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:3.1.26.3 (RNase III)
1,015 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The splice junction sequence of td mRNA from T4-infected cells has been determined (5'....GGU-CUA....3') and shown to be identical to that of the RNA ligation product encoded by the cloned gene [Belfort et al. Cell 41 (1985) 375-382]. The RNA processing functions, T4 RNA ligase, T4 polynucleotide kinase, and the host prr gene product appear not to be essential for exon ligation; neither are the host endoribonucleases RNase III, RNase P and RNase E required for intron excision. While these results are consistent with the autocatalytic splicing mechanism demonstrated in vitro [Chu et al. J. Biol. Chem. 260 (1985) 10680-10688], they leave unanswered the question of which protein(s), if any, might stimulate the in vivo reaction. Analysis of the products of the cloned td gene has led to identification of two td-encoded polypeptides, namely a polypeptide corresponding to the exon-I-coding sequence (NH2-TS), and the catalytically active thymidylate synthase (TS). Kinetic and nucleotide sequence data provide evidence that NH2-TS is the product of the primary transcript and that TS is encoded by spliced mRNA. These results suggest that splicing may provide a switch controlling the relative expression of NH2-TS and TS, two proteins with markedly different temporal appearances despite their identical transcriptional and translational start sites.
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PMID:RNA splicing and in vivo expression of the intron-containing td gene of bacteriophage T4. 242 90

Bacteriophage lambda transcripts were synthesized in vitro using lambda b2 DNA and Escherichia coli RNA polymerase. RNA molecules initiating with ATP were labeled exclusively at the 5'-end by transcribing in the presence of [gamma-32P]ATP. They were analyzed by electrophoresis in 3.5% polyacrylamide, 7.5 M urea gels followed by autoradiography. The previously known 6 S rho-independent transcript and rho-dependent 9 S and 8 S transcripts were observed. A new rho-dependent 5 S transcript was also detected. The 5 S RNA was not the result of cleavage of larger RNAs by ribonuclease III. Analysis of partial ribonuclease T1 digestion products of isolated 5'-end-labeled transcripts showed that the 5 S RNA is synthesized from promoter pR, the promoter for the 9 S and 8 S RNA species. A similar analysis of transcripts labeled with 32P exclusively at the 3'-terminus by the post-transcriptional addition of a [32P]pCp moiety with T4 RNA ligase revealed the positions of guanosine nucleotides proximal to the 3'-hydroxyl end. This information, and inspection of the known DNA nucleotide sequence downstream from pr, allowed us to conclude that the 5 S RNA is a mixture of molecules 112 and 113 nucleotides long terminating in the middle of gene cro. The nucleotide sequence at the 3'-end of the 5 S RNA is similar to that of other rho-dependent transcripts and lacks the oligo(U) found at the terminus of rho-independent transcripts.
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PMID:Characterization of a rho-dependent termination site within the cro gene of bacteriophage lambda. 644 59

A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem affinity purification (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing- or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3' TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identified by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids.
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PMID:Isolation of a U-insertion/deletion editing complex from Leishmania tarentolae mitochondria. 1257 27

We have examined the expression of pnp encoding the 3'-5'-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5' ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.
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PMID:Organization and expression of the polynucleotide phosphorylase gene (pnp) of Streptomyces: Processing of pnp transcripts in Streptomyces antibioticus. 1512 78

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.
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PMID:Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins. 1696 61

RNA editing in the sleeping sickness parasite Trypanosoma brucei remodels mitochondrial transcripts by the addition and deletion of uridylates as specified by guide RNAs. Editing is catalyzed by at least three distinct approximately 20S multiprotein editosomes, all of which contain KREPB4, a protein with RNase III and Pumilio motifs. RNAi repression of KREPB4 expression in procyclic forms (PFs) strongly inhibited growth and in vivo RNA editing, greatly diminished the abundance of 20S editosomes, reduced cellular levels of editosome proteins, and generated approximately 5-10S editosome subcomplexes. Editing TUTase, exoUase, and RNA ligase activities were largely shifted from approximately 20S to approximately 5-10S fractions of cellular lysates. Insertion and deletion endonuclease activities in approximately 20S fractions were strongly reduced upon KREPB4 repression but were not detected in the 5-10S subcomplex fraction. Abundance of MRP1 and RBP16 proteins, which appear to be involved in RNA processing but are not components of the 20S editosome, was unaltered upon KREPB4 repression. These data suggest that KREPB4 is important for the structural integrity of approximately 20S editosomes, editing endonuclease activity, and the viability of PF T. brucei cells.
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PMID:An essential role of KREPB4 in RNA editing and structural integrity of the editosome in Trypanosoma brucei. 1736 11

Replication of viroids, small non-protein-coding plant pathogenic RNAs, entails reiterative transcription of their incoming single-stranded circular genomes, to which the (+) polarity is arbitrarily assigned, cleavage of the oligomeric strands of one or both polarities to unit-length, and ligation to circular RNAs. While cleavage in chloroplastic viroids (family Avsunviroidae) is mediated by hammerhead ribozymes, where and how cleavage of oligomeric (+) RNAs of nuclear viroids (family Pospiviroidae) occurs in vivo remains controversial. Previous in vitro data indicated that a hairpin capped by a GAAA tetraloop is the RNA motif directing cleavage and a loop E motif ligation. Here we have re-examined this question in vivo, taking advantage of earlier findings showing that dimeric viroid (+) RNAs of the family Pospiviroidae transgenically expressed in Arabidopsis thaliana are processed correctly. Using this methodology, we have mapped the processing site of three members of this family at equivalent positions of the hairpin I/double-stranded structure that the upper strand and flanking nucleotides of the central conserved region (CCR) can form. More specifically, from the effects of 16 mutations on Citrus exocortis viroid expressed transgenically in A. thaliana, we conclude that the substrate for in vivo cleavage is the conserved double-stranded structure, with hairpin I potentially facilitating the adoption of this structure, whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands. These results have deep implications on the underlying mechanism of both processing reactions, which are most likely catalyzed by enzymes different from those generally assumed: cleavage by a member of the RNase III family, and ligation by an RNA ligase distinct from the only one characterized so far in plants, thus predicting the existence of at least a second plant RNA ligase.
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PMID:Processing of nuclear viroids in vivo: an interplay between RNA conformations. 1805 30

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.
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PMID:Roles of DCL4 and DCL3b in rice phased small RNA biogenesis. 2197 20

Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5' and 3' termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.
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PMID:Viroid replication: rolling-circles, enzymes and ribozymes. 2199 52

Streptococcus mutans is a major cariogenic pathogen that resides in multispecies oral microbial biofilms. The VicRK 2-component system is crucial for bacterial adaptation, virulence, and biofilm organization and contains a global and vital response regulator, VicR. Notably, we identified an antisense vicR RNA (ASvicR) associated with an adjacent RNase III-encoding (rnc) gene that was relevant to microRNA-size small RNAs (msRNAs). Here, we report that ASvicR overexpression significantly impeded bacterial growth, biofilm exopolysaccharide synthesis, and cariogenicity in vivo. Transcriptome analysis revealed that the ASvicR RNA mainly regulated carbohydrate metabolism. In particular, overproducing ASvicR demonstrated a reduction in galactose and glucose metabolism by monosaccharide composition analysis. The results of high-performance gel permeation chromatography revealed that the water-insoluble glucans isolated from ASvicR presented much lower molecular weights. Furthermore, direct evidence showed that total RNAs were disrupted by rnc-encoded RNase III. With the coexpression of T4 RNA ligase, putative msRNA1657, which is an rnc-related messenger RNA, was verified to bind to the 5'-UTR regions of the vicR gene. Furthermore, ASvicR regulation revealed a sponge regulatory-mediated network for msRNA associated with adjacent RNase III-encoding genes. There was an increase in ASvicR transcript levels in clinical S. mutans strains from caries-free children, while the expression of ASvicR was decreased in early childhood caries patients; this outcome may be explored as a potential strategy contributing to the management of dental caries. Taken together, our findings suggest an important role of ASvicR-mediated sponge regulation in S. mutans, indicating the characterization of lactose metabolism by a vital response regulator in cariogenicity. These findings have a number of implications and have reshaped our understanding of bacterial gene regulation from its transcriptional conception to the key roles of regulatory RNAs.
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PMID:Carbohydrate Metabolism Regulated by Antisense vicR RNA in Cariogenicity. 3246 73


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