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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mitochondrial genome of eukaryotic cells is maintained by a mechanism distinct from that employed in the nucleus. Mitochondrial DNA replication at the leading-strand origin is coupled to transcription through the formation of an RNA-DNA hybrid known as an R-loop. In vivo and in vitro evidence has implicated an RNA processing enzyme, RNase MRP, in primer maturation. In our investigation of mammalian RNase MRP, we have analyzed its specific endoribonuclease activity on model R-loops. We demonstrate here that human RNase MRP cleaves this distinctly configured substrate at virtually all of the major DNA replication sites previously mapped in vivo. We further show that the processed RNA products remain stably base-paired to the template DNA strand and are functional for initiating DNA synthesis on a closed circular plasmid. Thus, in vitro initiation of leading-strand mtDNA synthesis requires only the actions of RNA polymerase and RNase MRP for the generation of replication primers.
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PMID:Initiation of mitochondrial DNA replication by transcription and R-loop processing. 980 33

Overexpression of P-glycoprotein (Pgp) or MDR1 mRNA has been shown to be a negative prognostic factor for clinical outcome in acute myeloid leukemia (AML). However, resistance to chemotherapy also occurs in the absence of Pgp overexpression. Therefore, besides Pgp expression, we have assessed the expression of MRP, a novel drug transporter gene, along with the functional multidrug-resistant (MDR) phenotype of leukemic cells. These MDR parameters are correlated with clinical outcome in individual patients. We found functional changes in fresh leukemic cells from de novo or relapsed patients similar to those reported for tumor cell lines with the MDR phenotype. These changes were reduced drug accumulation as assessed with radiolabeled doxorubicin (factor 1.6), daunomycin (factor 1.13), and vincristine (factor 1.6) in patients who were refractory to the combination treatment of 1-beta-D-arabinofuranosylcytosine (ara-C) and daunomycin or mitoxantrone as opposed to patients who had complete responses. Also, the intracellular distribution of doxorubicin fluorescence (nuclear/cytoplasmic ratio), as assessed with laser scan microscopy, was reduced 1.4-fold in blasts from refractory patients. Based on historically known clinical response to single-agent daunomycin or ara-C in the group of responding de novo AML patients, we have set a threshold level such that a defined part of the samples that had the highest drug accumulation or nuclear to cytoplasmic ratios were above this threshold value. This allowed discrimination between patients responding to daunomycin from those who were refractory to this drug. By using this threshold level, in the refractory group clinical resistance corresponded with high sensitivity with a resistant phenotype. A similar threshold was set for the data of the in vitro ara-C sensitivity test. By combining both assays for all individual patients, clinical refractoriness as well as sensitivity could be predicted with high accuracy. There appeared to be no stringent relationship between the functional MDR phenotype with expression of either Pgp (fluorescence-activated cell sorting analysis) or MRP mRNA (RNase protection). However, by combining both parameters the functional MDR phenotype correlated with the overexpression of either one or both of the parameters in 94% of the samples studied. It is concluded that this combined overexpression in conjunction with functional changes for MDR drugs and ara-C reveal a correlation of MDR phenotype with clinical resistance to combination chemotherapy in AML patients and hereby may adequately predict clinical MDR in individual AML patients.
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PMID:Functional multidrug resistance phenotype associated with combined overexpression of Pgp/MDR1 and MRP together with 1-beta-D-arabinofuranosylcytosine sensitivity may predict clinical response in acute myeloid leukemia. 981 90

In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40. Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized. Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29. Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P. Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP. Rpp14, by contrast, exhibits no significant homology to any known yeast gene. Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.
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PMID:Rpp14 and Rpp29, two protein subunits of human ribonuclease P. 1002 67

The eukaryotic nucleolus contains a large number of small RNA molecules that, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. One of the snoRNPs that has been shown to possess enzymatic activity is the RNase MRP. RNase MRP is an endoribonuclease involved in the formation of the 5' end of 5.8S rRNA. In this study the association of the hPop1 protein with the RNase MRP complex was investigated. The hPop1 protein seems not to be directly bound to the RNA component, but requires nt 1-86 and 116-176 of the MRP RNA to associate with the RNase MRP complex via protein-protein interactions. UV crosslinking followed by ribonuclease treatment and immunoprecipitation with anti-Th/To antibodies revealed three human proteins of about 20, 25, and 40 kDa that can associate with the RNase MRP complex. The 20- and 25-kDa proteins appear to bind to stem-loop I of the MRP RNA whereas the 40-kDa protein requires the central part of the MRP RNA (nt 86-176) for association with the RNase MRP complex. In addition, we show that the human RNase P proteins Rpp30 and Rpp38 are also associated with the RNase MRP complex. Expression of Vesicular Stomatitis Virus- (VSV) tagged versions of these proteins in HeLa cells followed by anti-VSV immunoprecipitation resulted in coprecipitation of both RNase P and RNase MRP complexes. Furthermore, UV crosslinking followed by anti-Th/To and anti-Rpp38 immunoprecipitation revealed that the 40-kDa protein we detected in UV crosslinking is probably identical to Rpp38.
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PMID:RNA-protein interactions in the human RNase MRP ribonucleoprotein complex. 1019 68

RNase MRP is a ribonucleoprotein particle involved in the processing of pre-rRNA. The RNase MRP particle is structurally highly related to the RNase P particle, which is involved in pre-tRNA processing. Their RNA components fold into a similar secondary structure and they share several protein subunits. We have identified and characterised human and mouse cDNAs that encode proteins homologous to yPop4p, a protein subunit of both the yeast RNase MRP and RNase P complexes. The human Pop4 cDNA encodes a highly basic protein of 220 amino acids. Transfection experiments with epitope-tagged hPop4 protein indicated that hPop4 is localised in the nucleus and accumulates in the nucleolus. Immunoprecipitation assays using extracts from transfected cells expressing epitope-tagged hPop4 revealed that this protein is associated with both the human RNase MRP and RNase P particles. Polyclonal rabbit antibodies raised against recombinant hPop4 recognised a 30 kDa protein in total HeLa cell extracts and specifically co-immunoprecipitated the RNA components of the RNase MRP and RNase P complexes. Finally we showed that anti-hPop4 immunoprecipitates possess RNase P enzymatic activity. Taken together, these data show that we have identified a protein that represents the human counterpart of the yeast Pop4p protein.
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PMID:hPop4: a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes. 1035 75

Much remains to be learned about drug resistance in the biology of RCC and its metastases. We measured MDR-1/P-glycoprotein expression in 19 tumor samples from patients with metastatic RCC by RNase protection and quantitative PCR assays. The median level of the 16 tumor metastases was 4.9 (range: 0.10 to 156.2) relative to the level of 10 assigned to a reference cell line, SW620, which has been characterized as expressing a minimum level of MDR-1. Since these levels were lower than expected for RCC, we asked whether the metastases possessed a phenotype different from primary RCC and examined MDR-1 expression in 5 paired cell lines derived from primary and metastatic RCC. In 8/10 lines, MDR-1 expression was >10. Relative to the level in the primary line, MDR-1 expression was decreased (3 to 50-fold) in 3 metastatic lines, was increased in 1, and unchanged in 1. MRP mRNA expression was lower in the metastatic lines while EGFR expression was variable. IC50 values for 6 compounds (including 4 standard agents and one new Phase 1 agent) were determined for the paired lines. Rhodamine and calcein efflux assays were performed as measures of P-glycoprotein and MRP function. Rhodamine efflux correlated with MDR-1 mRNA expression (r = 0.87) and with the IC50s (r = 0.60) for paclitaxel in the paired cell lines. In contrast, calcein efflux did not correlate with MRP expression. Lastly, MDR-1 expression correlated with cytokeratin 8 (CK8) protein levels, a measure of cellular differentiation. In sum, these data suggest renal cell carcinoma (RCC) metastases have altered MDR-1 expression potentially due to altered differentiation relative to the primary tumor. Thus, the drug resistance phenotype of primary RCC tumors may not reflect that of their metastases.
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PMID:Intrinsic drug resistance in primary and metastatic renal cell carcinoma. 1037 90

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors.
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PMID:Localization in the nucleolus and coiled bodies of protein subunits of the ribonucleoprotein ribonuclease P. 1044 65

RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to have roles in both mitochondrial DNA replication and nuclear 5.8S rRNA processing. SNM1 encodes an essential 22.5-kDa protein that is a component of yeast RNase MRP. It is an RNA binding protein that binds the MRP RNA specifically. This 198-amino-acid protein can be divided into three structural regions: a potential leucine zipper near the amino terminus, a binuclear zinc cluster in the middle region, and a serine- and lysine-rich region near the carboxy terminus. We have performed PCR mutagenesis of the SNM1 gene to produce 17 mutants that have a conditional phenotype for growth at different temperatures. Yeast strains carrying any of these mutations as the only copy of snm1 display an rRNA processing defect identical to that in MRP RNA mutants. We have characterized these mutant proteins for RNase MRP function by examining 5.8S rRNA processing, MRP RNA binding in vivo, and the stability of the RNase MRP RNA. The results indicate two separate functional domains of the protein, one responsible for binding the MRP RNA and a second that promotes substrate cleavage. The Snm1 protein appears not to be required for the stability of the MRP RNA, but very low levels of the protein are required for processing of the 5.8S rRNA. Surprisingly, a large number of conditional mutations that resulted from nonsense and frameshift mutations throughout the coding regions were identified. The most severe of these was a frameshift at amino acid 7. These mutations were found to be undergoing translational suppression, resulting in a small amount of full-length Snm1 protein. This small amount of Snm1 protein was sufficient to maintain enough RNase MRP activity to support viability. Translational suppression was accomplished in two ways. First, CEN plasmid missegregation leads to plasmid amplification, which in turn leads to SNM1 mRNA overexpression. Translational suppression of a small amount of the superabundant SNM1 mRNA results in sufficient Snm1 protein to support viability. CEN plasmid missegregation is believed to be the result of a prolonged telophase arrest that has been recently identified in RNase MRP mutants. Either the SNM1 gene is inherently susceptible to translational suppression or extremely small amounts of Snm1 protein are sufficient to maintain essential levels of MRP activity.
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PMID:Mutagenesis of SNM1, which encodes a protein component of the yeast RNase MRP, reveals a role for this ribonucleoprotein endoribonuclease in plasmid segregation. 1052 74

The exosome is a protein complex consisting of a variety of 3'-to-5' exonucleases that functions both in 3'-to-5' trimming of rRNA precursors and in 3'-to-5' degradation of mRNA. To determine additional exosome functions, we examined the processing of a variety of RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P, RNase MRP, and SRP RNAs, and 5S rRNAs in mutants defective in either the core components of the exosome or in other proteins required for exosome function. These experiments led to three important conclusions. First, exosome mutants accumulate 3'-extended forms of the U4 snRNA and a wide variety of snoRNAs, including snoRNAs that are independently transcribed or intron derived. This finding suggests that the exosome functions in the 3' end processing of these species. Second, in exosome mutants, transcripts for U4 snRNA and independently transcribed snoRNAs accumulate as 3'-extended polyadenylated species, suggesting that the exosome is required to process these 3'-extended transcripts. Third, processing of 5.8S rRNA, snRNA, and snoRNA by the exosome is affected by mutations of the nuclear proteins Rrp6p and Mtr4p, whereas mRNA degradation by the exosome required Ski2p and was not affected by mutations in RRP6 or MTR4. This finding suggests that the cytoplasmic and nuclear forms of the exosome represent two functionally different complexes involved in distinct 3'-to-5' processing and degradation reactions.
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PMID:Yeast exosome mutants accumulate 3'-extended polyadenylated forms of U4 small nuclear RNA and small nucleolar RNAs. 1061 Dec 22

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNA(Met) binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNA(AAC)(Val) (tRNA(Val*)) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd(-) phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd(-) phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNA(Met) levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd(-) phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNA(Tyr) that cannot be processed by RNase P had a Gcd(-) phenotype. Interestingly, the Gcd(-) phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Delta cells have a Gcd(-) phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNA(Val*) showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNA(Met) binding to the ribosome.
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PMID:Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. 1071 74


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