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PubMed1986

Small cytoplasmic RNAs from human placental free mRNPs. Structure and their effect on in vitro protein synthesis.

A new family of small cytoplasmic RNA species (scRNAs) was found to be associated with human term-placental free messenger ribonucleoprotein particles (mRNPs). Placental scRNAs strongly inhibit translation of both homologous and heterologous mRNAs in a cell-free rabbit reticulocyte system. scRNAs could be resolved into at least four different RNA species. One of the RNA molecules, scRNA species 1, was the most potent protein synthesis inhibitor found among the placental scRNAs. The nucleotide sequence of the scRNA species 1 was determined. In spite of its short length, scRNA species 1 still exhibited a very strong inhibitory effect on the in vitro protein synthesis. scRNAs were found to be complexed with proteins in the form of scRNPs. Proteins of these complexes enhanced the inhibitory effect of scRNAs on in vitro translation. Experiments provided evidence that inhibition of in vitro protein synthesis by the scRNAs is not dependent upon mRNA concentration. However, inhibition can be overcome by increasing the ratio lysate/scRNAs, thus suggesting that scRNAs act on some essential component of the cell-free system. The degree of inhibition is decreased when scRNAs are added after the start of translation, suggesting that scRNAs (or scRNPs) interfere with the initiation stage of translation, probably acting on an initiation factor(s). Placental scRNAs are unique in their size, being smaller than other known scRNAs. Their association with free cytoplasmic repressed mRNPs in human placenta suggests that scRNAs play a role in the regulation of mRNP metabolism and, consequently, in the control of mRNA translation.

Base Sequence↗
PubMed1991

Effect of heparin contained in preparations of small cytoplasmic RNAs on cell-free translation.

It has been reported that small cytoplasmic RNA (scRNA) from human placenta inhibits translation of most mRNAs in both wheat germ extracts and reticulocyte lysates (Lorberboum, H., Digweed, M., Erdmann, V. A., Servadio, Y., Weinstein, D., De Groot, N., and Hochberg, A. A. (1986) Eur. J. Biochem. 155, 279-287). We have investigated the mechanism by which scRNA preparations inhibit mRNA translation in vitro. We demonstrate that the inhibitory agent(s) is not sensitive to treatment with various ribonucleases but that translational inhibition is sensitive to incubation with 1 N NaOH at 95 degrees C or to treatment with heparinase. Based on these findings and on the ability of heparin to inhibit cell-free translation by itself, we conclude that the presence of heparin in preparations of scRNA from human placenta is responsible for effects which have previously been attributed to inhibitory RNA molecules. Since heparin is also frequently used in the isolation of translationally inhibitory scRNAs from other sources, we suggest that the sensitivity of these preparations to ribonucleases and heparinase should be examined.

Animals↗
PubMed2004

Zinc ion dependent B-cell epitope, associated with primary Sjogren's syndrome, resides within the putative zinc finger domain of Ro60kD autoantigen: physical and immunologic properties.

The Ro/La ribonucleoprotein (RNP) complex is composed of the proteins Ro60kD, Ro52kD, and La48kD that are in association with one small cytoplasmic RNA (YRNA). Specific protein-RNA and protein-protein interactions are thought to occur through the RNP and zinc-finger secondary structure elements of the Ro60kD protein. The aim of our study was to investigate the antigenic properties of the zinc finger domain of the Ro60KD autoantigen and its contribution to the formation of Ro/La RNP complex. It was found that the peptide VSLVCEKLCNEKLLKKARIHPFHILIA (Zif-1), which corresponds to the natural sequence of the zinc finger domain (301-327), and the peptide C(Acm)NEKLLKKARIC(Acm), analogous to the intermediate loop 310-319 (Zif-3) of the same domain of Ro60KD, are recognized by the majority of anti-Ro/SSA and anti-La/SSB positive sera (82.6% and 77.1%, respectively) in the absence of zinc ions. The same sera failed to react with Zif-1 peptide in the presence of Zn2+. In contrast, the addition of zinc ions was necessary for the binding of Zif-1 to recombinant Ro52KD as shown by direct binding experiments of the recombinant protein with synthetic peptides. Our data suggest the zinc finger domain of Ro60kD contains a B-cell epitope with high specificity for primary Sjogren's syndrome. Furthermore, depending on the presence of zinc ions, the zinc finger domain of the Ro60KD protein can exist in two different conformational states favoring either an interaction with the Ro52KD protein or binding with autoantibodies.

Autoantibodies↗
PubMed2005

Application of the BC1 RNA gene promoter for short hairpin RNA expression in cultured neuronal cells.

BC1 RNA is a neuronal cell-specific non-messenger RNA transcribed by RNA polymerase III (Pol III). We previously reported that the transcription of BC1 RNA is controlled both by intragenic promoters for Pol III and by a 5'-flanking region containing several unique cis-elements that are possible members of the Pol II transcription system. In this study, we chose beta-secretase (BACE1) as a target and applied the promoter to produce a short hairpin RNA (shRNA) for RNA interference (RNAi) in cultured neuronal cells. A plasmid vector in which the promoter was linked to a target sequence functioned in rodent NG108-15 cells and suppressed BACE1 protein expression, but did not function in non-neuronal NIH3T3 cells. It was also effective in rat primary hippocampal neurons. We further showed that the promoter can be active in human neuroblastoma SH-SY5Y cells and reduced expression of targeted protein, although the BC1 RNA gene is a rodent-specific gene. The use of this vector-based system for shRNA expression may be an important component of future development of neuronal cell-selective RNAi in both transgenic and therapeutic applications.

Amyloid Precursor Protein Secretases↗
PubMed2005

Structural insights into RNA quality control: the Ro autoantigen binds misfolded RNAs via its central cavity.

The Ro 60 kDa autoantigen is a major target of the immune response in patients with systemic lupus erythematosus. In vertebrate cells, Ro binds misfolded small RNAs and likely functions in RNA quality control. In eukaryotes and bacteria, Ro also associates with small RNAs called Y RNAs. We present structures of unliganded Ro and Ro complexed with two RNAs at 1.95 and 2.2 A resolution, respectively. Ro consists of a von Willebrand factor A domain and a doughnut-shaped domain composed of HEAT repeats. In the complex, a fragment of Y RNA binds on the outer surface of the HEAT-repeat ring, and single-stranded RNA binds in the toroid hole. Mutagenesis supports a binding site for misfolded RNAs that encompasses both sites, with a single-stranded end inserted into the toroid cavity. Our experiments suggest that one role of Y RNAs may be to regulate access of other RNAs to Ro.

Amino Acid Motifs↗
PubMed2005

Ro's role in RNA reconnaissance.

The Ro 60 kDa autoantigen binds misfolded RNAs and likely functions in small RNA quality control. In this issue of Cell, Stein et al. (2005) present crystal structures of Ro alone and bound to both double- and single-stranded RNA, revealing two distinct RNA binding sites that suggest how Ro may distinguish between native and misfolded small RNAs.

Animals↗
PubMed1999

A variety of RNA polymerases II and III-dependent promoter classes is repressed by factors containing the Krüppel-associated/finger preceding box of zinc finger proteins.

KRAB/FPB (Krüppel-associated/finger preceding box) domains are small, portable transcriptional repression motifs, encoded by hundreds of vertebrates C2-H2-type zinc finger genes. We report that KRAB/FPB domains feature an unprecedented, highly promiscuous DNA-binding dependent transcriptional repressing activity. Indeed, template bound chimeric factors containing KRAB/FPB modules actively repress in vivo the transcription of distinct promoter classes that depend on different core elements, recruit distinct basal transcriptional apparatuses and are transcribed either by RNA polymerase II or III. The promoter types repressed in transient assays in a dose- and DNA-binding dependent, but position- and orientation-independent manner, by GAL4-KRAB/FPB fusions include an RNA polymerase II-dependent small nuclear RNA promoter (U1) as well as RNA polymerase III-dependent class 2 (adenovirus VA1), class 3 (human U6) and atypical (human 7SL) promoters. Down-modulation of all of these templates depended on factors containing the A module of the KRAB/FPB domain. Data provide further insights into the properties and mode of action of this widespread repression motif, and support the notion that genes belonging to distinct classes may be repressed in vivo by KRAB/FPB containing zinc finger proteins. The exquisitely DNA-binding dependent transcriptional promiscuity exhibited by KRAB/FPB domains may provide a unique model system for studying the mechanism by which a promoter recruited repression motif can down-modulate a large variety of promoter types.

Animals↗
PubMed2002

Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato.

We investigated the occurrence and properties of small sense and antisense RNAs and aberrant transcripts in transgenic tomato plants exhibiting post-transcriptional silencing of the endogenous polygalacturonase (PG) sense gene and a truncated homologous transgene. Small antisense RNAs, about 23 nucleotides in size, were present in the leaves and immature green fruit where the transgene was transcribed in the absence of transcripts from the fruit ripening-specific endogenous PG gene. This suggests that the small antisense RNAs were produced from the transgene, which was supported by their failure to hybridise with the 3' half of the endogenous PG gene, the region that was excluded from the transgene. The features of two aberrant RNAs, corresponding to the 5' and 3' halves of the endogenous gene, strongly suggest that they arise by endonucleolytic cleavage of endogenous PG mRNA. The region where cleavage occurs is associated with the preferential production of small RNAs from the 3' end of the transgene. These results suggest a model where small antisense RNAs generated from the 3' end of the transgene anneal to the endogenous mRNA, resulting in cleavage at or near the annealing regions.

Blotting, Northern↗
PubMed1985

Synthesis of small nuclear ribonucleoprotein particles by the malarial parasite Plasmodium falciparum.

Sera from patients with autoimmune diseases have been used to identify small nuclear ribonucleoprotein particles (snRNPs) present in higher eukaryotic cells and also in dinoflagellates. Previously these sera have not detected crossreactive snRNP protein antigens of other lower eukaryotes such as yeast, Tetrahymena, or Dictyostelium. We report that anti-Sm, anti-U1-RNP, and anti-La/SS-B human antisera react with specific snRNP protein antigens synthesized by the protozoan Plasmodium falciparum, the human malarial parasite. These results suggest that the structure and antigenicity (and thus probably the function) of snRNPs have been widely conserved in eukaryote evolution.

Animals↗
PubMed2005

Down-regulation of 7SL RNA expression and impairment of vesicular protein transport pathways by Leishmania infection of macrophages.

The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.

Animals↗
PubMed1988

A 52-kD protein is a novel component of the SS-A/Ro antigenic particle.

Anti-SS-A/Ro autoantibodies are found in the sera of patients with Sjogren's syndrome (SS) and SLE. In the course of analyzing 61 SS patients for their autoantibody profiles, we found that 42 were positive for anti-SS-A by double diffusion in agarose and demonstrated precipitin lines identical to that produced by a prototype anti-SS-A serum. Further analysis of these SS-A antibody-positive sera by Western blotting of cell extracts revealed that 21 sera reacted with two proteins of 60 and 52 kD, 13 sera reacted with 52-kD protein, two detected only 60 kD, while six were nonreactive. Affinity-purified anti-60-kD and anti-52-kD antibodies reacted exclusively with their corresponding antigens. Partial proteolysis of these proteins did not reveal common degradation fragments. Thus the 52- and 60-kD proteins were found to be antigenically and apparently structurally distinct from each other. They were also distinct from 48-kD SS-B/La protein. In immunoprecipitation using labeled cell extracts, affinity-purified anti-52-kD antibodies brought down the 52-kD protein as well as the 60-kD band. In [32P]orthophosphate-labeled HeLa cell extract both antibodies precipitated the same spectrum of small RNAs (hYl-5). In indirect immunofluorescence, anti-52-kD and anti-60-kD antibodies immunolocalized in similar subcellular structures and showed similar punctate nuclear staining patterns. Western blot analysis revealed that both proteins were present in lymphocytic as well as epithelial human cell lines tested. The data above define a new antigen of 52 kD which is another component of the SS-A particle and is associated in complex formation with the previously reported 60-kD protein.

Autoantibodies↗
PubMed1994

Compilation of small RNA sequences.

This is an update containing small RNA sequences deposited in GenBank recently. Over four hundred small RNA sequences are available in this and earlier complications.

Animals↗
PubMed1995

An upstream U-snRNA gene-like promoter is required for transcription of the Arabidopsis thaliana 7SL RNA gene.

The genes transcribed by RNA polymerase (pol) III can be placed into four distinct groups based on the nature and position of their promoter elements. In the higher eukaryotes equivalent genes usually belong to the same sub-type of pol III promoters and there are few examples of genes which have changed promoter type during evolution. In this work we demonstrate that the promoter of the Arabidopsis thaliana 7SL RNA gene is located upstream of the coding region and is identical to the promoters of pol III-specific plant U-small nuclear RNA (U-snRNA) genes. Sequence analysis of two different 7SL genes from A. thaliana revealed that both genes contain two sequence elements in their 5' flanking regions identical in sequence and position to the highly conserved USE and TATA elements of the pol III-transcribed plant U-snRNA genes. Mutational analysis of these elements in the At7SL-2 gene indicates that the USE and TATA elements are both necessary and account for > or = 90% of the transcriptional activity of this gene in transfected plant protoplasts. Within the coding region of both genes there is a sequence element which is a 10/11 nt match to the consensus B-box element of tRNA genes, however, this element is not important for gene activity. These findings distinguish the plant genes from the human 7SL gene, which has both internal and upstream promoter elements and its upstream elements are different from those found in the human U-snRNA genes.

Arabidopsis↗
PubMed2004

RNA silencing in plants by the expression of siRNA duplexes.

In animal cells, stable RNA silencing can be achieved by vector-based small interfering RNA (siRNA) expression system, in which Pol III RNA gene promoters are used to drive the expression of short hairpin RNA, however, this has not been demonstrated in plants. Whether Pol III RNA gene promoter is capable of driving siRNA expression in plants is unknown. Here, we report that RNA silencing was achieved in plants through stable expression of short hairpin RNA, which was driven by Pol III RNA gene promoters. Using glucuronidase (GUS) transformed tobacco as a model system, the results demonstrated that 21 nt RNA duplexes, targeting at different sites of GUS gene, were stably expressed under the control of either human H1 or Arabidopsis 7SL RNA gene promoter, and GUS gene was silenced in 80% of siRNA transgenics. The severity of silencing was correlated with the abundance of siRNA expression but independent of the target sites and uridine residue structures in siRNA hairpin transcripts. Thus, the specific expression of siRNA provides a new system for the study of siRNA silencing pathways and functional genomics in plants. Moreover, the effectiveness of the human H1 promoter in a plant background suggested a conserved mechanism underlying Pol III complex functionality.

Gene Expression Regulation, Plant↗