Ro Ribonucleoproteins Research Articles

Are the Ro RNP‐associated Y RNAs concealing microRNAs? Y RNA‐derived miRNAs may be involved in autoimmunity

Here we discuss the hypothesis that the RNA components of the Ro ribonucleoproteins (RNPs), the Y RNAs, can be processed into microRNAs (miRNAs). Although Ro RNPs, whose main protein components Ro60 and La are targeted by the immune system in several autoimmune diseases, were discovered many years ago, their function is still poorly understood. Indeed, recent data show that miRNA-sized small RNAs can be generated from Y RNAs. This hypothesis leads also to a model in which Ro60 acts as a modulator in the Y RNA-derived miRNA biogenesis pathway. The implications of these Y RNA-derived miRNAs, which may be specifically produced under pathological circumstances such as in autoimmunity or during viral infections, for the enigmatic function of Ro RNPs are discussed.

Purification of antigenically intact Ro ribonucleoproteins; biochemical and immunological evidence that the 52-kD protein is not a Ro protein.

Anti-Ro sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functions are unknown and whose exact composition remains controversial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide (p52) has been proposed to be a stable component of the Ro RNPs. To confirm the immunological studies supporting this hypothesis, we have biochemically purified Ro RNPs from HeLa cells using non-denaturing conditions. Ro RNPs segregated into three distinct populations, one of which only contained hY5 RNA (RohY5 RNPs). No p52 co-purified with Ro RNPs. Despite the absence of p52, purified Ro RNPs had biochemical and immunological properties identical to those of unfractionated Ro RNPs. Many anti-Ro sera only recognize p52 in immunoblots, and are said to be monospecific anti-p52. Preincubation with purified RohY5 RNPs (free of p52) of all human anti-Ro (including so-called monospecific anti-p52) sera abolished their capacity to immunoprecipitate Ro RNPs from unfractionated HeLa cell extracts. Conversely, preincubation of anti-Ro sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target intact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrary to previous reports, p52 protein is not a stable component of antigenically intact Ro RNPs.

Crystal Structure of Rsr, an Ortholog of the Antigenic Ro Protein, Links Conformational Flexibility to RNA Binding Activity

Ro ribonucleoproteins are a class of antigenic ribonucleoproteins associated with rheumatic autoimmune diseases like systemic lupus erythematosus and Sjögrens syndrome in humans. Ro ribonucleoproteins are mostly composed of the 60-kDa Ro protein and small cytoplasmic RNAs, called Y RNAs, of unknown function. In eukaryotes, where Ro has been found to associate with damaged or mutant RNAs, it has been suggested that Ro may play a role in RNA quality control. In the radiation-resistant bacterium Deinococcus radiodurans and some eukaryotes, Ro has also been implicated in cell survival following UV damage. Here we present the first high resolution structure of a prokaryotic Ro ortholog, Rsr from D. radiodurans. The structure has been solved to 1.9 A resolution and shows distinct differences when compared with the eukaryotic apo- and RNA-bound Ro structures. Rsr is composed of two domains: a helical RNA binding domain and a mixed "von Willebrand factor A-like" domain containing a divalent metal binding site. Although the individual domains of Rsr are similar to the eukaryotic Ro, significantly large differences are seen at the interface of the two domains. Since this interface communicates with the conserved central cavity of Ro, which is implicated in RNA binding, changes at this interface could potentially influence RNA binding by Ro. Although the apo-Rsr protein is monomeric, Rsr binds Y RNA to form multimers of approximately 12 molecules of a 1:1 Rsr-Y RNA complex. Rsr binds D. radiodurans Y RNA with low nanomolar affinity, comparable with previously characterized eukaryotic Ro orthologs.

Autoantibody-associated congenital heart block: the clinical perspective.

Congenital heart block (CHB) can occur in association with structural heart disease, such as atrioventricular septal defects, left atrial isomerism, and abnormalities of the great arteries, with tumors, such as mesotheliomas, or as an isolated defect. In 1928, Aylward reported the occurrence of CHB in two children whose mother "suffered from Mikulicz's disease." This curious clinical observation was further solidified by the 1970s, with reports of CHB in children whose mothers had autoimmune diseases and that the maternal sera contained antibodies to Ro ribonucleoproteins. It was subsequently reported that many mothers also had antibodies to La. Other abnormalities affecting the skin, liver, and blood elements were associated with anti-Ro/La antibodies in the maternal and fetal circulation, and are now grouped under the heading of neonatal lupus syndromes. Neonatal lupus was termed because the cutaneous lesions of the neonate resembled those seen in systemic lupus erythematosus.

Heterogeneity of human Ro ribonucleoproteins (RNPS): nuclear retention of Ro RNPS containing the human hY5 RNA in human and mouse cells.

Ro ribonucleoproteins (RNPs) are autoantigens that result from the association of a 60-kDa protein (Ro60) with a small RNA (hY1, hY3, hY4 or hY5 in humans, mY1 or mY3 in mice). Previous studies localized Ro RNPs to the cytoplasm. Because Ro RNPs containing hY5 RNA (Ro(hY5) RNPs) have unique biochemical and immunological properties, their intracellular localization was reassessed. Subcellular distribution of mouse and human Ro RNPs in intact and hY-RNA transfected cells was assessed by immunoprecipitation and Northern hybridization. Human Ro(hY1--4) RNPs as well as murine Ro(mY1, mY3) RNPs are exclusively cytoplasmic. Ro RNPs containing an intact hY5 RNA, but not those containing a mutated form of hY5 RNA, are found in the nuclear fractions of human and mouse cells. Ro(hY5) RNPs are stably associated with transcriptionally active La protein and are known to associate with RoBPI, a nuclear autoantigen. Our results demonstrate that Ro(hY5) RNPs are specifically present in the nucleus of cultured human and murine cells. The signal for nuclear localization of Ro(hY5) RNPs appears to reside within the hY5 sequence itself. In conclusion, we suggest that the unique localization and interactions of primate-specific Ro(hY5) RNPs reflect functions that are distinct from the predicted cytoplasmic function(s) of more conserved Ro RNPs.

La transcription des gènes de Bax et du ligand de Fas contribue à la redistribution de la ribonucléoprotéine Ro60 dans les kératinocytes humains irradiés par UVA

Objectifs. Les ribonucléoprotéines Ro sont des marqueurs sériques de formes photosensibles de lupus comme le lupus érythémateux cutané subaigu, dans lequel les lésions à répétition de la peau sont déclenchées par l'exposition au soleil. Nous avons étudié le rôle de l'apoptose dans l'expression de Ro60. Méthodes. Nous avons utilisé les kératinocytes humains irradiés par les UVA. Résultats. Nous montrons que dans des cellules irradiées par les UVA, l'augmentation de l'expression de la ribonucléoprotéine Ro a pour origine une redistribution de l'antigène suite à l'activation des gènes de Bax et du ligand de Fas. Fas ligand and Bax gene transcription contributes to Ro60 ribonucleoprotein redistribution in UV-A irradiated human keratinocytes. Objectives. Ro ribonucleoproteins are of particular interest because they are serologic markers of photosensitive variants of lupus such as the subacute cutaneous lupus erythematosus (SCLE), in which the polycyclic skin lesions are triggered by exposure to the sun. We study the role of apoptosis in the expression of Ro antigen. Methods. We used UV-A irradiated keratinocytes. Results. We demonstrate in cultured human UVA-irradiated keratinocytes that the enhnaced expression of Ro60 ribonucleoprotein is caused by antigenic redistribution consecutive to Fas-L and Bax gene activation.

Analysis of the molecular composition of Ro ribonucleoprotein complexes

Human Ro ribonucleoproteins (RNPs) are composed of one of the four small Y RNAs and at least two proteins, Ro60 and La; association of additional proteins including the Ro52 protein and calreticulin has been suggested, but clear-cut evidence is still lacking. Partial purification of Ro RNPs from HeLa S100 extracts allowed characterization of several subpopulations of Ro RNPs with estimated molecular masses of between 150 and 550 kDa. The majority of these complexes contained Ro60 and La, whereas only a small proportion of Ro52 appeared to be associated with Ro RNPs. To identify novel Y RNA-associated proteins in vitro, binding of cytoplasmic proteins to biotinylated Y RNAs was investigated. In these reconstitution experiments, several proteins with estimated molecular masses of 80, 68, 65, 62, 60 and 53 kDa, the latter two being immunologically distinct from Ro60 and Ro52, respectively, appeared to bind specifically to Y RNAs. Furthermore, autoantibodies to these proteins were found in sera from patients with systemic lupus erythematosus. The proteins bound preferentially to Y1 and Y3 RNA but, with the exception of the 53-kDa protein, only weakly to Y4 RNA and not at all to Y5 RNA. Coprecipitation of the 80, 68, 65, and 53-kDa proteins by antibodies to Ro60 and La was observed, suggesting that at least a proportion of the novel proteins may reside on the same particles as La and/or Ro60. Finally, the binding sites for these proteins on Y1 RNA were clearly distinct from the Ro60-binding site involving a portion of the large central loop 2, which was found to be indispensable for binding of the 80, 68, 65 and 53-kDa proteins, as well as the stem 3-loop 3 and stem 2-loop 1 regions. Interestingly, truncation of the La-binding site resulted in decreased binding of the novel proteins (but not of Ro60), indicating La to be required for efficient association. Taken together, these results suggest the existence of further subpopulations of Ro RNPs or Y RNPs, consistent with the heterogeneous characteristics observed for these particles in the biochemical fractionation experiments.

Interaction cloning and characterization of RoBPI, a novel protein binding to human Ro ribonucleoproteins

Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA. RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb RoBPI mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs. RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs. Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs.

The levels of the RoRNP-associated Y RNA are dependent upon the presence of ROP-1, the Caenorhabditis elegans Ro60 protein.

The Ro ribonucleoproteins (RoRNP) consist of at least one major protein of 60 kD, Ro60, and one small associated RNA, designated Y RNA. Although RoRNP have been found in all vertebrate species examined so far, their function remains unknown. The Caenorhabditis elegans rop-1 gene previously has been identified as encoding a Ro60 homologue. We report here the phenotypic characterization of a C. elegans strain in which rop-1 has been disrupted. This is the first report regarding the inactivation of a major RoRNP constituent in any organism. The rop-1 mutant worms display no visible defects. However, at the molecular level, the disruption of rop-1 results in a dramatic decrease in the levels of the ROP-1-associated RNA (CeY RNA). Moreover, transgenic expression of wild-type rop-1 partially rescues the levels of CeY RNA. Considering that transgenes are poorly expressed in the germline, the fact that the rescue is only partial is most likely related to the high abundance of the CeY RNA in the adult germline and in embryos. The developmental expression pattern and localization of CeY RNA suggest a role for this molecule during embryogenesis. We conclude that, under laboratory culture conditions, ROP-1 does not play a crucial role in C. elegans.

Ontogeny of Ro hYRNAs in Human Heart

The Ro complex is composed of three ribonucleoproteins of 60, 54, and 52 kDa. The Ro60 is associated with the 1-5 hYRNAs. Autoantibodies against Ro are found in sera from lupus patients. Lupus frequently presents in female patients during childbearing years and transplacental passage of maternal anti-Ro/La autoantibodies has been implicated in triggering neonatal lupus and congenital heart block (CHB). Since expression of Ro ribonucleoproteins occurs before the onset of CHB, epitope spreading of Ro hYRNAs could arise during cardiac ontogeny, thus maternal autoantibodies would damage a previously developed AV node. To explore the cardiac ontogeny of Ro hYRNAs, embryonic and adult tissues were obtained from legal autopsies. In situ hybridization using oligonucleotides for hY1, hY3, hY4, hY5, and Ro60 was performed on sections of cardiac tissue; their corresponding cDNAs were amplified by RT-PCR. The principal results were as follows: expression of hY4/5RNAs was greater in fetal tissues from 8-12 weeks of development. The subcellular distribution of hYRNAs in embryonic tissues was nucleocytoplasmic, whereas in the adult heart it was cytoplasmic. In conclusion, hYRNAs expression occurs during early cardiac development.

Gene encoding human Ro-associated autoantigen Y5 RNA.

Ro ribonucleoproteins are composed of Y RNAs and the Ro 60 kDa protein. While the Ro 60 kDa protein is implicated in an RNA discard pathway that recognizes 3'-extended 5S rRNAs, the function of Y RNAs remains unknown [O'Brien,C.A. and Wolin,S.L. (1995) Genes Dev. 8,2891-2903]. Y5 RNA occupies a large fraction of Ro 60 kDa protein in human Ro RNPs, contains an atypical 3'-extension not found on other Y RNAs, and constitutes an RNA antigen in certain autoimmune patients [Boulanger et al. (1995) Clin. Exp. Immunol. 99, 29-36]. An overabundance of Y RNA retroposed pseudogenes has previously complicated the isolation of mammalian Y RNA genes. The source gene for Y5 RNA was isolated from human DNA as well as from Galago senegalis DNA. Authenticity of the hY5 RNA gene was demonstrated in vivo and its activity was compared with the hY4 RNA gene that also uses a type 3 promoter for RNA polymerase III. The hY5 RNA gene was subsequently found to reside within a few hundred thousand base pairs of other Y RNA genes and the linear order of the four human Y RNA genes on chromosome 7q36 was determined. Phylogenetic comparative analyses of promoter and RNA structure indicate that the Y5 RNA gene has been subjected to positive selection during primate evolution. Consistent with the proposal of O'Brien and Harley [O'Brian,C.A. and Wolin,S.L. (1992) Gene 116, 285-289], analysis of flanking sequences suggest that the hY5 RNA gene may have originated as a retroposon.

The Caenorhabditis elegans rop-1 gene encodes the homologue of the human 60-kDa Ro autoantigen

As a first step toward establishing a genetic system for the elucidation of the cellular role(s) of the Ro ribonucleoproteins (RoRNP), we have cloned the gene encoding the homologue of the human 60-kDa Ro protein (Ro60) in Caenorhabditis elegans (Ce). This Ce gene is present as a single copy and contains a 643-codon open reading frame interrupted by three introns. The encoded protein, Rop1p, shares 40% identity and 63% overall similarity with both the human and amphibian Ro60. Recombinant protein has been produced in Escherichia coli and used to elicit anti-Rop1p antibodies. Immunological analysis indicated that the Ro60 epitopes have been poorly conserved. Gene-fusion expression studies in transgenic nematodes will provide a new avenue of research to shed light on the function of these particles.

The human Y4 small cytoplasmic RNA gene is controlled by upstream elements and resides on chromosome 7 with all other hY scRNA genes.

Ro ribonucleoproteins (RNP) constitute a class of evolutionarily conserved small cytoplasmic (sc) RNPs whose functions are unknown. In human cells four distinctive scRNAs designated hY1, hY3, hY4 and hY5 are synthesized by RNA polymerase III (pol III) and accumulate as components of Ro scRNPs. The previously isolated hY1 and hY3 genes contain upstream sequences similar to the class III promoters for U6 and 7SK snRNAs. Additional mammalian Y scRNA genes have been refractory to cloning due to interference from numerous hY-homologous pseudogenes and studies of hY RNA genes have been sparse. Although homologs of hY1 and hY3 RNAs exist in rodent cells, the smaller Y4 and Y5 RNAs do not which has allowed us to localize the hY4 scRNA gene to human chromosome 7 by assaying for its transcript in rodent X human somatic cell hybrids (SCH). A chromosome 7-enriched yeast artificial chromosome (YAC) library was then screened and the authentic hY4 sequence was isolated by strepavidin--biotin-mediated hybrid-selection followed by poly(dA)-tailing and hemispecific PCR. The region upstream of the hY4 sequence contains a TATAAAA motif centered at -26, a candidate proximal sequence element at -63, and three octamer-like sequences located between -260 and -200. hY4 RNA is readily detectable on Northern blots after transient transfection of the hY4 gene into mouse cells but not after transfection of a construct in which the 5' flanking region was deleted. SCHs and chromosome 7-enriched YACs were used to demonstrate that all four hY RNA genes reside on human chromosome 7.

Xenopus Ro ribonucleoproteins: members of an evolutionarily conserved class of cytoplasmic ribonucleoproteins.

Ro small ribonucleoproteins consist of a 60-kDa protein and possibly additional proteins complexed with several small RNA molecules. The RNA components of these particles, designated Y RNAs, are about 100 nt long. Although these small ribonucleoproteins are abundant components of a variety of vertebrate species and cell types, their subcellular location is controversial, and their function is completely unknown. We have identified and characterized the Ro RNPs of Xenopus laevis. Three of the four distinct Xenopus Y RNAs appear to be related to the previously sequenced human hY3, hY4, and hY5 RNAs. The fourth Xenopus Y RNA, xY alpha, does not appear to be a homologue of any of the human Y RNAs. Each of the human and Xenopus Y RNAs possesses a conserved stem that contains the binding site for the 60-kDa Ro protein. Xenopus and human 60-kDa Ro proteins are 78% identical in amino acid sequence, with the conservation extending throughout the entire protein. When human hY3 RNA is mixed with Xenopus egg extracts, the human RNA assembles with the Xenopus Ro protein to form chimeric Ro ribonucleoproteins. By analyzing RNA extracted from manually enucleated oocytes and germinal vesicles, we have determined that Y RNAs are located in the oocyte cytoplasm. By examining the distribution of mouse Ro ribonucleoproteins in cytoplast and karyoplast fractions derived from L-929 cells, we have determined that Ro ribonucleoprotein particles also primarily reside in the cytoplasm of mammalian cells.

A subset of hY RNAs is associated with erythrocyte Ro ribonucleoproteins.

The Ro autoantigen is a mammalian cellular ribonucleoprotein (RNP) of unknown function. We have demonstrated that hY1 and hY4 Ro RNAs are associated with erythrocyte Ro RNPs and represent a subset of the four hY RNAs found in HeLa cell and leukocyte Ro RNPs. We have cloned and sequenced hY4 RNA, the only hY RNA not sequenced previously, from a polymerase chain reaction amplified erythrocyte hY cDNA library. Sequencing of the erythrocyte hY RNAs in conjunction with Northern blot analysis confirms that the erythrocyte hY RNAs contain the same sequences as the respective HeLa cell RNAs of similar mobility. Ribonuclease inhibition activity has been found in erythrocytes and this activity inhibits the degradation of hY3 and hY5 in leukocyte lysates thereby favoring the possibility that the presence of hY1 and hY4 in erythrocytes is the result of differential expression of the hY RNAs in erythrocyte precursors.

Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells.

Monoclonal antibodies were raised against homogeneous Ro and La antigens, two proteins associated with Ro and La ribonucleoproteins (RNPs). The specificity of the monoclonal antibodies was proven by immunoblot analysis and by immunoprecipitation. The anti-Ro antibody reacted with a Mr 95,000 protein in a mouse lymphoma cell extract and with a Mr 60,000 polypeptide in extracts from human spleen. The anti-La antibody recognized a Mr 50,000 polypeptide in the mouse L5178y cell extract. The two monoclonal antibodies precipitated RNPs that contained the typical RNA species of Ro or La RNPs. The localization of Ro and La antigen was performed by direct immunofluorescence microscopy. It was found that the anti-Ro antibody reacted with a fibrous network that behaves like cytokeratin, one of the intermediate filament systems. The anti-La antibody reacted with nuclear structures that gave a speckled-type pattern.