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PubMed2005

Elucidation of the small RNA component of the transcriptome.

Small RNAs play important regulatory roles in most eukaryotes, but only a small proportion of these molecules have been identified. We sequenced more than two million small RNAs from seedlings and the inflorescence of the model plant Arabidopsis thaliana. Known and new microRNAs (miRNAs) were among the most abundant of the nonredundant set of more than 75,000 sequences, whereas more than half represented lower abundance small interfering RNAs (siRNAs) that match repetitive sequences, intergenic regions, and genes. Individual or clusters of highly regulated small RNAs were readily observed. Targets of antisense RNA or miRNA did not appear to be preferentially associated with siRNAs. Many genomic regions previously considered featureless were found to be sites of numerous small RNAs.

Arabidopsis↗
PubMed1993

A novel small RNA virus isolated from the cotton bollworm, Helicoverpa armigera.

A small RNA virus with novel characteristics has been isolated from laboratory-bred larvae of Helicoverpa armigera. Infection by the H. armigera stunt virus causes severe retardation of larval development and subsequent death. Its particles are isometric, 38 nm in diameter, and have a buoyant density of 1.296 g/ml in caesium chloride. The viral capsid has two major non-glycosylated protein components with M(r)s of 65,000 and 6000, and contains a genome composed of two non-polyadenylated single-stranded RNA molecules with lengths of 2.4 kb and 5.5 kb. The 5' termini of these RNAs are capped; their 3' termini are unblocked. In vitro translations of the viral RNAs showed synthesis of large proteins of sizes near the maximum coding capacity of each strand along with synthesis of numerous smaller proteins; no evidence for processing of precursors was seen. The physicochemical properties of the virus are most similar to those of the Nudaurelia omega virus, a provisional member of the Tetraviridae, although no antigenic relationship was observed between the two viruses. The bipartite genome and distinct capsid structure of these two viruses indicate the existence of a previously unrecognized virus group.

Amino Acid Sequence↗
PubMed1986

Isolation and sequencing of a genomic clone for mouse brain specific small RNA.

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).

Animals↗
PubMed2002

A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli.

A small RNA, RyhB, was found as part of a genomewide search for novel small RNAs in Escherichia coli. The RyhB 90-nt RNA down-regulates a set of iron-storage and iron-using proteins when iron is limiting; it is itself negatively regulated by the ferric uptake repressor protein, Fur (Ferric uptake regulator). RyhB RNA levels are inversely correlated with mRNA levels for the sdhCDAB operon, encoding succinate dehydrogenase, as well as five other genes previously shown to be positively regulated by Fur by an unknown mechanism. These include two other genes encoding enzymes in the tricarboxylic acid cycle, acnA and fumA, two ferritin genes, ftnA and bfr, and a gene for superoxide dismutase, sodB. Fur positive regulation of all these genes is fully reversed in an ryhB mutant. Our results explain the previously observed inability of fur mutants to grow on succinate. RyhB requires the RNA-binding protein, Hfq, for activity. Sequences within RyhB are complementary to regions within each of the target genes, suggesting that RyhB acts as an antisense RNA. In sdhCDAB, the complementary region is at the end of the first gene of the sdhCDAB operon; full-length sdhCDAB message disappears and a truncated message, equivalent in size to the region upstream of the complementarity, is detected when RyhB is expressed. RyhB provides a mechanism for the cell to down-regulate iron-storage proteins and nonessential iron-containing proteins when iron is limiting, thus modulating intracellular iron usage to supplement mechanisms for iron uptake directly regulated by Fur.

Bacterial Proteins↗
PubMed2003

Global analysis of small RNA and mRNA targets of Hfq.

Hfq, a bacterial member of the Sm family of RNA-binding proteins, is required for the action of many small regulatory RNAs that act by basepairing with target mRNAs. Hfq binds this family of small RNAs efficiently. We have used co-immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic microarrays to identify members of this small RNA family. This approach was extremely sensitive; even Hfq-binding small RNAs expressed at low levels were readily detected. At least 15 of 46 known small RNAs in E. coli interact with Hfq. In addition, high signals in other intergenic regions suggested up to 20 previously unidentified small RNAs bind Hfq; five were confirmed by Northern analysis. Strong signals within genes and operons also were detected, some of which correspond to known Hfq targets. Within the argX-hisR-leuT-proM operon, Hfq appears to compete with RNase E and modulate RNA processing and degradation. Thus Hfq immunoprecipitation followed by microarray analysis is a highly effective method for detecting a major class of small RNAs as well as identifying new Hfq functions.

Escherichia coli↗
PubMed2003

Localized expression of small RNA inhibitors in human cells.

Several types of small RNAs have been proposed as gene expression repressors with great potential for use in gene therapy. RNA polymerase III (pol III) provides an ideal means of expressing small RNAs in cells because its normal products are small, highly structured RNAs that are found in a variety of subcellular compartments. We have designed cassettes that use human pol III promoters for the high-level expression of small RNAs in the cytoplasm, nucleoplasm, and nucleolus. The levels and subcellular destinations of the transcripts are compared for transcripts expressed using the U6 small nuclear RNA (snRNA), 5S ribosomal RNA (rRNA), and the 7SL RNA component of the signal recognition particle. The most effective location for a particular inhibitory RNA is not necessarily predictable; thus these cassettes allow testing of the same RNA insert in multiple subcellular locations. Several small interfering RNA (siRNA) inserts were tested for efficacy. An siRNA insert that reduces lamin expression when transcribed from the U6 snRNA promoter in the nucleus has no effect on lamin expression when transcribed from 5S rRNA and 7SL RNA-based cassettes and found in the nucleolus and cytoplasm. To test further the generality of U6-driven siRNA inhibitors, siRNAs targeting HIV were tested by co-transfection with provirus in cell culture. Although the degree of HIV-1 inhibition varied among inserts, results show that the U6 cassette provides a means of expressing an siRNA-like inhibitor of HIV gene expression.

Base Sequence↗
PubMed2005

Regulation of endothelial nitric oxide synthase by small RNA.

Repeats (27-nt) in intron 4 have been shown to play a cis-acting role in endothelial nitric oxide synthase (eNOS) promoter activity. We hypothesize that the 27-nt repeats could be the source of small nuclear RNA specifically regulating eNOS expression. In this study, we used synthesized 27-nt RNA duplex and found that the eNOS gene transcriptional efficiency was reduced 63% (0.047 +/- 0.009 vs. 0.126 +/- 0.015, P < 0.01) by nuclear run-on assay. In endothelial cells transfected with the 27-nt small RNA duplex, we found that the eNOS mRNA and protein levels were decreased by >64% (P < 0.01). Conversely, a randomly selected 27-nt from luciferase gene had no effect on the eNOS expression. Furthermore, this eNOS silencing effect appeared to be reversible under the stimulation of vascular endothelial growth factor (10 ng/ml), which is known to up-regulate eNOS expression. Using in situ hybridization and Northern blotting, we observed the presence of endogenous eNOS intron 4-derived 27-nt small RNA, which was confined to the nucleus. In summary, we demonstrated that intron-based microRNAs in eNOS can induce significant gene specific transcriptional suppression, which could be an effective negative feedback regulator for gene expression.

Cells, Cultured↗
PubMed1999

Detection of Epstein-Barr virus (EBV) in hepatocellular carcinoma tissue: a novel EBV latency characterized by the absence of EBV-encoded small RNA expression.

In this study, we investigated the presence of Epstein-Barr virus (EBV) in liver tissue from 35 patients with hepatocellular carcinoma (HCC). EBV DNA was detected in 13 patients (37%) by Southern blot hybridization. In 10 of these patients, EBV DNA was present in tumor tissue only, whereas in the other 3, it was detected in both tumor and nontumor tissues. The quantity of EBV DNA detected was equivalent to 1-10 viral DNA molecules/100 cells. EBV-determined nuclear antigen was detected in 7-13% of the carcinoma cells in three tumor tissue samples that contained approximately one copy of the EBV genome/10 cells. A single terminal fragment of EBV DNA was identified in these tissues, suggesting that the EBV-infected cells in HCC represent clonal proliferation. Western blotting and reverse transcription-polymerase chain reaction analyses demonstrated that these three tumor tissue specimens were positive for EBV-determined nuclear antigen 1 and BamHI A transcripts but were negative for the other latent EBV products, including EBV-encoded small RNA. The results indicated that there is a high EBV load in HCC tissue and that all of the HCC tissue examined showed a novel pattern of EBV latency characterized by absence of EBV-encoded small RNA expression.

Adult↗
PubMed1982

Small RNA molecules related to the Alu family of repetitive DNA sequences.

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.

Animals↗
PubMed1996

[Detection of a new small RNA, induced by heat shock, in wheat seed ribosomes].

Changes in the small cytoplasmic RNA (scRNA) composition have been examined in wheat embryos exposed to heat shock conditions. A novel scRNA of about 135 nucleotides in length termed as 5.3S RNA, has been detected for the first time. With a rise in temperature from 26 degrees C up to 37 degrees C, the amount of 5.3S RNA increases 5 to 6 times. It was found that 5.3S RNA is localized predominantly on ribosomes and also in the fraction of native 40S ribosomal subunits known to contain translation preinitiation complexes. Upon dissociation of 80S monoribosomes under high ionic strength conditions (0.5 M KCl), 5.3S RNA remains to be bound to the 40S subunit, thus indicating their strong and specific interaction. 5.3S RNA is neither a precursor of the 5S rRNA nor a discrete fragment of high molecular weight ribosomal RNA. It was supposed that 5.3S RNA is transcribed by RNA polymerase III and involved in protein synthesis regulation in plant cells under heat shock conditions at the level of translation initiation.

Base Sequence↗
PubMed2006

Down-regulation of porins by a small RNA bypasses the essentiality of the regulated intramembrane proteolysis protease RseP in Escherichia coli.

Adaptation to extracytoplasmic stress in Escherichia coli depends on the activation of sigmaE, normally sequestered by the membrane protein RseA. SigmaE is released in response to stress through the successive RseA cleavage by DegS and the RIP protease RseP. SigmaE and proteases that free it from RseA are essential. We isolated a multicopy suppressor that alleviated RseP and DegS requirement. The suppressor encodes a novel small RNA, RseX. Its activity required the RNA-binding protein Hfq. We used the property that small RNAs are often involved in RNA-RNA interactions to capture RseX putative partners; ompA and ompC mRNA, which encode two major outer membrane proteins, were identified. RseX activity was shown to confer an Hfq-dependent coordinate OmpA and OmpC down-regulation. Because RseP is shown to be no longer essential in a strain lacking OmpA and OmpC, we conclude that RseP, which is required for normal sigmaE activation, prevents toxicity due to the presence of two specific outer membrane proteins that are down-regulated by RseX.

Bacterial Outer Membrane Proteins↗
PubMed1996

Structural investigations on small RNA molecules using different force field methods.

The structures of two kinds of small RNA hairpin loops were investigated. As an example of four-membered loops the family of GNNA-loops was chosen, and the influence of a variation of the two middle bases on the rather uncommon G-A base pair was examined. Comparison of the resulting structures showed surprisingly little dependence on the sequence for the overall structure and for the G-A base pair geometry. As an example of three-membered loops a systematic investigation on the loop-sequences UUU, AUU, UUA, GUU, UUG was carried out. Here, not only the loop sequence but also the influence of the closing pair was examined. Again the structural variation with different sequences was very small. Both calculations were used to compare the AMBER 4.0 and the JUMNA IV force field programs. Though both programs show a quite different approach to modeling RNA structures the resulting geometries are comparable.

Base Sequence↗
PubMed1998

Combinatorial selection of a small RNA that induces amplification of IncFII plasmids in Escherichia coli.

Cellular RNAs play fundamental roles as genetic messages, structural components and, in some cases, as catalytic agents. The ability to create vast combinatorial libraries of random RNA sequences has previously been exploited in vitro to identify RNA aptamers with desirable binding specificities, and to isolate RNAs with novel catalytic properties. Despite the advantages of in vitro selections from RNA libraries, there is no way to predict if the identified RNAs can function in living cells. We are therefore exploring random RNA expression libraries in Escherichia coli to search for small RNAs with novel functions. Here we describe selections that identified a small RNA (approximately 260 nucleotides) capable of altering the copy-number control circuitry of IncFII plasmids. The novel RNA appears to function by annealing to a region of the mRNA encoding the plasmid replicator protein. The resulting RNA-RNA hybrid permits translation of the replicator protein, but blocks base-pairing with a natural negative regulatory RNA. Implications of this in vivo selection strategy are discussed.

Anti-Bacterial Agents↗
PubMed1981

Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA.

Transcription of ColE1 DNA by RNA polymerase in vitro starts at two sites in a region required for maintenance of the plasmid. Certain transcripts that start at one of the sites can be cleaved by RNase H and then act as primers for DNA replication. Transcription from the other site produces a RNA approximately 108 nucleotides long (species I or RNA I). Transcripts analogous to the primer and RNA I of ColE1 are produced when p15A or small derivatives of two other ColE1-compatible plasmids, CloDF13 and RSF1030, are used as template. If purified RNA I is added to the transcription reaction containing RNase H, formation of primer is inhibited. Each RNA I can inhibit primer formation by the plasmid that specifies it but has no effect on primer formation by heterologous templates. Thus, the inhibition of primer formation by RNA I is incompatibility specific. Because RNA I does not inhibit initiation or propagation of transcription or the processing of preformed precursors, the step that is sensitive to inhibition is probably formation of the hybrid between the primer precursor and the template. This hybrid is the required substrate for RNase H. Experiments with recombinant plasmids show the region that determines the specificity of response to RNA I to be greater than 300 base pairs upstream of the origin of DNA replication.

Base Composition↗
PubMed1995

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Transcription of downstream genes in the early operons of phage lambda requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of lambda N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.

Bacterial Proteins↗
PubMed2004

Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA.

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.

Bacterial Proteins↗
PubMed1982

Hybrid selection of small RNAs by using simian virus 40 DNA: evidence that the simian virus 40-associated small RNA is synthesized by specific cleavage from large viral transcripts.

The simian virus 40 (SV40)-associated small RNA (SAS-RNA), approximately 64 nucleotides, is virally encoded within a region of the viral late (+) DNA strand which encodes no known protein. The SAS-RNA arises in abundance late in SV40 lytic infection. Previous data indicate that the synthesis of the SAS-RNA may be under the control of the normal late viral promoter; i.e., inhibition of transcription from the late promoter results in cessation of SAS-RNA synthesis. The synthesis of SAS-RNA was examined to determine whether the SAS-RNA is the product of cleavage from noncoding regions of nuclear late transcripts or an independent transcription product like 5S RNA, or the adenovirus VA-RNAs. The data described below suggest that SAS-RNA is cleaved from large late transcripts. In vitro transcription of DNA fragments containing the SAS-RNA coding region yielded no SAS-RNA synthesis; this result was supported by DNA sequence analysis, which indicated no promoter-like regions either within or flanking the SAS-RNA coding region. In support of a cleavage mechanism, the SAS-RNA has a 3'-phosphate end, an occurrence which is indicative of nuclease cleavage. In addition, 5'-end labeling of the SAS-RNA was possible only after calf alkaline phosphatase treatment; this indicates that the SAS-RNA is not capped. Hybrid selection analysis was used to demonstrate that separation of the SAS-RNA coding region from the normal late promoter resulted in elimination of SAS-RNA synthesis. This was demonstrated in SV40-transformed cells in which integration of a single copy of SV40 breaks the continuity of the late coding region, so that the SAS-RNA coding region is physically separated from the normal late promoter. The lack of SAS-RNA synthesis indicates that the SAS-RNA coding region cannot function as a primary transcription unit. The same result and conclusion were obtained by using a permissive cell line transformed by SV40 (COS-1 cells); here it was found that the integrated SAS-RNA coding region was not expressed even during a viable lytic infection in which the SAS-RNA could be expressed from the infecting viral genomes. The simplest conclusion drawn from the data is that the SAS-RNA is cleaved from larger late transcripts which initiate at the normal late promoter. This conclusion suggests that many of the small RNAs found in normal eucaryotic cells may be synthesized by specific cleavage rather than by primary transcription. In the course of these studies several small cellular RNAs were detected, due to their specific hybrid selection, by using SV40 DNA. Primary mapping and characterization data of these RNAs are also presented.

Animals↗
PubMed1989

Epstein-Barr virus small RNA (EBER) genes: unique transcription units that combine RNA polymerase II and III promoter elements.

The Epstein-Barr virus-encoded small RNA (EBER) genes appear to comprise an interesting subset of class III genes different from any previously identified, including U6 and 7SK. EBER genes have functional A and B box intragenic control regions. In addition, they contain three upstream elements that together stimulate in vivo expression 50-fold and resemble sites associated with typical class II promoters. DNAase I footprinting analyses using purified proteins or oligonucleotide competition demonstrate that nucleotides -40 to -55 bind activating transcription factor (ATF) or a related protein, while nucleotides -56 to -77 bind Sp1 protein or a related protein. The element between positions -23 and -28 resembles a TATA box. EBERs are unusual RNA polymerase III transcripts shown to be controlled by ATF- and Sp1-like promoter elements, suggesting mechanisms for their high level expression in EBV-transformed lymphocytes.

Base Sequence↗