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Showing papers on "RNA published in 2002"


Journal ArticleDOI
TL;DR: Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority (≈68%) of CLL cases.
Abstract: Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue-specific gene regulation MiRs are transcribed as short hairpin precursors (≈70 nt) and are processed into active 21- to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via base-pairing interactions Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL) Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority (≈68%) of CLL cases

5,113 citations


Journal Article
01 Jan 2002-Nature
TL;DR: A conserved biological response to double-stranded RNA, known variously as RNA interference (RNAi) or post-transcriptional gene silencing, mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
Abstract: A conserved biological response to double-stranded RNA, known variously as RNA interference (RNAi) or post-transcriptional gene silencing, mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. RNAi has been cultivated as a means to manipulate gene expression experimentally and to probe gene function on a whole-genome scale.

2,503 citations


Journal ArticleDOI
20 Sep 2002-Science
TL;DR: It is shown that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function.
Abstract: In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells.

2,229 citations


Journal ArticleDOI
TL;DR: This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at the Comparative RNA Web (CRW) Site.
Abstract: Background: Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, wellorganized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields. Results: We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems. Conclusions: This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site [http://www.rna.icmb.utexas.edu] . In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site.

1,676 citations


Journal ArticleDOI
20 Sep 2002-Science
TL;DR: This work shows that Arabidopsis thaliana miRNA 39 (also known as miR171), a 21-ribonucleotide species that accumulates predominantly in inflorescence tissues, is produced from an intergenic region in chromosome III and functionally interacts with mRNA targets encoding several members of the Scarecrow-like (SCL) family of putative transcription factors.
Abstract: Micro-RNAs (miRNAs) are regulatory molecules that mediate effects by interacting with messenger RNA (mRNA) targets. Here we show that Arabidopsis thaliana miRNA 39 (also known as miR171), a 21-ribonucleotide species that accumulates predominantly in inflorescence tissues, is produced from an intergenic region in chromosome III and functionally interacts with mRNA targets encoding several members of the Scarecrow-like (SCL) family of putative transcription factors. miRNA 39 is complementary to an internal region of three SCL mRNAs. The interaction results in specific cleavage of target mRNA within the region of complementarity, indicating that this class of miRNA functions like small interfering RNA associated with RNA silencing to guide sequence-specific cleavage in a developmentally controlled manner.

1,648 citations


Journal ArticleDOI
TL;DR: This work has shown that the use of siRNAs to silence genes in vertebrate cells was only reported a year ago, and the emerging literature indicates that most vertebrate genes can be studied with this technology.
Abstract: Among the 3 billion base pairs of the human genome, there are approximately 30,000-40,000 protein-coding genes, but the function of at least half of them remains unknown. A new tool - short interfering RNAs (siRNAs) - has now been developed for systematically deciphering the functions and interactions of these thousands of genes. siRNAs are an intermediate of RNA interference, the process by which double-stranded RNA silences homologous genes. Although the use of siRNAs to silence genes in vertebrate cells was only reported a year ago, the emerging literature indicates that most vertebrate genes can be studied with this technology.

1,620 citations


Journal ArticleDOI
TL;DR: A large subset of Drosophila microRNAs is shown to be perfectly complementary to several classes of sequence motif previously demonstrated to mediate negative post-transcriptional regulation, suggesting a more general role for micro RNAs in gene regulation through the formation of RNA duplexes.
Abstract: Micro RNAs are a large family of noncoding RNAs of 21-22 nucleotides whose functions are generally unknown. Here a large subset of Drosophila micro RNAs is shown to be perfectly complementary to several classes of sequence motif previously demonstrated to mediate negative post-transcriptional regulation. These findings suggest a more general role for micro RNAs in gene regulation through the formation of RNA duplexes.

1,446 citations


Journal ArticleDOI
TL;DR: This work reports a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression and demonstrates robust inhibition of several endogenous genes of diverse functions in mammalian cells.
Abstract: Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic double-stranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells.

1,388 citations


Journal ArticleDOI
TL;DR: It is found that U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific β-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis.
Abstract: Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific β-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy.

1,317 citations


Journal ArticleDOI
31 Oct 2002-Nature
TL;DR: It is reported here that mRNAs encoding enzymes involved in thiamine (vitamin B1) biosynthesis in Escherichia coli can bindThiamine or its pyrophosphate derivative without the need for protein cofactors and provides an example of a 'riboswitch' whose evolutionary origin might pre-date the emergence of proteins.
Abstract: Although proteins fulfil most of the requirements that biology has for structural and functional components such as enzymes and receptors, RNA can also serve in these capacities. For example, RNA has sufficient structural plasticity to form ribozyme and receptor elements that exhibit considerable enzymatic power and binding specificity. Moreover, these activities can be combined to create allosteric ribozymes that are modulated by effector molecules. It has also been proposed that certain messenger RNAs might use allosteric mechanisms to mediate regulatory responses depending on specific metabolites. We report here that mRNAs encoding enzymes involved in thiamine (vitamin B(1)) biosynthesis in Escherichia coli can bind thiamine or its pyrophosphate derivative without the need for protein cofactors. The mRNA-effector complex adopts a distinct structure that sequesters the ribosome-binding site and leads to a reduction in gene expression. This metabolite-sensing regulatory system provides an example of a 'riboswitch' whose evolutionary origin might pre-date the emergence of proteins.

1,224 citations


Journal ArticleDOI
TL;DR: It is demonstrated that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-α) and that a higher frequency of cured cells could support both sub genomic and full-length HCV replication.
Abstract: Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-α) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.

Journal ArticleDOI
TL;DR: This review summarizes ongoing investigations of the enzyme family and their substrates, focusing on biological function as well as biochemical mechanism.
Abstract: ▪ Abstract ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify the information encoded in the genome, for example, by altering codons in mRNAs. The functions of ADARs in known substrates suggest that the enzymes serve to fine-tune and optimize many biological pathways, in ways that we are only starting to imagine. ADARs are also interesting in regard to the remarkable double-stranded structures of their substrates and how enzyme specificity is achieved with little regard to sequence. This review summarizes ongoing investigations of the enzyme family and their substrates, focusing on biological function as well as biochemical mechanism.

Journal ArticleDOI
04 Jul 2002-Nature
TL;DR: This paper showed that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates, and they also showed the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.
Abstract: RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

Journal ArticleDOI
TL;DR: RyhB provides a mechanism for the cell to down-regulate iron-storage proteins and nonessential ironcontaining proteins when iron is limiting, thus modulating intracellular iron usage to supplement mechanisms for iron uptake directly regulated by Fur.
Abstract: 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 ironcontaining proteins when iron is limiting, thus modulating intracellular iron usage to supplement mechanisms for iron uptake directly regulated by Fur.

Journal ArticleDOI
TL;DR: It is shown that siRNAs produced in plants from a green fluorescent protein (GFP) transgene are in short and long size classes, whereas those from endogenous retroelements are only in the long class, revealing an unexpected level of complexity in the RNA silencing pathway in plants that may also apply in animals.
Abstract: RNA silencing is a eukaryotic genome defence system that involves processing of double-stranded RNA (dsRNA) into 21-26 nt, short interfering RNA (siRNA). The siRNA mediates suppression of genes corresponding to the dsRNA through targeted RNA degradation. In some plant systems there are additional silencing processes, involving systemic spread of silencing and RNA-directed methylation/transcriptional suppression of homologous genomic DNA. We show here that siRNAs produced in plants from a green fluorescent protein (GFP) transgene are in short (21-22 nt) and long (24-26 nt) size classes, whereas those from endogenous retroelements are only in the long class. Viral suppressors of RNA silencing and mutations in Arabidopsis indicate that these classes of siRNA have different roles. The long siRNA is dispensable for sequence-specific mRNA degradation, but correlates with systemic silencing and methylation of homologous DNA. Conversely, the short siRNA class correlates with mRNA degradation but not with systemic signalling or methylation. These findings reveal an unexpected level of complexity in the RNA silencing pathway in plants that may also apply in animals.

Journal ArticleDOI
14 Feb 2002-Nature
TL;DR: The results indicate that non-coding RNAs have an active role in genomic imprinting, by inserting a polyadenylation signal that truncates 96% of the RNA transcript, that Air RNA is required for silencing.
Abstract: In genomic imprinting, one of the two parental alleles of an autosomal gene is silenced epigenetically by a cis-acting mechanism. A bidirectional silencer for a 400-kilobase region that contains three imprinted, maternally expressed protein-coding genes (Igf2r/Slc22a2/Slc22a3) has been shown by targeted deletion to be located in a sequence of 3.7 kilobases, which also contains the promoter for the imprinted, paternally expressed non-coding Air RNA. Expression of Air is correlated with repression of all three genes on the paternal allele; however, Air RNA overlaps just one of these genes in an antisense orientation. Here we show, by inserting a polyadenylation signal that truncates 96% of the RNA transcript, that Air RNA is required for silencing. The truncated Air allele maintains imprinted expression and methylation of the Air promoter, but shows complete loss of silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster on the paternal chromosome. Our results indicate that non-coding RNAs have an active role in genomic imprinting.

Journal ArticleDOI
TL;DR: The simplicity of the U6 expression cassette and its widespread transcription in human cell types suggest that this mode of siRNA delivery could be useful for suppressing expression of a wide range of genes.
Abstract: In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a double-stranded RNA (dsRNA) corresponding to exon sequences in the mRNA (refs 1,2). The use of this "RNA interference" (RNAi) in mammalian studies had lagged well behind its utility in lower animals because uninterrupted RNA duplexes longer than 30 base pairs trigger generalized cellular responses through activation of dsRNA-dependent protein kinases. Recently it was demonstrated that RNAi can be made to work in cultured human cells by introducing shorter, synthetic duplex RNAs (approximately 20 base pairs) through liposome transfection. We have explored several strategies for expressing similar short interfering RNA (siRNA) duplexes within cells from recombinant DNA constructs, because this might allow long-term target-gene suppression in cells, and potentially in whole organisms. Effective suppression of target gene product levels is achieved by using a human U6 small nuclear RNA (snRNA) promoter to drive nuclear expression of a single RNA transcript. The siRNA-like parts of the transcript consists of a 19 base pair siRNA stem with the two strands joined by a tightly structured loop and a U1-4 3' overhang at the end of the antisense strand. The simplicity of the U6 expression cassette and its widespread transcription in human cell types suggest that this mode of siRNA delivery could be useful for suppressing expression of a wide range of genes.

Journal ArticleDOI
TL;DR: It is proposed that Arabidopsis small RNAs participate in a wide range of post-transcriptional and epigenetic events.
Abstract: A large set of endogenous small RNAs of predominantly 21 to 24 nucleotides was identified in Arabidopsis. These small RNAs resembled micro-RNAs from animals and were similar in size to small interfering RNAs that accumulated during RNA silencing triggered by multiple types of inducers. Among the 125 sequences identified, the vast majority (90%) arose from intergenic regions, although small RNAs corresponding to predicted protein-coding genes, transposon-like sequences, and a structural RNA gene also were identified. Evidence consistent with the derivation of small RNAs of both polarities, and from highly base-paired precursors, was obtained through the identification and analysis of clusters of small RNA loci. The accumulation of specific small RNAs was regulated developmentally. We propose that Arabidopsis small RNAs participate in a wide range of post-transcriptional and epigenetic events.

Journal ArticleDOI
TL;DR: The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans and the depletion rate-dependent appearance of 3' mRNA cleavage fragments argues for the existence of a two-step mRNA degradation mechanism.
Abstract: Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3'-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3' mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.

Journal ArticleDOI
12 Sep 2002-Nature
TL;DR: The spliceosome is identified as the most complex cellular machine so far characterized, containing at least 30 proteins with known or putative roles in gene expression steps other than splicing, and its components comprise all previously known splicing factors and 58 newly identified components.
Abstract: The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify approximately 145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

Journal ArticleDOI
TL;DR: A vector-based siRNA expression system that can induce RNAi in mammalian cells is reported, which might allow therapeutic applications by means of vector-mediated RNAi and facilitate a wide range of functional analysis of mammalian genes.
Abstract: The first evidence for gene disruption by double-stranded RNA (dsRNA) came from careful analysis in Caenorhabditis elegans. This phenomenon, called RNA interference (RNAi), was observed subsequently in various organisms, including plants, nematodes, Drosophila, and protozoans. Very recently, it has been reported that in mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (small inhibitory RNAs, siRNAs) exhibit an RNAi effect. This is because siRNAs are not recognized by the well-characterized host defense system against viral infections, involving dsRNA-dependent inhibition of protein synthesis. However, the current method for introducing synthetic siRNA into cells by lipofection restricts the range of applications of RNAi as a result of the low transfection efficiencies in some cell types and/or short-term persistence of silencing effects. Here, we report a vector-based siRNA expression system that can induce RNAi in mammalian cells. This technical advance for silencing gene expression not only facilitates a wide range of functional analysis of mammalian genes but might also allow therapeutic applications by means of vector-mediated RNAi.

Journal ArticleDOI
09 Aug 2002-Science
TL;DR: The results show that it is possible to synthesize an infectious agent by in vitro chemical-biochemical means solely by following instructions from a written sequence.
Abstract: Full-length poliovirus complementary DNA (cDNA) was synthesized by assembling oligonucleotides of plus and minus strand polarity. The synthetic poliovirus cDNA was transcribed by RNA polymerase into viral RNA, which translated and replicated in a cell-free extract, resulting in the de novo synthesis of infectious poliovirus. Experiments in tissue culture using neutralizing antibodies and CD155 receptor-specific antibodies and neurovirulence tests in CD155 transgenic mice confirmed that the synthetic virus had biochemical and pathogenic characteristics of poliovirus. Our results show that it is possible to synthesize an infectious agent by in vitro chemical-biochemical means solely by following instructions from a written sequence.

Journal ArticleDOI
11 Jul 2002-Nature
TL;DR: It is concluded that DNA- and protein-based life was preceded by a simpler life form based primarily on RNA, referred to as the 'RNA world', during which the genetic information resided in the sequence of RNA molecules and the phenotype derived from the catalytic properties of RNA.
Abstract: All life that is known to exist on Earth today and all life for which there is evidence in the geological record seems to be of the same form — one based on DNA genomes and protein enzymes. Yet there are strong reasons to conclude that DNA- and protein-based life was preceded by a simpler life form based primarily on RNA. This earlier era is referred to as the 'RNA world', during which the genetic information resided in the sequence of RNA molecules and the phenotype derived from the catalytic properties of RNA.

Journal ArticleDOI
TL;DR: It is reported that siRNAs inhibit virus production by targeting the mRNAs for either the HIV-1 cellular receptor CD4, the viral structural Gag protein or green fluorescence protein substituted for the Nef regulatory protein.
Abstract: RNA interference silences gene expression through short interfering 21 23-mer double-strand RNA segments that guide mRNA degradation in a sequence-specific fashion. Here we report that siRNAs inhibit virus production by targeting the mRNAs for either the HIV-1 cellular receptor CD4, the viral structural Gag protein or green fluorescence protein substituted for the Nef regulatory protein. siRNAs effectively inhibit pre- and/or post-integration infection events in the HIV-1 life cycle. Thus, siRNAs may have potential for therapeutic intervention in HIV-1 and other viral infections.

Journal ArticleDOI
16 May 2002-Nature
TL;DR: The data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export, and is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II.
Abstract: The essential yeast proteins Yra1 and Sub2 are messenger RNA export factors that have conserved counterparts in metazoans, designated Aly and UAP56, respectively1,2,3,4,5,6,7. These factors couple the machineries that function in splicing and export of mRNA1,2,3,4,5,6,7. Here we show that both Yra1 and Sub2 are stoichiometrically associated with the heterotetrameric THO complex8, which functions in transcription in yeast8,9,10,11. We also show that Sub2 and Yra1 interact genetically with all four components of the THO complex (Tho2, Hpr1, Mft1 and Thp2). Moreover, these components operate in the export of bulk poly(A)+ RNA as well as of mRNA derived from intronless genes. Both Aly and UAP56 associate with human counterparts of the THO complex. Together, these data define a conserved complex, designated the TREX (‘transcription/export’) complex. The TREX complex is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II. Our data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export.

Journal ArticleDOI
TL;DR: This finding, along with related observations, supports the hypothesis that metabolic monitoring through RNA-metabolite interactions is a widespread mechanism of genetic control.

Journal ArticleDOI
19 Apr 2002-Cell
TL;DR: Small nucleolar RNAs represent an abundant, evolutionarily ancient group of noncoding RNAs which possess impressively diverse functions ranging from 2'-O-methylation and pseudouridylation of various classes of RNAs, through nucleolytic processing of rRNAs to the synthesis of telomeric DNA.

Journal ArticleDOI
TL;DR: No simple correlation between mRNA half-lives and ORF size, codon bias, ribosome density, or abundance is found, but the decay rates of mRNAs encoding groups of proteins that act together in stoichiometric complexes were generally closely matched.
Abstract: Posttranscriptional processing of mRNA is an integral component of the gene expression program. By using DNA microarrays, we precisely measured the decay of each yeast mRNA, after thermal inactivation of a temperature-sensitive RNA polymerase II. The half-lives varied widely, ranging from ∼3 min to more than 90 min. We found no simple correlation between mRNA half-lives and ORF size, codon bias, ribosome density, or abundance. However, the decay rates of mRNAs encoding groups of proteins that act together in stoichiometric complexes were generally closely matched, and other evidence pointed to a more general relationship between physiological function and mRNA turnover rates. The results provide strong evidence that precise control of the decay of each mRNA is a fundamental feature of the gene expression program in yeast.

Journal ArticleDOI
TL;DR: It is shown that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (≈500 nt), and this response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of ≈22-nt small RNAs.
Abstract: In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, ≈22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (≈500 nt). This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of ≈22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable “knock-down” cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.

Journal ArticleDOI
TL;DR: The characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs) and the possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease.
Abstract: RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. Here, we report the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). We identify two putative RNA-binding proteins, the Drosophila homolog of the fragile X mental retardation protein (FMRP), dFXR, and VIG (Vasa intronic gene), through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease.