cs) allele, prp16-302. Remarkably, each suppressor contains a single nucleotide deletion of 1 of the 6 residues that lie immediately upstream of a sequence in U6 that interacts with the 5' splice site. Analysis of site-directed mutations revealed that nucleotide substitutions in the adjacent U2-U6 helix I structure also suppress prp16-302, albeit more weakly. The U6 suppressors tested also partially reverse the phenotype of two other cs alleles, prp16-1 and prp16-301, but not the four temperature-sensitive alleles tested. Finally, overexpression of each cs allele exacerbates its recessive growth phenotype and confers a dominant negative cs phenotype. We propose that the snRNA suppressors function by destabilizing an interaction between the U2-U6 complex and a hypothetical factor (X), which is trapped by cs mutants of PRP16. The phenotypes of overexpressed prp16 alleles are consistent with the model that this trapped interaction inhibits the dissociation of Prp16 from the spliceosome. We discuss the intriguing possibility that factor X is Prp16 itself.

Adenosine Triphosphatases↗

PubMed2001

Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae.

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.

Adenosine Triphosphatases↗

PubMed2005

Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features.

We have investigated electrostatic and surface features of an essential region of the catalytic core of the spliceosome, the eukaryotic precursor messenger (pre-m)RNA splicing apparatus. The nucleophile for the first of two splicing reactions is the 2'-hydroxyl (OH) of the ribose of a specific adenosine within the intron. During assembly of the spliceosome's catalytic core, this adenosine is positioned by pairing with a short region of the U2 small nuclear (sn)RNA to form the pre-mRNA branch site helix. The solution structure of the spliceosomal pre-mRNA branch site [Newby,M.I. and Greenbaum,N.L. (2002) Nature Struct. Biol., 9, 958-965] showed that a phylogenetically conserved pseudouridine (psi) residue in the segment of U2 snRNA that pairs with the intron induces a markedly different structure compared with that of its unmodified counterpart. In order to achieve a more detailed understanding of the factors that contribute to recognition of the spliceosome's branch site helix and activation of the nucleophile for the first step of pre-mRNA splicing, we have calculated surface areas and electrostatic potentials of psi-modified and unmodified branch site duplexes. There was no significant difference between the total accessible area or ratio of total polar:nonpolar groups between modified and unmodified duplexes. However, there was substantially greater exposure of nonpolar area of the adenine base, and less exposure of the 2'-OH, in the psi-modified structure. Electrostatic potentials computed using a hybrid boundary element and finite difference nonlinear Poisson-Boltzmann approach [Boschitsch, A.H. and Fenley, M.O. (2004) J. Comput. Chem., 25, 935-955] revealed a region of exceptionally negative potential in the major groove surrounding the 2'-OH of the branch site adenosine. These surface and electrostatic features may contribute to the overall recognition of the pre-mRNA branch site region by other components of the splicing reaction.

Adenosine↗