Interaction of snRNAs with rapidly sedimenting nuclear sub-structures (hnRNPs) from HeLa cells.

We have shown previously (Liautard et al., 1982, J. Mol. Biol., 162, 623-643) that digestion with micrococcal nuclease under drastic conditions of a pure U1 snRNP, as well as a mixture containing U2, U1, U4, U5 and U6 snRNPs, gives rise to resistant RNA fragments derived from all but U6 snRNAs. As an attempt to elucidate the way in which snRNPs are attached to their native structure, the same approach was applied to hnRNP which are known to contain snRNP (Guimont-Ducamp et al., 1977, Biochimie, 59, 755-758). Micrococcal nuclease digestion of hnRNPs yielded a population of 15-50 nucleotides long resistant fragments of snRNAs. Sequence analyses showed that all fragments previously identified in core snRNPs were also present. Only U2 and U5 snRNAs were further protected as a result of their association with the hnRNP complex (from the cap to nucleotide 32 for U2 and from nucleotide 22 to nucleotide 70 for U5). No additional protected fragment derived from U1, U4 and U6 snRNAs was found. This finding confirms that the 5' terminal region of U1 snRNP remains available for base-pairing interaction with the premessenger RNA, as predicted by the model of Lerner et al. (Nature, 1980, 283, 220-224).     

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5′ splice sites at exon/intron boundaries. U1 snRNAs associate with 5′ splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the ‘Smith antigen’, or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs.     

Localization and structure of snRNPs during mitosis. Immunofluorescent and biochemical studies

The distribution of U snRNAs during mitosis was studied by indirect immunofluorescence microscopy with snRNA cap-specific anti-m3G antibodies. Whereas the snRNAs are strictly nuclear at late prophase, they become distributed in the cell plasm at metaphase and anaphase. They re-enter the newly formed nuclei of the two daughter cells at early telophase, producing speckled nuclear fluorescent patterns typical of interphase cells. While the snRNAs become concentrated at the rim of the condensing chromosomes and at interchromosomal regions at late prophase, essentially no association of the snRNAs was observed with the condensed chromosomes during metaphase and anaphase. Independent immunofluorescent studies with anti-(U1)RNP autoantibodies, which react specifically with proteins unique to the U1 snRNP species, showed the same distribution of snRNP antigens during mitosis as was observed with the snRNA-specific anti-m3G antibody. Immunoprecipitation studies with anti-(U1)RNP and anti-Sm autoantibodies, as well as protein analysis of snRNPs isolated from extracts of mitotic cells, demonstrate that the snRNAs remain associated in a specific manner with the same set of proteins during interphase and mitosis. The concept that the overall structure of the snRNPs is maintained during mitosis also applies to the coexistence of the snRNAs U4 and U6 in a single ribonucleoprotein complex. Particle sedimentation studies in sucrose gradients reveal that most of the snRNPs present in sonicates of mitotic cells do not sediment as free RNP particles, but remain associated with high molecular weight (HMW) structures other than chromatin, most probably with hnRNA/RNP.     

Polypeptide components of Drosophila small nuclear ribonucleoprotein particles.

In eukaryotes splicing of pre-mRNAs is mediated by the spliceosome, a dynamic complex of small nuclear ribonucleoprotein particles (snRNPs) that associate transiently during spliceosome assembly and the splicing reaction. We have purified snRNPs from nuclear extracts of Drosophila cells by affinity chromatography with an antibody specific for the trimethylguanosine (m3G) cap structure of snRNAs U1-U5. The polypeptide components of Drosophila snRNPs have been characterized and shown to consist of a number of proteins shared by all the snRNPs, and some proteins which appear to be specific to individual snRNP particles. On the basis of their apparent molecular weight and antigenicity many of these common and particle specific Drosophila snRNP proteins are remarkably conserved between Drosophila and human spliceosomes. By probing western blots of the Drosophila snRNP polypeptides with a number of antisera raised against human snRNP proteins, Drosophila polypeptides equivalent to many of the HeLa snRNP-common proteins have been identified, as well as candidates for a number of U1, U2 and U5-specific proteins.     

The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body

The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.     

Immunoprecipitation distinguishes non-overlapping groups of snRNPs in Schizosaccharomyces pombe.

The large number of snRNAs in the fission yeast Schizosaccharomyces pombe can be divided into four non-overlapping groups by immunoprecipitation with antibodies directed against mammalian snRNP proteins. 1) Of the abundant snRNAs, anti-Sm sera precipitate only the spliceosomal snRNAs U1, U2, U4, U5 and U6. Surprisingly, three Sm-sera tested distinguish between U2, U4 and U5 and U1 from S.pombe; one precipitating only U1 and two precipitating U2, U4 and U5 but not U1. 2) A group of 11 moderately abundant snRNAs are not detectably precipitated by human anti-Sm sera, but are specifically precipitated by monoclonal antibody H57 specific for the human B/B' polypeptides. From Aspergillus nidulans this antibody also precipitates at least 12 snRNAs. 3) Anti-(U3)RNP sera do not precipitate the above snRNAs, but precipitate at least 6 further snRNAs, including the homologues of U3. Both the anti-(U3)RNP sera and H57 also efficiently precipitate a number of discrete non-capped RNAs. 4) A small number of additional snRNAs are not detectably precipitated by any anti-serum tested to date, further analysis may identify antisera specific for these snRNPs. Western blots of purified snRNP proteins were used to identify the S.pombe proteins responsible for these immunoprecipitations. Several Sm-sera decorate a 16.3kD protein which may be a D protein homologue, monoclonal H57 decorates a further protein of 16kD and an anti-(U3)RNP serum decorates the homologue of the 36kD U3-specific protein, fibrillarin.     

5′-terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices : Double-label immunofluorescent studies with anti-m3G antibodies and with anti-RNP and anti-Sm autoantibodies

Antibodies specific for 2,2,7-trimethylguanosine (m3G), which do not cross-react with m7G-capped RNA molecules were used to study, by immunofluorescence microscopy, the reactivity of the m3G-containing cap structures of the snRNAs U1 to U5 in situ. In interphase cells, immunofluorescent sites were restricted to the nucleus, whilst nucleoli were free of fluorescence. This indicates that the 5' terminal of most of the nucleoplasmic snRNAs are not protected by an m3G cap-recognizing protein and that the snRNA caps are not necessarily required for the binding of snRNPs to subnuclear structures. The snRNAs in the nucleoplasm appeared as distinct units in the light microscope, and this allowed the comparison of the distribution of snRNP proteins by double label studies with anti-RNP or anti-Sm antibodies within the same cell. The three antibody classes produced superimposable fluorescent patterns. Taking into account that the various IgGs react with antigenic sites on snRNAs or snRNP proteins not shared by all the snRNP species, these data suggest that U1 snRNP particles are distributed in the same way as the other snRNPs in the nucleus. Qualitatively the same results were obtained with DNase-treated nuclear matrices indicating that intact snRNPs are part of the nuclear matrix. Our data are consistent with proposals that the various snRNPs may be involved in processing of hnRNA and that this may take place at the nuclear matrix.     

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.     

Structural requirements for the function of a yeast chromosomal replicator

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.     

Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.     

Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing

Small nuclear ribonucleoprotein particles (snRNPs) from eucaryotic cells can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7-trimethyl-guanosine (m3G), which is present in the 5'-terminal caps of the snRNAs. While the snRNPs U1, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNPs from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti-m3G, its co-chromatography with U4 RNP on anti-m3G affinity columns suggests either that discrete snRNPs U4 and U6 are intimately associated in nuclear extracts or that both RNAs are organized in one ribonucleoprotein particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNPs in sucrose gradients. Gel fractionation of RNAs shows identical distributions of snRNAs U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNPs U1 or U2. Physical association between snRNPs U4 and U6 during sedimentation is shown by their co-precipitation with anti-m3G IgG from the gradient fractions. Finally, experimental evidence is provided that snRNAs U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by our finding that anti-m3G IgG co-precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNPs at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Nucleocytoplasmic distribution of snRNPs and stockpiled snRNA-binding proteins during oogenesis and early development in Xenopus laevis

The distribution of small nuclear ribonucleoprotein particles containing U snRNAs (U snRNPs) during oogenesis and early development in Xenopus was analyzed with a lupus antibody (anti-Sm) that reacts with snRNA-binding proteins. Fully grown oocytes and embryos prior to gastrulation were found to be relatively depleted of U snRNPs in their nuclei and to contain an excess of snRNA-binding proteins stored in the cytoplasm. During late blastula-early gastrula, or after microinjection of U snRNAs into the cytoplasm of a mature oocyte, the proteins migrate into the nucleus. Dot hybridization analysis showed that small previtellogenic oocytes already contain a maximal amount of U1 (and U2) snRNAs, which then decreases to about 20% of that value in fully mature oocytes, even though the cell's volume has increased enormously. Thus fully grown oocytes and eggs accumulate snRNA-binding proteins for use during early development, but this is not coupled with the accumulation of U snRNA.     

High level of complexity of small nuclear RNAs in fungi and plants

The complexity of the trimethylguanosine-capped, small nuclear RNA (snRNA) populations in a number of organisms has been examined using immunoprecipitation and two-dimensional gels. From the fungi Aspergillus nidulans and Schizosaccharomyces pombe, over 30 major snRNAs can be resolved. The most abundant of these correspond to the putative analogues of vertebrate U1, U2, U4 and U5, which have been reported to be precipitated by anti-Sm antibodies, but other snRNAs are little less abundant than the major Sm-precipitable species. A similarly high level of complexity of snRNAs is detected in pea plants. In Candida albicans, the snRNAs are somewhat less numerous (about 22 major species) and are substantially less abundant than those of the above fungi, features shared with another budding yeast, Saccharomyces cerevisiae. Ten species of human snRNA have been reported; on two-dimensional gels, a number of additional snRNAs can be resolved from human cells. Each fungus, as well as pea plants, contains snRNAs substantially larger than any reported from vertebrates or detected in the human RNA used here. It appears that many eukaryotes contain substantially more species of snRNA than was previously believed.     

cDNA cloning of U1, U2, U4 and U5 snRNA families expressed in pea nuclei.

Differences observed between plant and animal pre-mRNA splicing may be the result of primary or secondary structure differences in small nuclear RNAs (snRNAs). A cDNA library of pea snRNAs was constructed from anti-trimethylguanosine (m3(2,2,7)G immunoprecipitated pea nuclear RNA. The cDNA library was screened using oligo-deoxyribonucleotide probes specific for the U1, U2, U4 and U5 snRNAs. cDNA clones representing U1, U2, U4 and U5 snRNAs expressed in seedling tissue have been isolated and sequenced. Comparison of the pea snRNA variants with other organisms suggest that functionally important primary sequences are conserved phylogenetically even though the overall sequences have diverged substantially. Structural variations in U1 snRNA occur in regions required for U1-specific protein binding. In light of this sequence analysis, it is clear that the dicot snRNA variants do not differ in sequences implicated in RNA:RNA interactions with pre-mRNA. Instead, sequence differences occur in regions implicated in the binding of small ribonucleoproteins (snRNPs) to snRNAs and may result in the formation of unique snRNP particles.     

The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases.

The primary structure of the 200 kDa protein of purified HeLa U5 snRNPs (U5-200kD) was characterized by cloning and sequencing of its cDNA. In order to confirm that U5-200kD is distinct from U5-220kD we demonstrate by protein sequencing that the human U5-specific 220 kDa protein is homologous to the yeast U5-specific protein Prp8p. A 246 kDa protein (Snu246p) homologous to U5-200kD was identified in Saccharomyces cerevisiae. Both proteins contain two conserved domains characteristic of the DEXH-box protein family of putative RNA helicases and RNA-stimulated ATPases. Antibodies raised against fusion proteins produced from fragments of the cloned mammalian cDNA interact specifically with the HeLa U5-200kD protein on Western blots and co-immunoprecipitate U5 snRNA and to a lesser extent U4 and U6 snRNAs from HeLa snRNPs. Similarly, U4, U5 and U6 snRNAs can be co-immunoprecipitated from yeast splicing extracts containing an HA-tagged derivative of Snu246p with HA-tag specific antibodies. U5-200kD and Snu246p are thus the first putative RNA helicases shown to be intrinsic components of snRNPs. Disruption of the SNU246 gene in yeast is lethal and leads to a splicing defect in vivo, indicating that the protein is essential for splicing. Anti-U5-200kD antibodies specifically block the second step of mammalian splicing in vitro, demonstrating for the first time that a DEXH-box protein is involved in mammalian splicing. We propose that U5-200kD and Snu246p promote one or more conformational changes in the dynamic network of RNA-RNA interactions in the spliceosome.     

5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs

Immune precipitation assays with antibodies specific for 2,2,7-trimethylguanosine (m2,2,7(3)G) have been used to study the accessibility of the 5'-terminal m2,2,7(3)G-containing caps of eucaryotic small nuclear RNAs (snRNAs) either as naked RNAs or in intact small nuclear ribonucleoprotein (snRNPs). The antibody selectively precipitates snRNA species U1a, U1b, U2, U4, and U5 from total deproteinized RNA isolated from Ehrlich ascites cells. Binding by the antibody occurs via the m2,2,7(3)G moiety of the snRNAs' caps, since complex formation with the antibody can be completely abolished by excess nucleoside m2,2,7(3)G. The specificity of the antibody is further demonstrated by the complete absence of reaction with deproteinized snRNA species U6, the 5' terminus of which does not contain m2,2,7(3)G. Most importantly, the cap structures of the snRNAs U1a, U1b, U2, U4, and U5 are also accessible for anti-m2,2,7(3)G IgGs when intact snRNPs are reacted with the antibody. In this case, snRNP species U6 is coprecipitated, suggesting that there are intermolecular interactions between this and other snRNPs. Our data demonstrate that the 5'-terminal regions of the above snRNAs are not protected by the snRNP proteins. This finding is of special interest for snRNP species U1, and is discussed in terms of a model which proposes that the 5'-terminal region of U1 participates in the proper alignment of splice junctions in eucaryotic pre-mRNAs (Lerner, M. R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224).