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The U1 small nuclear ribonucleoprotein particle (snRNP) has an important function in the early formation of the spliceosome, the multicomponent complex in which pre-mRNA splicing takes place. The nuclear localization signals of two of the three U1 snRNP-specific proteins, U1-70K and U1A, have been mapped. Both proteins are transported actively to the nucleus. Here we show by microinjection of Xenopus laevis oocytes that the third U1 snRNP-specific protein, U1C, passively enters the nucleus. Furthermore, we show that in both X. laevis oocytes and cultured HeLa cells mutant U1C proteins that are not able to bind to the U1 snRNP do not accumulate in the nucleus, indicating that nuclear accumulation of U1C is due to incorporation of the protein into the U1 snRNP.

The Ro ribonucleoprotein particle (RoRNP) is the target of a variety of specific anti-protein autoantibodies produced by patients with systemic autoimmune diseases. RoRNPs, the function of which remains elusive, appear to be rather heterogeneous in terms of their molecular composition. Among several Ro proteins, a protein of 52 kD (Ro52) has been identified, but its association with RoRNPs remains questionable. In this study, we first mapped the Ro52 regions that are accessible at the surface of the isolated protein, by means of antibodies raised in rabbits, against 39 overlapping synthetic peptides covering the whole Ro52 molecule and using several different methods, including various ELISA formats and Western blotting (based on the use of recombinant Ro52) and immunoprecipitation of in vitro translated Ro52. Then, the whole set of anti-peptide antibodies was used to attempt to immunoprecipitate RoRNPs from a cell extract of K562 cells. RoRNPs, especially Ro(hY3)RNPs, were effectively immunoprecipitated by certain anti-peptide antibodies, indicating that Ro52 protein is associated with at least a subset of RoRNP particles. As expected, a number of epitopes available on the isolated Ro52 molecule are no longer accessible when Ro52 is associated with the RoRNP. Conversely, neotopes, among them at least one corresponding to a previously characterized epitope recognized by patients' antibodies, are only present at the surface of the particle and not detectable on the isolated Ro52 protein.

In this study we have used a number of monoclonal antibodies with various anti-Sm specificities originating from MRL/lpr mice to map B cell epitopes of the Sm-B/B' and Sm-D1 proteins. Selection of Sm-B subfragments reactive with the Sm-B/B'-specific monoclonal antibody ANA125 from a DNaseI fragment expression library revealed that the epitope recognized by this monoclonal antibody is located between amino acids 146 and 158: GRGTVAAAAAAAT. The epitopes recognized by two distinct Sm-D1-specific monoclonal antibodies, 7.13 and ANA127, appeared to be located in the carboxy-terminal region of the protein as revealed by immunoprecipitation of in vitro translated deletion mutants of Sm-D1. These epitopes are probably identical and not simply composed of a GR repeat, which is a characteristic feature of this part of the protein. Immunoprecipitation of in vitro translated deletion mutants of both Sm-B and Sm-D1 was also employed to determine the sequence requirements for recognition by two monoclonal antibodies that are cross-reactive with several Sm proteins, Y12 and ANA128. The epitope recognized by these two monoclonal antibodies is probably also identical and composed by the juxtaposition of several regions in the folded protein. The low, but significant, level of immunoprecipitation of truncated versions of both Sm-B and Sm-D1, suggests that the Sm domain, which is shared by all Sm proteins, in particular the amino-terminal part of the Sm1 motif of Sm-B and Sm-D1, plays an important role in formation of the cross-reactive epitope and might contribute to cross-reactivity with other Sm proteins. The results of immunoprecipitation experiments with cellular extracts show that the epitopes recognized by all anti-Sm monoclonal antibodies used in this study are accessible in the assembled snRNPs.

The La (SS-B) autoantigen is a 47-kDa protein which binds to the 3' termini of nascent RNA polymerase III transcripts and to a number of viral leader RNAs. The La protein plays a direct role in the termination of RNA polymerase III transcription and recent findings have suggested an additional role in several aspects of translation of (viral) mRNAs. In this study we have addressed the intracellular trafficking of the La protein and characterized cis-acting elements involved in nuclear import and retention in Xenopus laevis oocytes by microinjection of in vitro translated La protein. The steady-state distribution of recombinant human La protein was, like the endogenous Xenopus La protein, mainly nuclear. Nuclear import of La appeared to be energy-dependent and is governed by a nuclear localization signal (NLS) located in the extreme C-terminal part of the protein, resembling the consensus bipartite NLS. Another sequence element in La, which completely corresponds to the bipartite NLS consensus, appeared to be nonfunctional in nuclear import of the La protein. Nuclear accumulation of La was found to be mediated by retention in the nuclear compartment. The N-terminal RNA binding domain of La is not involved in this retention, but sequence elements in the central region of the polypeptide (amino acids 165 to 337) appear to be required. Amino acids 266-269 as well as 313-337 were found to be of major importance for retention in the nucleus.

Immunization of Balb/c mice with a homogeneously purified recombinant human La/SS-B protein resulted in activation of an autoreactive B cell secreting a novel monoclonal anti-La antibody termed La4B6. La4B6 reacted with La protein from a variety of sources including human, bovine, rat and mouse. ATP blocked the binding of La4B6 to recombinant La protein. The human epitope was identified as consisting of the amino acid sequence SKGRRFKGKGKGN, which includes the proposed ATP-binding site of the La protein. In the human and bovine La protein, the epitope exists as a continuous amino acid sequence. In rat and mouse the epitope was found to consist of the amino acid sequence SKG interrupted by a species-specific insert of 16 amino acids, and followed by the second half of the epitope, the amino acid sequence RRFKGKGKGN. Our data suggest that in the case of the rat and mouse La proteins the two separated parts of the epitope are able to form a conformational epitope which looks similar to the continuous human epitope.

We investigated the role of humoral factors in the pathogenesis of Coxsackie B1 virus-induced murine myositis (CB1-myositis). At 2, 4 and 8 weeks after inoculation, serum was studied for circulating immune complexes (CIC) (Raji-cell assay), haemolytic complement activity (CH50 titre) and anticytoplasmic autoantibodies (Western blotting, immunoprecipitation) in relation to degree of mononuclear cell infiltration and muscle fibre necrosis. At 2, 4 and 8 weeks, cell infiltration correlated positively to muscle fibre necrosis. From 2 weeks on, moderate quantities of CIC were found in nearly all CB1-inoculated mice, but without correlation to histological changes. Except for a positive correlation of CH50 titre to muscle necrosis at 4 weeks (r = 0.60; P = 0.02), CH50 titres did not correlate to muscle lesions. Anticytoplasmic and other known autoantibody specificities were absent. In conclusion, first, in CB1-myositis CIC occurred from 2 weeks on, but no correlative evidence was found for their involvement in pathogenesis, neither for that of complement nor for anticytoplasmic autoantibodies. Secondly, cell infiltration correlated positively to muscle necrosis, underscoring the importance of cellular mechanisms. Thus, our data do not support, or conclusively exclude, a role for humoral processes in CB1-myositis.

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2 alpha to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2 alpha shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.