SEARCH · PubMed Health

Results for “Enhancer RNAs”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,063 records · Page 59Linked to original sources

PubMed1999

Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization.

BACKGROUND: Ribozymes can function as allosteric enzymes that undergo a conformational change upon ligand binding to a site other than the active site. Although allosteric ribozymes are not known to exist in nature, nucleic acids appear to be well suited to display such advanced forms of kinetic control. Current research explores the mechanisms of allosteric ribozymes as well as the strategies and methods that can be used to create new controllable enzymes. RESULTS: In this study, we exploit the modular nature of certain functional RNAs to engineer allosteric ribozymes that are activated by flavin mononucleotide (FMN) or theophylline. By joining an FMN- or theophylline-binding domain to a hammerhead ribozyme by different stem II elements, we have identified a minimal connective bridge comprised of a G.U wobble pair that is responsive to ligand binding. Binding of FMN or theophylline to its allosteric site induces a conformational change in the RNA that stabilizes the wobble pair and ultimately favors the active form of the catalytic core. These ligand-sensitive ribozymes exhibit rate enhancements of more than 100-fold in the presence of FMN and of approximately 40-fold in the presence of theophylline. CONCLUSIONS: An adaptive strategy for modular rational design has proven to be an effective approach to the engineering of novel allosteric ribozymes. This strategy was used to create allosteric ribozymes that function by a mechanism involving ligand-induced structure stabilization. Conceivably, similar engineering strategies and allosteric mechanisms could be used to create a variety of novel allosteric ribozymes that function with other effector molecules.

Allosteric Regulation↗

PubMed2006

Unr, a cytoplasmic RNA-binding protein with cold-shock domains, is involved in control of apoptosis in ES and HuH7 cells.

Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein involved in the regulation of messenger RNA stability and internal initiation of translation. We have used Unr-deficient murine embryonic stem (ES) cells to analyse Unr role in cell proliferation and response to stress. Disruption of both unr gene copies had no effect on ES cell proliferation. However, after ionizing radiation (IR), clonogenic survival of unr(-/-) ES cells was approximately 3-fold enhanced as compared to unr(+/+) cells. We further determined that IR-induced apoptosis was decreased in unr(-/-) ES cells, and that reintroduction of the unr gene in unr(-/-) cells restored normal IR-induced apoptosis. Three pro-apoptotic genes, p53, caspase-3 and Gadd45gamma, were downregulated in unr(-/-) ES cells, indicating that Unr, as other cytoplasmic RNA-binding proteins, regulates a complex genetic program, promoting cell death after IR. In contrast, in the human hepatoma cell line HuH7, Unr knockdown using unr-specific small interfering RNAs induced apoptosis, both in untreated and gamma-irradiated cells. Thus, our results establish that Unr acts as a positive or negative regulator of cell death, depending on the cell type. Manipulating the level of Unr may constitute a specific approach to sensitize cancer cells to anticancer treatments.

Animals↗

PubMed1989

Evidence for specificity in the encapsidation of Sindbis virus RNAs.

We investigated the interaction of the capsid protein of Sindbis virus with Sindbis viral RNAs and defined a region of the genome that is required for binding in vitro and for packaging in vivo. The binding studies were performed with purified capsid protein immobilized on nitrocellulose and 32P-labeled RNAs transcribed in vitro from viral and nonspecific cDNAs. Genomic and defective interfering (DI) RNAs bound capsid protein significantly better than either the subgenomic (26S) RNA or nonspecific RNAs. Transcripts prepared from either truncated or deleted cDNAs were used to define the segment required for binding. This segment, which is represented twice in DI RNA, lies between nucleotides 746 and 1226 of the genomic RNA and is within the coding region of the nonstructural protein nsP1. Insertion of a domain covering these sequences into a nonviral RNA was able to convert it from a background level of binding to an activity that was 80% that of the Sindbis virus DI RNA. We analyzed DI RNA transcripts in detail because they could be studied not only for the ability to bind capsid protein in vitro but also for the ability to be replicated and packaged in vivo in the presence of helper virion RNA. The results obtained with three DI RNAs are reported. One (CTS14), which has one copy of the binding domain, bound efficiently to capsid protein in vitro and was packaged in vivo as measured by amplification on passaging. In contrast, a DI RNA (CTS1) which lacked this region did not bind to capsid protein and was not detected on passaging. By using lipofectin (P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J.P. Northrop, G. M. Ringold, and M. Danielson, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987) to enhance RNA uptake, we were able to demonstrate that CTS1 RNA was replicated in the transfected cells. It was replicated to the same level as another DI RNA (CTS253) which has only the 3' 279 nucleotides of the binding domain and these are located near the 3' terminus of the RNA. CTS253 bound capsid protein to an intermediate level but was amplified on passaging. The binding studies and the in vivo packaging data, taken together, provide strong support for the conclusion that there is a specific capsid recognition domain in Sindbis virus RNA that plays a role in nucleocapsid assembly.

Capsid↗

PubMed2003

Mechanism of RNA recombination in carmo- and tombusviruses: evidence for template switching by the RNA-dependent RNA polymerase in vitro.

RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligation-based RNA recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.

Base Sequence↗

PubMed1996

Enhancement of nucleocytoplasmic export of HTLV-1 Rex mRNA through cis and trans interactions of the mRNA with the complex of Rex protein and Rex-responsive element.

p27 rex of HTLV-1 promotes nucleocytoplasmic export of viral mRNAs through binding of the Rex-response element (RexRE) present at the 3' end of the viral transcripts in cis with respect to the ORFs of the viral mRNAs. We have found that expression of the RexRE in trans, as a separate RNA, still allows Rex protein to promote export of viral mRNAs lacking the RexRE. The data suggest the formation of a ternary complex between Rex protein, RexRE and upstream elements of viral mRNA and hence the existence of secondary sites of interaction between Rex protein and viral RNAs.

Animals↗

PubMed1989

Ribonucleoside analysis by reversed-phase high-performance liquid chromatography.

Over the past fifteen years we have developed and refined the analytical chromatographic methodologies using reversed-phase high-performance liquid chromatography and UV-photodiode array detection (RPLC-UV) for the detection and measurement of the major and modified nucleosides in nucleic acids and biological fluids. RPLC-UV nucleoside analysis as it has now evolved is a powerful new research tool to aid investigators in the fields of biochemical and biomedical research. This RPLC-UV nucleoside method can resolve more than 65 nucleosides in a single analysis with "run-to-run" peak retention variations of less than 1%. A complete nucleoside composition can be obtained from as little as 0.5 micrograms RNA. Identification and confirmation of nucleosides can be made from the highly reproducible retention times and from the characteristic UV spectrum from a few picomoles (ca. 1 ng) of nucleoside. In this paper we introduce standard RPLC-UV methodologies for the analysis of nucleosides and nucleoside composition of RNAs. The chromatographic protocols and standard nucleoside columns are presented and the essential requirements necessary in the HPLC instrumentation are described. Three optimized RPLC systems were developed, each with particular emphasis placed on resolution, speed, or sensitivity. In addition, three unfractionated tRNAs were selected as sources of reference nucleosides and for assessment of the performance of the chromatography. From these tRNAs, a large array of nucleosides were characterized which are used in standardization and calibration of the method. Also discussed is the use of a diode-array detector for enhancement of the reliability of nucleoside identification and accuracy of measurement. An extended enzymatic hydrolysis protocol for the liberation of exotically modified nucleosides in tRNAs is also described. Chromatographic retention times and UV spectra for a large number of ribonucleosides are tabulated. The RPLC-UV ribonucleoside analytical protocols are capable of quantifying 31 nucleosides. Approximately 1 microgram of an isoaccepting tRNA, or 20 micrograms of unfractionated tRNA are needed for quantitative analysis. With this amount of tRNA, the percent relative error of measurement of the four major nucleosides is less than 2%, and for the modified nucleosides about 5%. As little as 0.2 micrograms of pure isoaccepting tRNA can be analyzed, but at the expense of precision as at this low sample size a 20-30% relative error for modified nucleosides is to be expected. For quantitation of the modified nucleosides in rRNA, which contains much less modification than tRNAs, 10-100 micrograms of sample are needed per injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Single base bulges in small RNA hairpins enhance ethidium binding and promote an allosteric transition.

A set of four RNA hairpin helices has been prepared by in vitro transcription with T7 RNA polymerase. The hairpins all contain the same nine base pair helix, but with an extra A, C, or U residue forming a bulge at one position; the fourth hairpin is a perfect helix with no bulge. The helix with a bulged A duplicates six base pairs of a helix in the 16S rRNA known to have an unusually high affinity for ethidium bromide [J. M. Kean, S. A. White, and D. E. Draper, Biochemistry 24, 5062 (1985)]. Binding and chemical cleavage studies with ethidium or the reagent methidiumpropylEDTA-Fe(II) [MPE-Fe(II)] showed that the sequence CpG is a preferred intercalation site whether or not a bulge is present; all three bulged bases enhance intercalation at the CpG sequence by an order of magnitude; and intercalation in a bulged helix results in a concerted conformational change involving the entire helix backbone, again dependent on the presence of a bulge but independent of the particular base. These results suggest that an extra sugar-phosphate residue in an RNA helix backbone has a dramatic effect on the ability of the RNA to adopt new conformations. This effect could be an important reason for the conservation of bulges at certain positions in ribosomal and other RNAs.

Allosteric Site↗

PubMed2000

RNA aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour.

We have probed the effects of altering buffer conditions on the behaviour of two aptamer RNAs for the bacterio-phage MS2 coat protein using site-specific substitution of 2'-deoxy-2-aminopurine nucleotides at key adenosine positions. These have been compared to the wild-type operator stem-loop oligonucleotide, which is the natural target for the coat protein. The fluorescence emission spectra show a position and oligonucleotide sequence dependence which appears to reflect local conformational changes. These are largely similar between the differing oligonucleotides and deviations can be explained by the individual features of each sequence. Recognition by coat protein is enhanced, unaffected or decreased depending on the site of substitution, consistent with the known protein-RNA contacts seen in crystal structures of the complexes. These data suggest that the detailed conformational dynamics of aptamers and wild-type RNA ligands for the same protein target are remarkably similar.

Base Sequence↗

PubMed2026

Molecular Pathways, Target Landscape, and Translational Models in Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a substantial global health burden and the greatest unmet medical need for cardiovascular diseases. It is marked by pronounced clinical heterogeneity and complex multi-system pathophysiology with limited therapeutic options. Progress in developing effective therapeutics is constrained by the inadequacy of experimental models to fully recapitulate the multifactorial nature of the disease. Recent evidence underscores the significant involvement of inflammatory, oxidative, and mitochondrial pathways in the pathogenesis of HFpEF, with non-coding RNAs and epigenetic regulation serving as crucial modulators and prospective therapeutic targets. This review maps the HFpEF target landscape, while critically assessing the mechanistic contributions, translational fidelity, and limitations of existing in vivo and in vitro models. Further, advances are noted among the in vitro technologies, including human cardiac organoids and engineered heart tissues integrated with high-throughput multi-omics and computational modeling, enabling in-depth examination of HFpEF mechanisms. Finally, we underscore the necessity of integrative, systems-level approaches and multi-marker strategies to enhance translational relevance, improve risk stratification, and accelerate development of mechanism-based therapies. Collectively, this review supports phenotypic-guided and mechanism-informed therapeutic development for HFpEF, and provides a roadmap for next generation model development and therapeutic innovation.

Humans↗

PubMed1991

Expression of insulin-like growth factor II and receptors for insulin-like growth factor II, insulin-like growth factor I and insulin in isolated and cultured rat hepatocytes.

Insulin-like growth factor II is believed to play an important role in fetal growth and development. The insulin-like growth factor II gene expression is tissue specific and developmentally regulated. We have previously shown an enhanced level of insulin-like growth factor II messenger RNA and protein in human hepatocellular carcinomas. This led to the suggestion that hepatocytes might be involved in insulin-like growth factor II expression. However, previous studies based on in situ hybridization only showed insulin-like growth factor II messenger RNA in liver sinusoidal cells. This paper reports on the analysis of the expression of insulin-like growth factor II and insulin-like growth factor II, insulin-like growth factor I and insulin receptor messenger RNAs in vivo in isolated rat hepatocytes at various stages of development and in vitro in adult rat hepatocytes primary culture. Our study indicates that isolated rat hepatocytes synthesize insulin-like growth factor II messenger RNA with a switch between fetal and adult messenger RNA profiles occurring 21 days after birth. In addition, adult rat hepatocytes in culture expressed insulin-like growth factor II messenger RNA and protein. Insulin-like growth factor II, insulin-like growth factor I and insulin receptor messenger RNAs were also detected. Therefore these results are consistent with the hypothesis that insulin-like growth factor II acts as an autocrine growth factor for hepatocytes in addition to having a paracrine effect. They also indicate that primary culture of hepatocytes is a good model for further studies on insulin-like growth factor II gene regulation.

Animals↗

PubMed1998

Coding sequences enhance internal initiation of translation by hepatitis A virus RNA in vitro.

Hepatitis A virus (HAV), unlike other picornaviruses, has a slow-growth phenotype in permissive cell lines and in general does not induce host cell cytopathology. Although there are no published reports of productive infection of HeLa cells by HAV, HAV RNA appears to be readily translated in HeLa cells when transcribed by T7 RNA polymerase provided by a recombinant vaccinia virus. The 5' noncoding region of HAV was fused to poliovirus (PV) coding sequences to determine the effect on translation efficiency in HeLa cell extracts in vitro. Conditions were optimized for utilization of the HAV internal ribosome entry segment (IRES). Transcripts from chimeric constructs fused precisely at the initiation codon were translated very poorly. However, chimeric RNAs which included 114 or more nucleotides from the HAV capsid coding sequences downstream of the initiation codon were translated much more efficiently than those lacking these sequences, making HAV-directed translation efficiency similar to that directed by the PV IRES. Sixty-six nucleotides were insufficient to confer increased translation efficiency. The most 5'-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an inhibitory effect on translation efficiency. Constructs lacking these terminal sequences, or those in which the PV 5'-terminal sequences replaced those from HAV, translated three- to fourfold better than those with the intact HAV 5'-terminal end.

Cell Extracts↗

PubMed2004

Vascular endothelial growth factor increases messenger RNAs encoding cyclooxygenase-II and membrane-associated prostaglandin E synthase in rat luteal cells.

Vascular endothelial growth factor (VEGF) is known to be necessary for the vascularization of the developing corpus luteum. Our recent data suggested that cyclooxygenase-II (COX-II) may play a role in the formation of vascular plexuses in developing corpora lutea of the rat. Here we examined the relationship between VEGF and the expression of prostaglandin (PG)- metabolizing enzymes in rat ovarian luteal cells. VEGF treatment caused a dose-dependent increase in the expression of COX-II and membrane-associated PGE synthase (mPGES) mRNA in cultured rat luteal cells. However, pretreatment of the luteal cells with a selective COX-II inhibitor, NS-398, abolished the VEGF-enhanced mPGES mRNA expression. VEGF also increased PGE2 secretion. Conversely, PGE2 dose-dependently stimulated VEGF mRNA expression. Furthermore, VEGF induced VEGF mRNA expression, but this effect was abolished by NS-398 pretreatment. These findings suggest that VEGF enhances PGE2 production by stimulating COX-II and mPGES expression in rat corpus luteum and that the effect of VEGF on luteal cells may be partially mediated by this stimulation of PGE2 production.

Animals↗

PubMed2003

RNA crystallization.

RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to synthesize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

Base Sequence↗

PubMed2002

The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs.

The Escherichia coli host factor I, Hfq, binds to many small regulatory RNAs and is required for OxyS RNA repression of fhlA and rpoS mRNA translation. Here we report that Hfq is a bacterial homolog of the Sm and Sm-like proteins integral to RNA processing and mRNA degradation complexes in eukaryotic cells. Hfq exhibits the hallmark features of Sm and Sm-like proteins: the Sm1 sequence motif, a multisubunit ring structure (in this case a homomeric hexamer), and preferential binding to polyU. We also show that Hfq increases the OxyS RNA interaction with its target messages and propose that the enhancement of RNA-RNA pairing may be a general function of Hfq, Sm, and Sm-like proteins.

Amino Acid Sequence↗

PubMed2006

Pairwise coupling analysis of helical junction hydrogen bonding interactions in luteoviral RNA pseudoknots.

A 28-nucleotide mRNA pseudoknot that overlaps the P1 and P2 genes of sugarcane yellow leaf virus (ScYLV) stimulates -1 ribosomal frameshifting. The in vitro frameshifting efficiency is decreased >or=8-fold upon substitution of the 3'-most loop 2 nucleotide (C27) with adenosine, which accepts a hydrogen bond from the 2'-OH group of C14 in stem S1. The solution structures of the wild-type (WT) and C27A ScYLV RNA pseudoknots show that while the RNAs adopt virtually identical overall structures, there are significant structural differences at the helical junctions of the two RNAs. Specifically, C8(+) in loop L1 in the C8(+).(G12.C28) L1-S2 major groove base triple is displaced by approximately 2.3 A relative to the accepting stem 2 base pair (G12.C28) in the C27A RNA. Here, we use a double mutant cycle approach to analyze the pairwise coupling of the C8(+).(G12.C28)...C27.(C14-G7) and ...A27.(C14-G7) hydrogen bonds in the WT and C27A ScYLV RNAs, respectively, and compare these findings with previous results from the beet western yellows virus (BWYV) RNA. We find that the pairwise coupling free energy (delta(AB)(i)) is favorable for the WT RNA (-0.7 +/- 0.1 kcal/mol), thus revealing that formation of these two hydrogen bonds is positively cooperative. In contrast, delta(AB)(i) is 0.9 +/- 0.4 kcal/mol for the poorly functional C27A ScYLV RNA, indicative of nonadditive hydrogen bond formation. These results reveal that cooperative hydrogen bond formation across the helical stem junction in H-type pseudoknots correlates with enhanced frameshift stimulation by luteoviral mRNA pseudoknots.

Hydrogen Bonding↗

PubMed1997

Facilitation or inhibition of the estradiol-induced gonadotropin surge in the immature rat by progesterone: regulation of GnRH and LH messenger RNAs and activation of GnRH neurons.

UNLABELLED: We have developed and extensively characterized immature female rat models to demonstrate inhibition or facilitation of the estradiol (E2)-induced gonadotropin surge by progesterone (P). We show here that the surge of free alpha-subunit is regulated similarly by P in these models. To investigate the possibility that P alters the biosynthesis of GnRH and/or LH, we measured levels of LH subunit mRNAs by Northern blot hybridization and GnRH mRNA by a solution hybridization-RNase protection assay. In the P inhibition model, alpha-subunit mRNA was significantly decreased when P was administered together with E2 for 32 or 48 h, and LHbeta, at 29 h. In the facilitation model, neither alpha-subunit nor LHbeta mRNA increased with premature and enhanced release of LH and free alpha-subunit. Levels of GnRH mRNA in E2-treated rats were significantly higher on the afternoon of the LH surge than on that or the following morning. There was no effect of P on GnRH mRNA levels, however, before, during, or after the LH surge in either paradigm. The time course of activation of GnRH neurons in P-facilitated rats was determined by double-label immunocytochemistry for GnRH and cFos. When serum LH concentrations were basal there was no expression of cFos in GnRH neurons. LH secretion in P-facilitated rats was initiated at 14.00 h and remained elevated until at least 19.00 h. During this time 63-78% of GnRH neurons were cFos positive. Both serum LH concentrations and the percentage of cFos-activated GnRH neurons were significantly lower in control rats treated with E2 alone than in those treated also with P. IN CONCLUSION: 1) suppression of LH and free alpha-subunit secretion by P can be accounted for at least partly by suppression of alpha-subunit mRNA levels; 2) P facilitation is not associated with changes in LH subunit or GnRH mRNA levels; 3) the large proportion of cFos-positive GnRH neurons in P-facilitated rats closely parallels increases in serum LH concentrations but is not accompanied by changes in GnRH mRNA levels. It is likely, therefore, that P acts in the facilitation model to trigger release of pre-existing GnRH stores by altering synthesis or activity of neuro-transmitters/neuropeptides involved in GnRH regulation and/or release of LH stores by altering, for example, pituitary responsiveness to GnRH (including self-priming) and components of the LH secretory apparatus. Similar possibilities may also obtain for the blockade of the gonadotropin surge in the inhibition model.

Animals↗

PubMed2001

Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays.

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.

Base Sequence↗

PubMed2003

Small RNA: can RNA interference be exploited for therapy?

CONTEXT: RNA interference (RNAi) is the sequence-specific gene-silencing induced by double-stranded RNA (dsRNA), and gives information about gene function quickly, easily, and inexpensively. The use of RNAi for genetic-based therapies is widely studied, especially in viral infections, cancers, and inherited genetic disorders. RNAi has been used to make tissue-specific knockdown mice for studying gene function in a whole animal. Combined with genomics data, RNAi-directed gene-silencing could allow functional determination of any gene expressed in a cell or pathway. The term RNAi came from the discovery that the injection of dsRNAs into Caenorhabditis elegans interferes with the expression of specific genes containing a complementary region to the delivered dsRNA. Although stalled for a time by the non-gene-specific interferon response elicited by dsRNA molecules longer than about 30 nucleotides in mammalian cells, Tom Tuschl's group found that transfection of synthetic 21-nucleotide small-interfering RNA (siRNA) duplexes were highly selective and sequence-specific inhibitors of endogenous genes. STARTING POINT: siRNA expression has been studied with siRNA from plasmid and viral vectors that efficiently deliver siRNAs into both dividing and non-dividing cells, stem cells, zygotes, and their differentiated progeny. A collection of RNA interference vectors that suppress 50 human de-ubiquitinating enzymes allowed Thijn Brummelkamp and colleagues to study this gene family and to identify de-ubiquitinating enzymes in cancer-relevant pathways (Nature 2003; 424: 797-801). These researchers found that the familial cylindromatosis tumour suppressor gene (CYLD), previously of unknown function, could enhance the activation of the transcription factor NF-kappaB, leading to increased resistance to apoptosis. They have now started to investigate the use of CYLD inhibitors in clinical trials. WHERE NEXT: The ability to efficiently and stably produce and deliver sufficient amounts of siRNA to the proper target tissues require refinement before this new technology can be tried clinically. Initial in-vivo studies reported effective transgene suppression in adult mice by chemically synthesised siRNAs. More recently many researchers have used plasmid and viral vectors for transcription of short-hairpin RNAs, both in vitro and in vivo. With these expression systems, gene expression was more stably inhibited than with the transient knockdown recorded with chemically synthesised siRNA. Human trials exploiting these latest findings are likely to soon follow.

Animals↗