Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (approximately 70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.
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PMID:Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. 1195 43

Keeping in view the fact that molecular basis of Burkitt's lymphoma (BL) is poorly understood, we attempted to explore the small interfering RNA (siRNA) mediated c-myc gene regulation using BL-derived EB-3 cell line as archetype cellular model. Such a study revealed that EB-3 cells possess 4-fold higher expression of Dicer gene coupled with 2-fold higher activity of RNA polymerase III than that observed in normal human lymphocytes. siRNAs derived from EB-3 cells had the inherent capacity to suppress c-myc gene expression in normal cells but not in native cells. Based on these findings we have proposed a novel RNA-mediated c-myc gene regulation pathway that may be responsible for BL.
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PMID:Defective RNA-mediated c-myc gene silencing pathway in Burkitt's lymphoma. 1469 25

Micro-RNAs (miRNAs) are small, noncoding RNAs of 18-25 nt in length that negatively regulate their complementary mRNAs at the posttranscriptional level. Previous work has shown that some RNase III-like enzymes such as Drosha and Dicer are known to be involved in miRNA biogenesis in animals. However, the mechanism of plant miRNA biogenesis still remains poorly understood. In this article, the process of Arabidopsis miR163 biogenesis was examined. The results revealed that two types of miR163 primary transcripts (pri-miR163s) are transcribed from a single gene by RNA polymerase II and that miR163 biogenesis requires at least three cleavage steps by RNase III-like enzymes at 21-nt-long intervals. The first step is from pri-miR163 to long miR163 precursor (premiR163), the second step is from long pre-miR163 to short premiR163, and the last step is from short pre-miR163 to mature miR163 and the remnant. It is interesting that, during the process, four small RNAs including miR163 are released. By using dcl1 mutants, it was demonstrated that Arabidopsis Dicer homologue Dicer-like 1 (DCL1) catalyzes at least the first and second cleavage steps and that double-stranded RNA-binding domains of DCL1 are involved in positioning of the cleavage sites. Our result is direct evidence that DCL1 is involved in processing of pri- and pre-miRNA.
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PMID:Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. 1531 13

MicroRNAs (miRNAs) represent a family of small noncoding RNAs that are found in plants and animals (for recent reviews, see ). miRNAs are expressed in a developmentally and tissue-specific manner and regulate the translational efficiency and stability of partial or fully sequence-complementary mRNAs. miRNAs are excised in a stepwise process from double-stranded RNA precursors that are embedded in long RNA polymerase II primary transcripts (pri-miRNA). Drosha RNase III catalyzes the first excision event, the release in the nucleus of a hairpin RNA (pre-miRNA), which is followed by export of the pre-miRNA to the cytoplasm and further processing by Dicer to mature miRNAs. Here, we characterize the human DGCR8, the DiGeorge syndrome critical region gene 8, and its Drosophila melanogaster homolog. We provide biochemical and cell-based readouts to demonstrate the requirement of DGCR8 for the maturation of miRNA primary transcripts. RNAi knockdown experiments of fly and human DGCR8 resulted in accumulation of pri-miRNAs and reduction of pre-miRNAs and mature miRNAs. Our results suggest that DGCR8 and Drosha interact in human cells and reside in a functional pri-miRNA processing complex.
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PMID:The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. 1558 61

Plants encode subunits for a fourth RNA polymerase (Pol IV) in addition to the well-known DNA-dependent RNA polymerases I, II, and III. By mutation of the two largest subunits (NRPD1a and NRPD2), we show that Pol IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3. The existence of this distinct silencing polymerase may explain the paradoxical involvement of an RNA silencing pathway in maintenance of transcriptional silencing.
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PMID:RNA polymerase IV directs silencing of endogenous DNA. 1569 15

Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing.
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PMID:RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing. 1620 82

A virus-associated RNA (VAI) of adenoviruses is a cytoplasmic non-coding RNA and it plays an important role for viral replication in infected cells. VAI RNA transcripts, produced by RNA polymerase III (pol III), form tightly structured stems, which confer resistance to cellular defense systems. We demonstrate here that small RNAs of approximately 22 nucleotides are produced from a terminal stem region but not from an apical stem of VAI RNA. We determined the processing sites of VAI RNA by S1 nuclease mapping and further confirmed that the processed small RNA can act as small interfering RNAs (siRNAs) or as microRNAs (miRNAs) in transient transfection assays and during viral infection. Our data demonstrate that non-coding RNAs synthesized by pol III can be substrates for Dicer, and diced small RNAs might regulate cellular phenomena as siRNAs and miRNAs.
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PMID:Sequence-specific interference by small RNAs derived from adenovirus VAI RNA. 1647 8

MicroRNAs (miRNAs) are a group of recently discovered small RNAs produced by the cell using a unique process, involving RNA polymerase II, Microprocessor protein complex, and the RNAase III/Dicer endonuclease complex, and subsequently sequestered in an miRNA ribonucleoprotein complex. The biological functions of miRNAs depend on their ability to silence gene expression, primarily via degradation of the target mRNA and/or translational suppression, mediated by the RNA-induced silencing complex (RISC). First discovered in Caenorhabditis elegans (lin-4), miRNAs have now been identified in a wide array of organisms, including plants, zebrafish, Drosophila, and mammals. The expression of miRNAs in multicellular organisms exhibits spatiotemporal, and tissue- and cell-specificity, suggesting their involvement in tissue morphogenesis and cell differentiation. More than 200 miRNAs have been identified or predicted in mammalian cells. Recent studies have demonstrated the importance of miRNAs in embryonic stem cell differentiation, limb development, adipogenesis, myogenesis, angiogenesis and hematopoiesis, neurogenesis, and epithelial morphogenesis. Overexpression (gain-of-function) and inactivation (loss-of-function) are currently the primary approaches to studying miRNA functions. Another family of small RNAs related to miRNAs is the small interfering RNAs (siRNAs), generated by Dicer from long double-stranded RNAs (dsRNAs), and produced from an induced transgene, a viral intruder, or a rogue genetic element. siRNAs silence genes via either mRNA degradation, using the RISC, or DNA methylation. siRNAs are actively being applied in basic, functional genetic studies, particularly in the generation of gene knockdown animals, as well as in gene knockdown studies of cultured cells. These studies have provided invaluable information on the specific function(s) of individual genes. siRNA technology also presents exciting potential as a therapeutic approach in disease prevention and treatment, as suggested by a recent study targeting apolipoprotein B (ApoB) in primates. Further elucidation of how miRNAs and other small RNAs interact with known and yet-to-be identified gene regulatory pathways in the cell should provide us with a more in-depth understanding of the mechanisms regulating cellular function and differentiation, and facilitate the application of small RNA technology in disease control and treatment.
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PMID:MicroRNAs and cell differentiation in mammalian development. 1684 91

The definition of hormone function was classically accomplished by ablation-replacement studies. However, as our knowledge of the complexity of hormones and growth factors has grown, it has become increasingly difficult to clearly define the necessity and function of many of the hormones, growth factors, and regulatory proteins under investigation. The use of homologous recombination within mouse embryonic stem cell lines allows functional gene ablation and has been used extensively during the past 15 yr to define specific gene function. The use of similar methodologies in livestock species has yet to yield an efficient approach. In contrast, the parallel development of our understanding of naturally occurring RNA interference, along with the development of efficient virus-based vectors for gene transfer, holds great potential for effectively "knocking down" specific gene function. Short-hairpin (sh) RNA-encoding cassettes, typically consisting of inverted repeats separated by a loop sequence and followed by a short poly(T) string to terminate transcription, are inserted downstream of an RNA polymerase III promoter within the viral vector of choice. Several viral vectors are useful for delivery of shRNA expression cassettes, each with particular attributes. Adenovirus- and lentivirus-derived vectors provide a high rate of infectivity in most mammalian cell types, with lentiviral vectors allowing stable integration into the host genome if the study of long-term effects is needed. Upon transcription, a shRNA is generated, and the loop is recognized by the processing enzyme Dicer, generating guide sequences. Guide sequences are incorporated into the RNA-induced silencing complex, which targets mRNA for degradation if recognized by the guide sequence. For each mRNA of interest, design and testing of a number of shRNA, along with adequate controls, are required to identify the most efficient construct before proceeding to in vivo use. This technology may become the method of choice for defining gene function in livestock.
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PMID:Ribonucleic acid interference: a new approach to the in vivo study of gene function. 1704 Sep 49

Methods most commonly used for producing small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) are chemical synthesis and intracellular expression from engineered vectors. For shRNAs, chemical synthesis is very costly and construction of vectors is laborious. Synthesis by phage RNA polymerases from their natural promoters results in a 5 -terminal triphosphate that can trigger an interferon (IFN) response. Moreover, due to the requirement of phage promoters for 5 - GPuPuPu sequences for transcription initiation, shRNA transcripts may have extra 5 -nucleotides that can constrain the sequences that can be targeted. Also, the 3 ends may have an additional n + 1 nucleotide not encoded by the template. Here we present a novel approach for synthesizing functional shRNAs via rolling circle transcription (RCT) of small (approximately 70 nt) single-stranded DNA circles using T7 RNA polymerase, which avoids these issues. Due to internal pairing, these circles are dumbbell-shaped. RCT produces large transcripts (>10 kb in length) consisting of multimers (>150 copies) of shRNAs in the absence of promoter, terminator, or primer sequences. Dumbbells targeting red fluorescent protein (DsRed), human tumor necrosis factor-alpha (TNF-alpha) and hepatitis C virus (HCV) internal ribosome entry site (IRES) were prepared and transcribed. The resulting long transcripts are substrates for Dicer. When introduced into 293FT and Huh7 cells, the multimeric transcripts inhibited their target genes at levels similar to an equivalent mass of monomeric shRNAs, indicating that they can enter the RNAi pathway. Thus, rolling circle transcription of small DNA dumbbells provides a new source of biologically active interfering RNA.
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PMID:RNA interference from multimeric shRNAs generated by rolling circle transcription. 1715 10


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