Pre-mRNA splicing by complementation with purified human U1, U2, U4/U6 and U5 snRNPs.

The four major nucleoplasmic small nuclear ribonucleoprotein particles U1, U2, U4/U6 and U5 can be extensively purified from HeLa cells by immunoaffinity chromatography using a monoclonal anti-trimethylguanosine antibody. The snRNP particles in active splicing extracts are selectively bound to the immunoaffinity matrix, and are then gently eluted by competition with an excess of free nucleoside. Biochemical complementation studies show that the purified snRNPs are active in pre-mRNA splicing, but only in the presence of additional non-snRNP protein factors. All the RNPs that are necessary for splicing can be purified in this manner. The active snRNPs are characterized with respect to their polypeptide composition, and shown to be distinct from several other activities implicated in splicing.     

Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents.

A procedure is described for the purification of the individual major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 from HeLa cells. The salient feature of the method is the combined usage of antibodies against 2,2,7-trimethylguanosine (m3G) and 6-methyladenosine (m6A) for differential immune affinity chromatography of the snRNPs. While anti-m3G affinity columns allow the separation of snRNPs U1, U2 and U5 from U4/U6 RNPs, anti-m6A antibodies selectively react with snRNPs U2 and U4/U6. Our technique further incorporates immune affinity chromatography of snRNPs with antibodies against snRNP proteins in addition to ion exchange chromatography. The procedure avoids the usage of denaturing agents, so as to maintain the native structure of the particles. This is mainly provided for by the possibility of eluting the anti-m3G and anti-m6A bound snRNPs with excess of the respective nucleosides. We have so far identified 12 polypeptides as constituents of the major snRNPs U1 to U6. Seven proteins of approximate mol. wts 29 kd (B'), 28 kd (B), 16 kd (D), 15.5 kd (D'), 12 kd (E), 11 kd (F) and 9 kd (G) were present in each of the individual snRNPs U1, U2, U5 and U4/U6. In addition to the common proteins, U1 RNPs contain three unique polypeptides of mol. wts 70 kd, 34 kd (A) and 22 kd (C). U2 RNPs are characterized by the presence of a 33-kd and a 28.5-kd protein, denoted A' and B". We could not detect any unique polypeptide confined to the purified snRNPs U5 or U4/U6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for three distinct D proteins, which react differentially with anti-Sm autoantibodies, in the cores of the major snRNPs U1, U2, U4/U6 and U5.

Electrophoresis of the mixture of proteins from purified snRNPs U1, U2, U4/U6 and U5 on SDS-polyacrylamide gels that had been allowed to polymerise in the presence of high TEMED concentrations have revealed the presence of proteins in the snRNPs that previously had eluded detection. The most striking case is that of protein D, heretofore generally observed as a single broad band; in high-TEMED gels, this splits into three clearly-separated bands, identified as three distinct proteins. We have denoted these proteins D1 (16 kDa), D2 (16.5 kDa) and D3 (18 kDa). Chemical and immunological studies have shown that D1 is identical with the common snRNP protein D, whose structure was recently resolved by cDNA cloning (Rokeach et al. (1988), Proc. Natl. Acad. Sci. USA, 85, 4832-4836) and that D2 and D3 are clearly distinct from D1 and very probably from each other. In addition to D1, proteins D2 and D3 are present in purified U1, U2, U4/U6 and U5 snRNPs isolated from HeLa cells, so these also belong to the group of common snRNP proteins. They are also found in snRNPs isolated from mouse cells, indicating that the role of these proteins in the structure and/or function of UsnRNPs has been conserved in evolution. Interestingly, patients with systemic lupus erythematosus produce populations of anti-Sm autoantibodies that react differentially with the D proteins; some recognise all of them and others only a subset. The high-TEMED gels allow improved resolution not only of the D proteins, but also of some of the U5-specific proteins contained in 20S U5 snRNPs, in particular the 15-kDa protein. In addition, under these conditions, the common G protein, previously observed as a single band, appears as a doublet. Whether the additional band represents a distinct common snRNP protein or a post-translationally modified version of G is not yet known.     

Antibodies specific for N6-methyladenosine react with intact snRNPs U2 and U4/U6

Antibodies specific for N6-methyladenosine (m6A) were elicited in rabbits and used to study the accessibility in intact snRNPs of the m6A residues present in the snRNAs U2, U4 and U6. The antibody quantitatively precipitates snRNPs U2 and U4/U6 from total nucleoplasmic snRNPs U1-U6 isolated from HeLa cells, which demonstrates that the m6A residues of the respective snRNAs are not protected by snRNP proteins in the snRNP particles. While the anti-m6A IgG does not react at all with U5 RNPs lacking m6A, a significant amount of U1 RNPs was co-precipitated despite the fact that U1 RNA does not contain m6A either. Since anti-m6A IgG does not react with purified U1 RNPs and co-precipitation of U1 RNPs is dependent on the presence of U2 RNPs but not of U4/U6 RNPs, these data indicate an interaction between snRNPs U1 and U2 in vitro. The anti-m6A precipitation pattern described above was also observed with snRNPs isolation from mouse Ehrlich ascites tumor cells, indicating similar three-dimensional arrangements of snRNAs in homologous snRNP particles from different organisms.     

Assembly and nuclear transport of the U4 and U4/U6 snRNPs

We have analyzed the assembly of the spliceosomal U4/U6 snRNP by injecting synthetic wild-type and mutant U4 RNAs into the cytoplasm of Xenopus oocytes and determining the cytoplasmic-nuclear distribution of U4 and U4/U6 snRNPs by CsCl density gradient centrifugation. Whereas the U4 snRNP was localized in both the cytoplasmic and nuclear fractions, the U4/U6 snRNP was detected exclusively in the nuclear fraction. Cytoplasmic-nuclear migration of the U4 snRNP did not depend on the stem II nor on the 5' stem-loop region of U4 RNA. Our data provide strong evidence that, following the cytoplasmic assembly of the U4 snRNP, the interaction of the U4 snRNP with U6 RNA/RNP occurs in the nucleus; furthermore, cytoplasmic-nuclear transport of the U4 snRNP is independent of U4/U6 snRNP assembly.     

Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera

Small nuclear ribonucleoprotein particles (snRNPs) of the U-snRNP class from Ehrlich ascites tumor cells were purified in a one-step procedure by affinity chromatography with antibodies specific for 2,2,7-trimethylguanosine (m23.2.7G), which is part of the 5'-terminal cap structure of snRNAs U1-U5. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess nucleoside m23.2.7G; this guarantees maintenance of their native structure. The snRNPs U1, U2, U4, U5 and U6 can be recovered quantitatively from nuclear extracts by this procedure. Co-isolation of U6 snRNP must be due to interactions between this and other snRNPs, as anti-m23.2.7G antibodies do not react with deproteinized U6 snRNA. We have so far defined nine proteins of approximate mol. wts. 10 000, 12 000, 13 000, 16 000, 21 000, 28 000, 32 000, 34 000 and 75 000. Purified snRNPs react with anti-(U1)RNP and with anti-Sm antisera from patients with mixed connective tissue disease and from MRL/l mice. As determined by the protein blotting technique, six of the snRNP polypeptides, characterized by apparent mol. wts. 13 000, 16 000, 21 000, 28 000, 34 000 and 75 000, bear antigenic determinants for one or the other of the above autoantibody classes. This suggests strongly that the U-snRNPs produced by the procedure described here are indeed representative of the snRNPs in the cell. With highly purified snRNPs available, investigation of possible enzymic functions of the particles may now be undertaken.     

Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs.

Several small nuclear RNAs (snRNAs), including the spliceosomal U1, U2, U4 and U5 snRNAs, are associated with Sm proteins. These eight small proteins form a heteromeric complex that binds to snRNAs and plays a major role in small nuclear ribonucleoprotein (snRNP) biogenesis and transport. These proteins are also a major target for autoantibodies in the human disease systemic lupus erythematosus. By sequence comparison I have shown that all the known Sm proteins share a common structural motif which might explain their immunological cross-reactivity. Database searches using this motif uncovered a large number of Sm-like proteins from plants, animals and fungi. These proteins have been grouped in at least 13 different subfamilies. Genes encoding divergent yeast members were cloned and used to produce tagged fusion proteins. Some of these proteins are canonical Sm proteins as they associate with the yeast U1, U2, U4/U6 and U5 snRNAs. Surprisingly, one Sm-like protein was found to be a component of the U6 snRNP. These findings have implications for the structure of the Sm protein complex, spliceosomal snRNP evolution, snRNA transport and modification as well as the involvement of Sm proteins in systemic lupus erythematosus.     

Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA

Communication between U1 and U2 snRNPs is critical during pre-spliceosome assembly; yet, direct connections have not been observed. To investigate this assembly step, we focused on Prp5, an RNA-dependent ATPase of the DExD/H family. We identified homologs of Saccharomyces cerevisiae Prp5 in humans (hPrp5) and Schizosaccharomyces pombe (SpPrp5), and investigated their interactions and function. Depletion and reconstitution of SpPrp5 from extracts demonstrate that ATP binding and hydrolysis by Prp5 are required for pre-spliceosome complex A formation. hPrp5 and SpPrp5 are each physically associated with both U1 and U2 snRNPs; Prp5 contains distinct U1- and U2-interacting domains that are required for pre-spliceosome assembly; and, we observe a Prp5-associated U1/U2 complex in S. pombe. Together, these data are consistent with Prp5 being a bridge between U1 and U2 snRNPs at the time of pre-spliceosome formation.     

The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 degrees C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Delta extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17-spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Delta and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.     

A new cyclophilin and the human homologues of yeast Prp3 and Prp4 form a complex associated with U4/U6 snRNPs.

We have purified three new human U4/U6-snRNP proteins from HeLa cells. The three proteins formed a tightly bound complex and behaved as a single species throughout the purification. All three proteins have been identified by peptide sequencing, and full-length cDNA sequences have been obtained for all of them. Two of the proteins are homologues of the Saccharomyces cerevisiae splicing factors Prp3 and Prp4, and the third protein is a cyclophilin. Both the human and S. cerevisiae Prp4 proteins have seven repeats of the WD motif and likely fold into structures very similar to those of the beta subunits of G proteins. The human Prp3 protein is highly basic and is closely related to S. cerevisiae Prp3 only in its carboxyl-terminal half. The human homologues of Prp3 and Prp4 are part of a stable complex in the absence of RNA. The third protein in the complex is a new cyclophilin. Cyclophilins have been proposed to act as chaperones in a variety of cellular processes, and we discuss some possible roles of this U4/U6 snRNP-associated cyclophilin.     

A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs

We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.     

Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.

The 25S [U4/U6.U5] tri-snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre-mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri-snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6.U5] tri-snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri-snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm-like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA-binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre-mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri-snRNPs and the U2 snRNP are co-precipitated with protein A-tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2.U4/U6.U5] tetra-snRNP complex.     

A monoclonal antibody specific for snRNPs U1 and U2

A monoclonal antibody (D-5) is described which selectively precipitates snRNPs U1 and U2. The antibody was derived from a mouse immunized with extracts from chick embryonic nuclei. By immunoblotting on either total proteins from purified snRNPs U1-U6, U2-U6 or U1 only, we could demonstrate that the monoclonal antibody cross-reacts with the U1 RNP specific polypeptide A and the U2 RNP specific polypeptide B", thereby establishing that the two snRNP proteins share at least one epitope. D-5 precipitates snRNPs U1 and U2 from a variety of species, including man, chicken, mouse, rat kangaroo and Xenopus laevis. It will thus be a useful tool for studying structure function relationships of the two snRNP species in different cell systems.     

Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF.

We have used protection against ribonuclease H to investigate the mechanisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5' splice sites. The initial binding of U1 snRNPs to alternative consensus splice sites was indiscriminate, and on a high proportion of pre-mRNA molecules both sites were occupied simultaneously. When the sites were close, this inhibited splicing. We propose that double occupancy leads to the use of the downstream site for splicing and that this is the cause of the proximity effect seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity of U1 snRNPs for the downstream site. This prediction was tested both by cleaving the 5' end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstream 5' splice site use by splicing factor SF2/ASF appears to be mediated by an increase in the strength of U1 snRNP binding to all sites indiscriminately.     

Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.U5 trimeric particle. Comparative analysis of minor and major tri-snRNPs by using immunoprecipitation experiments revealed that their protein compositions are very similar, if not identical. Not only U5-specific proteins but, surprisingly, all tested U4/U6- and major tri-snRNP-specific proteins were detected in the minor tri-snRNP complex. Significantly, the major tri-snRNP-specific proteins 65K and 110K, which are required for integration of the major tri-snRNP into the U2-dependent spliceosome, were among those proteins detected in the minor tri-snRNP, raising an interesting question as to how the specificity of addition of tri-snRNP to the corresponding spliceosome is maintained. Moreover, immunodepletion studies demonstrated that the U4/U6-specific 61K protein, which is involved in the formation of major tri-snRNPs, is essential for the association of the U4atac/U6atac di-snRNP with U5 to form the U4atac/U6atac.U5 tri-snRNP. Subsequent immunoprecipitation studies demonstrated that those proteins detected in the minor tri-snRNP complex are also incorporated into U12-dependent spliceosomes. This remarkable conservation of polypeptides between minor and major spliceosomes, coupled with the absence of significant sequence similarity between the functionally analogous snRNAs, supports an evolutionary model in which most major and minor spliceosomal proteins, but not snRNAs, are derived from a common ancestor.     

Identification of an RNA-dependent ATPase activity in mammalian U5 snRNPs.

Nuclear pre-mRNA splicing requires ATP at several steps from spliceosome assembly to product release. Here, we demonstrate that an integral component of the 20S U5 snRNP is an RNA-dependent ATPase. The ATPase activity of 20S U5 and 25S [U4/U6.U5] snRNPs purified by glycerol gradient centrifugation is strongly stimulated by homopolymeric RNA but not ssDNA. Purified 12S Ul and U2 snRNPs do not exhibit ATPase activity. Moreover, the U5-associated NTPase specifically hydrolyzes ATP and dATP. The additional purification of 20S U5 snRNPs by Mono Q chromatography does not affect the efficiency of ATP hydrolysis. Both U5 and tri-snRNPs bind ATP stoichiometrically in an RNA-independent manner. A candidate ATPase was identified by UV-irradiation of purified snRNPs with radiolabeled ATP. In the presence of homopolymeric RNA, the 200 kDa U5-specific protein is the major crosslinked protein, even in Mono Q-purified U5 snRNPs. The correlation between RNA-dependent ATPase activity in the U5 snRNP and the RNA-dependent onset of this crosslink strongly suggests that the 200 kDa protein is an RNA-dependent ATPase. Furthermore, both the formation of the crosslink and ATPase activity appear with a similar substrate specificity for ATP.     

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.