Three novel functional variants of human U5 small nuclear RNA.

We have identified and characterized three new variants of U5 small nuclear RNA (snRNA) from HeLa cells, called U5D, U5E, and U5F. Each variant has a 2,2,7-trimethylguanosine cap and is packaged into an Sm-precipitable small nuclear ribonucleoprotein (snRNP) particle. All retain the evolutionarily invariant 9-base loop at the top of stem 1; however, numerous base changes relative to the abundant forms of U5 snRNA are present in other regions of the RNAs, including a loop that is part of the yeast U5 minimal domain required for viability and has been shown to bind a protein in HeLa extracts. U5E and U5F each constitute 7% of the total U5 population in HeLa cells and are slightly longer than the previously characterized human U5 (A, B, and C) species. U5D, which composes 5% of HeLa cell U5 snRNAs, is present in two forms: a full-length species, U5DL, and a shorter species, U5DS, which is truncated by 15 nucleotides at its 3' end and therefore resembles the short form of U5 (snR7S) in Saccharomyces cerevisiae. We have established conditions that allow specific detection of the individual U5 variants by either Northern blotting (RNA blotting) or primer extension; likewise, U5E and U5F can be specifically and completely degraded in splicing extracts by oligonucleotide-directed RNase H cleavage. All variant U5 snRNAs are assembled into functional particles, as indicated by their immunoprecipitability with anti-(U5) RNP antibodies, their incorporation into the U4/U5/U6 tri-snRNP complex, and their presence in affinity-purified spliceosomes. The higher abundance of these U5 variants in 293 cells compared with that in HeLa cells suggests possible roles in alternative splicing.     

A protein that specifically recognizes the 3' splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein

Using a protein blotting method for the detection of nucleic acid binding proteins, we have identified in HeLa cell nuclear extracts an intron binding protein (IBP) that selectively recognizes the 3' splice site region of mammalian pre-mRNAs. The binding site was accurately delineated using oligonucleotides complementary to human beta-globin pre-mRNA. It spans the 3' splice site AG dinucleotide and the crucial polypyrimidine stretch upstream, but includes neither the branchpoint nor the lariat structure. Although the technique used here shows that the binding specificity is an intrinsic property of IBP and does not depend on snRNA-pre-mRNA interactions, it comigrates with U5 snRNP and is immunoprecipitated by anti-Sm antibody. This strongly suggests that IBP belongs to U5 snRNP. We propose that it is involved in one of the earliest steps of the splicing reaction by mediating the interaction of U5 snRNP with the 3' splice site.     

The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro.

Stable association of the eight common Sm proteins with U1, U2, U4 or U5 snRNA to produce a spliceosomal snRNP core structure is required for snRNP biogenesis, including cap hypermethylation and nuclear transport. Here, the assembly of snRNP core particles was investigated in vitro using both native HeLa and in vitro generated Sm proteins. Several RNA-free, heteromeric protein complexes were identified, including E.F.G, B/B'.D3 and D1.D2.E.F.G. While the E.F.G complex alone did not stably bind to U1 snRNA, these proteins together with D1 and D2 were necessary and sufficient to form a stable U1 snRNP subcore particle. The subcore could be chased into a core particle by the subsequent addition of the B/B'.D3 protein complex even in the presence of free competitor U1 snRNA. Trimethylation of U1 snRNA's 5' cap, while not observed for the subcore, occurred in the stepwise-assembled U1 snRNP core particle, providing evidence for the involvement of the B/B' and D3 proteins in the hypermethylation reaction. Taken together, these results suggest that the various protein heterooligomers, as well as the snRNP subcore particle, are functional intermediates in the snRNP core assembly pathway.     

In vitro assembly of the mouse U14 snoRNP core complex and identification of a 65-kDa box C/D-binding protein.

The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) that have been categorized into two major families based on evolutionarily conserved sequence elements. U14 snoRNA is a member of the larger, box C/D snoRNA family and possesses nucleotide box C and D consensus sequences. In previous studies, we have defined a U14 box C/D core motif that is essential for intronic U14 snoRNA processing. These studies also revealed that nuclear proteins that recognize boxes C/D are required. We have now established an in vitro U14 snoRNP assembly system to characterize protein binding. Electrophoretic mobility-shift analysis demonstrated that all the sequences and structures of the box C/D core motif required for U14 processing are also necessary for protein binding and snoRNP assembly. These required elements include a base paired 5',3' terminal stem and the phylogenetically conserved nucleotides of boxes C and D. The ability of other box C/D snoRNAs to compete for protein binding demonstrated that the box C/D core motif-binding proteins are common to this family of snoRNAs. UV crosslinking of nuclear proteins bound to the U14 core motif identified a 65-kDa mouse snoRNP protein that requires boxes C and D for binding. Two additional core motif proteins of 55 and 50 kDa were also identified by biochemical fractionation of the in vitro-assembled U14 snoRNP complex. Thus, the U14 snoRNP core complex is a multiprotein particle whose assembly requires nucleotide boxes C and D.     

Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro.

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.     

Recognition of U1 and U2 small nuclear RNAs can be altered by a 5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle (snRNP) B" protein and through interactions with U2 snRNP-A'' protein.

We have investigated the sequence elements influencing RNA recognition in two closely related small nuclear ribonucleoprotein particle (snRNP) proteins, U1 snRNP-A and U2 snRNP-B". A 5-amino-acid segment in the RNA-binding domain of the U2 snRNP-B" protein was found to confer U2 RNA recognition when substituted into the corresponding position in the U1 snRNP-A protein. In addition, B", but not A, was found to require the U2 snRNP-A' protein as an accessory factor for high-affinity binding to U2 RNA. The pentamer segment in B" that conferred U2 RNA recognition was not sufficient to allow the A' enhancement of U2 RNA binding by B", thus implicating other sequences in this protein-protein interaction. Sequence elements involved in these interactions have been localized to variable loops of the RNA-binding domain as determined by nuclear magnetic resonance spectroscopy (D. Hoffman, C.C. Query, B. Golden, S.W. White, and J.D. Keene, Proc. Natl. Acad. Sci. USA, in press). These findings suggest a role for accessory proteins in the formation of RNP complexes and pinpoint amino acid sequences that affect the specificity of RNA recognition in two members of a large family of proteins involved in RNA processing.     

Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs

The U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) involved in pre-mRNA splicing contain seven Sm proteins (B/B', D1, D2, D3, E, F, and G) in common, which assemble around the Sm site present in four of the major spliceosomal small nuclear RNAs (snRNAs). These proteins share a common sequence motif in two segments, Sm1 and Sm2, separated by a short variable linker. Crystal structures of two Sm protein complexes, D3B and D1D2, show that these proteins have a common fold containing an N-terminal helix followed by a strongly bent five-stranded antiparallel beta sheet, and the D1D2 and D3B dimers superpose closely in their core regions, including the dimer interfaces. The crystal structures suggest that the seven Sm proteins could form a closed ring and the snRNAs may be bound in the positively charged central hole.     

The box C/D motif directs snoRNA 5'-cap hypermethylation

The 5′-cap structure of most spliceosomal small nuclear RNAs (snRNAs) and certain small nucleolar RNAs (snoRNAs) undergoes hypermethylation from a 7-methylguanosine to a 2,2,7-trimethylguanosine structure. 5′-Cap hypermethylation of snRNAs is dependent upon a conserved sequence element known as the Sm site common to most snRNAs. Here we have performed a mutational analysis of U3 and U14 to determine the cis-acting sequences required for 5′-cap hypermethylation of Box C/D snoRNAs. We have found that both the conserved sequence elements Box C (termed C′ in U3) and Box D are necessary for cap hypermethylation. Furthermore, the terminal stem structure that is formed by sequences that flank Box C (C′ in U3) and Box D is also required. However, mutation of other conserved sequences has no effect on hypermethylation of the cap. Finally, the analysis of fragments of U3 and U14 RNAs indicates that the Box C/D motif, including Box C (C′ in U3), Box D and the terminal stem, is capable of directing cap hypermethylation. Thus, the Box C/D motif, which is important for snoRNA processing, stability, nuclear retention, protein binding, nucleolar localization and function, is also necessary and sufficient for cap hypermethylation of these RNAs.     

The Drosophila melanogaster Cajal body

Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB-specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.     

Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5'', 3'' terminal stem.

U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA. Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function. In this study, we have further defined three structural elements required for U14 accumulation. The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem. The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin. New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified. An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C. The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem. The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.     

Cytoplasmic assembly and nuclear accumulation of mature small nuclear ribonucleoprotein particles

The assembly pathway of small nuclear ribonucleoprotein (snRNP) particles in the cytoplasm of L929 mouse fibroblasts was analyzed by observing the nuclear accumulation of snRNP proteins. Immunoprecipitations of nuclear and cytoplasmic fractions after a pulse label and chase indicate that the snRNP D, E, F, and G proteins assemble first, followed by the small nuclear RNA (snRNA), then the snRNP B protein and, in the case of the U1 snRNP, the A and C proteins. The snRNP B' protein is not detected in the L929 cells. The U1-specific A and C proteins can enter the nucleus in the absence of snRNP assembly, suggesting that these proteins exchange on the mature nuclear snRNP particles. Two-dimensional electrophoresis using nonequilibrium pH gradient electrophoresis identifies the A, B, B", C, D, E, F, and G proteins in a distribution similar to that reported previously by immunoprecipitation (Sauterer, R. A., and Zieve, G. W. (1989) J. Biol. Chem., submitted for publication). The D protein appears in multiple isoelectric variants in the cytoplasm and shifts toward more basic variants during maturation. Kinetic experiments analyzed by two-dimensional electrophoresis indicate a quantitative maturation of the cytoplasmic B protein into nuclear particles. Quantitative densitometry of immunoprecipitated stable nuclear snRNPs labeled with [35S] methionine corrected for the published methionine content of the A, B, C, D, and E proteins indicates that the mature nuclear U1 snRNP probably contains four copies of D, two copies each of B, C, and A, and one copy of E.     

The C-terminal RG dipeptide repeats of the spliceosomal Sm proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti-Sm autoantibodies

The Sm proteins B/B', D1, D2, D3, E, F, and G are components of the small nuclear ribonucleoproteins U1, U2, U4/U6, and U5 that are essential for the splicing of pre-mRNAs in eukaryotes. D1 and D3 are among the most common antigens recognized by anti-Sm autoantibodies, an autoantibody population found exclusively in patients afflicted with systemic lupus erythematosus. Here we demonstrate by protein sequencing and mass spectrometry that all arginines in the C-terminal arginine-glycine (RG) dipeptide repeats of the human Sm proteins D1 and D3, isolated from HeLa small nuclear ribonucleoproteins, contain symmetrical dimethylarginines (sDMAs), a posttranslational modification thus far only identified in the myelin basic protein. The further finding that human D1 individually overexpressed in baculovirus-infected insect cells contains asymmetrical dimethylarginines suggests that the symmetrical dimethylation of the RG repeats in D1 and D3 is dependent on the assembly status of D1 and D3. In antibody binding studies, 10 of 11 anti-Sm patient sera tested, as well as the monoclonal antibody Y12, reacted with a chemically synthesized C-terminal peptide of D1 containing sDMA, but not with peptides containing asymmetrically modified or nonmodified arginines. These results thus demonstrate that the sDMA-modified C terminus of D1 forms a major linear epitope for anti-Sm autoantibodies and Y12 and further suggest that posttranslational modifications of Sm proteins play a role in the etiology of systemic lupus erythematosus.     

Drosophila SNF/D25 combines the functions of the two snRNP proteins U1A and U2B' that are encoded separately in human, potato, and yeast.

The plant and vertebrate snRP proteins U1A and U2B' are structurally closely related, but bind to different U snRNAs. Two additional related snRNP proteins, the yeast U2B' protein and Drosophila SNF/D25 protein, are analyzed here. We show that the previously described yeast open reading frame YIB9w encodes yeast U2B' as judged by the fact that the protein encoded by YIB9w bindsto stem-loop IV of yeast U2 snRNA in vitro and is part of the U2 snRNP in vivo. In contrast to the human U2B' protein, specific binding of yeast U2B' to RNA in vitro can occur in the absence of an accessory U2A' protein. The Drosophila SNF-D25 protein, unlike all other U1A/U2B' proteins studied to date, is shown to be a component of both U1 and U2 snRNPs. In vitro, SNF/D25 binds to U1 snRNA on itsown and to U2 snRNA in the presence of either the human U2A' protein or of Drosophila nuclear extract. Thus, its RNA-binding properties are the sum of those exhibited by human or potato U1A and U2B' proteins. Implications for the role of SNF/D25 in alternative splicing, and for the evolution of the U1A/U2B' protein family, are discussed.     

Chemical cross-linking of Sm and RNP antigenic proteins

Nuclear extracts, competent for in vitro premessenger RNA splicing, were chemically cross-linked with thiol-reversible reagents in order to study the organization of proteins within ribonucleoprotein particles (RNPs) containing uridine-rich small nuclear RNAs (UsnRNPs). The distribution of select UsnRNP antigens within cross-linked complexes was determined by Western blotting of diagonal two-dimensional gels. On the basis of calculations from the molecular weights of cross-linked complexes containing UsnRNP common proteins B', B, and D, it is proposed that each of these proteins was associated with UsnRNP common proteins E and G. In addition, D' is proposed to be positioned close to D. The spatial distribution of UsnRNP common proteins was such that B' and B could not be cross-linked to D. The data also suggested that the 63-kDa U1 snRNP specific protein was cross-linked to other U1-specific proteins, particularly C, but not to the UsnRNP common proteins. We propose that part of the UsnRNP core of common proteins contains at least two asymmetrical copies of B':B:D:D':E:G with stoichiometries of 2:1:1:1:1:1 and 1:2:1:1:1:1.     

Rrp47p is an exosome-associated protein required for the 3' processing of stable RNAs

Related exosome complexes of 3'-->5' exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg(2+)-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3'-->5' exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3' processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Delta strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3'-extended nuclear pre-mRNAs or the cytoplasmic 3'-->5' mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.     

Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. They consist of uridylate-rich small nuclear RNA complexed with several proteins. snRNP particles U1, U2, U4/U6, and U5 all contain a common protein core consisting of proteins B'/B, D, D', E, F, and G. In addition to this core, U1 snRNP particles contain proteins 70K, A, and C, whereas U2 snRNP particles contain proteins A' and B". Almost any of the small nuclear RNA-associated polypeptides is targeted by autoantibodies in the sera from patients with SLE or related connective tissue diseases. We immunized a genetically non-autoimmune mouse with recombinant human B" protein and obtained three mAb reactive with native U2 snRNP particles. Two of these mAb particles cross-reacted with U1 snRNP, 9A9 and 11A1, via epitopes present on the U2 snRNP B" protein as well as on the U1 snRNP-specific A protein. A third mAb 4g3, reacted exclusively with U2 snRNP via a unique epitope on protein B". Two epitopes mapped at the carboxy-terminal region of the B" protein, whereas binding of the third mAb involved both amino- and carboxy-terminal amino acids of the B" protein. Epitope mapping, employing a DNAse I fragment library of the B" cDNA, revealed that the three mAb-reactive sites were discontinuous. Autoantibodies in sera from patients with SLE and other connective tissue diseases competed for binding with the mAb, implying that the mAb define a major autoantibody-reactive region on protein B".     

RNA-protein organization of U1, U5 and U4-U6 small nuclear ribonucleoproteins in HeLa cells

Small nuclear ribonucleoproteins (snRNPs) containing U1 and U5 snRNAs from HeLa cells have been fractionated using a combination of isopycnic centrifugation in cesium chloride and ion-exchange chromatography on DEAE-Sepharose. The procedure is based on the extreme stability conferred upon snRNPs by Mg2+ enabling them to withstand the very high ionic strength that prevails in cesium chloride. U1 snRNP prepared by this method contains all nine major proteins (68K, A, B, B', C, D, E, F, G) corresponding to those previously identified by immunoprecipitation and is therefore precipitable by anti-RNP and anti-Sm antibodies. U5 snRNP purified in this way contains the common D to G proteins and is also enriched in a 25 X 10(3) Mr protein that may be U5 snRNP-specific. The core-resistant U5 snRNA sequence (nucleotide 84 to 3' OH) covered by D to G proteins is extended by only six nucleotides. A similar situation is seen in U4-U6 snRNP, which we have obtained in a sufficiently pure form to examine protected sequences. However, the core-resistant sequence of U4 (nucleotide 116 to 3' OH) in U4-U6 snRNP is extended by 37 nucleotides, suggesting that the protein composition of this particle could be more complex than that of U5 snRNP. The ribonucleoprotein organization of snRNPs is summarized and discussed in view of our current knowledge on snRNA sequences protected by proteins.     

Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome

Autoantibodies directed against the U2 small nuclear ribonucleoprotein (snRNP) have been found in the serum of a patient with scleroderma-polymyositis overlap syndrome. This specificity, called anti-(U2)-RNP, is distinct from all previously described autoantibodies, including those that precipitate related snRNPs: anti-Sm antibodies, which react with the entire set of U1, U2, U4, U5, and U6 snRNPs, and anti-(U1)RNP antibodies, which recognize only U1 snRNPs. From HeLa cell extracts, anti-(U2)RNP immunoprecipitates predominantly one 32P-labeled RNA species, identified as U2 small nuclear RNA, and six [35S]methionine-labeled protein bands, A' (Mr = 32,000), B (Mr = 28,000), D (Mr = 16,000), E (Mr = 13,000), F (Mr = 12,000), and G (Mr = 11,000). Protein blot analysis reveals that the A' protein carries (U2)RNP antigenic determinant(s) and therefore represents a polypeptide unique to the U2 snRNP; the B protein associated with U2 snRNPs may also be unique. Like U1 and the other Sm snRNPs, U2 snRNPs occupy a nuclear, non-nucleolar location and are antigenically conserved from insects to man. An antibody specific for the U2 snRNP will be useful in deciphering the function of this particle.     

C-reactive protein reacts with the U1 small nuclear ribonucleoprotein

C-reactive protein (CRP) was found to produce a small, discrete, speckled fluorescence pattern in the nucleus of HEp-2 cells. Double staining with anti-RNP serum and CRP produced very similar staining patterns. By counterimmunoelectrophoresis CRP was bound to extractable nuclear antigens found in rabbit thymus extract. The reactive components of the extract were only partially sensitive to treatment with RNase. CRP immunoprecipitated the U1 RNA species from [32P]labeled HeLa cells and the protein bands of the Sm/RNP complex from [35S]-methionine-labeled HeLa cells. By blotting, CRP bound to several discrete bands in a calcium-dependent, PC-inhibitable manner. Two of the bands comigrated with the 70K protein band associated with the U1 snRNP, and its major breakdown product. Binding to these bands was inhibited by both EDTA and PC indicating that CRP binds these proteins through the PC-binding site. Binding to the 70K protein of the U1 snRNP was confirmed by reactivity with the recombinant 70K protein in a dot blot. These findings indicate the CRP binds to the U1-RNP snRNP particle. Considering the ability of CRP to inhibit antibody responses to its ligands and its ability to activate C and promote phagocytosis it is suggested that CRP may play a role in the regulation of autoantibody responses to nuclear Ag.