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)

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

SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.
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PMID:[RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells]. 1596 75

The absB locus of Streptomyces coelicolor encodes a homolog of bacterial RNase III. We cloned and overexpressed the absB gene product and purified a decahistidine-tagged version of the protein. We show here that AbsB is active against double-stranded RNA transcripts derived from synthetic DNAs but is inactive with single-stranded homopolymers. We thus designate the absB product RNase IIIS. Using T7 RNA polymerase and a cloned template containing the rpsO-pnp intergenic region, we synthesized an RNA substrate representing a portion of the read-through transcript normally produced in S. coelicolor. This transcript contains the sequences that form the putative rpsO terminator and those that form an intergenic stem-loop structure thought to be the site for RNase IIIS processing of the read-through transcript. We show that RNase IIIS does cleave that model transcript, with primary and secondary cleavage sites in an internal loop in the stem-loop structure. We have identified the primary and secondary cleavage sites by primer extension and demonstrate the further processing of the initial cleavage products. Thus, as is the case in Escherichia coli, the read-through transcript from rpsO-pnp is cleaved by RNase IIIS in S. coelicolor. However, the cleavage sites are different in the two systems. The positions of the cleavage sites in the stem-loop of the S. coelicolor transcript are more akin to those identified in the processing of bacteriophage T7 mRNAs. A kinetic assay for RNase IIIS was developed, and kinetic parameters for the reaction utilizing the model transcript from rpsO-pnp were determined.
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PMID:The absB gene encodes a double strand-specific endoribonuclease that cleaves the read-through transcript of the rpsO-pnp operon in Streptomyces coelicolor. 1607 42

Consecutive homologous codons that are rarely used in E. coli are known to inhibit translation to varying degrees. As few as two consecutive rare arginine codons exhibit a profound inhibition of translation when they are located in the 5' portion of a gene in E. coli. We have previously shown that nine consecutive rare CUA leucine codons cause almost complete inhibition of translation when they are placed after the 13th codon of a test message (although they do not inhibit translation when they are placed in the middle of the message). In the present work, we report that five consecutive rare CUA leucine codons exhibit approximately a threefold inhibition of translation when they are similarly placed after the 13th codon of a test message, compared to five consecutive common CUG leucine codons, in a T7 RNA polymerase-driven system. Further, by removing RNase III processing sites at the 3' ends of the mRNAs, we have manipulated the stability of the mRNAs encoding the test and control messages to see if decreasing mRNA stability might have an effect on the extent of translation inhibition by the rare leucine codons. However, the inhibition with the less stable mRNAs was similar to that with the stable mRNAs, approximately 3.4-fold, indicating that mRNA stability per se does not have a major influence on the effects of rare codons in this system.
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PMID:Inhibition of translation by consecutive rare leucine codons in E. coli: absence of effect of varying mRNA stability. 1701 24

Nascent transcripts encoded by the putL and putR sites of phage HK022 bind the transcript elongation complex and suppress termination at downstream transcription terminators. We report here that the chemical stability of putL RNA is considerably greater than that of the typical Escherichia coli message because the elongation complex protects this RNA from degradation. When binding to the elongation complex was prevented by mutation of either putL or RNA polymerase, RNA stability decreased more than 50-fold. The functional modification conferred by putL RNA on the elongation complex is also long-lived: the efficiency of terminator suppression remained high for at least 10 kb from the putL site. We find that RNase III rapidly and efficiently cleaved the transcript just downstream of the putL sequences, but such cleavage changed neither the stability of putL RNA nor the efficiency of antitermination. These results argue that the continuity of the RNA that connects put sequences to the growing point is not required for persistence of the antiterminating modification in vivo.
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PMID:Protection of antiterminator RNA by the transcript elongation complex. 1723 21

The majority of human microRNA (miRNA) loci are located within intronic regions and are transcribed by RNA polymerase II as part of their hosting transcription units. The primary transcripts are cleaved by Drosha to release approximately 70 nt pre-miRNAs that are subsequently processed by Dicer to generate mature approximately 22 nt miRNAs. It is generally believed that intronic miRNAs are released by Drosha from excised introns after the splicing reaction has occurred. However, our database searches and experiments indicate that intronic miRNAs can be processed from unspliced intronic regions before splicing catalysis. Intriguingly, cleavage of an intron by Drosha does not significantly affect the production of mature mRNA, suggesting that a continuous intron may not be required for splicing and that the exons may be tethered to each other. Hence, Drosha may cleave intronic miRNAs between the splicing commitment step and the excision step, thereby ensuring both miRNA biogenesis and protein synthesis from a single primary transcript. Our study provides a novel example of eukaryotic gene organization and RNA-processing control.
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PMID:Processing of intronic microRNAs. 1725 51

In Saccharomyces cerevisiae, the double-stranded-RNA-specific RNase III (Rnt1p) is required for the processing of pre-rRNA and coprecipitates with transcriptionally active rRNA gene repeats. Here we show that Rnt1p physically interacts with RNA polymerase I (RNAPI) and its deletion decreases the transcription of the rRNA gene and increases the number of rRNA genes with an open chromatin structure. In contrast, depletion of ribosomal proteins or factors that impair RNAPI termination did not increase the number of open rRNA gene repeats, suggesting that changes in the ratio of open and closed rRNA gene chromatin is not due to a nonspecific response to ribosome depletion or impaired termination. The results demonstrate that defects in pre-rRNA processing can influence the chromatin structure of the rRNA gene arrays and reveal links among the rRNA gene chromatin, transcription, and processing.
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PMID:Deletion of Rnt1p alters the proportion of open versus closed rRNA gene repeats in yeast. 1799 94

microRNAs (miRNAs) are extensively involved in developmental programming. Some miRNAs are highly conserved, while others are lineage specific. All miRNAs maturate through a series of processing steps. Here we review recent progresses in the studies of early steps in miRNA biogenesis, focusing on animal systems. The miRNA maturation pathways are surprisingly diverse, involving transcription by RNA polymerase II or III, cleavage by the Drosha nuclease or the spliceosome, and sometimes modifications by the adenosine deaminase ADAR. The relationship between the diversity in miRNA biogenesis and the apparently rapid evolution of miRNA genes and functions is discussed.
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PMID:MicroRNA biogenesis: there's more than one way to skin a cat. 1877 99

microRNAs (miRNAs) are generated from long primary (pri-) RNA polymerase II (Pol II)-derived transcripts by two RNase III processing reactions: Drosha cleavage of nuclear pri-miRNAs and Dicer cleavage of cytoplasmic pre-miRNAs. Here we show that Drosha cleavage occurs during transcription acting on both independently transcribed and intron-encoded miRNAs. We also show that both 5'-3' and 3'-5' exonucleases associate with the sites where co-transcriptional Drosha cleavage occurs, promoting intron degradation before splicing. We finally demonstrate that miRNAs can also derive from 3' flanking transcripts of Pol II genes. Our results demonstrate that multiple miRNA-containing transcripts are co-transcriptionally cleaved during their synthesis and suggest that exonucleolytic degradation from Drosha cleavage sites in pre-mRNAs may influence the splicing and maturation of numerous mRNAs.
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PMID:Primary microRNA transcripts are processed co-transcriptionally. 1917 42


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