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PubMed1996

Human lupus anti-spliceosome A protein autoantibodies bind contiguous surface structures and segregate into two sequential epitope binding patterns.

High levels of anti-spliceosomal autoantibodies are commonly found in systemic lupus erythematosus (lupus) sera. We have evaluated the binding of lupus autoantibodies to octapeptides of nuclear ribonucleoprotein (nRNP) A, identified patterns of binding that vary between sera, quantified the proportion of anti-nRNP that binds individual peptides, and related these data to the partial tertiary structure of nRNP A. Anti-nRNP A-positive lupus sera share binding to eight antigenic groups of octapeptides from nRNP A. The four shared antigenic sites in the known nRNP A tertiary structure are contiguous on its surface and include surface loops between beta sheets and/or alpha helices. Anti-peptide Abs account for a significant portion of the human autoimmune response to nRNP A. Lupus sera are also obviously divisible into two subsets by their binding to nRNP A-derived peptides. Eight of the thirteen anti-nRNP A sera tested bind most of the eight common antigenic regions of nRNP A. All five sera in the other subset bind to two and only two groups of nRNP A octapeptides. Of this subset, absorption experiments show that as much as 75% of the anti-nRNP found in a patient serum is bound to these two octapeptides from nRNP A. The 70K spliceosome protein contains similar peptides that are also bound by these sera (OR=13.5, p<0.001). Lupus autoantibodies bind to peptides of nRNP A in discernibly consistent patterns that identify serologic subsets and in a manner that provides useful insights into the autoimmunity and antigenic structure of the nRNP A spliceosomal protein.

Amino Acid Sequence↗

PubMed1996

A spontaneous duplication in U6 spliceosomal RNA uncouples the early and late functions of the ACAGA element in vivo.

U6 RNA enters the spliceosome base paired with U4 RNA, but dissociates from U4 RNA before the catalytic steps of splicing. We have identified a cold-sensitive lethal mutation in U4 RNA (U4-cs1) that blocks the splicing pathway after U4/U6 complex formation, but before the first catalytic step of splicing. Remarkably, selection for suppressors of the cold-sensitive growth of the U4-cs1 strain yielded a tandem duplication of the highly conserved ACAGA sequence of U6 RNA (U6-Dup). The ACAGA sequence plays an essential role in spliceosome assembly and in the second catalytic step of pre-mRNA splicing; one or both of these roles involves direct base pairing to the pre-mRNA 5' splice site. In a U4-cs1/U6-Dup double-mutant strain grown at low temperature, the upstream ACAGA sequence of U6 RNA is required for suppression of the U4 mutation, whereas the downstream ACAGA sequence is required for other essential functions. Based on the sequence requirements for function of the upstream ACAGA element of U6-Dup, we propose that it pairs with the pre-mRNA 5' splice site during incorporation of the U4/U6 complex into the spliceosome and that the subsequent dissociation of U4 RNA exposes the downstream ACAGA sequence, which functions in the catalytic steps. The properties of this mutant U4/U6 complex provide compelling in vivo evidence that U6 RNA normally base pairs with the 5' splice site before disruption of its pairing with U4 RNA.

Base Composition↗

PubMed1998

PRP16, a DEAH-box RNA helicase, is recruited to the spliceosome primarily via its nonconserved N-terminal domain.

Dynamic rearrangement of RNA structure is crucial for intron recognition and formation of the catalytic core during pre-mRNA splicing. Three of the splicing factors that contain sequence motifs characteristic of the DExD/DExH-box family of RNA-dependent ATPases (Prp16, Prp22, and the human homologue of Brr2) recently have been shown to unwind RNA duplexes in vitro, providing biochemical evidence that they may direct structural rearrangements on the spliceosome. Notably, however, the unwinding activity of these proteins is sequence nonspecific, raising the question of how their functional specificity is determined. Because the highly conserved DExD/DExH-box domain in these proteins is typically flanked by one or more nonconserved domains, we have tested the hypothesis that the nonconserved regions of Prp16 determine the functional specificity of the protein. We found that the nonconserved N-terminal domain of Prp16 is (1) essential for viability, (2) required for the nuclear localization of Prp16, and (3) capable of binding to the spliceosome specifically at the step of Prp16 function. Moreover, this domain can interact with the rest of the protein to allow trans-complementation. Based on these results, we propose that the spliceosomal target of the unwinding activity of Prp16, and possibly other DExD/DExH-box splicing factors as well, is defined by factors that specifically interact with the nonconserved domains of the protein.

Adenosine Triphosphatases↗

PubMed1987

Specific accessory sequences in Saccharomyces cerevisiae introns control assembly of pre-mRNAs into spliceosomes.

In experiments involving deletion and rearrangement of intron sequences two small regions of the intron in the yeast CYH2 ribosomal protein gene were found to play important roles in splicing of the pre-mRNA. One element lies downstream of the 5' splice site, and the other is upstream of the branchpoint sequence UACUAAC. Deletion of the element upstream of the branchpoint prevents spliceosome formation and blocks splicing in vivo and in vitro. Deletion of the element downstream of the 5' splice site does not on its own block splicing but rescues spliceosome formation and splicing of pre-mRNA lacking the element upstream of the branchpoint. These elements correspond to two regions of sequence complementarity which are a conserved feature of the introns in yeast pre-mRNAs. Mixing and matching of the elements from the ACT1 and CYH2 gene introns showed that these elements can cooperate in an intron-specific fashion to control spliceosome assembly.

Base Sequence↗

PubMed1987

Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes.

Electrophoretic separation of ribonucleoprotein particles in a nondenaturing gel was used to analyze the splicing of mRNA precursors. Early in the reaction, a complex formed consisting of the U2 small nuclear ribonucleoprotein particle (snRNP) bound to sequences upstream of the 3' splice site. This complex is modeled as a precursor of a larger complex, the spliceosome, which contains U2, U4/6, and U5 snRNPs. Conversion of the U2 snRNP-precursor RNA complex to the spliceosome probably involves binding of a single multi-snRNP particle containing U4/6 and U5 snRNPs. The excised intron was released in a complex containing U5, U6, and probably U2 snRNPs. Surprisingly, U4 snRNP was not part of the intron-containing complex, suggesting that U4/6 snRNP disassembles and assembles during splicing. Subsequently, the reassembled U4/6 snRNP would associate with U5 snRNP and participate in de novo spliceosome formation. U1 snRNP was not detected in any of the splicing complexes.

HeLa Cells↗

PubMed1990

Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus.

A monoclonal antibody raised against mammalian spliceosomes specifically recognizes a non-snRNP factor required for spliceosome assembly. This splicing factor is highly concentrated in discrete regions within the nucleus, in a pattern that is a distinct subset of that seen with anti-snRNP antibodies. These observations are evidence that spliceosome assembly could be compartmentalized within the nucleus.

Animals↗

PubMed1988

Identification of a functional mammalian spliceosome containing unspliced pre-mRNA.

Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5' cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.

Animals↗

PubMed1990

Protein composition of mammalian spliceosomes assembled in vitro.

This paper reports an analysis of the protein composition of highly purified mammalian spliceosomes isolated by a two-step large-scale affinity chromatography procedure. Splicing complexes were assembled in vitro on biotinylated pre-mRNA, fractionated by gel filtration, and then affinity-purified by binding to avidin-agarose. The purified spliceosomes are unexpectedly complex, containing at least 50 proteins that range in molecular mass from less than 14 to 200 kDa. Three complexes that assemble in the absence of ATP were also purified and characterized. These include a complex enriched in the small nuclear ribonucleoprotein particle U1 and non-specific complexes assembled either on pre-mRNA or an RNA lacking splice sites. Comparison between these complexes and the spliceosome revealed a distinct set of pre-mRNA-specific proteins and a set of proteins that bind to pre-mRNA only in the presence of ATP. Proteins in these two classes, many of which do not correspond in size to known small nuclear ribonucleoprotein particle proteins, are strong candidates for functional splicing components.

Animals↗

PubMed1991

Requirement of protein factors and ATP for the disassembly of the spliceosome after mRNA splicing reaction.

Pre-mRNA splicing reaction occurs in a large ribonucleoprotein complex called the spliceosome. After the splicing reaction, the spliceosome is disassembled to release the splicing products including spliced mRNA. Here we show that protein factors in a HeLa nuclear extract or a DEAE-cellulose fraction as well as ATP are required to release the splicing products form the spliceosome in which the splicing reaction has already completed.

Adenosine Triphosphate↗

PubMed1987

Spliceosome assembly in yeast.

Precursors to mRNA become substrates for splicing by being assembled into a complex multisubunit structure, the spliceosome. To study the assembly of the yeast spliceosome, intermediate complexes were separated by electrophoresis on nondenaturing polyacrylamide gels. Four splicing-dependent complexes, A1, A2-1, A2-2, and B, were observed. The order of assembly of these complexes was determined to be B----A2-1----A1----A2-2. The assembly process can be blocked at complex A1 by addition of 5 mM EDTA or by carrying out the assembly process in heat-inactivated rna2 extracts. The snRNA composition of the complexes was determined by hybridization with probes for five yeast snRNAs. snR14 (U4) was only found in complex A2-1, snR6 (U6) and snR7 (U5) were in complexes A1, A2-1, and A2-2, whereas snR20 (U2) was in all four of the complexes. snR19 (U1) was not present in any of the complexes. Hybridization with these probes was also employed to detect snRNPs present in yeast splicing extracts. We found that snR6, snR7, and snR14 were present together in a large complex. This complex underwent an ATP-dependent dissociation to give snR7 and snR6-snR14 complexes. snR19 and snR20 are present in distinct RNPs but the mobility of these is not affected by ATP. A mechanism for spliceosome assembly is proposed.

Animals↗

PubMed1987

Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

Cell Nucleus↗

PubMed1987

U1 small nuclear ribonucleoproteins are required early during spliceosome assembly.

U1 small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within U1 RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of U1 snRNPs with the spliceosome. U1-specific antibodies detected U1 association with precursor RNA early during assembly. Removal of the 5' terminal sequences of U1 RNA by oligo-directed cleavage or removal of U1 snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or U1-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of U1 RNA. The dependency of Sm precipitability of precursor RNA upon the presence of U1 snRNPs suggests that U1 snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of U1 depressed U1 snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of U1 snRNPs with the assembling spliceosome in the absence of U1 RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that U1 snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.

Adenoviridae↗

PubMed1988

Early commitment of yeast pre-mRNA to the spliceosome pathway.

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.

Chromosome Deletion↗

PubMed1997

Developmental expression of the murine spliceosome-associated protein mSAP49.

We have isolated the mouse homologue of human spliceosome-associated protein SAP49, mSAP49. mSAP49 contains two RNA recognition motifs (RRM) in the N terminus of the predicted amino acid sequence, and a highly basic C terminus rich in glycine/proline. mSAP49 displayed a plastic of expression in cardiac development. In the adult mouse, mSAP49 is widely distributed, although it was found at relatively lower levels in the heart. In situ hybridization analysis of mSAP49 mRNA distribution in staged mouse embryos showed that mSAP49 onset occurs later in the heart than in other embryonic tissues. While mSAP49 expression was found at day 10.0 postconception (pc) in the optic eminence, optic vesicle, hindbrain, and somites, it was not in cardiac structures. mSAP49 was detected in the ventricles at day 11.5, and at day 13.5 it was also detected in the atria. Northern analysis showed that mSAP49 mRNA displayed a peak of expression in the heart at days 14.0-15.0 pc, and its abundance decayed in the adult. This dynamic pattern of cardiac expression suggests that mSAP49 may be contributing to a change in the ratio of spliceosome components during cardiac growth and development, which may have consequences for tissue-specific splicing, RNA stabilization, or translation.

Amino Acid Sequence↗

PubMed1995

Autoimmune response to the spliceosome. An immunologic link between rheumatoid arthritis, mixed connective tissue disease, and systemic lupus erythematosus.

OBJECTIVE: To assess the significance of autoantibodies to RA33, the A2 protein of the heterogeneous nuclear ribonucleoproteins (hnRNP), and to the related hnRNP proteins A1, B1, and B2 in rheumatic diseases. METHODS: Using a partially purified preparation of hnRNP-A and hnRNP-B proteins, sera from 303 patients with various rheumatic diseases were investigated by immunoblotting. For the analysis of cross-reactivities, autoantibodies were affinity purified by blot elution. RESULTS: Anti-A2/RA33 was found in 35% of rheumatoid arthritis (RA) patients, 38% of mixed connective tissue disease (MCTD) patients, 23% of systemic lupus erythematosus (SLE) patients, and, apart from single exceptions, not in patients with other rheumatic diseases. All anti-A2/RA33-positive sera were also reactive with B1 and B2, and anti-A2/RA33 antibodies cross-reacted with both proteins. Antibodies to hnRNP-A1 were found less frequently; moreover, the majority of anti-A1-positive sera also contained anti-A2/RA33 antibodies. In anti-A1, anti-A2/RA33 double-positive sera, cross-reactivity between the 2 antibodies was generally observed. In SLE patients, the presence of anti-A2/RA33 was correlated with the presence of anti-(U1) small nuclear RNP (snRNP) and anti-Sm (P < 0.0001 and P < 0.005, respectively), but there was no evidence for cross-reactivity between antibodies to hnRNP and antibodies to snRNP antigens. CONCLUSION: Since both hnRNPs and snRNPs are essential components of the spliceosome, the data show that the immune systems of patients with RA, SLE, and MCTD react to this functional complex. However, compared with MCTD and SLE patients, RA patients have a more restricted immune response to the spliceosome: they react to hnRNP proteins, particularly to hnRNP-A2/RA33, but not to snRNPs.

Arthritis, Rheumatoid↗

PubMed1992

A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.

Adenosine Triphosphatases↗

PubMed1994

Assembly and disassembly of spliceosomes along a specific pre-messenger RNP fiber.

Transcriptionally active Balbiani ring (BR) genes in the salivary glands of the dipteran Chironomus tentans were studied by immunoelectron microscopy to establish the distribution of spliceosome components along a specific pre-messenger ribonucleoprotein (pre-mRNP) fiber. The BR genes are 35-40 kb in size with three introns close to the 5' end and one close to the 3' end; a very large middle portion lacks introns. As a rule the 5' introns are spliced concomitant with transcription in the promoter proximal third of the gene, while the 3' intron is spliced post-transcriptionally. The BR genes with growing pre-mRNPs were visualized in situ, while completed and released pre-mRNPs were isolated from the nucleoplasm and studied unfolded on a grid surface. An anti-snRNP antibody (Y12) bound mainly to the promoter proximal third of the BR gene (86%) and only to a minor extent to the middle and distal thirds (7 and 7% respectively). An antibody to an hnRNP protein reacted with the proximal, middle and distal regions to an increasing extent (17, 38 and 45% respectively), reflecting the increase in size of the growing transcription product. In the nucleoplasmic pre-mRNP particle only one end of the RNP fiber was labeled by Y 12, presumably the 3' end; the anti-hnRNP antibody decorated the entire RNP fiber. Thus, the snRNPs do not associate along the whole pre-mRNP fiber but rather bind to the 5' and 3' ends, i.e. the regions containing the introns. The results also imply that the spliceosomes both assemble and disassemble rapidly on the pre-mRNP fiber.

Animals↗

PubMed1995

Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants.

In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation.

Amino Acid Sequence↗