Role for a bidentate ribonuclease in the initiation step of RNA interference
TL;DR: Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals, and has a distinctive structure, which includes a helicase domain and dualRNase III motifs.
Abstract: RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.
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TL;DR: Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.
32,946 citations
Cites background from "Role for a bidentate ribonuclease i..."
...…cleavage and that Dicer progressively chops 2004), indicating that there is no obligate link between up an RNA double strand, irrespective of its sequence the identity of the polymerase and downstream miRNA (Zamore et al., 2000; Bernstein et al., 2001; Elbashir et al., processing or function....
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TL;DR: 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
Abstract: RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
10,451 citations
Cites background from "Role for a bidentate ribonuclease i..."
...The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNA...
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TL;DR: Two founding members of the microRNA family were originally identified in Caenorhabditis elegans as genes that were required for the timed regulation of developmental events and indicate the existence of multiple RISCs that carry out related but specific biological functions.
Abstract: MicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. The two founding members of the microRNA family were originally identified in Caenorhabditis elegans as genes that were required for the timed regulation of developmental events. Since then, hundreds of microRNAs have been identified in almost all metazoan genomes, including worms, flies, plants and mammals. MicroRNAs have diverse expression patterns and might regulate various developmental and physiological processes. Their discovery adds a new dimension to our understanding of complex gene regulatory networks.
6,282 citations
Cites background from "Role for a bidentate ribonuclease i..."
...Both miRNAs and siRNAs depend on Dicer for their maturatio...
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TL;DR: The two RNase III proteins, Drosha and Dicer, may collaborate in the stepwise processing of miRNAs, and have key roles in miRNA-mediated gene regulation in processes such as development and differentiation.
Abstract: Hundreds of small RNAs of approximately 22 nucleotides, collectively named microRNAs (miRNAs), have been discovered recently in animals and plants. Although their functions are being unravelled, their mechanism of biogenesis remains poorly understood. miRNAs are transcribed as long primary transcripts (pri-miRNAs) whose maturation occurs through sequential processing events: the nuclear processing of the pri-miRNAs into stem-loop precursors of approximately 70 nucleotides (pre-miRNAs), and the cytoplasmic processing of pre-miRNAs into mature miRNAs. Dicer, a member of the RNase III superfamily of bidentate nucleases, mediates the latter step, whereas the processing enzyme for the former step is unknown. Here we identify another RNase III, human Drosha, as the core nuclease that executes the initiation step of miRNA processing in the nucleus. Immunopurified Drosha cleaved pri-miRNA to release pre-miRNA in vitro. Furthermore, RNA interference of Drosha resulted in the strong accumulation of pri-miRNA and the reduction of pre-miRNA and mature miRNA in vivo. Thus, the two RNase III proteins, Drosha and Dicer, may collaborate in the stepwise processing of miRNAs, and have key roles in miRNA-mediated gene regulation in processes such as development and differentiation.
5,191 citations
TL;DR: It is shown that many 21- and 22-nt expressed RNAs, termed microRNAs, exist in invertebrates and vertebrates and that some of these novel RNAs are highly conserved, which suggests that sequence-specific, posttranscriptional regulatory mechanisms mediated by smallRNAs are more general than previously appreciated.
Abstract: In Caenorhabditis elegans, lin-4 and let-7 encode 22- and 21-nucleotide (nt) RNAs, respectively, which function as key regulators of developmental timing. Because the appearance of these short RNAs is regulated during development, they are also referred to as small temporal RNAs (stRNAs). We show that many 21- and 22-nt expressed RNAs, termed microRNAs, exist in invertebrates and vertebrates and that some of these novel RNAs, similar to let-7 stRNA, are highly conserved. This suggests that sequence-specific, posttranscriptional regulatory mechanisms mediated by small RNAs are more general than previously appreciated.
4,484 citations
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TL;DR: It is shown that ‘loss-of-function’ phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs, which coincides with a marked reduction in the level of cognate cellular messenger RNAs.
Abstract: In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that 'loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.
3,208 citations
"Role for a bidentate ribonuclease i..." refers background in this paper
...In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppresse...
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TL;DR: The 25-nucleotide antisense RNA detected in transgene-induced PTGS is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.
Abstract: Posttranscriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.
3,202 citations
"Role for a bidentate ribonuclease i..." refers background in this paper
...In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppresse...
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TL;DR: It is found that RNAi is ATP dependent yet uncoupled from mRNA translation, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.
3,034 citations
TL;DR: It is shown that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates and the possibility that one natural function of RNAi is transposon silencing is discussed.
1,386 citations
"Role for a bidentate ribonuclease i..." refers background in this paper
...Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNA...
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TL;DR: A database based on hidden Markov model profiles (HMMs), which combines high quality and completeness, and a large number of previously unannotated proteins from the Caenorhabditis elegans genome project were classified.
Abstract: Databases of multiple se- quence alignments are a valuable aid to protein sequence classification and analysis. One of the main challenges when constructing such a data- base is to simultaneously satisfy the conflicting demands of completeness on the one hand and quality of alignment and domain definitions on the other. The latter properties are best dealt with by manual approaches, whereas complete- ness in practice is only amenable to automatic methods. Herein we present a database based on hidden Markov model profiles (HMMs), which combines high quality and completeness. Our database, Pfam, consists of parts A and B. Pfam-Ais curated and contains well-character- ized protein domain families with high quality alignments, which are maintained by using manually checked seed alignments and HMMs to find and align all members. Pfam-B contains sequence families that were generated auto- matically by applying the Domainer algorithm to cluster and align the remaining protein sequences after removal of Pfam-A domains. By using Pfam, a large number of previously unannotated proteins from theCaenorhabditis elegans genome project were classified. We have also identified many novel family member- ships in known proteins, including new kazal, Fibronectin type III, and response regulator receiver domains. Pfam-Afamilies have perma- nent accession numbers and form a library of HMMs available for searching and automatic annotation of new protein sequences. Proteins: 28:405-420, 1997. r1997 Wiley-Liss, Inc.
1,283 citations