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PubMed1993

Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III.

Vaults are large cytoplasmic ribonucleoprotein particles with a sedimentation value of about 150 S. These particles contain a unique small RNA (vault RNA (vRNA)). We have determined the sequence of the RNA associated with vaults purified from both rat and bullfrog. The rat vRNA is 141 bases in length, whereas the bullfrog vRNA is present as two highly related species of 89 and 94 bases. Despite the differences in length the predicted secondary structures of the three vRNAs are clearly related. All of the vRNAs contain sequences related to the internal promoter elements necessary for transcription by RNA polymerase III. The gene for the rat vRNA was isolated and sequenced from a rat genomic library, and its transcription by RNA polymerase III was verified using an in vitro transcription assay. The rat vRNA gene was efficiently transcribed in vitro, producing a single transcript of about 140 bases. Unlike most RNA polymerase III genes, the rat vRNA is present as a single copy gene and has a distinct tissue-specific expression pattern.

Animals↗
PubMed1987

Characterization of the small RNA of the bacteriophage phi 29 DNA packaging machine.

The prohead connector of the bacteriophage luminal diameter 29 DNA packaging machine was reconstructed with the small RNA that regulates DNA packaging in vitro. The complete sequence of the 120 nucleotide RNA proved its origination from the promoter PE1(A1) of the left early region of phi 29 DNA, the end packaged first during assembly. The prohead RNA was clearly distinct from eubacterial 5S rRNA in sequence and composition.

Bacillus subtilis↗
PubMed2025

A Small RNA Derived From the 5' End of the IS200 tnpA Transcript Regulates Multiple Virulence Regulons in Salmonella typhimurium.

The insertion sequence IS200 is widely distributed in Eubacteria. Despite its prevalence, IS200 does not appear to be mobile and as such is considered an ancestral component of bacterial genomes. Previous work in Salmonella enterica revealed that the IS200 tnpA transcript is processed to form a small, highly structured RNA (5'tnpA) that participates in the posttranscriptional control of invF expression, encoding a key transcription factor in this enteropathogen's invasion regulon. To further examine the scope of 5'tnpA transcript integration into Salmonella gene expression networks, we performed comparative RNA-seq, revealing the differential expression of over 200 genes in a Salmonella SL1344 5'tnpA disruption strain. This includes the genes for the master regulators of both invasion and flagellar regulons (HilD and FlhDC, respectively), plus genes involved in cysteine biosynthesis and an operon (phsABC) encoding a thiosulfate reductase complex. These expression changes were accompanied by an 80-fold increase in Salmonella invasion of HeLa cells. Follow-up experimentation suggested an additional direct target of 5'tnpA to be the small RNA PinT, which has previously been shown to be a negative regulator of invasion genes through its inhibitory action on key transcription factors governing the Salmonella pathogenicity island 1 regulon. This study provides a powerful new example of bacterial transposon domestication that is based not on the production/use of a regulatory protein or regulatory DNA sequences, but on the function of a transposon-derived small RNA.

Salmonella typhimurium↗
PubMed2006

Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

DNA Methylation↗
PubMed2006

[Comparison between the suppression of tissue inhibitor of metalloproteinase-1 gene expression by recombinant adeno-associated virus carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 in rat hepatic stellate cells].

OBJECTIVES: Elevated tissue inhibitor of metalloproteinase-1 (TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses (AAV) carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 (rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo), and then to compare the suppression of TIMP-1 gene expression on rat hepatic stellate cell (HSC)-T6 cells infected by these two types of viruses in vitro. METHODS: Antisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector (pdl6-95/neo) and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells. RESULTS: The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/ANTI-TIMP-1/neo and pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TIMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells (P less than 0.01), and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly (60%), while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANTI-TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells (P more than 0.05). CONCLUSION: RNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integration.

Animals↗
PubMed1994

Analysis of Epstein-Barr virus-encoded small RNA 1 expression in benign lymphoepithelial salivary gland lesions.

Salivary gland enlargement resulting from benign lymphoepithelial lesions may be seen in patients with Sjögren's syndrome (SS) and in the early stages of human immunodeficiency virus (HIV) infection. Although the pathogenesis of these lesions is thought to differ in SS and HIV infection, Epstein-Barr virus (EBV) has been suggested as a pathogenetic agent in both cases. We have assessed the presence of latent EBV infection in a series of 15 lymphoepithelial salivary gland lesions using RNA-RNA in situ hybridization with digoxigenin-labeled riboprobes complementary to the abundantly-expressed EBV-encoded small RNA 1 (EBER1). Two of four benign lymphoepithelial lesions (BLEL) from patients seropositive for HIV expressed EBER1 in lymphocyte nuclei in a fashion similar to that described previously in HIV-associated persistent generalized lymphadenopathy (PGL). By contrast, EBER1 was not expressed in any of four BLEL from patients with SS, or seven lymphoepithelial cysts (LEC) and BLEL from patients with neither HIV infection nor SS. These data suggest that latent EBV infection does not play a major pathogenetic role in the lymphoepithelial salivary gland lesions associated with SS or those seen in patients without systemic disease. In the case of HIV-associated salivary gland lesions, the data are consistent with earlier proposals that these BLEL result from the involvement of intraparotid lymph nodes by PGL.

Adult↗
PubMed2002

Specific inhibition of bcr-abl gene expression by small interfering RNA.

Small interfering RNAs (siRNAs) were designed to target the bcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl-positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti-bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl-positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl-positive and normal CD34(+) cells, respectively. Finally, anti-bcr-abl siRNA inhibited BCR-ABL-dependent, but not cytokine-dependent, proliferation in a bcr-abl-positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells.

Animals↗
PubMed2004

The small RNA regulators of Escherichia coli: roles and mechanisms*.

Small noncoding RNAs have been found in all organisms, primarily as regulators of translation and message stability. The most exhaustive searches have taken place in E. coli, resulting in identification of more than 50 small RNAs, or 1%-2% of the number of protein-coding genes. One large class of these small RNAs uses the RNA chaperone Hfq; members of this class act by pairing to target messenger RNAs. Among the members of this class are DsrA and RprA, which positively regulate rpoS translation, OxyS, which negatively regulates rpoS translation and fhlA translation, RyhB, which reapportions iron use in the cell by downregulating translation of many genes that encode Fe-containing proteins, and Spot 42, which changes the polarity of translation in the gal operon. The promoters of these small RNAs are tightly regulated, frequently as part of well-understood regulons. Lessons learned from the study of small RNAs in E. coli can be applied to finding these important regulators in other organisms.

Bacterial Outer Membrane Proteins↗
PubMed1994

Herpesvirus saimiri small RNA and interleukin-4 mRNA AUUUA repeats compete for sequence-specific factors including a novel 70K protein.

A highly oncogenic strain of the lymphotropic tumour virus herpesvirus saimiri (HVS; strain 484-77) expresses four small RNAs (HSUR1 to 4) in high copy numbers in transformed T cells. In HSUR1 and HSUR2 the 5' terminal regions contain conserved AUUUA sequence repeats. The same AUUUA repeats occur in the 3' non-coding regions of growth factor, lymphokine and protooncogene mRNAs, and the sequence is involved in rapid mRNA degradation. We report here that by using a highly specific u.v. cross-linking method we identified a novel 70K binding factor with AUUUA sequence specificity. Non-radiolabelled competition and V8 protease analysis show that the protein can form a complex with the 3' non-coding region of interleukin-4 mRNA and bind the AUUUA repeats of a HVS small RNA. We also detected an AUUUA-specific minor 32K human protein with the same electrophoretic mobility as a marmoset factor implicated in growth factor mRNA destabilization. The findings are consistent with the hypothesis that the viral small RNAs can compete for factors involved in rapid degradation of growth factor mRNAs and may contribute to viral oncogenesis.

Base Sequence↗
PubMed1998

Selection with SELEX Method of Small RNA Molecules Specifically Binding to Starch.

A 73-base DNA library with T7 promoter was chemically synthesized according to the sequence specified by the 2.5S RNA component of glycogen branching holoenzyme (EC 2.4.1.18) from rabbit muscle, in which 20 consecutive bases corresponding to a stem-loop structure were completely randomized. The in vitro transcribed RNA library was subjected to 9 successive rounds of selection with SELEX method, resulting in a sharp increase of the percentage of the starch-binding RNA from less than 0.01% for the first round pool to 31% for the ninth-round pool. This demonstrates the existence of small RNA species that can specifically bind to starch. Structural and functional analysis of them is in progress.

Journal Article↗
PubMed1997

Use of adenoviral VAI small RNA as a carrier for cytoplasmic delivery of ribozymes.

The in vivo effectiveness of therapeutic RNAs, like antisense molecules and ribozymes, relies on several features: RNA molecules need to be expressed at high levels in the correct cellular compartment as stable and active molecules. The exploitation of "natural" small RNA coding genes as expressing cassettes gives high chances to fulfill these requirements. We have investigated the utilization of the adenoviral VAI RNA as a cytoplasmatic carrier for expressing ribozymes against HIV-1. The conserved 5' leader sequence of HIV was chosen as a target, because it is present in all the viral transcripts and is highly conserved. Hammerhead ribozymes were substituted to different portions of the VAI RNA and the resulting chimera were tested in the in vivo system of Xenopus laevis oocytes for their level of accumulation, cellular compartmentalization, and assembly in specific ribonucleoparticles containing the La antigen. Interesting differences in the activity of the different chimera were found in both in vitro cleavage assays and S100 extracts of injected oocytes where the catalytic activity of the ribozymes in the RNP context can be analyzed.

Adenoviruses, Human↗
PubMed1995

[Expression of Epstein-Barr virus encoded small RNA-1 and latent membrane protein-1 in midline T-cell lymphomas].

Nine cases of midline T-cell lymphoma (MTL) were studied for expression of Epstein-Barr virus (EBV) encoded small RNA-1 (EBER-1) and EBV latent membrane protein-1 (LMP-1) using in situ hybridization and immunohistochemistry respectively. EBER-1 was detected in nuclei of tumor cells in 8/9 cases of MTL and LMP-1 was found on membrane and in cytoplasm of tumor cells in 7/9 cases of MTL. The results show: (1) There is a strong association between EBV and MTL. EBV may play an important role in the pathogenesis of MTL. (2) The detection frequency of EBV in MTL is higher than peripheral T-cell lymphoma from other sites of the body. (3) EBER-1 in situ hybridization and LMP-1 immunohistochemistry are sensitive and reliable in detection of EBV in MTL and the latter is more economic and convenient.

Herpesvirus 4, Human↗
PubMed1986

Efficient and accurate in vitro processing of simian virus 40-associated small RNA.

Nuclei were isolated from simian virus 40 (SV40)-infected cells with a hypotonic, detergent-free buffer and incubated in vitro in a high-ionic-strength buffer containing [alpha-32P]UTP. The labeled viral RNAs produced were analyzed by gel electrophoresis together with 3-h-labeled viral RNAs extracted from SV40-infected cells. The in vitro-synthesized RNA contained a major RNA species of 62 to 64 nucleotides that appeared on the gel at the same position as in vivo-synthesized SV40-associated small RNA (SAS-RNA). Analyses of the in vitro-synthesized 62- to 64-nucleotide RNA by hybridization to restriction fragments and by the use of an SAS-RNA deletion mutant clearly identified it as SAS-RNA. The intensity of the band of the in vitro-synthesized SAS-RNA increased with an increase in the labeling time or when a short pulse was followed by a chase. Moreover, the SAS-RNA band disappeared when ITP replaced GTP in the transcription reaction mixture. These results indicate that SAS-RNA is processed from a precursor molecule and that an RNA secondary structure could be an element recognized by the processing enzyme.

Animals↗
PubMed2004

Selective inhibition of HIV-1 reverse transcriptase (HIV-1 RT) RNase H by small RNA hairpins and dumbbells.

We present here the design of a novel class of RNA inhibitors of the RNase H domain of HIV-1 RT, a ribonuclease activity that is essential for viral replication in vivo. Specifically, we show that small RNA hairpins and dumbbells can selectively inhibit the RNase H activity of HIV-1 RT without affecting other cellular RNases H (e.g., E. coli and human RNase H). These results suggest that the inhibitors do not interact with the nucleic acid binding site of RT RNase H, as this region should be well conserved among the various enzymes. The most potent inhibitors displayed IC50 values in the 3-8 microM range. Remarkably, the DNA polymerase activity, an intrinsic property of HIV RT, was not inhibited by the hairpin and dumbbell aptamers, a property not previously observed for any nucleic acid aptamer directed against RT RNase H. The results described here suggest a noncompetitive binding mechanism, as outlined in the differential inhibitory characteristics of each of the nucleic acid aptamers against the bacterial, human, and viral RNase H homologues.

Base Sequence↗
PubMed2006

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing.

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections.

Arabidopsis↗
PubMed1989

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment.

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.

Base Sequence↗
PubMed2006

Analysis of prognostic factors of nasopharyngeal carcinoma: impact of in situ hybridization for Epstein-Barr virus encoded small RNA 1.

OBJECTIVES: To evaluate the impact of clinical, histopathological, and molecular biological parameters on the prognosis of nasopharyngeal carcinoma (NPC). STUDY DESIGN AND SETTING: The clinical records of 48 patients with a diagnosis of NPC were retrospectively reviewed. In situ hybridization for Epstein-Barr virus encoded small RNA 1 (EBER-ISH) was applied for 30 paraffin-embedded specimens available. Prognostic factors of NPC were meticulously analyzed. RESULTS: The EBER-ISH signal was shown to be highly significant as a favorable prognostic factor (P = 0.007). Although EBV was more commonly associated with Type III NPC, EBER-ISH-positive Type I-II NPC had also significantly better survival rate than EBER-ISH-negative Type I-II NPC (P = 0.036). CONCLUSIONS: In addition to the WHO histopathological grade, the 1997 UICC staging, nodal status, and distant metastasis at presentation, the EBER-ISH signal was shown to be significant as a prognostic factor. SIGNIFICANCE: This is the first report to describe the EBER-ISH as an independent prognostic factor of NPC regardless of histopathology. EBM RATING: B-2b.

Adult↗
PubMed1999

Sequence analysis of the small RNA segment of guinea pig-passaged Pichinde virus variants.

The established animal model for Lassa fever is based on the new world arenavirus Pichinde (PIC). Natural isolates of PIC virus are attenuated in guinea pigs, but serial guinea pig passage renders them extremely virulent in that host. We have compared the nucleotide sequences of the small RNA segments of two attenuated, low-passage variants of the PIC virus Munchique strain (CoAn 4763) and two virulent, high-passage derivatives. Missense mutations in the glycoprotein precursor (GPC) gene at codons GPC-119, GPC-140, and GPC-164 and the nucleoprotein gene (NP) codons NP-35 and NP-374 were most closely associated with virulence. Codon GPC-140 is predicted to represent a region of peak hydrophilicity of the glycoprotein 1 (GP1); it is conceivable that mutations at this site could influence virulence by altering B cell epitopes or virus attachment protein conformation.

Animals↗