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Results for “RNA, Small Cytoplasmic”

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PubMed1996

[Presence of autoantibodies in patients infected with HIV].

The presence of serum autoantibodies directed against single-stranded DNA (ssDNA), RNA, histones, nuclear antigen SS-A, mitochondria and cardiolipin were investigated in 30 HIV-negative drug addicts (from years 1986-87), 30 addicts actually infected with HIV and 31 AIDS patients presenting with clinical symptoms indicating autoimmune disorders. Positive results were found in 12 (40%) drug addicts from years 1986-87, 5 (16.7%) actually infected and 16 (51.6%) AIDS patients. Autoantibodies were more often detectable in patients with thrombocytopenia 8/12 (66.7%) than in remaining 8 out of 19 subjects (42.1%) who presented dermatitis, hepatitis, pancarditis, ulcerative colitis and polyneuropathy. The prevalence of autoantibodies in all investigated groups were significantly higher than in controls-1/20 (3.3%).

Acquired Immunodeficiency Syndrome↗

PubMedDate not supplied

[Participation of small RNAs, associated with poly(A)+RNA in cytoplasm in hormonal regulation of genome expression].

The fraction of small RNA (sacc-RNA), associated with cytoplasmic rat liver poly(A)+RNA by non-covalent, possibly complementary interactions, has been isolated and studied. Fingerprint analysis data reveal that the specific changes occur in the population of sacc-RNA in response to glucocorticoid treatment. The close similarity of oligonucleotide composition of sacc-RNA and RNA-component of small nuclear RNP-acceptor of glucocorticoid hormones has been found. The tightly boynd peptides were observed in the fraction of sacc-RNA. The hypothesis of the involvement of the small RNA in the hormonal regulation at post-transcriptional stages of gene expression in the cytoplasm has been forward.

Animals↗

PubMed1998

The Zn2+ binding domain of the human Ro 52 kDa protein is a target for conformation-dependent autoantibodies.

The Ro 52 kDa protein was originally identified using autoimmune sera directed against the Ro/SS-A antigen, also known as Ro ribonucleoprotein particles (Ro RNPs). In most human cells these consist of one of four small human cytoplasmic (hY) RNA molecules complexed with the Ro 60 kDa protein. This protein interacts directly with the hY RNAs, whereas the Ro 52 kDa protein is thought to associate via protein-protein interactions. The Ro RNPs are present in all mammalian cells, but their intracellular location and function remain unclear. The primary structure shows that the Ro 52 kDa protein is a member of a small family of proteins sharing two features, a leucine zipper region and an aminoterminal cysteine-histidine rich region with two different putative zinc finger motifs. To study if the Ro 52 kDa protein actually binds Zn2+, both the full-length Ro 52 kDa clone and various subclones were expressed as recombinant proteins and assayed for Zn2+ binding in vitro. A fragment containing the first cysteine-histidine cluster at residues 14-54 and other larger overlapping fragments were found to bind Zn2+. In this report we also demonstrate that the Zn2+ binding domain is a target for conformation-dependent anti-Ro 52 kDa autoantibodies. The antigenicity is dramatically increased by the same reducing conditions that promote Zn2+ binding. This is in contrast to the previously described immunodominant Ro 52 kDa domain.

Autoantibodies↗

PubMed1984

Autoantibodies reactive with small ribonucleoprotein antigens: a convergence of molecular biology and clinical immunology.

Autoantibodies to nuclear antigens (ANA) occur in patients with systemic lupus erythematosus (SLE) and other multisystem autoimmune diseases. Although heterogeneous, there are 2 major groups, autoantibodies to DNA and autoantibodies to non-DNA antigens, the latter including ANAs to the soluble or "extractable nuclear antigens" (ENA). This review discusses those ENAs which are ribonucleoproteins (RNPs) consisting of small RNA molecules (80-400 nucleotides) attached to non-histone proteins: these are called small nuclear (sn) or small cytoplasmic (sc) ribonucleoproteins according to their location in the cell and at least some are known to play an important role in nuclear metabolism. ENAs can be immunoprecipitated from crude preparations of nuclei by sera from patients with multisystem autoimmune diseases and, after removal of the associated proteins, the RNA components can be analyzed by gel electrophoresis. This shows 3 main categories of small RNAs: the U group comprising U1-U6 snRNAs, the Ro group comprising small nucleocytoplasmic RNAs, and the La group comprising several species of cellular snRNAs as well as the Ro scRNAs. La, in addition, includes small RNAs encoded by adenovirus (VA I, VA II), Epstein-Barr virus (EBER 1, EBER 2) and vesicular stomatitis virus (leader RNA). In the case of each group, the RNAs themselves are not antigenic but become so when associated with proteins, most of which are uncharacterized. The U snRNAs, located in the nucleus, are transcribed by RNA polymerase II and appear to be involved in the splicing of introns from mRNA. Sera from patients with mixed connective tissue disease (MCTD) react with RNPs containing U1 RNA and sera from patients with SLE react with U RNPs containing U1, U2, U4, U5 and U6 RNAs, collectively known as the Sm antigen. The Ro RNAs are transcribed by RNA polymerase III and have no known function. Sera from patients with primary Sjögren's syndrome and some cases of SLE react with Ro scRNPs. The La RNAs are also transcribed by RNA polymerase III and are located mostly in the nucleus; functionally the protein associated with the La RNAs appears to be important in RNA polymerase III transcription. Sera from patients with primary Sjögren's syndrome react with the heterogeneous group of both cellular and viral RNAs which constitute the La RNP antigen. Sera of patients with SLE, scleroderma, polymyositis and dermatomyositis also react with RNPs relevant to nuclear metabolism and further definition of these RNPs is awaited. Many advances can be expected from the convergence of molecular biology and clinical immunology exemplified by the current studies on ENAs.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Antinuclear↗

PubMed2000

Analysis of the molecular composition of Ro ribonucleoprotein complexes. Identification of novel Y RNA-binding proteins.

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.

Autoantibodies↗

PubMed2003

The Ro autoantigen binds misfolded U2 small nuclear RNAs and assists mammalian cell survival after UV irradiation.

The Ro 60 kDa autoantigen, an RNA binding protein, is a major target of the immune response in patients with systemic lupus erythematosus. As mice lacking Ro develop a lupus-like syndrome, Ro may be important for preventing autoimmunity. However, the cellular function of Ro, which binds small cytoplasmic RNAs of unknown function called Y RNAs, has been enigmatic. Ro has been proposed to function in 5S rRNA quality control based on experiments in Xenopus laevis oocytes, and a Ro ortholog enhances survival of the eubacterium Deinococcus radiodurans after ultraviolet irradiation. To test the general importance of these two observations for Ro function, we investigated the role of Ro in mammalian cells. We report that, in mouse embryonic stem (ES) cells, Ro binds variant spliceosomal U2 snRNAs. Expression of mouse U2 snRNAs in Xenopus oocytes reveals that binding occurs in nuclei and appears to involve recognition of misfolded RNA. Moreover, mouse ES cells lacking Ro exhibit decreased survival after ultraviolet irradiation. In irradiated cells, both Ro and a Y RNA accumulate in nuclei. We propose that Ro plays a general role in small RNA quality control and that this function is important for cell survival after ultraviolet irradiation.

Animals↗

PubMed2005

Fragile X mental retardation protein (FMRP) binds specifically to the brain cytoplasmic RNAs BC1/BC200 via a novel RNA-binding motif.

Fragile X mental retardation protein (FMRP), the protein responsible for the fragile X syndrome, is an RNA-binding protein involved in localization and translation of neuronal mRNAs. One of the RNAs known to interact with FMRP is the dendritic non-translatable brain cytoplasmic RNA 1 BC1 RNA that works as an adaptor molecule linking FMRP and some of its regulated mRNAs. Here, we showed that the N terminus of FMRP binds strongly and specifically to BC1 and to its potential human analog BC200. This region does not contain a motif known to specifically recognize RNA and thus constitutes a new RNA-binding motif. We further demonstrated that FMRP recognition involves the 5' stem loop of BC1 and that this is the region that exhibits complementarity to FMRP target mRNAs, raising the possibility that FMRP plays a direct role in BC1/mRNA annealing.

Base Sequence↗

PubMed2001

The Saccharomyces cerevisiae small GTPase, Gsp1p/Ran, is involved in 3' processing of 7S-to-5.8S rRNA and in degradation of the excised 5'-A0 fragment of 35S pre-rRNA, both of which are carried out by the exosome.

Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗

PubMed1985

Nucleoprotein autoantibodies in lupus erythematosus.

Autoantibodies against deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) proteins are commonly detected in patients with lupus erythematosus (LE). Antibodies against native DNA are frequently detected in a subset of LE patients with a high prevalence of renal disease. Single-stranded DNA antibodies are also commonly detected in patients with systemic lupus erythematosus (SLE) but recent evidence indicates that approximately 25% of patients with benign, cutaneous (discoid) lupus also possess single-stranded DNA IgM autoantibodies. LE patients also frequently possess antibodies directed against a variety of ribonuclear proteins (RNP). These RNA protein autoantibodies are generally divided into two groups. One group is termed snRNPs (small nuclear ribonuclear protein); the other is termed scRNPs (small cytoplasmic ribonuclear protein). Anti-RNA protein autoantibodies occur as frequently in patients with SLE as do native DNA antibodies. Furthermore, in contradistinction to nDNA antibodies, lupus patients generally make large quantities (detected by gel precipitin techniques) of anti-RNP antibodies. The anti-RNP antibodies are directed against proteins that bind with specific RNA nucleotides. The best evidence at present indicates that these RNA proteins containing the specific RNA nucleotides are involved in RNA processing and post-translational activities such as protein synthesis. Furthermore, these SLE autoantibodies are now being employed, together with other autoantibody systems detected in other connective tissue diseases, to define the biological role of the respective RNA proteins.

Autoantibodies↗

PubMed1993

Expression and functional conservation of the human La (SS-B) nuclear autoantigen in murine cell lines.

We have studied the cellular expression and functional conservation of the human La autoantigen across species by introducing genes encoding human La antigen into cultured murine cell lines. In transfected murine fibroblasts and lymphoid cell lines human La was expressed as a predominantly nuclear antigen, with a typical pattern of nuclear speckling. Radiolabeled human La was of the predicted molecular mass (48 kDa) when expressed in murine cells and was associated intracellularly with murine 60 kDa Ro antigen. Under certain conditions of cell lysis the association between human La and murine Ro was disrupted, whereas human La and human Ro complexes remained intact, suggesting the possibility of species-specific protein interaction between La/Ro antigens. Expression of human La from the genomic gene construct was associated in some transfectants with the presence of additional high molecular weight bands reactive with anti-La monoclonal antibodies. Cell surface La determinants were not detected on any of the transfected cells. Human La protein associated with many of the same radiolabeled RNAs as the murine La antigen in transfected murine cells, indicating intact RNA-binding function of La protein across species. The findings indicate that despite 23% non-identity within the primary structure of human and murine La, the human molecule is functionally highly conserved across species.

Animals↗

PubMed1998

Involvement of the Xenopus laevis Ro60 autoantigen in the alternative interaction of La and CNBP proteins with the 5'UTR of L4 ribosomal protein mRNA.

In vertebrates the synthesis of ribosomal proteins is co-ordinately regulated at the translational level. The 5'-untranslated region (5'UTR) of this class of mRNAs contains conserved regions that are necessary and sufficient for translational regulation. Recently, we found that two proteins, the Xenopus laevis La autoantigen and the cellular nucleic acid binding protein (CNBP), are able to bind in vitro a pyrimidine tract at the 5' end and a downstream region, respectively. These regions are considered the common cis-acting elements of translational regulation. It was previously observed that the binding of both these putative trans-acting factors to their RNA sequences is assisted by a protease-sensitive factor(s) that dissociates from the complex after its formation. Here we provide evidence that the requirement for an ancillary factor assisting La binding to the pyrimidine tract of ribosomal protein mRNAs is typical of this RNA, and secondly that it may involve an RNA recognition motif of the La protein not clearly characterized previously. We also show that the Ro60 autoantigen is involved in the common factor activity necessary for the binding of La and CNBP proteins to their respective sequences. In addition, our findings suggest that an RNA also participates in this process. We show that CNBP can multimerise and that it binds to the 5'UTR as a dimer. Both La and CNBP compete for the interaction with the factor, and their binding to the 5'UTR is mutually exclusive. Our results from the binding analysis of mutations in the 5'UTR, which are known to disrupt the translational control in vivo, suggest a model in which the protein interactions and the 5'UTR RNA structure may co-operate in regulating the translational fate of ribosomal protein mRNAs.

Amino Acid Sequence↗

PubMed1994

Sera from some patients with antibody-associated paraneoplastic encephalomyelitis/sensory neuronopathy recognize the Ro-52K antigen.

Screening a small cell lung cancer cDNA library with serum from a patient with antibody-associated paraneoplastic sensory neuronopathy (Anti-Hu syndrome) resulted in the isolation of a cDNA clone encoding the Ro-52 kD antigen. The Ro-52 kD antigen is one of the major antigens recognized by the sera of patients with Sjögren's syndrome and systemic lupus erythematosus. Further investigation revealed that the sera of only a small percentage (4%) of patients with the Hu syndrome reacted with Ro-52 kD antigen. The cross-reactivity may result from a domain of high homology between Ro-52 kD and HuD antigen. These results emphasize the necessity of using recombinant antigens whenever possible when assaying for assaying for specific antibodies.

Amino Acid Sequence↗

PubMed2003

The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses.

The Fragile X syndrome, which results from the absence of functional FMRP protein, is the most common heritable form of mental retardation. Here, we show that FMRP acts as a translational repressor of specific mRNAs at synapses. Interestingly, FMRP associates not only with these target mRNAs, but also with the dendritic, non-translatable RNA BC1. Blocking of BC1 inhibits the interaction of FMRP with its target mRNAs. Furthermore, BC1 binds directly to FMRP and can also associate, in the absence of any protein, with the mRNAs regulated by FMRP. This suggests a mechanism where BC1 could determine the specificity of FMRP function by linking the regulated mRNAs and FMRP. Thus, when FMRP is not present, loss of translational repression of specific mRNAs at synapses could result in synaptic dysfunction phenotype of Fragile X patients.

Animals↗

PubMed2000

Role of SRP19 in assembly of the Archaeoglobus fulgidus signal recognition particle.

Previous studies have shown that SRP19 promotes association of the highly conserved signal peptide-binding protein, SRP54, with the signal recognition particle (SRP) RNA in both archaeal and eukaryotic model systems. In vitro characterization of this process is now reported using recombinantly expressed components of SRP from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidis. A combination of native gel mobility shift, filter binding, and Ni-NTA agarose bead binding assays were used to determine the binding constants for binary and ternary complexes of SRP proteins and SRP RNA. Archaeal SRP54, unlike eukaryotic homologues, has significant intrinsic affinity for 7S RNA (K(D) approximately 15 nM), making it possible to directly compare particles formed in the presence and absence of SRP19 and thereby assess the precise role of SRP19 in the assembly process. Chemical modification studies using hydroxyl radicals and DEPC identify nonoverlapping primary binding sites for SRP19 and SRP54 corresponding to the tips of helix 6 and helix 8 (SRP19) and the distal loop and asymmetric bulge of helix 8 (SRP54). SRP19 additionally induces conformational changes concentrated in the proximal asymmetric bulge of helix 8. Selected nucleotides in this bulge become modified as a result of SRP19 binding but are subsequently protected from modification by formation of the complete complex with SRP54. Together these results suggest a model for assembly in which bridging the ends of helix 6 and helix 8 by SRP19 induces a long-range structural change to present the proximal bulge in a conformation compatible with high-affinity SRP54 binding.

Archaeal Proteins↗

PubMed2005

Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0.

In many Gram-negative bacteria, the GacS/GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS/GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acyl-homoserine lactones, and which autoinduces the Gac/Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS/A-controlled cell population density-dependent regulation in P. fluorescens.

Bacterial Proteins↗

PubMed1994

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes.

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.

Animals↗

PubMed1997

The SRP9/14 subunit of the human signal recognition particle binds to a variety of Alu-like RNAs and with higher affinity than its mouse homolog.

The heterodimeric subunit, SRP9/14, of the signal recognition particle (SRP) has previously been found to bind to scAlu and scB1 RNAs in vitro and to exist in large excess over SRP in anthropoid cells. Here we show that human and mouse SRP9/14 bind with high affinities to other Alu-like RNAs of different evolutionary ages including the neuron-specific BC200 RNA. The relative dissociation constants of the different RNA-protein complexes are inversely proportional to the evolutionary distance between the Alu RNA species and 7SL RNA. In addition, the human SRP9/14 binds with higher affinity than mouse SRP9/14 to all RNAs analyzed and this difference is not explained by the additional C-terminal domain present in the anthropoid SRP14. The conservation of high affinity interactions between SRP9/14 and Alu-like RNAs strongly indicates that these Alu-like RNPs exist in vivo and that they have cellular functions. The observation that human SRP9/14 binds better than its mouse counterpart to distantly related Alu RNAs, such as recently transposed elements, suggests that the anthropoid-specific excess of SRP9/14 may have a role in controlling Alu amplification rather than in compensating a defect in SRP assembly and functions.

Amino Acid Sequence↗

PubMed2004

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain.

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop-loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop-loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.

Alu Elements↗