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PubMed2005

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway.

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.

Adenosine Triphosphate↗

PubMed2006

U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes.

The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5' end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5' end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.

Cross-Linking Reagents↗

PubMed2006

Phase distribution of spliceosomal introns: implications for intron origin.

BACKGROUND: The origin of spliceosomal introns is the central subject of the introns-early versus introns-late debate. The distribution of intron phases is non-uniform, with an excess of phase-0 introns. Introns-early explains this by speculating that a fraction of present-day introns were present between minigenes in the progenote and therefore must lie in phase-0. In contrast, introns-late predicts that the nonuniformity of intron phase distribution reflects the nonrandomness of intron insertions. RESULTS: In this paper, we tested the two theories using analyses of intron phase distribution. We inferred the evolution of intron phase distribution from a dataset of 684 gene orthologs from seven eukaryotes using a maximum likelihood method. We also tested whether the observed intron phase distributions from 10 eukaryotes can be explained by intron insertions on a genome-wide scale. In contrast to the prediction of introns-early, the inferred evolution of intron phase distribution showed that the proportion of phase-0 introns increased over evolution. Consistent with introns-late, the observed intron phase distributions matched those predicted by an intron insertion model quite well. CONCLUSION: Our results strongly support the introns-late hypothesis of the origin of spliceosomal introns.

Animals↗

PubMed2005

Pseudouridine modification in Caenorhabditis elegans spliceosomal snRNAs: unique modifications are found in regions involved in snRNA-snRNA interactions.

BACKGROUND: Pseudouridine (Psi) is an abundant modified nucleoside in RNA and a number of studies have shown that the presence of Psi affects RNA structure and function. The positions of Psi in spliceosomal small nuclear RNAs (snRNAs) have been determined for a number of species but not for the snRNAs from Caenorhabditis elegans (C. elegans), a popular experimental model system of development. RESULTS: As a prelude to determining the function of or requirement for this modification in snRNAs, we have mapped the positions of Psi in U1, U2, U4, U5, and U6 snRNAs from worms using a specific primer extension method. As with other species, C. elegans U2 snRNA has the greatest number of Psi residues, with nine, located in the 5' half of the U2 snRNA. U5 snRNA has three Psis, in or near the loop of the large stem-loop that dominates the structure of this RNA. U6 and U1 snRNAs each have one Psi, and two Psi residues were found in U4 snRNA. CONCLUSION: The total number of Psis found in the snRNAs of C. elegans is significantly higher than the minimal amount found in yeasts but it is lower than that seen in sequenced vertebrate snRNAs. When the actual sites of modification on C. elegans snRNAs are compared with other sequenced snRNAs most of the positions correspond to modifications found in other species. However, two of the positions modified on C. elegans snRNAs are unique, one at position 28 on U2 snRNA and one at position 62 on U4 snRNA. Both of these modifications are in regions of these snRNAs that interact with U6 snRNA either in the spliceosome or in the U4/U6 small nuclear ribonucleoprotein particle (snRNP) and the presence of Psi may be involved in strengthening the intermolecular association of the snRNAs.

Animals↗

PubMed1995

Colocalization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes.

It was previously demonstrated that monoclonal antibody CC-3 binds to a phosphorylation dependent epitope present on a 255 kDa nuclear protein (p255). We show here that in interphase cells, p255 distributes to typical nuclear speckles that correspond to the localization of spliceosome components as revealed by antibodies to the m3G cap of snRNAs or to the non-snRNP splicing factor SC-35. Immunofluorescence and immunoblot studies indicated that p255 is resistant to extraction with non-ionic detergents, nucleases and high ionic strength buffers and may thus be defined biochemically as a nuclear matrix phosphoprotein. To determine the nature of the association of p255 with the nuclear structure, its distribution was studied at different stages of the cell cycle and after the cells were treated with nucleases or heat shocked. We found that the antigen diffused into the cytoplasm during metaphase but was reorganized into cytoplasmic speckles during anaphase-telophase transition, where it colocalized with SC-35. Nuclear matrix preparations that were digested with DNases and RNases showed that interphasic p255 still localized to nuclear speckles even though snRNA and snRNP antigens were removed. Heat-shocked cells labelled with monoclonal antibody CC-3 exhibited more rounded and less interconnected speckles, identical to those decorated by anti-SC-35 antibody under such conditions. These results indicate that p255 and SC-35 are present in the same nuclear structures, to which they are more tightly bound than the snRNP antigens. They further suggest that both proteins are implicated in spliceosome assembly or attachment.

3T3 Cells↗

PubMed2004

Modified nucleotides at the 5' end of human U2 snRNA are required for spliceosomal E-complex formation.

U2 snRNA, a key player in nuclear pre-mRNA splicing, contains a 5'-terminal m3G cap and many internal modifications. The latter were shown in vertebrates to be generally required for U2 function in splicing, but precisely which residues are essential and their role in snRNP and/or spliceosome assembly is presently not clear. Here, we investigated the roles of individual modified nucleotides of HeLa U2 snRNA in pre-mRNA splicing, using a two-step in vitro reconstitution/complementation assay. We show that the three pseudouridines and five 2'O-methyl groups within the first 20 nucleotides of U2 snRNA, but not the m3G cap, are required for efficient pre-mRNA splicing. Individual pseudouridines were not essential, but had cumulative effects on U2 function. In contrast, four of five 2'O-methylations (at positions 1, 2, 12, and 19) were individually required for splicing. The in vitro assembly of 17S U2 snRNPs was not dependent on the presence of modified U2 residues. However, individual internal modifications were required for the formation of the ATP-independent early spliceosomal E complex. Our data strongly suggest that modifications within the first 20 nucleotides of U2 play an important role in facilitating the interaction of U2 with U1 snRNP and/or other factors within the E complex.

Base Sequence↗

PubMedDate not supplied

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs.

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.

Cell Nucleolus↗

PubMed2005

Genetic analysis reveals a role for the C terminus of the Saccharomyces cerevisiae GTPase Snu114 during spliceosome activation.

Snu114 is the only GTPase required for mRNA splicing. As a homolog of elongation factor G, it contains three domains (III-V) predicted to undergo a large rearrangement following GTP hydrolysis. To assess the functional importance of the domains of Snu114, we used random mutagenesis to create conditionally lethal alleles. We identified three main classes: (1) mutations that are predicted to affect GTP binding and hydrolysis, (2) mutations that are clustered in 10- to 20-amino-acid stretches in each of domains III-V, and (3) mutations that result in deletion of up to 70 amino acids from the C terminus. Representative mutations from each of these classes blocked the first step of splicing in vivo and in vitro. The growth defects caused by most alleles were synthetically exacerbated by mutations in PRP8, a U5 snRNP protein that physically interacts with Snu114, as well as in genes involved in snRNP biogenesis, including SAD1 and BRR1. The allele snu114-60, which truncates the C terminus, was synthetically lethal with factors required for activation of the spliceosome, including the DExD/H-box ATPases BRR2 and PRP28. We propose that GTP hydrolysis results in a rearrangement between Prp8 and the C terminus of Snu114 that leads to release of U1 and U4, thus activating the spliceosome for catalysis.

Alleles↗

PubMed1999

Monoclonal antibody specific to a subclass of polyproline-Arg motif provides evidence for the presence of an snRNA-free spliceosomal Sm protein complex in vivo: implications for molecular interactions involving proline-rich sequences of Sm B/B' proteins.

The human spliceosomal Sm B/B' proteins are essential for the biogenesis of the snRNP particles. B/B' proteins contain several clusters of the PPPPGM/IR sequence, which occurs within the C-terminus of Sm B/B'. This sequence is very similar to the PPPPPGHR sequence of the cytoplasmic tail of the CD2 receptor and closely resembles the class II of SH3 ligands, suggesting a similarly important role. We report that a monoclonal antibody (3E10) against the PPPPPGHR sequence recognizes spliceosomal Sm B/B' proteins. Proteins that are specifically immunoprecipitated by 3E10 include Sm B, B', D1, D2, D3, E, F, and G. However, unlike Y12 and other anti-Sm immunoprecipitates, 3E10 immunoprecipitates appear to lack the U1 snRNP-specific proteins A and C and U snRNAs. These findings indicate that 3E10 recognizes a subset of Sm protein core and suggest the presence of snRNA-free Sm protein complex(es) in vivo. We propose that the epitope binding for 3E10 may become unaccessible upon interactions of Sm proteins and their subsequent incorporation into the core particles. The Sm proline-rich sequences may have an important role in mediating protein-protein interactions necessary for the proper snRNP core assembly or function, or both. To our knowledge, 3E10 is the first well characterized mAb specific for a subclass of polyproline-arg motif recognizing Sm B/B' and CD2 proteins. 3E10 antibody can be used to further characterize the nature of protein components in the snRNA-free Sm subcore protein complex(es) that are formed during the snRNP core assembly steps.

Amino Acid Sequence↗

PubMedDate not supplied

Induction of anti-RA33 hnRNP autoantibodies and transient spread to U1-A snRNP complex of spliceosome by idiotypic manipulation with anti-RA33 antibody preparation in mice.

OBJECTIVE: Anti-RA33 antibodies occur in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD) and target the A2/B1 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex 4 which forms part of the spliceosome. The aim of the present study was to evaluate the immune response and pathological features induced in mice immunized with anti-RA33 antibodies or patient-derived recombinant single-chain variable fragments (scFv) of anti-RA33 antibodies. METHODS: In the first set of the experiment, two strains of mice (C57BL/6J and BALB/c) were immunized with IgG preparations obtained from two patients with RA and one normal donor. One of the patients had high titer anti-RA33 antibodies; the other one showed weak borderline reactivity. In the second set of the experiment three groups of C57BL/6J mice were immunized, respectively, with affinity-purified (AP) anti-RA33 antibodies, scFv of anti-RA33 antibodies and normal human IgG. The immunological response induced in immunized mice was studied by immunoblotting and line immunoassay (LIA). The presence of arthritis, serositis or myositis was assessed six-months following initial immunization. RESULTS: While anti-RA33 antibodies developed in only two of the mice immunized with different human IgG fractions, anti-RA33 antibodies were clearly detected in 7 sera of 13 mice immunized with AP anti-RA33 antibodies three months after the boost immunization and, moreover, also in 2 sera of 13 mice immunized with scFv of anti-RA33 antibodies. In contrast, mice immunized with normal human IgG did not develop anti-RA33 antibodies. Interestingly, transient autoantibody production against another nuclear autoantigen, U1 snRNP, was observed in 3 C57BL/6J mice immunized with scFv and in 1 mouse immunized with AP autoantibodies. However, these immunological responses were not associated with pathological findings. CONCLUSIONS: Active immunization of naive mice with AP anti-RA33 antibodies and scFv of anti-RA33 antibodies resulted on the one hand in the production of murine anti-RA33 antibodies and led, on the other hand, to transient "autoantibody spread" to snRNP component of the spliceosome and other nuclear autoantigens. This "autoantibody spread" probably reflected disregulation of the idiotypic anti-idiotypic cascade.

Animals↗

PubMed1995

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.

Base Sequence↗

PubMed1996

The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome.

Specific recognition of the 5' splice site (5'SS) by the spliceosome components was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP particles into spliceosome-like complexes and actively participates in a trans-splicing reaction. Short-range cross-liking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron. The GU:p220 interaction can be detected in the functional splicing complex B. Although p220 has been known to contact several nucleotides around the 5' splice junction, the p220:GU dinucleotide interaction described here is remarkably specific. Consistent with the high conservation of the GU, even minor modifications of this element affect recognition of the 5'SS RNA by p220. Substitution of uridine at the GU with base analogues containing a large methyl or iodo group, but not a smaller flouro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional participation in splicing.

Base Sequence↗

PubMed1996

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.

Animals↗

PubMed1996

Intramolecular structure in yeast introns aids the early steps of in vitro spliceosome assembly.

rp5l B pre-mRNA, like many Saccharomyces cerevisiae primary transcripts, contains a secondary structure within its intron sequence. The structure is required for optimal in vivo splicing efficiency and includes two complementary regions near the 5' splice site and the branchpoint (UB1 and DB1, respectively). An intron-containing RNA was probed in vitro with RNase T1 and dimethyl sulfate (DMS), and is folded as expected. We have also examined in vitro splicing of rp5l B pre-mRNA, by analyzing the formation of splicing complexes as well as splicing products. Similar analyses were done with mutant pre-mRNAs containing shortened or lengthened UB1/DB1 base pairing regions. Our experiments indicate that the secondary structure acts at an early step of spliceosome assembly to aid the formation of U1 snRNP-containing commitment complexes. pre-mRNAs were probed with DMS in vivo and the folding takes place inside cells. The effect of the different UB1/DB1 interactions on in vivo splicing efficiency was also analyzed. The results are consistent with the idea that the intramolecular interaction takes place prior to or at the beginning of spliceosome assembly.

Base Sequence↗

PubMed1996

Transcriptional pulse-chase analysis reveals a role for a novel snRNP-associated protein in the manufacture of spliceosomal snRNPs.

Vertebrate spliceosomal snRNAs associate with a conserved set of proteins, the Sm proteins, via a conserved RNA sequence, the Sm site. Assembly of this complex is required for the accumulation of stable snRNPs, hypermethylation of the 5' cap structure and nuclear import of the resultant particles. The function of individual core snRNP proteins is poorly understood, in part because of the difficulty of selectively inactivating individual polypeptides in vivo. Using a transcriptional pulse-chase method we have defined for the first time the steps of snRNP biogenesis in Saccharomyces cerevisiae. We describe a novel component of spliceosomal snRNPs, Brr1, which is distinct in sequence from Sm core proteins and yet which shares many of their properties, as well as a genetic interaction with the yeast homolog of Sm D1 core protein. Through a kinetic analysis of snRNP formation in wild-type and brr1 mutant cells we demonstrate specific defects in a subset of steps in the brr1 mutant: newly synthesized snRNAs are destabilized and 3'-end processing is slowed, whereas the cap hypermethylation reaction is unaffected. Notably, the stability of mature particles, as measured by promoter shut-off experiments, is normal in the absence of the Brr1 snRNP protein.

Amino Acid Sequence↗

PubMed1987

Two spliceosomes can form simultaneously and independently on synthetic double-intron messenger RNA precursors.

We have investigated the formation of splicing complexes in vitro on mRNA precursors (pre-mRNAs) containing two introns. Sucrose gradient sedimentation analysis revealed that the double-intron substrate becomes associated with 60S structures, which are larger than the 50S splicing complexes we previously observed with single-intron pre-mRNA precursors. We have demonstrated that the 60S complex represents the assembly of two single splicing complexes on the individual introns by conversion of the 60S double splicing complexes into single 50S spliceosomes by oligodeoxynucleotide directed RNase H cleavage of the double-intron pre-mRNAs within the middle exon. In addition, we have observed by native gel electrophoresis a transient double 'pre-splicing' complex analogous to the 35S 'pre-splicing' complex previously found with single-intron pre-mRNAs. Our results indicate that splicing complexes can form independently and simultaneously on the individual introns of multi-intron pre-mRNAs and that the assembly of these multiple spliceosomes proceeds with the same stepwise pathway observed for single-intron RNAs.

Cell Nucleus↗

PubMed1999

Identification, characterization and crystal structure analysis of the human spliceosomal U5 snRNP-specific 15 kD protein.

The U5 small ribonucleoprotein particle (snRNP) contains various proteins involved in catalytic activities mediating conformational rearrangements of the spliceosome. We have isolated and characterized the evolutionarily highly conserved human U5 snRNP-specific protein U5-15kD. The crystal structure of U5-15kD determined at 1.4 A resolution revealed a thioredoxin-like fold and represents the first structure of a U5 snRNP-specific protein known so far. With respect to human thioredoxin the U5-15kD protein contains 37 additional residues causing structural changes which most likely form putative binding sites for other spliceosomal proteins or RNA. Moreover, a novel intramolecular disulfide bond replaces the canonical one found in the thioredoxin family. Even though U5-15kD appears to lack protein disulfide isomerase activity, it is strictly required for pre-mRNA splicing in vivo as we demonstrate by genetic depletion of its ortholog in Saccharomyces cerevisiae. Our data suggest that the previously reported involvement of its Schizosaccharomyces pombe ortholog Dim1p in cell cycle regulation is a consequence of its essential role in pre-mRNA splicing.

Amino Acid Sequence↗

PubMed1989

Complementation of in vitro-assembled spliceosomes.

We describe the development and application of a system of in vitro-assembled splicing complexes that can be used for the identification of protein splicing factors which become associated with the spliceosome at the end of the assembly process ("late" splicing components). A splicing reaction performed in the presence of polyvinyl alcohol is interrupted after 15 to 20 minutes, before the appearance of splicing intermediates and products in significant amounts. Following low-speed centrifugation, a pellet is obtained containing splicing complexes that can be solubilized with 0.6 M-KCl. These complexes can be rapidly complemented for splicing in the presence of ATP and Mg2+ with protein factors that are present in HeLa cell nuclear extracts or in chromatographic extract fractions. Biochemical features of the complementation reactions, and conditions for reversible uncoupling of the two splicing steps, are described and discussed. These conditions are used to generate fully assembled spliceosomes in which splicing of the pre-mRNA can occur in the presence of ATP and Mg2+, but in the absence of nuclear extract ("autonomous splicing").

Adenosine Triphosphate↗