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)

Ribonuclease III from Escherichia coli has been purified to apparent homogeneity by affinity chromatography on immobilized double-stranded RNA. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate gave one band of protein with a molecular weight of approximately 25,000. Chromatography on Sephadex G-100 is consistent with a molecular weight of 50,000, suggesting that the native enzyme is a dimer. RNase III cuts some single-stranded RNAs, such as bacteriophage T7 early RNA, at specific sites in vivo. This RNA is cut as these same sites by the purified enzyme under all conditions tested. However, at low ionic strength relatively small increases in enzyme concentration produce cuts as secondary sites. At high ionic strength, the enzyme's preference for the sites cut in vivo is more pronounced and secondary cuts are made only at very high enzyme concentrations. Secondary cuts are shown to occur at specific sites and are made in a variety of RNAs even from sources other than E. coli. By cutting RNAs at secondary sites it should be possible to generate RNA fragments which would be useful in a number of studies.
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PMID:RNase III cleavage of single-stranded RNA. Effect of ionic strength on the fideltiy of cleavage. 93 8

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.
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PMID:Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates. 137 63

A double-stranded ribonuclease has been purified more than 90-fold to near homogeneity from the yeast, Saccharomyces cerevisiae. The enzyme shows a high specificity for double-stranded RNA as its substrate. It has a molecular weight of 27000 as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme degrades dsRNA optimally at 30 degrees C; it is stimulated by KCl and by the -SH reagent, dithiothreitol. In contrast to RNase III from Escherichia coli, the yeast enzyme is inhibited by divalent cations. Physiological studies have demonstrated that in vivo levels of the enzyme activity fell during the latter part of the exponential growth phase but rose during stationary phase. The specific activity of the enzyme in nitrogen-starved yeast cells was 2-3-fold higher than in non-starved cells. The enzyme could be detected in yeast strains containing both, one or none of the species of cytoplasmic dsRNA (L and MdsRNAs) and may, therefore, have some wider role.
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PMID:Purification and properties of a double-stranded ribonuclease from the yeast Saccharomyces cerevisiae. 636 60

The double-stranded RNA segments of infectious pancreatic necrosis virus were extracted from virions by a method which avoids proteinase. In contrast to proteinase-treated RNA, such segments (i) exhibited a lower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels and agarose gels, (ii) had a slightly lower buoyant density, and (iii) demonstrated a marked tendency toward aggregation as observed by electron microscopy. A small amount of protein tightly bound to the RNA could account for the above properties, and a 110,000-dalton protein was liberated from purified virion RNA by sequential digestion with RNase III and RNase A. The amount of radioactivity associated with RNA from virions labeled in vivo with [35S]methionine suggested that an average of 1.4 molecules was bound per RNA segment. Interactions between RNA segments seen in electron micrographs appeared to occur only among the ends of the segments, suggesting these were the exclusive sites of protein attachment.
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PMID:Evidence that infectious pancreatic necrosis virus has a genome-linked protein. 714 73

Cloned RNase III gene in a T7 RNA polymerase promoter system was expressed in Escherichia coli cells lacking endogenous RNase III, and the over-expressed recombinant RNase III was purified to homogeneity using ion exchange, exclusion and affinity column chromatography. The overexpressed RNase III was found to separate with the membrane fraction after sonication, which was solubilized, fractionated with (NH)2SO4 and the active fractions used for further purification. The properties of the purified recombinant RNase III were studied using the synthetic RNA substrate, 3[H]poly[A].poly[U], and the natural substrates, 7S and p10Sa RNAs, and compared with the partially purified RNase III from wild-type E. coli cells. The recombinant RNase III showed maximal activity at 37 degrees C and at a pH range of 6.9 to 7.4, which was similar to the RNase III purified from the wild-type cells. Recombinant RNase III efficiently hydrolyzed 3[H].poly[A].poly[U] in the presence of Mg2+. However, the recombinant RNase III cleaved natural RNA substrates efficiently and accurately in the presence of Mn2+. A concentration of Mn2+ ranging from 150 to 300 microM was found to be optimal; concentrations higher than 0.5 mM were inhibitory. Other divalent cations did not support RNase III activity. Monovalent cations, Na+, K+ and NH4+ at 20 mM were equally effective in stimulating RNase III activity although they were not absolutely required for the activity. The thermal stability of the recombinant RNase III was examined at two temperatures, 37 degrees and 50 degrees C. Incubation of RNase III at 37 degrees C for 30 min did not affect activity, but it lost almost 50% of its activity when incubated at 50 degrees C for 30 min. Thus, the recombinant RNase III prefers Mn2+ for the cleavage of natural substrates and exhibits several properties similar to the wild-type RNase III.
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PMID:Expression, purification and properties of recombinant E. coli ribonuclease III. 879 39

Intercellular exchange of protein and RNA-containing microparticles is an increasingly important mode of cell-cell communication. Here we investigate if mesenchymal stem cells (MSCs) known for secreting therapeutic paracrine factors also secrete RNA-containing microparticles. We observed that human embryonic stem cell (hESC)-derived MSC conditioned medium contained small RNAs (less than 300 nt) encapsulated in cholesterol-rich phospholipid vesicles as evidenced by their RNase sensitivity only in the presence of a sodium dodecyl sulfate-based cell lysis buffer, phospholipase A2 and a chelator of cholesterol, cyclodextrin and the restoration of their lower than expected density by detergent or phospholipase A2 treatment. MicroRNAs (miRNAs) such as hsa-let-7b and hsa-let-7g were present in a high precursor (pre)- to mature miRNA ratio by microarray analysis and quantitative reverse transcription-polymerase chain reaction. The pre-miRNAs were cleaved to mature miRNA by RNase III in vitro. High performance liquid chromatography-purified RNA-containing vesicles have a hydrodynamic radius of 55-65 nm and were readily taken up by H9C2 cardiomyocytes. This study suggests that MSCs could facilitate miRNA-mediated intercellular communication by secreting microparticles enriched for pre-miRNA.
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PMID:Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs. 1985 Jul 15

Emerging evidence indicates that microRNAs, a class of small and well-conserved noncoding RNAs, participate in many physiological and pathological processes. RNase III endonuclease DICER is one of the key enzymes for microRNA biogenesis. Here, we found that DICER was downregulated in tumor samples of colorectal cancer (CRC) patients at both mRNA and protein levels. Importantly, intestinal epithelial cell (IEC)-specific deletion of Dicer mice got more tumors after azoxymethane and dextran sulfate sodium (DSS) administration. Interestingly, IEC-specific deletion of Dicer led to severe chronic inflammation and epithelium layer remodeling in mice with or without DSS administration. Microarray analysis of 3 paired Dicer deletion CRC cell lines showed that miR-324-5p was one of the most significantly decreased miRNAs. In the intestinal epithelium of IEC-specific deletion of Dicer mice, miR-324-5p was also found to be markedly reduced. Mechanistically, miR-324-5p directly bound to the 3'untranslated regions (3'UTRs) of HMG-box containing 3 (HMGXB3) and WAS protein family member 2 (WASF-2), two key proteins participated in cell motility and cytoskeleton remodeling, to suppress their expressions. Intraperitoneal injection of miR-324-5p AgomiR (an agonist of miR-324-5p) curtailed chronic inflammation and cytoskeleton remodeling of colorectal epithelium and restored intestinal barrier function in IEC-specific deletion of Dicer mice induced by DSS. Therefore, our study reveals a key role of a DICER/miR-324-5p/HMGXB3/WASF-2 axis in tumorigenesis of CRC by regulation of cytoskeleton remodeling and maintaining integrity of intestinal barriers.
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PMID:Dicer suppresses cytoskeleton remodeling and tumorigenesis of colorectal epithelium by miR-324-5p mediated suppression of HMGXB3 and WASF-2. 2891 52