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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↗
PubMed1989

Metabolism of small RNAs in cultured human cells.

There are gaps in what is known about the metabolism of some mammalian small RNA species. Our present observations can be summarized as follows. The level of radiolabeled mature U1 RNA doubled between 2 and 24 hr of label chase, while that of all other small RNA species tested did not change. These results are compatible with the possibility that the nucleotide precursor pool for U1 RNA transcription may be partly segregated, or that there may be a second pathway of U1 RNA formation. Precursors of nucleolar U3 RNA were detected whose electrophoretic mobilities are equivalent to those of transcripts approximately 14 and approximately 8 nucleotides longer than the mature species, and which are apparently cytoplasmic. The ladder of U2 RNA precursors showed a gap, suggesting that some of the cleavages during U2 RNA processing are endonucleolytic. We detected an apparent U5 RNA precursor whose electrophoretic mobility is equivalent to that of a species approximately 1 nucleotide longer than mature U5 RNA. There was a predominant band in the middle of the ladder of U4 RNA precursors (apparently approximately 3 nucleotides longer than mature U4 RNA) which suggests that U4 RNA maturation may pause briefly at that intermediate. There are apparently two additional species of mature hY3 RNA, which are less abundant and are about 1 and 2 bases longer than the major hY3 RNA species. An apparent hY3 RNA precursor was detected, which may be approximately 2-3 nucleotides longer than the main mature hY3 RNA species.

Cells, Cultured↗
PubMed2001

Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs.

Antisense-mediated gene silencing (ASGS) and posttranscriptional gene silencing (PTGS) with sense transgenes markedly reduce the steady-state mRNA levels of endogenous genes similar in transcribed sequence. RNase protection assays established that silencing in tobacco plants transformed with plant-defense-related class I sense and antisense chitinase (CHN) transgenes is at the posttranscriptional level. Infection of tobacco plants with cucumber mosaic virus strain FN and a necrotizing strain of potato virus Y, but not with potato virus X, effectively suppressed PTGS and ASGS of both the transgenes and homologous endogenes. This suggests that ASGS and PTGS share components associated with initiation and maintenance of the silent state. Small, ca. 25-nt RNAs (smRNA) of both polarities were associated with PTGS and ASGS in CHN transformants as reported for PTGS in other transgenic plants and for RNA interference in Drosophila. Similar results were obtained with an antisense class I beta-1,3-glucanase transformant showing that viral suppression and smRNAs are a more general feature of ASGS. Several current models hold that diverse signals lead to production of double-stranded RNAs, which are processed to smRNAs that then trigger PTGS. Our results provide direct evidence for mechanistic links between ASGS and PTGS and suggest that ASGS could join a common PTGS pathway at the double-stranded RNA step.

Chitinases↗
PubMed1999

Sequence-dependent conformational differences of small RNAs revealed by native gel electrophoresis.

In this study, we use native polyacrylamide gel electrophoresis and one-dimensional NMR spectroscopy to analyze small RNA hairpins containing a UUCG tetraloop. The aggregation state of one RNA 16-mer (5'-CGGCUUCGGUCGACCA-3') in the presence of Mg(2+) was confirmed by laser light scattering. Although it is widely known in the RNA field that some RNAs tend to aggregate, especially when present at high concentrations, the sequence elements responsible for this effect are rarely identified. In this work, we show that Mg(2+)-induced aggregation of the 16-mer RNA hairpin is sensitive to the presence of the 3'-terminal base and a specific 2'-hydroxyl group. Our study highlights the fact that even small changes in a particular RNA sequence can increase its tendency to undergo Mg(2+)-dependent aggregation in an unpredictable manner. Our analysis also shows that native gel electrophoresis is a sensitive probe of RNA conformation with the capability to detect differences apparently caused by subtle base stacking effects at the ends of helices.

Base Sequence↗
PubMed1992

Epstein-Barr virus-encoded small RNAs (EBERs) do not modulate interferon effects in infected lymphocytes.

The recent derivation of otherwise isogenic Epstein-Barr virus (EBV) recombinants carrying or lacking the EBV small RNA (EBER) genes enabled us to test whether EBERs are similar to adenovirus VA RNAs in modulating interferon (IFN) effects on virus infection. EBER-positive and -negative EBV recombinants did not differ in their sensitivity to alpha interferon (IFN-alpha)- or IFN-gamma-mediated inhibition of lymphocyte growth transformation. In addition, EBERs did not decrease the inhibitory effects of IFN on vesicular stomatitis virus replication in EBV-transformed lymphocytes. EBER deletion also did not render EBV-transformed B lymphocytes susceptible to an IFN effect on cell proliferation or EBV replication.

B-Lymphocytes↗
PubMed1987

The effects of protein synthesis inhibition, and of mutations rna1.1 and rna82.1, on the synthesis of small RNAs in yeast.

Strains of Saccharomyces cerevisiae have been constructed that possess temperature-sensitive defects in tRNA precursor (pre-tRNA) splicing and which also lack the processing endonuclease that acts at the 3'-terminus of 5 S rRNA and 35 S rRNA precursors (pre-rRNAs). The unspliced pre-tRNAs accumulated by such strains at the nonpermissive temperature are identical in structure to those accumulated by pre-tRNA splicing-defective strains with a functional pre-5 S RNA processing enzyme. The pre-RNA processing activity is therefore not obligatorily involved in maturation of several yeast tRNAs. However, gels of the pulse-labelled RNAs of RNA82+ and rna82.1 strains provide evidence that this enzyme acts upon a few small unstable transcripts that are not 5 S RNA forms. The most prominent of these transcripts on gels was, in wild-type strains, an RNA 145 +/- 2 nucleotides in length.

Cycloheximide↗
PubMed1985

Characterization of leader-related small RNAs in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription.

Mouse hepatitis virus (MHV), a murine coronavirus, replicates in the cytoplasm and synthesizes 7 viral mRNAs containing an identical stretch of leader RNA sequences at the 5'-end of each RNA. The leader-coding sequences at the 5'-end of genomic RNA are at least 72 nucleotides in length and are joined to the viral mRNAs by a unique mechanism. Utilizing a leader-specific cDNA probe, we have detected several free leader RNA species ranging from 70 to 82 nucleotides in length. The predominant leader RNA was approximately 75 nucleotides. In addition, larger distinct leader-containing RNAs were also detected ranging from 130 to 250 nucleotides in length. The 70-82-nucleotide leader-related RNAs were present in both the cytosol and membrane fractions of infected cells. They were also detected only in the small RNA fractions but not associated with the replicative-intermediate RNA. These data suggest that the leader RNAs were associated with the membrane-bound transcription complex but at least part of them were dissociated from the RNA template. We have also identified a temperature-sensitive mutant, which synthesizes only leader RNA but not mRNAs at nonpermissive temperature, indicating that leader RNA synthesis is distinct from the transcription of mRNAs. These data support the leader-primed mechanism for coronavirus transcription and suggest that one or more free leader RNAs are used as primers of mRNA synthesis.

Animals↗
PubMed2007

Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis.

Strong viroid-caused pathogenesis was achieved in tomato cv. Rutgers by biolistic transfer of severe or lethal potato spindle tuber viroid (PSTVd) strains, while other tomato genotypes (e.g., Moneymaker) were tolerant. With reciprocal hybrids between sensitive and tolerant genotypes, we show that plant depression dominates over tolerance. Biolistic transfer of the most pathogenic PSTVd strain AS1 to Nicotiana benthamiana, which is considered to be a symptomless PSTVd host, led to a strong pathogenesis reaction and stunting, suggesting the presence of specific viroid pathogenesis-promoting target(s) in this plant species. Total levels of small siRNA-like PSTVd-specific RNAs were enhanced in strongly symptomatic tomato and N. benthamiana plants after biolistic infection with AS1 in comparison to the mild QFA strain. This indicates association of elevated levels of viroid-specific small RNA with production of strong symptoms. In symptom-bearing tomato leaves in comparison to controls, an RNase of approximately 18 kDa was induced and the activity of a nuclease of 34 kDa was elevated by a factor of seven in the vascular system. Sequence analysis of the nuclease cDNA designated TBN1 showed high homology with plant apoptotic endonucleases. The vascular-specific pathogenesis action is supported by light microscopic observations demonstrating a certain lack of xylem tissue and an arrest of the establishment of new vascular bundles in collapsed plants.

Amino Acid Sequence↗
PubMed2000

Construction of adenovirus for high level expression of small RNAs in mammalian cells. Application to a Bcl-2 ribozyme.

A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA sequences. A truncated human U6 gene containing convenient restriction sites has been shown to be expressed at high levels following electroporation into a series of human cell lines. This gene was ligated into a promoterless adenoviral plasmid, and we have generated high titer virus by homologous recombination with adenoviral Addl327 DNA in 293 cells. Recombinant adenovirus containing a hammerhead ribozyme sequence targeted toward the Bcl-2 mRNA has been used to transduce a panel of human tumor cell lines. We have demonstrated high level expression of the recombinant U6 gene containing the ribozyme and reduction of Bcl-2 protein in transduced cells. These plasmids are suitable for the development of adenoviral vectors designed to express both ribozymes and antisense RNA in human cells.

Adenoviridae↗
PubMedDate not supplied

[Great potential of small RNAs: RNA interference and microRNA].

Double-stranded RNA (dsRNA) induces a sequence-specific silencing in eukaryotic cells. This silencing process beggins when long dsRNA is cleaved to 21 to 26 long small RNA by means of the RNAse III-type enzyme Dicer. These small dsRNA are included into silencing effector complexes, that are targeted to complementary sequences. Small RNA dependent gene silencing can be achieved by distinct mechanisms based depending mainly on the nature of target sequences and on the proteins present in the effector complex. The route of interference RNA (RNAi) begins when Dicer yields small interference RNA (siRNA) that bind to complementary mRNA for its degradation, forming the RISC complex. siRNA are naturally formed from transposons and dsRNA viruses during its replication, as well as from other bidirectional transcribed repetitive sequences. Some of the enzymes thar are part of the RNAi machinery, including Dicer, are encoded by multigene families in many species, that also play a role in other mechanisms of RND-dependent gene silencing. MicroRNA's (miRNA) are other small RNA's that can induce gene silencing at the mRNA level. These are formed in a general manner when Dicer process hairpin structures resulting from the transcription of non-coding sequences from plant and animal genomes. miRNA's are integrated into a RISC-like complex, after which, depending on their degree of complementarity with target mRNA, can either repress translation or induce mRNA degradation. miRNA-dependent silencing is essential for the development of multicellular organisms. Artificial RNAi induction by means of siRNA or miRNA is being used as a tool to inactivate gene expression in culture cells and in living organisms. This review focuses on the progress in the understanding of the mechanisms involved in gene regulation by RNA in animals and details some current efforts to apply theses phenomena as a tool in research and in the therapeutic of human diseases.

Humans↗
PubMed2005

Labeling and characterization of small RNAs associated with the RNA interference effector complex RITS.

RNA interference (RNAi) is a gene silencing mechanism that acts at both the posttranscriptional and transcriptional levels. We have recently identified an RNA-containing complex, named RNA-induced transcriptional silencing (RITS), that directly links RNAi to transcriptional gene silencing in Schizosaccharomyces pombe. Here we review the affinity purification methods we use to isolate RITS and describe how to purify, detect, and analyze RNAs associated with this complex.

Blotting, Southern↗
PubMed1986

Localization of Epstein-Barr virus-encoded small RNAs by in situ hybridization.

Human B lymphocytes latently infected with Epstein-Barr virus (EBV) synthesize two low molecular weight RNAs designated EBER 1 and 2. Using an in situ hybridization technique we have localized EBER 1 and 2 within the nucleus of single EBV-harboring B lymphocytes from established and recently transformed cell lines. As controls, the locations of the small nuclear RNA, U1, and the small cytoplasmic RNA, 7SL, were examined in HeLa and EBV-harboring cells. Because of possible functional similarities between EBERs and the adenovirus-associated (VA) RNAs, VAI was also localized; it appeared to be in the nucleus and cytoplasm, implying that VAI may have a different role than that of the nuclear-localized EBERs.

Cell Nucleus↗
PubMed2004

Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis.

In many bacteria, iron homeostasis is controlled primarily by the ferric uptake regulator (Fur), a transcriptional repressor. However, some genes, including those involved in iron storage, are positively regulated by Fur. A Fur-repressed regulatory small RNA (sRNA), RyhB, has been identified in Escherichia coli, and it has been demonstrated that negative regulation of genes by this sRNA is responsible for the positive regulation of some genes by Fur. No RyhB sequence homologs were found in Pseudomonas aeruginosa, despite the identification of genes positively regulated by its Fur homolog. A bioinformatics approach identified two tandem sRNAs in P. aeruginosa that were candidates for functional homologs of RyhB. These sRNAs (PrrF1 and PrrF2) are >95% identical to each other, and a functional Fur box precedes each. Their expression is induced under iron limitation. Deletion of both sRNAs is required to affect the iron-dependent regulation of an array of genes, including those involved in resistance to oxidative stress, iron storage, and intermediary metabolism. As in E. coli, induction of the PrrF sRNAs leads to the rapid loss of mRNAs for sodB (superoxide dismutase), sdh (succinate dehydrogenase), and a gene encoding a bacterioferritin. Thus, the PrrF sRNAs are the functional homologs of RyhB sRNA. At least one gene, bfrB, is positively regulated by Fur and Fe(2+), even in the absence of the PrrF sRNAs. This work suggests that the role of sRNAs in bacterial iron homeostasis may be broad, and approaches similar to those described here may identify these sRNAs in other organisms.

Bacterial Proteins↗