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)

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages. Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.
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PMID:Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. 767 94

In Pseudomonas aeruginosa, arginine catabolism via the arginine deiminase pathway depends on the anaerobically inducible arcDABC operon, whose expression is further modulated by mRNA processing. Fusion of the cloned arc operon to an external tac promoter did not alter the processing pattern in P. aeruginosa and allowed heterologous expression in Escherichia coli. Processing within a specific region of the arcD mRNA was similar in P. aeruginosa and in E. coli. In E. coli, a conditional temperature-sensitive (ts) mutation in the gene specifying RNase E prevented cleavage of the arc mRNA at the non-permissive temperature, whereas mutations in the genes encoding RNase III or RNase P had no effect. We therefore speculate that in P. aeruginosa, an RNase E-like enzyme exists which is involved in the specific processing of the arc mRNA.
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PMID:Processing of the Pseudomonas arcDABC mRNA requires functional RNase E in Escherichia coli. 768 78

Alanine-substitution mutations were targeted to 14 amino acid residues within the double-stranded (ds) RNA binding motif (dsRBM) of the vaccinia virus E3 protein. Substitutions at six positions--Glu-124, Phe-135, Phe-148, Lys-167, Arg-168, and Lys-171--caused significant reductions in dsRNA binding. These six residues are conserved in the two dsRBMs for which structural information is available (Escherichia coli RNase III and Drosophila melanogaster staufen) and in many other members of the dsRBM protein family. Residues we show to be important for dsRNA binding by vaccinia virus E3 map to the same face of the dsRBM structure and are thus likely to compose part of the RNA binding site.
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PMID:Mutational analysis of the vaccinia virus E3 protein defines amino acid residues involved in E3 binding to double-stranded RNA. 864 94

A human RNase III gene encodes a protein of 160 kDa with multiple domains, a proline-rich, a serine- and arginine-rich, and an RNase III domain. The expressed purified RNase III domain cleaves double-strand RNA and does not cleave single-strand RNA. The gene is ubiquitously expressed in human tissues and cell lines, and the protein is localized in the nucleus of the cell. The levels of transcription and translation of the protein do not change during different phases of the cell cycle. However, a significant fraction of the protein in the nucleus is translocated to the nucleolus during the S phase of the cell cycle. That this human RNase III is involved in processing of pre-rRNA, but might cleave at sites different from those described for yeast RNase III, is shown by antisense inhibition of RNase III expression. Inhibition of human RNase III expression causes cell death, suggesting an essential role for human RNase III in the cell. The antisense inhibition technique used in this study provides an effective method for functional analysis of newly identified human genes.
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PMID:Human RNase III is a 160-kDa protein involved in preribosomal RNA processing. 1094 99

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

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control. On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (si)RNA-soaked M. incognita eggs. Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH(2))-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal. Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality. To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s, which recovered within 24h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M. incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-eri-1 transcript which encodes an RNAi-inhibiting siRNA exonuclease. We observe a marked up-regulation of Mi-eri-1 transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RNAi-based reverse genetic screens in plant parasitic nematodes.
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PMID:Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality. 2039 69

The RNase III enzyme Drosha is a key factor in microRNA (miRNA) biogenesis and as such indispensable for cellular homeostasis and developmental processes. Together with its co-factor DGCR8, it converts the primary transcript (pri-miRNA) into the precursor hairpin (pre-miRNA) in the nucleus. While the middle and the C-terminal domain are crucial for pri-miRNA processing and DGCR8 binding, the function of the N-terminus remains cryptic. Different studies have linked this region to the subcellular localization of Drosha, stabilization and response to stress. In this study, we identify alternatively spliced Drosha transcripts that are devoid of a part of the arginine/serine-rich (RS-rich) domain and expressed in a large set of human cells. In contrast to their expected habitation, we find two isoforms also present in the cytoplasm, while the other two isoforms reside exclusively in the nucleus. Their processing activity for pri-miRNAs and the binding to co-factors remains unaltered. In multiple cell lines, the endogenous mRNA expression of the Drosha isoforms correlates with the localization of endogenous Drosha proteins. The pri-miRNA processing efficiency is not significantly different between groups of cells with or without cytoplasmic Drosha expression. In summary, we discovered novel isoforms of Drosha with differential subcellular localization pointing toward additional layers of complexity in the regulation of its activity.
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PMID:Alternative splicing affects the subcellular localization of Drosha. 2718 95