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Results for “small RNA”

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At least 451 records · Page 25Linked to original sources
PubMed1992

Epitope regions on U1 small nuclear RNA recognized by anti-U1RNA-specific autoantibodies.

Autoantibodies specifically directed to U1RNA were found in patients suffering from systemic lupus erythematosus (SLE) overlap syndromes. To obtain more insight in the mechanism responsible for this U1RNA-specific antibody formation and to use the antibodies eventually as a tool to study U1RNA-protein (U1RNP) interactions, the B cell epitopes on U1RNA were mapped. Using in vitro synthesized domains of U1RNA, the main epitope regions were found in stemloops II and IV. Furthermore, 3'-end or 5'-end truncation of both stemloop II and stemloop IV showed that the conformation of the stemloops is critical for antibody recognition. Mutant studies on both stemloops indicated that in the case of stemloop II the stem is the main antigenic region, whereas in stemloop IV, the loop (E-loop) is a main target. The results of this study support the idea that the anti-U1RNA autoantibody could be the result of a process driven by the human U1RNP complex itself (antigen-driven process).

Antibody Affinity↗
PubMed2006

Downregulation of xIAP expression by small interfering RNA inhibits cellular viability and increases chemosensitivity to methotrexate in human hepatoma cell line HepG2.

The aim of this study was to investigate whether downregulating the expression of xIAP by RNAi (RNA interference) technology can induce the apoptosis of HepG2 cells, inhibit cellular viability and increase chemosensitivity of cancer cells. HepG2 cells were transfected with U6 promoter plasmids coding for short interfering RNAs (siRNAs) targeting xIAP. RT-PCR and western blot analysis were used to assess the mRNA and protein levels of xIAP expression. T he suppression efficiency o f xIAPby RNAi was evaluated using the MTT assay for cellular viability and Annexin V-PI binding assay for apoptosis. These results showed that siRNAs reduced cellular viability and increased cellular apoptosis. Moreover, downregulation of xIAP expression enhanced the chemosensitivity of HepG2 cells to methotrexate. These results suggest that the downregulation of xIAP by RNAi could potentially be used as a therapeutic strategy for human hepatocellular carcinoma.

Antimetabolites, Antineoplastic↗
PubMed2003

Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571).

Bcr-Abl proteins are effective inducers of the leukemic phenotype in chronic myeloid leukemia (CML) and distinct variants of acute lymphoblastic leukemia (ALL). Targeting bcr-abl by treatment with the selective tyrosine kinase inhibitor imatinib has proved to be highly efficient for controlling leukemic growth. However, it is unclear whether imatinib is sufficient to eradicate the disease because of primary or secondary resistance of leukemic cells. Therefore, targeting Bcr-Abl with an alternative approach is of great interest. We demonstrate that RNA interference (RNAi) with a breakpoint-specific short-interfering RNA (siRNA) is capable of decreasing Bcr-Abl protein expression and of antagonizing Bcr-Abl-induced biochemical activities. RNAi selectively inhibited Bcr-Abl-dependent cell growth. Furthermore, bcr-abl-homologous siRNA increased sensitivity to imatinib in Bcr-Abl-overexpressing cells and in a cell line expressing the imatinib-resistant Bcr-Abl kinase domain mutation His396Pro, thereby antagonizing 2 of the major mechanisms of resistance to imatinib.

Antineoplastic Agents↗
PubMed2005

Small inhibitory RNA--a tool for credentialing candidate genes.

The effect of a drug is related to the interaction of the numerous gene products that participate in a drug pathway. The clinical importance of genetic variability on the overall outcome of most drug pathways remains to be determined. As a result, there is a need to efficiently identify the genes that not only participate in the pathway but also play a significant role in regulating the effects of a drug. The emerging technique of specific gene silencing through RNA interference initiated by small inhibitory RNA may prove to be a useful and powerful tool in the credentialing of candidate genes in drug pathways.

Animals↗
PubMedDate not supplied

Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA.

RNA interference (RNAi) mediates sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA. RNAi is an effective tool for functional genomics in C. elegans, whereas biochemical characteristics of RNAi hamper its use in mammalian cells. We here analyze kinetic and quantitative aspects of mammalian RNAi in different cell lines using novel lentiviral constructs with double H1-shRNA expression cassettes located in the U3 region of the LTR. Using enhanced green fluorescence protein (EGFP) as a target gene for RNAi and red fluorescence protein (RFP) as surrogate marker for intracellular siRNA expression, we show that long-term siRNA expression mediates stable RNAi. Furthermore, RNAi-induced gene silencing varies from minimal to complete loss-of-function phenotypes within homogeneous and between different cell populations. Interestingly, the extent of gene silencing correlates to lentiviral integrations as well as siRNA expression levels in target cells. Finally, we demonstrate functional gene silencing of a cytokine receptor gene in normal CD34+ hematopoietic progenitor cells. These data suggest that the use of suitable lentiviral constructs to quantify siRNA expression in living cells may allow analysis of loss-of-function phenotypes in a dose-dependent manner in a wide variety of target cells.

Animals↗
PubMed2006

[Targeting hTERT gene influenced the viability on hMSCs by small hairpin RNA].

OBJECTIVE: To observe the effect of shRNA by targeting human telomerase reverse transcriptase (hTERT) mRNA in the human mesenchymal stem cells (hMSCs) lines and explore the safety of RNAi by targeting hTERT mRNA in clinical application. METHODS: According to the strategy of RNAi, which determined the specific base sequences to design shRNA plasmid. Plasmid shRNA1 involved in fluorescein gene and homologous to hTERT were designed. control shRNA2-a random sequences heterogeneous to mankind's gene were also constructed. METAFECTENE was used as the transfection reagent. The experiment was divided into 4 groups: A (shRNA1), B (shRNA2), C (metafectene) and D (normal culture medium ). At 24h, fluorescence expression was detected by confocal microscopy after administration of plasmid. At 24h, 48h and 72h cells viability were determined using the MTT assay. At 48h, the morphological change were examined by HE stain. At 48h, hTERT mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) and the telomerase activity of hMSCs were examined by PCR ELISA. RESULTS: Many cells gave out green fluorescent after treated by shRNA1 and shRNA2. It was observed that the viability of hMSCs hadn't change after treatment with different methods within 72h (P > 0.05). Cells, morphology and density had not change after treated by different factor. All cells didn't express hTERT mRNA and the telomere activity of hMSCs was negative. CONCLUSIONS: The results suggest that shRNA plasmid directed against hTERT can not influence the viability on hMSCs. The treatment with RNA interference may be a safety strategy for gene therapy in vivo.

Bone Marrow Cells↗
PubMed2003

A survey of small RNA-encoding genes in Escherichia coli.

Small RNA (sRNA) molecules have gained much interest lately, as recent genome-wide studies have shown that they are widespread in a variety of organisms. The relatively small family of 10 known sRNA-encoding genes in Escherichia coli has been significantly expanded during the past two years with the discovery of 45 novel genes. Most of these genes are still uncharacterized and their cellular roles are unknown. In this survey we examined the sequence and genomic features of the 55 currently known sRNA-encoding genes in E.coli, attempting to identify their common characteristics. Such characterization is important for both expanding our understanding of this unique gene family and for improving the methods to predict and identify sRNA-encoding genes based on genomic information.

Base Composition↗
PubMed1975

Nucleotide sequence of microvariant RNA: another small replicating molecule.

Microvariant RNA, a small self-replicating molecule (114 nucleotides long), has been isolated from Qbeta replicase reactions incubated in the absence of exogenous template. Its complete nucleotide sequence has been determined. Comparison with MDV-1 RNA, a somewhat larger endogenous Qbeta replicase product (220 nucleotides long) that had previously been characterized, revealed no significant sequence similarity. Since Qbeta replicase can mediate the synthesis of both of these disparate RNA molecules, primary sequence cannot be the sole determining factor in the processes of enzyme recognition and replication. This implies that the key is to be found in the secondary or tertiary structures. The availability of two different replicating molecules of defined sequence should aid in identifying these critical structural features.

Base Sequence↗
PubMed1989

Transcription and processing of Bacillus subtilis small cytoplasmic RNA.

The 271 nucleotides long scRNA (small cytoplasmic RNA) from Bacillus subtilis is structurally related to the Escherichia coli 4.5 S RNA (114 nucleotides), an essential molecule supposed to be involved in protein biosynthesis, but it possesses an additional moiety completely missing in the E. coli 4.5 S RNA. Both RNAs share a conserved hairpin with the eukaryotic 7SL RNAs, which mediate protein translocation as part of the signal recognition particle (SRP). We have cloned and sequenced the entire scRNA gene region from B. subtilis and have studied transcription and processing of the scRNA in B. subtilis by nuclease S1 mapping. This analysis revealed the scRNA gene to constitute a monofunctional transcription unit, expressed from a single promoter to a rho-independent terminator, yielding a precursor which extends the mature scRNA by approximately 40 nucleotides at both ends. Processing of the scRNA apparently involves only two endonucleolytic cuts and occurs first at the 5' end.

Bacillus subtilis↗
PubMed1990

Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA.

The cap structure of U6 small nuclear RNA (snRNA) is gamma-monomethyl phosphate and is distinct from other known RNA cap structures (R. Singh and R. Reddy, Proc. Natl. Acad. Sci. USA 86:8280-8283, 1989). Here we show that the information for capping the U6 snRNA in vitro is within the initial 25 nucleotides of the U6 RNA. The capping determinant in mammalian U6 snRNA is a bipartite element--a phylogenetically conserved stem-loop structure and an AUAUAC sequence, or a part thereof, following this stem-loop. Wild-type capping efficiency was obtained when the AUAUAC motif immediately followed the stem-loop and when the gamma-phosphate of the initiation nucleotide was in close proximity to the capping determinant. Incorporation of a synthetic stem-loop followed by an AUAUAC sequence is sufficient to covert a noncapped heterologous transcript into a capped transcript. Transcripts with the initial 32 nucleotides of Saccharomyces cerevisiae U6 snRNA are accurately capped in HeLa cell extract, indicating that capping machinery from HeLa cells can cap U6 snRNA from an evolutionarily distant eucaryote. The U6-snRNA-specific capping is unusual in that it is RNA sequence dependent, while the capping of mRNAs and other U snRNAs is tightly coupled to transcription and is independent of the RNA sequence.

Base Sequence↗
PubMed1987

A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA.

A small RNA of Bacillus subtilis bacteriophage phi 29 is shown to have a novel and essential role in viral DNA packaging in vitro. This requirement for RNA in the encapsidation of viral DNA provides a new dimension of complexity to the attendant protein-DNA interactions. The RNA is a constituent of the viral precursor shell of the DNA-packaging machine but is not a component of the mature virion. Studies of the sequential interactions involving this RNA molecule are likely to provide new insight into the structural and possible catalytic roles of small RNA molecules. The phi 29 assembly in extracts and phi 29 DNA packaging in the defined in vitro system were strongly inhibited by treatment with the ribonucleases A or T1. However, phage assembly occurred normally in the presence of ribonuclease A that had been treated with a ribonuclease inhibitor. An RNA of approximately 120 nucleotides co-purified with the phi 29 precursor protein shell (prohead), and this particle was the target of ribonuclease action. Removal of RNA from the prohead by ribonuclease rendered it inactive for DNA packaging. By RNA-DNA hybridization analysis, the RNA was shown to originate from a viral DNA segment very near the left end of the genome, the end packaged first during in vitro assembly.

Bacillus subtilis↗