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PubMed1991

Autoantibodies in male homosexuals and HIV infection.

We have used ELISA to study the frequency of autoantibodies to several antigens in the serum of 17 male homosexuals (MHS) negative for HIV (HIV-), 11 asymptomatic HIV seropositive MHS (HIV+) and patients with ARC (N = 15) or AIDS (N = 13), and compared them to 20 matched healthy heterosexual controls. Serum antibody binding to histones, cardiolipin, ss-A, ss-B and Sm was found to be significantly higher in each of the MHS groups studied as compared to controls (P less than 0.001), and was also increased in the HIV+ patients vs. the HIV- group (P less than 0.05). In contrast, no increase in autoantibodies to ss-DNA, ds-DNA, poly(I), poly(G) or RNP were found in any of the groups tested. These results enlarge the spectrum of autoimmunity previously reported in HIV infection and identify a similar pattern to a lesser degree, already present in HIV- MHS, suggesting a role for HIV or concomitant virus infections in its pathogenesis.

AIDS-Related Complex↗

PubMed1990

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents.

Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.

Cell Nucleolus↗

PubMed2002

Yeast hnRNP K-like genes are involved in regulation of the telomeric position effect and telomere length.

Mammalian heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA- and DNA-binding protein implicated in the regulation of gene expression processes. To better understand its function, we studied two Saccharomyces cerevisiae homologues of the human hnRNP K, PBP2 and HEK2 (heterogeneous nuclear RNP K-like gene). pbp2Delta and hek2Delta mutations inhibited expression of a marker gene that was inserted near telomere but not at internal chromosomal locations. The telomere proximal to the ectopic marker gene became longer, while most of the other telomeres were not altered in the double mutant cells. We provide evidence that telomere elongation might be the primary event that causes enhanced silencing of an adjacent reporter gene. The telomere lengthening could, in part, be explained by the inhibitory effect of hek2Delta mutation on the telomeric rapid deletion pathway. Hek2p was detected in a complex with chromosome regions proximal to the affected telomere, suggesting a direct involvement of this protein in telomere maintenance. These results identify a role for hnRNP K-like genes in the structural and functional organization of telomeric chromatin in yeast.

Amino Acid Sequence↗

PubMed2000

The single-stranded DNA- and RNA-binding proteins pur alpha and pur beta link BC1 RNA to microtubules through binding to the dendrite-targeting RNA motifs.

Neural BC1 RNA is distributed in neuronal dendrites as RNA-protein complexes (BC1 RNPs) containing Translin. In this study, we demonstrated that the single-stranded DNA- and RNA-binding protein pur alpha and its isoform, pur beta, which have been implicated in control of DNA replication and transcription, linked BC1 RNA to microtubules (MTs). The binding site was within the 5' proximal region of BC1 RNA containing putative dendrite-targeting RNA motifs rich in G and U residues, suggesting that in the cytoplasm of neurons, these nuclear factors are involved in the BC1 RNA transport along dendritic MTs. The pur proteins were not components of BC1 RNP but appeared to associate with MTs in brain cells. Therefore, it is suggested that they may transiently interact with the RNP during transport. In this respect, the interaction of pur proteins with BC1 RNA could be regulated by the Translin present within the RNP, because the binding mode of these two classes of proteins (pur proteins and Translin) to the dendrite-targeting RNA motifs was mutually exclusive. As the motifs are well conserved in microtubule-associated protein 2a/b mRNA as well, the pur proteins may also play a role(s) in the dendritic transport of a subset of mRNAs.

Animals↗

PubMed2006

Spinal muscular atrophy: the RNP connection.

Degenerated motor neurons in the spinal cord are the pathological hallmark of spinal muscular atrophy (SMA). SMA is caused by mutations in the ubiquitously expressed survival motor neuron 1 (SMN1) gene, which lead to reduced levels of functional SMN protein. Many different functions have been assigned to SMN, including assembly of ribonucleoproteins (RNPs), splicing, transcription and axonal mRNA transport. Recently, tissue from SMA patients and animal models has been used to determine which function of SMN is affected in SMA patients. A surprising picture has emerged: the impaired assembly of RNP subunits of the spliceosome seems to be responsible for SMA pathogenesis. Here, we present a model of how this defect might cause motor-neuron degeneration and consider potential therapies.

Alternative Splicing↗

PubMed1998

Catastrophic antiphospholipid syndrome. Clinical and laboratory features of 50 patients.

We analyzed the clinical and laboratory characteristics of 50 patients with catastrophic antiphospholipid syndrome (APS) (5 from our clinics and 45 from a MEDLINE computer-assisted review of the literature from 1992 through 1996). Thirty-three (66%) patients were female and 17 (34%) were male. Twenty-eight (56%) patients had primary APS, 15 (30%) had defined systemic lupus erythematosus (SLE), 6 (12%) had "lupus-like" syndrome, and 1 (2%) had rheumatoid arthritis. Mean age of patients in this series was 38 +/- 14 years (range, 11-74 yr). Three (6%) patients developed the clinical picture of catastrophic APS under the age of 15 years, and 11 (22%) were 50 years old or more. In 11 (22%) patients, precipitating factors contributed to the development of catastrophic APS (infections in 3, drugs in 3, minor surgical procedures in 3, anticoagulation withdrawal in 2, and hysterectomy in 1). The presentation of the acute multi-organ failure was usually complex, involving multiple organs simultaneously or in a very short period of time. The majority of patients manifested microangiopathy--that is, occlusive vascular disease affecting predominantly small vessels of organs, particularly kidney, lungs, brain, heart, and liver--with a minority of patients experiencing only large vessel occlusions. Thrombocytopenia was reported in 34 (68%) patients, hemolytic anemia in 13 (26%), disseminated intravascular coagulation in 14 (28%), and schistocytes in 7 (14%). The following antibodies were detected: lupus anticoagulant (94%), anticardiolipin antibodies (94%), anti-dsDNA (87% of patients with SLE), antinuclear antibodies (58%), anti-Ro/SS-A (8%), anti-RNP (8%), and anti-La/SS-B (2%). Anticoagulation was used in 70% of the patients, steroids in 70%, plasmapheresis in 40%, cyclophosphamide in 34%, intravenous gammaglobulins in 16%, and splenectomy in 4%. Most patients, however, received a combination of nonsurgical therapies. Death occurred in 25 of the 50 (50%) patients. In most, cardiac problems seemed to be the major cause of death. In several of these, respiratory failure was also present, usually due to acute respiratory distress syndrome and diffuse alveolar hemorrhage. Among the 20 patients who received the combination of anticoagulation, steroids, and plasmapheresis or intravenous gammaglobulins, recovery occurred in 14 (70%) patients. The use of ancrod and defibrotide appeared to be effective in the 2 respective patients in whom they were used.

Adolescent↗

PubMed1998

Specific telomerase RNA residues distant from the template are essential for telomerase function.

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5' end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.

Deoxyribonucleases, Type II Site-Specific↗

PubMed1996

Nuclear colocalization of the Ro 60 kDa autoantigen and a subset of U snRNP domains.

The Ro 60 kDa protein is an RNA binding molecule present both in the cytoplasm and in the nucleus. Cytoplasmic Ro 60 kDa is complexed to other proteins and to certain RNAs denoted hYRNAs. This RNA-protein complex is also known as the Ro/SSA antigen recognized by sera from patients with certain autoimmune disorders. Components interacting with the nuclear Ro 60 kDa protein fraction in mammalian cells have not been identified. To look for an association with previously known nuclear structures, rabbit antisera to the amino- and carboxy-terminal parts of the Ro 60 kDa protein were used in immunomorphological studies on HeLa cells. A strong speckled nuclear pattern and a weak cytoplasmic staining were detected. Double immunofluorescence staining with affinity purified anti-Ro 60 kDa antibodies and monoclonal antibodies recognizing the Sm and RNP antigens of the U snRNPs, displayed colocalization. Another U snRNP containing nuclear compartment, the coiled bodies, did not contain any Ro 60 kDa protein. Cells infected with a toga virus demonstrated redistribution of both U snRNP antigens and the Ro 60 kDa protein with retained colocalization. These results indicate a role for the nuclear fraction of the Ro 60 kDa protein in RNA processing.

Animals↗

PubMed2001

The yeast hnRNP-Like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p.

Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

Fungal Proteins↗

PubMed1990

[Pathogenic role of antinuclear antibodies in lupus disease].

The majority of anti-nuclear antibodies do not have a proven pathogenic role in systemic lupus erythematosus (SLE). The pathogenicity of native anti-DNA antibodies, suggested by clinical data, is difficult to confirm. It is linked to several factors: isotype and avidity of these antibodies, affinity for DNA, size of immune complexes. DNA, anti-DNA antibodies and cross-reactive anti-DNA idiotypes (16/6) have been isolated from human and NZB mouse glomeruli. Moreover, DNA has a particular affinity for the glomerular membrane due to its cross-reactivity with some constituents of this membrane. Anti-SSA (Ro) antibodies may play a role in some cases of nephropathy in SLE and participate in photosensitization. They are associated with neonatal lupus, with some cases of fetal death in black women and with the atrioventricular heart block. However, factors other than these antibodies are needed to induce such lesions. Anti-U1 RNP antibodies do not protect against kidney involvement in SLE; however, they can decrease suppressive function by penetrating into suppressor T cells. Anti-Sm antibodies may have particular immunoregulation properties. Some antinuclear antibodies may at least in part, be responsible for the lesions in SLE, in conjunction with other still-unknown factors.

Animals↗

PubMed1988

Two Ro (SS-A) autoantibody responses in systemic lupus erythematosus. Correlation of HLA-DR/DQ specificities with quantitative expression of Ro (SS-A) autoantibody.

Autoantibodies to Ro (SS-A), La (SS-B), and Sm/nuclear RNP were quantitated by enzyme-linked immunosorbent assay in 106 white patients with systemic lupus erythematosus. Two Ro autoantibody subgroups were identified that differed quantitatively, genetically, and clinically. The subgroup having anti-Ro only demonstrated significantly lower mean anti-Ro levels than did the subgroup with concomitant anti-La and showed a strong association with the linked HLA alleles DR2 and DQw1. The anti-Ro with anti-La subgroup was associated with the linked HLA alleles B8, DR3, DRw52, and DQw2 (DR3 was primary), and this subgroup consisted of patients with older ages at disease onset, sicca complex, and less renal involvement. Overall, the relative risk (RR) for having anti-Ro was highest in HLA-DR2/DR3 heterozygotes compared with non-DR2/DR3 heterozygotes (RR 15) and all other DR combinations (RR 7), suggesting a compound effect of 2 immune responses. Heterozygotes for HLA-DQw1/DQw2 demonstrated significantly higher mean levels of anti-Ro, which may be indicative of trans gene interaction at HLA-DQ. These data suggest the hypothesis that HLA genes exert their major effects on Ro/La autoantibody subsets of systemic lupus erythematosus.

Autoantibodies↗

PubMed1996

Clinical and immunological aspects of autoantibodies to RA33/hnRNP-A/B proteins--a link between RA, SLE and MCTD.

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are major constituents of the spliceosome. They are composed of approximately 30 different proteins which can bind to nascent pre-mRNA. Among these, the hnRNP-A/B proteins form a subgroup of highly related proteins consisting of two adjacent RNA binding domains (RBD) within the N-terminal parts, whereas the C-terminal halves contain almost 50% glycine residues. These proteins, in particular A2/RA33, are targeted by autoantibodies from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). In SLE anti-hnRNP antibodies frequently occur together with antibodies to U1 small nuclear RNP (U1-snRNP) and Sm, other proteins of the spliceosome. Preliminary epitope mapping studies have revealed major antibody binding sites in the RNA binding regions for all three diseases. Nevertheless, there is some indication of disease specific epitope recognition. Studies in animal models have demonstrated anti-RA33/hnRNP-A/B antibodies in lupus-prone mouse strains. Thus, autoantibodies to the spliceosomal hnRNP-A/B proteins are a common feature of RA, SLE, and MCTD. However, these diseases differ in their reactivities to other spliceosomal proteins, especially anti-U1 snRNP and Sm. Therefore, anti-RA33/hnRNP-A/B autoantibodies are not only valuable diagnostic markers but may also allow additional insights into the pathogenesis of rheumatic autoimmune diseases.

Animals↗

PubMed1997

Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.

An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.

Amino Acid Sequence↗

PubMed1989

Genetic studies of Ro (SS-A) and La (SS-B) autoantibodies in families with systemic lupus erythematosus and primary Sjögren's syndrome.

Using enzyme-linked immunosorbent assays, autoantibodies to Ro (SS-A) were detected in the sera of 21% of the first-degree relatives and 11% of the second-degree relatives of anti-Ro-positive probands with systemic lupus erythematosus (SLE) or primary Sjögren's syndrome, as compared with 3% of normal control subjects (P = 0.003 and P = 0.09, respectively). In a parallel study, anti-Ro occurred in 28% of the first-degree relatives of unselected members of families of SLE patients, regardless of the proband's serologic status, compared with 6% of the relatives from normal healthy families. Antibodies to La and to Sm/nuclear RNP were infrequent. Anti-Ro occurred in 41% of the relatives considered to have a dominant, non-HLA-linked "autoimmune trait" by virtue of having any autoimmune disorder and/or serologic abnormality (antinuclear antibodies, anti-single-stranded DNA, or biologic false-positive VDRL test result), as compared with only 2% of the healthy, seronegative relatives without the trait (P = 0.009). Moreover, HLA-DR2 and/or DR3 occurred in 90% of anti-Ro-positive subjects, regardless of their clinical status. These results demonstrate that the Ro autoantibody response occurs frequently in relatives of patients with SLE and is genetically mediated by both major histocompatibility complex and non-major histocompatibility complex effects.

Adult↗

PubMed2003

Gene duplication and other evolutionary strategies: from the RNA world to the future.

Beginning with a hypothetical RNA world, it is apparent that many evolutionary transitions led to the complexity of extant species. The duplication of genetic material is rooted in the RNA world. One of two major routes of gene amplification, retroposition, originated from mechanisms that facilitated the transition to DNA as hereditary material. Even in modern genomes the process of retroposition leads to genetic novelties including the duplication of protein and RNA coding genes, as well as regulatory elements and their juxtapositon. We examine whether and to what extent known evolutionary principles can be applied to an RNA-based world. We conclude that the major basic Neo-Darwinian principles that include amplification, variation and selection already governed evolution in the RNA and RNP worlds. In this hypothetical RNA world there were few restrictions on the exchange of genetic material and principles that acted as borders at later stages, such as Weismann's Barrier, the Central Dogma of Molecular Biology, or the Darwinian Threshold were absent or rudimentary. RNA was more than a gene: it had a dual role harboring, genotypic and phenotypic capabilities, often in the same molecule. Nuons, any discrete nucleic acid sequences, were selected on an individual basis as well as in groups. The performance and success of an individual nuon was markedly dependent on the type of other nuons in a given cell. In the RNA world the transition may already have begun towards the linkage of nuons to yield a composite linear RNA genome, an arrangement necessitating the origin of RNA processing. A concatenated genome may have curbed unlimited exchange of genetic material; concomitantly, selfish nuons were more difficult to purge. A linked genome may also have constituted the beginning of the phenotype/genotype separation. This division of tasks was expanded when templated protein biosynthesis led to the RNP world, and more so when DNA took over as genetic material. The aforementioned barriers and thresholds increased and the significance and extent of horizontal gene transfer fluctuated over major evolutionary transitions. At the dawn of the most recent transformation, a fast evolutionary transition that we will be witnessing in our life times, a form of Lamarckism is raising its head.

Animals↗

PubMed1998

TLS (translocated-in-liposarcoma) is a high-affinity interactor for steroid, thyroid hormone, and retinoid receptors.

Nuclear receptors for steroid hormones, thyroid hormone, retinoids, and vitamin D are thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription complex. In an effort to identify potential coregulators, receptor fusions with glutathione-S-transferase were used to isolate proteins in nuclear extracts capable of binding nuclear hormone receptors. Glutathione-S-transferase fusions with mouse retinoid X receptor-alpha enabled the selective isolation of a 65-kDa protein (p65) from nuclear extracts of rat and human cells. Binding of p65 to mouse retinoid X receptor-alpha was centered around the DNA-binding domain. p65 also bound regions encompassing the DNA-binding domain in estrogen, thyroid hormone, and glucocorticoid receptors. p65 was identified as TLS (translocated-in-liposarcoma), a recently identified member of the RNP family of nuclear RNA-binding proteins whose members are thought to function in RNA processing. The N-terminal half of TLS bound to thyroid hormone receptor with high affinity while the receptor was bound to appropriate DNA target sites. Functional studies indicated that the N-terminal half of TLS can interact with thyroid hormone receptor in vivo. TLS was originally discovered as part of a fusion protein arising from a chromosomal translocation causing human myxoid liposarcomas. TLS contains a potent transactivation domain whose translocation-induced fusion with a DNA-binding protein (CHOP) yields a powerful transforming oncogene and transcription factor. The transactivation and RNA-binding properties of TLS and the nature of its interaction with nuclear receptors suggest a novel role in nuclear receptor function.

Animals↗

PubMed2005

DNA looping-mediated repression by histone-like protein H-NS: specific requirement of Esigma70 as a cofactor for looping.

Transcription initiation by RNA polymerase (RNP) carrying the house-keeping sigma subunit, sigma70 (Esigma70), is repressed by H-NS at a number of promoters including hdeABp in Escherichia coli, while initiation with RNP carrying the stationary phase sigma, sigma38 (Esigma38), is not. We investigated the molecular mechanism of selective repression by H-NS to identify the differences in transcription initiation by the two forms of RNPs, which show indistinguishable promoter selectivities in vitro. Using hdeABp as a model promoter, we observed with purified components that H-NS, acting at a sequence centered at -118, selectively repressed transcription by Esigma70. This selective repression is attributed to the differences in the interactions between hdeABp and the two forms of RNPs, since no other factor is required for the repression. We observed that the two forms of RNPs could form an open initiation complex (RP(O)) at hdeABp, but that Esigma70 failed to initiate transcription in the presence of H-NS. Interestingly, KMnO4 assays and high-resolution atomic force microscopy (AFM) revealed that hdeABp DNA wrapped around Esigma70 more tightly than around Esigma38, resulting in the potential crossing over of the DNA arms that project out of Esigma70 . RP(O) but not out of Esigma38 . RP(O). Based on these observations, we postulated that H-NS bound at -118 laterally extends by the cooperative recruitment of H-NS molecules to the promoter-downstream sequence joined by wrapping of the DNA around Esigma70 . RP(O), resulting in effective sealing of the DNA loop and trapping of Esigma70. Such a ternary complex of H-NS . Esigma70 hdeABp was demonstrated by AFM. In this case, therefore, Esigma70 acts as a cofactor for DNA looping. Expression of this class of genes by Esigma38 in the stationary phase is not due to its promoter specificity but to the architecture of the promoter . Esigma38 complex.

Bacterial Proteins↗

PubMed2002

Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes.

Ro RNPs are evolutionarily conserved, small cytoplasmic RNA-protein complexes with an unknown function. In human cells, Ro RNPs consist of one of the four hY RNAs and two core proteins: Ro60 and La. Recently, the association of hnRNP I and hnRNP K with particles containing hY1 and hY3 RNAs has been described. The association of three other proteins, namely calreticulin, Ro52 and RoBPI, with (subsets of) the Ro RNPs is still controversial. To gain more insight into the composition and function of the Ro RNPs, we have immunopurified these particles from HeLa cell extracts using monoclonal antibodies against Ro60 and La. Using this approach, we have identified the RNA-binding protein nucleolin as a novel subunit of Ro RNP particles containing hY1 or hY3 RNA, but not hY4 and hY5 RNA. Using an in vitro hY RNA-binding assay we established that the internal pyrimidine-rich loop of hY1 and hY3 RNA is essential for the association of nucleolin. The binding is critically dependent on the presence of all four RNP motifs of nucleolin, but not of the C-terminal RGG-box. Moreover, we demonstrate that, in contrast to nucleolin and hnRNP K, nucleolin and hnRNP I can bind simultaneously to the internal pyrimidine-rich loop of hY1 RNA. We postulate that nucleolin functions in the biogenesis and/or trafficking of hY1 and hY3 RNPs through the nucleolus and subsequent transport to the cytoplasm.

Autoantigens↗