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PubMed1990

micF RNA binds to the 5' end of ompF mRNA and to a protein from Escherichia coli.

micF RNA regulates the levels of outer membrane protein F (OmpF) in Escherichia coli in response to temperature increase and other stress conditions by decreasing the levels of ompF mRNA (Andersen et al., 1989). A 93-nucleotide micF RNA was synthesized in vitro directly from polymerase chain reaction generated DNA which was designed to contain a functional T7 RNA polymerase promoter upstream of the micF RNA gene and an appropriate restriction site for transcription termination. A transcript (150 nucleotides) containing the ribosomal binding domain of ompF mRNA messenger was synthesized in vitro from the ompF gene cloned into a T7 expression vector. A stable duplex was formed between micF RNA and the 150-nucleotide 5' transcript of ompF mRNA after incubation at 37 degrees C in a physiological buffer. The melting curve of the duplex formed by micF RNA and 150-nucleotide transcript revealed a Tm of 56 degrees C and a delta Tm that spans about 20 degrees C; both are consistent with the proposed structure for the micF/ompF duplex. In addition, as determined by competition studies and UV cross-linking/label-transfer analyses, an E. coli protein was found to bind specifically to micF RNA. The protein also bound weakly to the 150-nucleotide ompF transcript. The data are the first to demonstrate the complex between micF RNA and the 5' end of ompF mRNA and suggest that in vivo a micF ribonucleoprotein (RNP) particle may participate in the destabilization ompF mRNA during thermoregulation of OmpF porin.

Bacterial Outer Membrane Proteins↗

PubMed1999

A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.

We describe the isolation and molecular characterization of seven distinct proteins present in human [U4/U6.U5] tri-snRNPs. These proteins exhibit clear homology to the Sm proteins and are thus denoted LSm (like Sm) proteins. Purified LSm proteins form a heteromer that is stable even in the absence of RNA and exhibits a doughnut shape under the electron microscope, with striking similarity to the Sm core RNP structure. The purified LSm heteromer binds specifically to U6 snRNA, requiring the 3'-terminal U-tract for complex formation. The 3'-end of U6 snRNA was also co-precipitated with LSm proteins after digestion of isolated tri-snRNPs with RNaseT(1). Importantly, the LSm proteins did not bind to the U-rich Sm sites of intact U1, U2, U4 or U5 snRNAs, indicating that they can only interact with a 3'-terminal U-tract. Finally, we show that the LSm proteins facilitate the formation of U4/U6 RNA duplices in vitro, suggesting that the LSm proteins may play a role in U4/U6 snRNP formation.

Amino Acid Sequence↗

PubMed1996

Hel-N1/Hel-N2 proteins are bound to poly(A)+ mRNA in granular RNP structures and are implicated in neuronal differentiation.

Human proteins Hel-N1 and Hel-N2 contain three RNA recognition motifs (RRMs), and are members of a family of proteins highly homologous to Drosophila ELAV, which is essential for neuronal differentiation. Both proteins bind to A+U-rich 3' untranslated regions of a variety of growth-related mRNAs in vitro. Here we demonstrate that in medulloblastoma cells derived from childhood brain tumors, Hel-N1 and Hel-N2 are mainly expressed in the cytoplasm, but are detectable in the nucleus. Both proteins are associated with polysomes and can be UV-crosslinked to poly(A)+ mRNA in cell extracts. In the cytoplasm the Hel-N1 protein family resides in granular structures that may contain multiple protein molecules bound to each mRNA. Evidence supporting this multimeric ribonucleoprotein (RNP) model includes in vitro reconstitution and competition experiments in which addition of a single RRM (RRM3) can alter complex formation. As in medulloblastoma cells, the Hel-N1 protein family is present in granular particles in the soma and the proximal regions of dendrites of cultured neurons, and colocalizes with ribosomes. In addition, we demonstrate that expression of the Hel-N1 protein family is up-regulated during neuronal differentiation of embryonic carcinoma P19 cells. Our data suggest that the Hel-N1 protein family is associated with the translational apparatus and implicated in both mRNA metabolism and neuronal differentiation. Furthermore, our findings open the possibility that these proteins participate in mRNA homeostasis in the dendrites and soma of mature neurons.

Animals↗

PubMed1987

Autoantibodies against small nuclear ribonucleoprotein components.

Sera from patients with systemic rheumatic diseases often contain antibodies against nuclear antigens. Among these are proteins complexed with the small nuclear RNA (snRNA) U1, U2, U4, U5 and U6. In human cells, at least 11 different polypeptides (called 70K, A, A', B', B", B, C, D, E, F and G) ranging in molecular weight from 70 to 11 kDa, are associated with the snRNA, constituting the snRNP particles. In this report characteristics of 3 classes of autoantibodies are described. Anti-(U1) RNP antibodies recognize the U1 specific 70K and/or A and/or C proteins. Anti-Sm antibodies recognize proteins associated with all U snRNA, primarily the B', B and D proteins. Anti-(U1,U2) RNP sera contain antibodies directed against the U2 specific A' or B" proteins. These antibodies are useful tools for studying the structure and function of U snRNP complexes. Using mutant U2 snRNA and monospecific antibodies directed against the U2 specific proteins, we analyzed the interactions between proteins and RNA in the U2 snRNP particle. We have also isolated recombinant clones containing cDNA of mRNA coding for a number of snRNA associated proteins. Possible directions of future research are discussed.

Antibodies, Antinuclear↗

PubMedDate not supplied

[Nuclear RNP, containing pre-mRNA. XIV. Nature of particles, containing 5'- and 3'-end structures].

Some characteristic peculiarities of the 5'-end and 3'-end structures of pre-mRNA isolated from nuclear RNP particles have been investigated: presence of triphosphorylated nucleotides on the 5'-ends, as a characteristic of the primary product of transcription; presence of modified 5'-ends -- blocked and methylated structures -- caps; presence of poly(A) blocks attached to the 3'-end of pre-mRNA during post-transcriptional transformation of the latter. It was shown that pre-mRNA isolated from nuclear RNP particles contained triphosphorylated nucleotides as well as a "cap" structure at the 5'-ends. On the 3'-ends of pre-mRNA from nuclear RNP particles isolated in the presence of a RNAse inhibitor, the presence of poly(A) blocks have been shown. These poly(A) structures are separated very easily from pre-mRNA during mild RNAase digestion. This is the reason why they are not detected in 30S monoparticles, containing pre-mRNA fragments connected with one informofer. Almost all poly(A) complexes in this condition are combined with proteins and have the sedimentation coefficient of about 14S. It was concluded that formation of nuclear pre-mRNA containing RNP-particles take place just after the onset of RNA synthesis. All processing steps occur with pre-mRNA packed in nuclear RNP particles.

Animals↗

PubMed2001

Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins.

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and alpha-actinin and colocalizes with vinculin/metavinculin and alpha-actinin at microfilament attachment sites, such as cell-cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

Actin Cytoskeleton↗

PubMed2000

MSSP promotes ras/myc cooperative cell transforming activity by binding to c-Myc.

BACKGROUND: MSSPs, myc single strand binding proteins, were originally identified as proteins recognizing a putative replication origin/transcriptional enhancer in the human c-Myc gene. The cDNAs encoding four of the family proteins, MSSP-1, MSSP-2, Scr2 and Scr3, were cloned. These proteins carry two copies of the putative RNA binding domains, RNP-A and RNP-B, and have been suggested to participate in DNA replication and cell cycle progression from the G1 to the S phase. RESULTS: We report that MSSP-1 and MSSP-2 bound directly to the C-terminal portion of c-Myc, along with Max, side by side. MSSP, c-Myc and Max formed a ternary complex in vivo, although MSSP did not directly associate with Max. The MSSP/Myc/Max ternary complex lost the binding activity to the E-box sequence-the recognition sequence of c-Myc/Max complex-thereby abrogating the E-box-dependent transcription activity of c-Myc. MSSP specifically stimulated the cooperative transforming activity of c-myc with ras, in a manner dependent upon the RNP sequences, while mssp itself showed no transforming activity in mouse NIH3T3 cells. The NIH3T3 transformants, together with ras, myc and mssp, grew to form very large colonies in soft agar, as compared to those with ras plus myc or ras alone. CONCLUSIONS: MSSP is a modulator of c-Myc and the c-Myc/MSSP complex may deregulate cell cycle controls and lead cells towards transforming pathways.

3T3 Cells↗

PubMed1997

C-myc deregulation during transformation induction: involvement of 7SK RNA.

The process of oncogenic transformation has been widely studied but is still poorly understood. We have focused on the mechanism of deregulation of the c-myc gene during transformation of a temperature-sensitive SV40-transformed mouse cell line. Run-on transcription assays showed that the two c-myc minor promoters, P1 and P3, are transiently activated following induction of transformation and that peak activation of both promoters is preceded by a large increase in transcription of a small RNA (7SK). To test the possibility that this RNA might participate in promoter activation, we transfected cells with sense and antisense oligodeoxynucleotides corresponding to different regions of the 7SK RNA predicted to be accessible within the RNP particle. Out of 14 oligos tested, inhibition of activation of P1 and/or P3 was observed with four antisense oligonucleotides corresponding to looped regions in the putative 7SK secondary structure. To identify c-myc promoter sequences which might serve as targets for 7SK activity, we carried out mobility-shift assays with either whole or 7SK-depleted cell extracts. The CT element of the c-myc promoter formed a 7SK-dependent complex which could be competed only with the same antisense 7SK oligo that suppressed P1 and P3 activation in vivo. Taken together these results suggest that 7SK RNP participates in transformation-dependent c-myc deregulation.

3T3 Cells↗

PubMed1987

Hypertrophic osteoarthropathy, cutaneous vasculitis, and mixed-type cryoglobulinemia in a patient with nasopharyngeal carcinoma.

We describe a 27-year-old woman with a primary nasopharyngeal carcinoma who developed hypertrophic osteoarthropathy and cutaneous vasculitis. Her serum contained antibodies to Epstein-Barr virus and U1 RNP antigens. Cryoproteins isolated from her serum contained antibodies to U1 RNP and a protein with a molecular weight of 32 kd which reacted specifically with antibodies to U1 RNP. HLA typing revealed HLA-B7 and DR1; these have been reported to be increased in Japanese patients with rheumatic diseases who have autoantibodies to U1 RNP. These findings indicate that some features of the paraneoplastic syndrome in this patient might have been caused by immune complexes, part of which were formed by specific autoantibodies produced under genetically controlled conditions of immune responsiveness.

Adult↗

PubMed2006

The vertebrate E1/U17 small nucleolar ribonucleoprotein particle.

Each of the many different box H/ACA ribonucleoprotein particles (RNPs) present in eukaryotes and archaea consists of four common core proteins and one specific H/ACA small RNA, which bears the sequence elements H (ANANNA) and ACA. Most of the H/ACA RNPs are small nucleolar RNPs (snoRNPs), which are localized in nucleoli, and are one of the two major classes of snoRNPs. Most H/ACA RNPs direct pseudouridine synthesis in pre-rRNA and other RNAs. One H/ACA small nucleolar RNA (snoRNA), vertebrate E1/U17 (snR30 in yeast), is required for pre-rRNA cleavage processing that generates mature 18S rRNA. E1 snoRNA is encoded in introns of protein-coding genes, and the evidence suggests that human E1 RNA undergoes uridine insertional RNA editing. The vertebrate E1 RNA consensus secondary structure shows several features that are absent in other box H/ACA snoRNAs. The available UV-induced RNA-protein crosslinking results suggest that the E1 snoRNP is asymmetrical in vertebrate cells, in contrast to other H/ACA snoRNPs. The vertebrate E1 snoRNP in cells is surprisingly complex: (i) E1 RNA contacts directly and specifically several proteins which do not appear to be any of the H/ACA RNP four core proteins; and (ii) multiple E1 RNA sites are needed for E1 snoRNP formation, E1 RNA stability, and E1 RNA-protein direct interactions.

Animals↗

PubMed1989

The mammalian analogue of the yeast PRP8 splicing protein is present in the U4/5/6 small nuclear ribonucleoprotein particle and the spliceosome.

HeLa cell nuclear extracts contain a protein reactive with antibodies against PRP8, a polypeptide essential for pre-mRNA splicing in yeast and a specific component of the yeast U5 small nuclear ribonucleoprotein (snRNP) [Lossky, M., Anderson, G. J., Jackson, S. P. & Beggs, J. (1987) Cell 51, 1019-1026]. The mammalian protein appears as a doublet at approximately 200 kDa, smaller than the 260-kDa yeast protein, and possesses an Sm epitope as determined by immunoblotting. Its association with a snRNP of the Sm class other than U1 or U2 is indicated by its immunoprecipitation by anti-Sm and anti-trimethylguanosine antibodies but not by anti-(U1) or anti-(U2) RNP sera. Gradient fractionation of splicing extracts demonstrates that the 200-kDa protein is a component of the U4/5/6 snRNP complex and of U5 snRNPs. It is also present in affinity-purified spliceosomes.

Antibodies, Monoclonal↗

PubMed1964

STUDIES ON GUINEA PIG OOCYTES. I. ELECTRON MICROSCOPIC OBSERVATIONS ON THE DEVELOPMENT OF CYTOPLASMIC ORGANELLES IN OOCYTES OF PRIMORDIAL AND PRIMARY FOLLICLES.

Oocytes in primordial and primary follicles of young adult guinea pig ovaries fixed in osmium tetroxide and embedded in Epon 812, have been observed by electron microscopy. The gradual differentiation of a series of cytoplasmic organelles has been correlated with the growth in size of the oocyte and the development of the follicular wall. The most immature primordial oocyte is morphologically a simple cell consisting of a large slightly eccentric nucleus, a few large spherical mitochondria, a profusion of granular cytoplasmic vesicles, and free RNP particles. At the primary follicle stage, abundant endoplasmic reticulum, clusters of mitochondria proliferating around a rosette formation, multiple Golgi complexes, vesicular aggregates forming cortical granules, and a profusion of microvilli have been differentiated. Concentrations of cytoplasmic organelles at the periphery of the oocyte in the primary follicle suggest that it is equipped for the absorption, utilization, and intracellular transport of material delivered to its surface membrane. The juxtaposition of components of the ultrastructure during the development and growth of this large cell appears to follow a precise pattern and provides an unusual opportunity to study the interrelationships of differentiating organelles.

Adult↗

PubMed2005

Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export.

A specific messenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules in the dipteran Chironomus tentans, can be visualized during passage through the nuclear pore complex (NPC). We have now examined the transport through the nuclear basket preceding the actual translocation through the NPC. The basket consists of eight fibrils anchored to the NPC core by nucleoprotein Nup153. On nuclear injection of anti-Nup153, the transport of BR granules is blocked. Many granules are retained on top of the nuclear basket, whereas no granules are seen in transit through NPC. Interestingly, the effect of Nup153 seems distant from the antibody-binding site at the base of the basket. We conclude that the entry into the basket is a two-step process: an mRMP first binds to the tip of the basket fibrils and only then is it transferred into the basket by a Nup153-dependent process. It is indicated that ribosomal subunits follow a similar pathway.

Animals↗

PubMed2005

Unusual features of fibrillarin cDNA and gene structure in Euglena gracilis: evolutionary conservation of core proteins and structural predictions for methylation-guide box C/D snoRNPs throughout the domain Eucarya.

Box C/D ribonucleoprotein (RNP) particles mediate O2'-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution.

Animals↗

PubMed1982

Interaction of 5S RNA-L5 protein complex with 40S subunits in rat liver ribosomes.

When rat liver 80S ribosomes or polysomes were dissociated into their subunits by EDTA-treatment, a 5S RNA-L5 protein complex was present in EDTA-derived small subunits, and released from the subunits by urea. The interaction of 5S RNP with the small subunits did not occur in the case of 80S couples formed from 40S and 60S subunits in the presence of Mg2+, indicating that the attachment of 5S RNP to EDTA-derived small subunits was not an artifact caused by the unfolding of the subunits. These results give direct evidence that 5S RNP is located at the interface between large and small subunits, and suggest that 5S RNP binds to the 40S subunit in the functioning polysomes, but not the 80S couples.

Animals↗

PubMed2004

Solution structure and domain architecture of the divisome protein FtsN.

Prokaryotic cell division occurs through the formation of a septum, which in Escherichia coli requires coordination of the invagination of the inner membrane, biosynthesis of peptidoglycan and constriction of the outer membrane. FtsN is an essential cell division protein and forms part of the divisome, a putative complex of proteins located in the cytoplasmic membrane. Structural analyses of FtsN by nuclear magnetic resonance (NMR) reveals an RNP-like fold at the C-terminus (comprising residues 243-319), which has significant sequence homology to a peptidoglycan-binding domain. Sequential deletion mutagenesis in combination with NMR shows that the remaining of the periplasmic region of FtsN is unfolded, with the exception of three short, only partially formed helices following the trans-membrane helix. Based on these findings we propose a model in which FtsN, anchored in the inner membrane, bridges over to the peptidoglycan layer, thereby enabling the coordination of the divisome and the murein-shaping machinery in the periplasm.

Amino Acid Sequence↗

PubMed1983

Ultrastructural and purification studies on human tumor nucleolar antigens and nucleolar particles.

The presence of common nucleolar antigens in a broad array of human malignant tumors has led to several lines of investigations. In addition to studies on an increasing number of benign and malignant neoplasms with a variety of antibodies designed to statistically evaluate the presence of nucleolar antigens, purification procedures and chemical analyses are being used to characterize the specific antigens. The localization of the nucleolar antigens in HeLa cells was studied by the postembedding immunoelectron microscopic procedure employing rabbit antibodies to nucleoli or nuclear Tris extracts of these cells. The products of the peroxidase-antiperoxidase complexes visualized by the reaction with diaminobenzidine in nucleoli were mainly found in the nucleolonemas which contain the dense nucleolar RNP components. When these nucleoli became compact after treatment of HeLa cells with adriamycin, the distribution of the immunoreactive products was altered along with distribution of the dense nucleolar components. The human nucleolar antigens were mainly localized to nucleolar regions containing the nucleolar RNP components. Improved purification of the antigens made it possible to provide a satisfactory amino acid analysis of one pI 6.3 antigen. Interestingly, some of the nucleolar antigen was found in miniparticle undescribed until now.

Amino Acids↗

PubMedDate not supplied

Hypothesis: systemic lupus erythematosus as a disease secondary to polyclonal lymphocyte activation.

Systemic lupus erythematosus (SLE) is characterized by the concomitance of B cell polyclonal activation and the production of a wide spectrum of autoantibodies showing a marked preference for nuclear antigens. We propose the hypothesis that the production of the most pathogenic antinuclear antibodies with high affinity and IgG isotype is driven by antigens liberated by the death of rapidly proliferating lymphoid cells. The disease could evolve in three steps: (1) polyclonal B cell activation and proliferation caused by unknown etiologic factors (virus?, mitogen? ...); (2) primary activation of natural antibody-producing clones leading to the appearance in the serum of large amounts of natural autoantibodies with public idiotype; and (3) secondary immunization against molecules synthesized in large quantities during lymphocyte proliferation, released by dying cells and subsequently presented in an immunogenic form to T cells. These molecules include nucleic acids (DNA, RNA), nucleic acid-binding proteins (histones, Ro, La), and parts of the RNA splicing machinery (RNP). Other autoantibody activities could relate to cross-reactivities or the binding properties of DNA-anti-DNA immune complexes.

Animals↗