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At least 253 records · Page 14Linked to original sources

PubMed2005

Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway.

Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading complex and show that the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5'-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2'-position of the 5'-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.

Animals↗

PubMed2004

RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei.

Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth inhibition. It also resulted in the loss of both proteins, even when only one of the MRP mRNAs was reduced, indicating a mutual dependence for stability. Elimination of the MRPs gave rise to substantially reduced levels of edited CyB and RPS12 mRNAs but little or no reduction of the level of edited Cox2, Cox3, and A6 mRNAs as measured by poisoned primer extension analyses. In contrast, edited NADH-dehydrogenase (ND) subunit 7 mRNA was increased 5-fold in MRP1+2 double knock-down cells. Furthermore, MRP elimination resulted in reduced levels of Cox1, ND4, and ND5 mRNAs, which are never edited, whereas mitoribosomal 12 S rRNA levels were not affected. These data indicate that MRP1 and MRP2 are not essential for RNA editing per se but, rather, play a regulatory role in the editing of specific transcripts and other RNA processing activities.

Animals↗

PubMed2006

[Inhibition of HPV16 E6 oncogene in cervical cancer by RNA interference].

OBJECTIVE: The efficiency of HPV16 E6 gene silenced by RNA interference in vitro and in vivo was assessed. METHODS: The specific siRNA of HPV16 E6 was designed and transfected into CaSki cells by liposome. Cell apoptotic rates and the changes in HPV16 E6 mRNA and protein before and after transfection were measured. Cervical cancer nude mice models were set up, siRNA was injected directly into subcutaneous tumor. The function of siRNA was evaluated by the changes in tumor volume, HPV16 E6 protein expression and apoptosis of tumor cells. RESULTS: In vitro research, the cell apoptotic rates were 7.7%, 11.8%, 37.4% and 12.6% respectively at 24 h, 48 h, 5th day and 9th day after transfection. The HPV16 E6 mRNA was reduced by 77%, 83%, 59% and 41% at 24 h, 48 h, 5th day and 9th day after transfection. The inhibition rates of E6 protein measured by Flow cytometry were 79.7%, 80.4%, 71.3% and 57.4% at 24 h, 48 h, 5th day and 9th day after transfection, which were confirmed by the results of Western blot. In vivo research, E6 siRNA administration groups had great power in inhibiting tumor growth, restraining E6 protein expression, increasing tumor necrosis and apoptosis. The result of repeated injections of siRNA was better than that of single injection. CONCLUSION: RNA interference with HPV16 E6 is specific and highly efficient in vitro and in vivo.

Animals↗

PubMed2004

RNA interference and chemically modified small interfering RNAs.

RNA interference (RNAi) is a powerful biological process for specific silencing of gene expression in diversified eukaryotic cells and has tremendous potential for functional genomics, drug discovery through in vivo target validation, and development of novel gene-specific medicine. The future success of this technology relies on identifying appropriate chemical modifications to improve stability, potency and in vivo cellular delivery. The present review summarizes the role of the chemist's toolbox in this emerging technology.

Animals↗

PubMed2004

[RNA interference in functional genomics and medical research].

RNA interference (RNAi) is a post-transcriptional gene silencing process by targeting mRNA for degradation in a sequence-specific manner. This powerful platform has enormous potential in functional genomics and medical research. As a tool to knock out expression of specific genes in a variety of organisms, RNAi was used to investigate gene function in a high throughput fashion. Highly conserved in evolution RNAi appears to have evolved as a cellular defense mechanism in plants and animals to suppress viral infection, transposon jumping and endogenous aberrant genes. Exploiting the natural mechanism, the researchers can shut down disease-causing genes and develop novel therapeutics against infection, tumor and other disease.

Gene Expression Regulation↗

PubMed2004

RNA interference: the molecular immune system.

Introduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo- and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21- to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.

AIDS Vaccines↗

PubMed2006

RNA interference in cancer.

In the recent years, RNA interference (RNAi) has emerged as a major regulatory mechanism in eukaryotic gene expression. The realization that changes in the levels of microRNAs are directly associated with cancer led to the recognition of a new class of tumor suppressors and oncogenes. Moreover, RNAi has been turned into a potent tool for artificially modulating gene expression through the introduction of short interfering RNAs. A plethora of individual inhibitory RNAs as well as several large collections of these reagents have been generated. The systems for stable and regulated expression of these molecules emerged as well. These tools have helped to delineate the roles of various cellular factors in oncogenesis and tumor suppression and laid the foundation for new approaches in gene discovery. Furthermore, successful inhibition of tumor cell growth by RNAi aimed at oncogenes in vitro and in vivo supports the enthusiasm for potential therapeutic applications of this technique. In this article we review the evidence of microRNA involvement in cancer, the use of short interfering RNAs in forward and reverse genetics of this disease, and as well as both the benefits and limitations of experimental RNAi.

Animals↗

PubMed2005

Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells.

Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats ( approximately 3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells.

Active Transport, Cell Nucleus↗

PubMed2005

Cross-inhibition to heterologous foot-and-mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-23 nucleotide duplexes of small interfering RNA (siRNA). Strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Here we described a significant cross-inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in BHK-21 cells by siRNAs targeted to various conserved regions (5'NCR, VP4, VPg, POL, and 3'NCR) of the viral genome. The results showed that siRNAs generated in vitro by human recombinant dicer enzyme gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection (hpi). The inhibition extended to at least 6 days post-infection. For serotype Asia1, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by approximately 10-fold in cells pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. The inhibition was specific to FMDV replication, as no reduction was observed in virus yield of pseudorabies virus, an unrelated virus. Moreover, we also demonstrated an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV.

3' Untranslated Regions↗

PubMed2002

Antisense-RNA regulation and RNA interference.

For a long time, RNA has been merely regarded as a molecule that can either function as a messenger (mRNA) or as part of the translational machinery (tRNA, rRNA). Meanwhile, it became clear that RNAs are versatile molecules that do not only play key roles in many important biological processes like splicing, editing, protein export and others, but can also--like enzymes--act catalytically. Two important aspects of RNA function--antisense-RNA control and RNA interference (RNAi)--are emphasized in this review. Antisense-RNA control functions in all three kingdoms of life--although the majority of examples are known from bacteria. In contrast, RNAi, gene silencing triggered by double-stranded RNA, the oldest and most ubiquitous antiviral system, is exclusively found in eukaryotes. Our current knowledge about occurrence, biological roles and mechanisms of action of antisense RNAs as well as the recent findings about involved genes/enzymes and the putative mechanism of RNAi are summarized. An interesting intersection between both regulatory mechanisms is briefly discussed.

Animals↗

PubMed2005

HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome.

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.

Anti-HIV Agents↗

PubMed2004

RNA interference silencing the transcriptional message: aspects and applications.

RNA interference (RNAi) is silencing of gene expression by double-stranded RNA (dsRNA) having complementary sequence to the target gene to be silenced. This phenomenon has transformed into a complete technology for functional genomic studies. Small interfering RNAs (siRNAs) are 21- to 23-nucleotide dsRNAs, in which the sense strand is the same as the target mRNA and the antisense strand is the complement of the target mRNA sequence. These are the effector molecules for inducing RNAi, leading to posttranscriptional gene silencing with RNA-induced silencing complex. Besides siRNA, which can be chemically synthesized, various other systems in the form of potential effector molecules for posttranscriptional gene silencing are available, such as short hairpin RNAs (shRNAs), long dsRNAs, short temporal RNAs, and micro RNAs (miRNAs). These effector molecules either are processed into siRNA such as in the case of shRNA or directly aid gene silencing as in the case of miRNA. RNAi for various unknown genes may facilitate to elucidate inherited genetic diseases and provide drug candidates for viral and oncogenic diseases. This can be achieved by targeting mRNA from oncogenic genes or mRNA for viral cellular receptor and viral structural proteins for RNAi. In this article, we evaluate various aspects and applications of RNAi technology and provide comprehensive information for the system currently available for inducing RNAi.

Agriculture↗

PubMed2004

RNA interference using boranophosphate siRNAs: structure-activity relationships.

In RNA interference (RNAi), short double-stranded RNA (known as siRNA) inhibits expression from homologous genes. Clinical or pre-clinical use of siRNAs is likely to require stabilizing modifications because of the prevalence of intracellular and extracellular nucleases. In order to examine the effect of modification on siRNA efficacy and stability, we developed a new method for synthesizing stereoregular boranophosphate siRNAs. This work demonstrates that boranophosphate siRNAs are consistently more effective than siRNAs with the widely used phosphorothioate modification. Furthermore, boranophosphate siRNAs are frequently more active than native siRNA if the center of the antisense strand is not modified. Boranophosphate modification also increases siRNA potency. The finding that boranophosphate siRNAs are at least ten times more nuclease resistant than unmodified siRNAs may explain some of the positive effects of boranophosphate modification. The biochemical properties of boranophosphate siRNAs make them promising candidates for an RNAi-based therapeutic.

Boron Compounds↗

PubMed2003

RNA interference in functional genomics and medicine.

RNA interference (RNAi) is the sequence-specific gene silencing induced by double-stranded RNA (dsRNA). Being a highly specific and efficient knockdown technique, RNAi not only provides a powerful tool for functional genomics but also holds a promise for gene therapy. The key player in RNAi is small RNA (approximately 22-nt) termed siRNA. Small RNAs are involved not only in RNAi but also in basic cellular processes, such as developmental control and heterochromatin formation. The interesting biology as well as the remarkable technical value has been drawing widespread attention to this exciting new field.

Animals↗

PubMed2005

[Detection of RNA interference in nasopharyngeal carcinoma cell lines using reporter genes].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) technique is now widely used in studies of gene function, signal transduction pathway, and gene therapy because it can effectively and specifically inhibit gene expression. This study was designed to synthesize small interfering RNA (siRNA) by in vitro transcription, and construct retrovirus vectors to express small hairpin RNA (shRNA), detect RNAi in nasopharyngeal carcinoma cell lines, and to develop a RNAi technique platform. METHODS: siRNAs targeting green fluorescent protein (GFP) and luciferase (Luc) were synthesized by in vitro transcription, while shRNAs targeting GFP and Luc were constructed from pSUPER.retro. Cervical cancer cell line HeLa, nasopharyngeal carcinoma cell lines CNE1, CNE2, and 5-8F were co-transfected with siRNAs or shRNAs and reporter gene pEGFP-N1 or pGL3. The expression of GFP was detected by fluorescent microscopy and Western blot. The activity of luciferase was measured by Luciferase Enzyme Assay System. RESULTS: siRNA duplexes with 3' UU overhangs and shRNA specifically silenced GFP expression, while antisense RNA and siRNA without 3' UU overhangs did not trigger RNA interference of GFP. Quantitative luciferase activity analysis showed that siRNA inhibited Luc expression in HeLa, CNE1, CNE2, and 5-8F cell lines with inhibition rates of 91.43%, 78.01%, 90.30%, and 62.85%, respectively. Similarly, the inhibition rate was 78.22% when shRNA targeting Luc was co-transfected into HeLa cell line. CONCLUSIONS: Both siRNAs and shRNAs can induce RNAi. 3' UU overhangs of siRNA may play a role in RNAi. RNAi can be triggered in both nasopharyngeal carcinoma cell lines and HeLa cell line.

Cell Line, Tumor↗

PubMed2006

[Construction of "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference].

OBJECTIVE: To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs). METHODS: According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing. RESULTS: The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment. CONCLUSION: The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.

Cloning, Molecular↗

PubMed2005

Suppression of hLRH-1 mediated by a DNA vector-based RNA interference results in cell cycle arrest and induction of apoptosis in hepatocellular carcinoma cell BEL-7402.

RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of mRNA. A DNA vector-based approach has been shown to be able to trigger RNA interference in mammalian cells successfully. LRH-1 is an orphan nuclear receptor predominantly expressed in tissues of endodermal origin, where it controls development and cholesterol homeostasis. In the present study, we demonstrated that the expression of hLRH-1 and cyclin E1 in BEL-7402 cells could be suppressed by up to approximately 80% via DNA vector-based RNA interference. The suppression of hLRH-1 resulted in cell cycle arrest mediated by the down-regulation of cyclin E1. Induction of apoptosis and down-regulation of Gadd45beta were also shown in hLRH-1 knock down BEL-7402 cells. These results, together with the findings that Gadd45beta remained unchanged in cyclin E1 RNAi cells, suggested that the induction of apoptosis by knock down of hLRH-1 was closely related to the down-regulation of Gadd45beta.

Antigens, Differentiation↗

PubMed2004

RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter.

Small interfering RNAs (siRNAs) represent RNA duplexes of 21 nucleotides in length that inhibit gene expression. We have used the human gene-external 7S K RNA promoter for synthesis of short hairpin RNAs (shRNAs) which efficiently target human lamin mRNA via RNA interference (RNAi). Here we demonstrate that orientation of the target sequence within the shRNA construct is important for interference. Furthermore, effective interference also depends on the length and/or structure of the shRNA. Evidence is presented that the human 7S K promoter is more active in vivo than other gene-external promoters, such as the human U6 small nuclear RNA (snRNA) gene promoter.

HeLa Cells↗