Autoantibodies to ribonucleoprotein particles containing U2 small nuclear RNA.

Autoantibodies exclusively precipitating U1 and U2 small nuclear ribonucleoprotein (snRNP) particles [anti-(U1,U2)RNP] were detected in sera from four patients with autoimmune disorders. When tested by immunoblotting, these sera recognized up to four different protein antigens in purified mixtures of U1-U6 RNP particles. With purified antibody fractions eluted from individual antigen bands on nitrocellulose blots, each anti-(U1,U2)RNP serum precipitated U2 RNP by virtue of the recognition of a U2 RNP-specific B" antigen (mol. wt. 28 500). Antibodies to the U2 RNP-specific A' protein (mol. wt. 31 000) were found in only one serum. The B" antigen differs slightly in mol. wt. from the U1-U6 RNA-associated B/B' antigens and can be separated from this doublet by two-dimensional gel electrophoresis, due to its more acidic pI. In immunoprecipitation assays, the purified anti-B" antibody specificity also reacts with U1 RNPs which is due to cross-reactivity of the antibody with the U1 RNA-specific A protein, as demonstrated by immunoblotting using proteins from isolated U1 RNPs as antigenic material. Thus the A antigen not only bears unique antigenic sites for anti-A antibodies contained in anti-(U1)RNP sera, it also shares epitopes with the U2 RNP-specific B" antigen.     

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.     

Autoantibodies to nuclear lamin B in a patient with thrombopenia

We report the characterization of novel nucleus specific autoantibodies in the serum of a patient with systemic lupus erythematosus. Immunofluorescent staining of cycling cells and absorption experiments localized the antigen to the nuclear envelope. Two-dimensional gel electrophoretic analysis of immunoprecipitated nuclear proteins show the antigen to be an acidic polypeptide (IP approximately 5.4) of 68 kDa molecular mass. It has been identified as lamin B, one of the three major nuclear envelope polypeptides of mammalian cells. Antibodies shown to be polyclonal immunoglobulin Gs, were directed against determinant(s) of the protein that have apparently been conserved during evolution. They do not appear to be related to other autoantibodies present in the serum (anti-DNA and anti-platelet). The nuclear specificity shown by these antibodies further demonstrates the antigenicity of proteins related to intermediate filament proteins in patients with autoimmune disorders.     

Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures.

Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.     

U1 small nuclear ribonucleoprotein studied by in vitro assembly

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody- precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA- protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.     

Mg2+ induces a sharp and reversible transition in U1 and U2 small nuclear ribonucleoprotein configurations.

When U1 and U2 small nuclear ribonucleoproteins (snRNPs) purified by a procedure which preserves their immunoprecipitability by autoimmune antibodies (Hinterberger et al., J. Biol. Chem. 258:2604-2613, 1983), were submitted to extensive digestion with micrococcal nuclease, we found that their degradation pattern was sharply dependent upon magnesium concentration, indicating that they undergo a profound structural modification. At low Mg2+ (less than or equal to 5 mM), both particles only exhibit a core-resistant structure previously identified as being common to all but U6 snRNAs (Liautard et al., J. Mol. Biol. 162: 623-643, 1982). At high Mg2+ (greater than or equal to 7 mM), U1 and U2 snRNPs behave differently from one another. In U1 snRNP, most U1 snRNA sequence is protected, except for the 10 5'-terminal nucleotides presumably involved in splicing and a short sequence between nucleotides 102 and 108. Another region spanning nucleotides 60 to 79 is only weakly protected. This structural modification was demonstrated to be reversible. In U2 snRNP, the U2 snRNA sequence remains exposed in its 5' part up to nucleotide 92, and the 3'-terminal hairpin located outside the core structure becomes protected.     

U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle.

U4 and U6 RNAs of mammalian cells possess extensive intermolecular sequence complementarity and hence have the potential to base pair. A U4/U6 RNA complex, detectable in nondenaturing polyacrylamide gels, is released when human small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 RNAs are dissociated with proteinase K in the presence of sodium dodecyl sulfate. The released RNA/RNA complex dissociates with increasing temperature, consistent with the existence of specific base-pairing between the two RNAs. Since U6 RNA is selectively released from intact snRNPs under the same conditions required to dissociate the U4/U6 RNA complex, the RNA-RNA interaction may be sufficient to maintain U4 and U6 RNAs in the same snRNP particle. The biological implications of these findings are discussed.     

Ser/Arg-rich protein-mediated communication between U1 and U2 small nuclear ribonucleoprotein particles

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5' splice site, can positively influence utilization of an upstream 3' splice site via exon definition in both trans- and cis-splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5' splice site on trans-splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5' splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient trans-splicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis-spliced intron. Weakening of the 3' splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between U1 and U2 snRNPs whether this interaction is across introns or exons.     

Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection

The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus.     

A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

Evolutionary conservation of the U7 small nuclear ribonucleoprotein in Drosophila melanogaster

The U7 snRNP involved in histone RNA 3' end processing is related to but biochemically distinct from spliceosomal snRNPs. In vertebrates, the Sm core structure assembling around the noncanonical Sm-binding sequence of U7 snRNA contains only five of the seven standard Sm proteins. The missing Sm D1 and D2 subunits are replaced by U7-specific Sm-like proteins Lsm10 and Lsm11, at least the latter of which is important for histone RNA processing. So far, it was unknown if this special U7 snRNP composition is conserved in invertebrates. Here we describe several putative invertebrate Lsm10 and Lsm11 orthologs that display low but clear sequence similarity to their vertebrate counterparts. Immunoprecipitation studies in Drosophila S2 cells indicate that the Drosophila Lsm10 and Lsm11 orthologs (dLsm10 and dLsm11) associate with each other and with Sm B, but not with Sm D1 and D2. Moreover, dLsm11 associates with the recently characterized Drosophila U7 snRNA and, indirectly, with histone H3 pre-mRNA. Furthermore, dLsm10 and dLsm11 can assemble into U7 snRNPs in mammalian cells. These experiments demonstrate a strong evolutionary conservation of the unique U7 snRNP composition, despite a high degree of primary sequence divergence of its constituents. Therefore, Drosophila appears to be a suitable system for further genetic studies of the cell biology of U7 snRNPs.     

Interaction domains and nuclear targeting signals in subunits of the U2 small nuclear ribonucleoprotein particle-associated splicing factor SF3a

Human splicing factor SF3a is a component of the mature U2 small nuclear ribonucleoprotein particle (snRNP) and its three subunits of 60, 66, and 120 kDa are essential for splicing in vitro and in vivo. The SF3a heterotrimer forms in the cytoplasm and enters the nucleus independently of the U2 snRNP. Here, we have analyzed domains required for in vitro interactions between the SF3a subunits. Our results indicate that the SF3a66-SF3a120 interaction is mediated by a 27-amino acid region in SF3a120 C-terminal to the second suppressor-of-white-apricot and prp21/spp91 domain and amino acids 108-210 of SF3a66. Neither of these sequences contains known structural motifs, suggesting that the interaction domains are novel. Moreover, an ～100-amino acid region, including the SURP2 domain of SF3a120 but extending into neighboring regions, is sufficient for binding to SF3a60. Analysis of determinants for nuclear import of SF3a demonstrates that SF3a120 provides the major nuclear localization signal and SF3a60 contributes to nuclear import.     

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 structure of the human U4/U6 small nuclear ribonucleoprotein particle-specific SnuCyp-20, a nuclear cyclophilin

The cyclophilin SnuCyp-20 is a specific component of the human U4/U6 small nuclear ribonucleoprotein particle involved in the nuclear splicing of pre-mRNA. It stably associates with the U4/U6-60kD and -90kD proteins, the human orthologues of the Saccharomyces cerevisiae Prp4 and Prp3 splicing factors. We have determined the crystal structure of SnuCyp-20 at 2.0-A resolution by molecular replacement. The structure of SnuCyp-20 closely resembles that of human cyclophilin A (hCypA). In particular, the catalytic centers of SnuCyp-20 and hCypA superimpose perfectly, which is reflected by the observed peptidyl-prolyl-cis/trans-isomerase activity of SnuCyp-20. The surface properties of both proteins, however, differ significantly. Apart from seven additional amino-terminal residues, the insertion of five amino acids in the loop alpha1-beta3 and of one amino acid in the loop alpha2-beta8 changes the conformations of both loops. The enlarged loop alpha1-beta3 is involved in the formation of a wide cleft with predominantly hydrophobic character. We propose that this enlarged loop is required for the interaction with the U4/U6-60kD protein.     

Pre-mRNA splicing in vitro requires intact U4/U6 small nuclear ribonucleoprotein

Selective cleavage of U4 or U6 RNA in a HeLa cell nuclear extract inhibits splicing of pre-mRNAs containing an adenovirus or a simian virus 40 intron. RNAs in the U4/U6 small nuclear ribonucleoprotein (snRNP) were specifically degraded with RNAase H and deoxyoligonucleotides. Two oligomers complementary to U4 RNA and two complementary to U6 RNA cleave their target RNAs and inhibit the appearance of both spliced products and reaction intermediates. Splicing is reconstituted by mixing an extract containing cleaved U4 or U6 RNA with one in which splicing has been inhibited by degrading U2 RNA. All four abundant snRNPs, containing U1, U2, U5, or U4 and U6 RNAs, are now implicated in pre-mRNA splicing. Possible interactions of the U4/U6 snRNP with other components of the splicing complex are discussed.     

Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5

Pre-mRNA binding to the yeast U2 small nuclear ribonucleoprotein (snRNP) during prespliceosome formation requires ATP hydrolysis, the highly conserved UACUAAC box of the branch point region of the pre-mRNA, and several factors. Here we analyzed the binding of a radiolabeled 2'-O-methyl oligonucleotide complementary to U2 small nuclear RNA to study interactions between the UACUAAC box, U2 snRNP, and Prp5p, a DEAD box protein necessary for prespliceosome formation. Binding of the 2'-O-methyl oligonucleotide to the U2 snRNP in yeast cell extract was assayed by gel electrophoresis. Binding was rapid, enhanced by ATP, and dependent on the integrity and conformation of the U2 snRNP. It was also stimulated by Prp5p that was found to associate physically with U2 snRNP. In vitro heat inactivation of the temperature-sensitive prp5-1 mutant extract decreased oligonucleotide binding to U2 and the ATP enhancement of binding by 3-fold. Furthermore, the temperature-sensitive prp5-1 mutation maps to the ATP-binding motif I within the helicase-like domain. Thus the catalytic activity of Prp5p likely promotes a conformational change in the U2 snRNP.     

Heterogeneous nuclear ribonucleoprotein H is required for optimal U11 small nuclear ribonucleoprotein binding to a retroviral RNA-processing control element: implications for U12-dependent RNA splicing

An RNA-processing element from Rous sarcoma virus, the negative regulator of splicing (NRS), represses splicing to generate unspliced RNA that serves as mRNA and as genomic RNA for progeny virions and also promotes polyadenylation of the unspliced RNA. Integral to NRS function is the binding of U1 small nuclear ribonucleoprotein (snRNP), but its binding is controlled by U11 snRNP that binds to an overlapping site. U11 snRNP, the U1 counterpart for splicing of U12-dependent introns, binds the NRS remarkably well and requires G-rich elements just downstream of the consensus U11 binding site. We present evidence that heterogeneous nuclear ribonucleoprotein (hnRNP) H binds to the NRS G-rich elements and that hnRNP H is required for optimal U11 binding in vitro. It is further shown that hnRNP H (but not hnRNP F) can promote U11 binding and splicing from the NRS in vivo when tethered to the RNA as an MS2 fusion protein. Interestingly, 17% of the naturally occurring U12-dependent introns have at least two potential hnRNP H binding sites positioned similarly to the NRS. For two such introns from the SCN4A and P120 genes, we show that hnRNP H binds to each in a G-tract-dependent manner, that G-tract mutations strongly reduce splicing of minigene RNA, and that tethered hnRNP H restores splicing to mutant RNA. In support of a role for hnRNP H in both splicing pathways, hnRNP H antibodies co-precipitate U1 and U11 small nuclear ribonucleoproteins. These results indicate that hnRNP H is an auxiliary factor for U11 binding to the NRS and that, more generally, hnRNP H is a splicing factor for a subset of U12-dependent introns that harbor G-rich elements.     

The U1 RNA-binding site of the U1 small nuclear ribonucleoprotein (snRNP)-associated A protein suggests a similarity with U2 snRNPs.

The site of interaction between human U1 RNA and one of its uniquely associated proteins, A, was examined with in vitro binding assays. The A protein bound directly to stem-loop II of U1 RNA in a region which exhibits sequence similarity to U2 RNA. The similarity with U2 RNA was in a region that has been shown to interact with a U2 RNA-associated protein. The A protein-binding site on U1 RNA overlapped a previously described epitope for an RNA-specific human autoantibody (S. L. Deutscher and J. D. Keene, Proc. Natl. Acad. Sci. USA 85:3299-3303, 1988), supporting the hypothesis that the anti-RNA antibody originated as an anti-idiotypic response to A protein-specific autoantibodies.