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PubMed2006

RNase R degrades non-stop mRNAs selectively in an SmpB-tmRNA-dependent manner.

The SmpB-tmRNA-mediated trans-translation system has two well-established activities: rescuing ribosomes stalled on aberrant mRNAs and marking the associated protein fragments for proteolysis. Although the causative non-stop mRNAs are known to be degraded, little is known about the enabling mechanism or the RNases involved in their disposal. We report that Escherichia coli has an enabling mechanism that requires RNase R activity and is dependent on the presence of SmpB protein and tmRNA, suggesting a requirement for active transtranslation in facilitating RNase R engagement and promoting non-stop mRNA decay. Interestingly, this selective transcript degradation by RNase R targets aberrant (non-stop and multiple-rare-codon containing) mRNAs and does not affect the decay of related messages containing in-frame stop codons. Most surprisingly, RNase II and PNPase do not play a significant role in tmRNA-facilitated disposal of aberrant mRNAs. These findings demonstrate that RNase R is a crucial component of the trans-translation-mediated non-stop mRNA decay process, thus providing a requisite activity well suited to complement the ribosome rescue and protein tagging functions of this unique quality control system.

Blotting, Northern↗
PubMed2005

Analysis of ABA hypersensitive germination2 revealed the pivotal functions of PARN in stress response in Arabidopsis.

Accumulating evidence suggests that mRNA degradation systems are crucial for various biological processes in eukaryotes. Here we provide evidence that an mRNA degradation system is associated with some plant hormones and stress responses in plants. We analysed a novel Arabidopsis abscisic acid (ABA)-hypersensitive mutant, ahg2-1, that showed ABA hypersensitivity not only in germination, but also at later developmental stages, and that displayed pleiotropic phenotypes. We found that ahg2-1 accumulated more endogenous ABA in seeds and mannitol-treated plants than did the wild type. Microarray experiments showed that the expressions of ABA-, salicylic acid- and stress-inducible genes were increased in normally grown ahg2-1 plants, suggesting that the ahg2-1 mutation somehow affects various stress responses as well as ABA responses. Map-based cloning of AHG2 revealed that this gene encodes a poly(A)-specific ribonuclease (AtPARN) that is presumed to function in mRNA degradation. Detailed analysis of the ahg2-1 mutation suggests that the mutation reduces AtPARN production. Interestingly, expression of AtPARN was induced by treatment with ABA, high salinity and osmotic stress. These results suggest that both upregulation and downregulation of gene expression by the mRNA-destabilizing activity of AtPARN are crucial for proper ABA, salicylic acid and stress responses.

Abscisic Acid↗
PubMed1996

Molecular and functional phenotypes of melanoma cells with abnormalities in HLA class I antigen expression.

Analysis of melanoma cell lines with abnormalities in HLA Class I antigen expression has identified two serological phenotypes caused by distinct molecular defects. One is characterized by lack of HLA Class I antigen expression which is not induced by IFN-gamma or by incubation at 25 degrees C for 24 hrs. This phenotype reflects structural changes in the beta(2)m gene which interfere with its transcription and/or translation or result in the synthesis of a defective beta(2)-mu polypeptide unable to associate with HLA Class I heavy chains. The other phenotype manifests very low HLA Class I antigen expression which is enhanced by IFN-gamma or by incubation at 25 degrees C for 24 hrs. This phenotype reflects abnormalities in TAP heterodimer expression, which cause defects in stable assembly and intracellular transport of the HLA Class I antigen trimolecular complex. Loss of HLA Class I antigens renders melanoma cells resistant to lysis by HLA Class I antigen-restricted cytotoxic T cells which specifically recognize melanoma associated antigens. Therefore, abnormalities in HLA Class I antigen expression may have a negative impact on the outcome of T cell based immunotherapy. Characterization of the molecular defects underlying loss of HLA Class I antigens may suggest approaches to restore their expression. Inclusion of these approaches in the protocols of T cell based immunotherapy may improve its efficacy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1982

The ppp(A2'p)nA and protein kinase systems in wild-type and interferon-resistant Daudi cells.

Daudi cells, a human lymphoblastoid line, are exceptionally sensitive to the growth inhibitory effects of interferon, 1 unit/ml being sufficient to inhibit cell growth. In addition, interferon treatment of these cells severely inhibits the incorporation of exogenous thymidine into DNA and causes cells to accumulate in the G1(G0) at the expense of the S phase of the cell cycle. The possible involvement of ppp(A2'p)nA(n = 2 to less than or equal to 4) in these effects has been investigated. No (less than 1 nM) ppp(A2'p)nA or (A2'p)nA or alternative products of the ppp(A2'p)nA synthetase [e.g. NAD (2'pA)2] were detected in interferon-treated cells. In addition no evidence was obtained for the occurrence of ppp(A2'p)nA-mediated ribosomal RNA cleavage in these cells even after several days of treatment with relatively high doses of interferon. A line of Daudi cells which is resistant to all three of the above effects of interferon was selected. The wild type and resistant lines were compared with respect to the ppp(A2'p)nA and interferon and double-stranded RNA (dsRNA)-mediated protein kinase systems. The resistant line was not receptor-negative as it responded to interferon by the production of elevated levels of the ppp(A2'p)nA synthetase similar to those observed in extracts from wild-type cells. There was no detectable difference between the lines in the levels of the (2'-5')phosphodiesterase responsible for the degradation of ppp(A2'p)nA. There was, however, about a twofold increase in the ppp(A2'p)nA-dependent endoribonuclease activity in response to interferon with extracts from the wild-type but not the resistant cells. In addition, although the dsRNA-dependent protein kinase activity increased in both types of cell there was a striking reduction in the level of protein phosphorylation in general in response to interferon with material from the wild-type but not the resistant cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Specificity and other properties of three ribonucleases of Tetrahymena pyriformis.

Three ribonucleases, RNase I, RNase II and RNase III, were purified from the 109,000 X g supernate of detergent-treated Tetrahymena pyriformis strain W. RNases I and II act optimally at pH 5.5-6.0 and are inhibited by increasing concentrations of salts of monovalent cations. RNase III acts optimally at pH 7.5 and is activated 1.5-fold by millimolar concentrations of ZnSO4 and 5-fold by 50 mM KCl. RNases II and III are activated approximately 100% in the presence of 3 M and 5 M urea respectively. All enzymes are heat-sensitive and acid-resistant. They are endonucleases forming 2',3'-cyclic products. Their base specificity, as tested against ribosomal RNAs of known sequence, is as follows: RNase I hydrolyzes preferentially YpN and secondarily GpN bonds, RNase II is highly specific for RpN bonds, though the preparation can also hydrolyze the UpU sequence. Finally the principal targets of RNase III are YpR sequences and secondarily YpY sequences. A shorthand visualization of base specificity of nucleases in the form of right isosceles triangles is presented. The triangles are constructed by subdividing each of the two perpendicular sides in as many units as the maximum number of times the most abundant dinucleotide appears in all substrates employed and plotting the frequency of hydrolysis of each dinucleotide sequence by the enzyme under study. The proximity of each dinucleotide sequence to the hypotenuse or to one of the perpendicular sides is indicative of its susceptibility or resistance to the enzyme's action.

Animals↗
PubMed1989

Poly(A) polymerase from Vigna unguiculata seedlings. A bifunctional enzyme responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities.

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes I and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclease activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63,000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymerizing activity required Mn2+ but not Mg2+. ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity.

Cations, Divalent↗
PubMed1990

A guanyloribonuclease of mouse liver cytosol.

The acid RNase activity of mouse liver cytosol has been resolved into two different enzymes named acid RNase I and acid RNase II respectively. Acid RNase I is a typical pancreatic-type enzyme hydrolyzing CpN and UpN bonds. Acid RNase II, however, hydrolyzes GpN bonds in non-hydrogen-bonded regions of the substrate.

Animals↗
PubMed1993

The 5' ends of RNA oligonucleotides in Escherichia coli and mRNA degradation.

The 5' ends of RNA oligonucleotides in Escherichia coli were identified to assess the contributions of specific endoribonucleases to the cleaving of bulk mRNA. About 60% of the total 5' ends have a 5' OH, and 40% a phosphate. Of those oligonucleotides with a 5'-OH end, 55% of the larger-sized molecules started with 5'-OH-A. With decreasing size there was a progressive decrease in its relative abundance, reaching 33% for the mononucleotide pool, close to its content in E. coli mRNA. In a mutant lacking RNase I* (a form of RNase I), the fraction starting with 5'-OH-A was even higher; 65-70% for oligonucleotides of any size, as well as the mononucleotides, whereas only 3-5% started with 5'-OH-U. Oligonucleotides with a 5'-P end were analyzed after pulse-labeling growing cells with 32Pi. Virtually all of them had a 5'-ppp-purine end which would result from transcription initiations, and there were four-times more G than A starts. The fraction of 5' ends with a monophosphate (5'-pN) was too low to measure. The known degradative enzymes of E. coli (RNases I, I*, M and R) release a 5'-OH oligonucleotide upon cleavage, whereas known processing endoribonucleases, e.g. RNases E, H, P and III, generate 5'-P oligonucleotides. Among these enzymes, RNase M is the only one known to enrich for 5'-OH-A ends, since its preference is for pyrimidine-A bonds [Cannistraro, V. J. & Kennell, D. (1989) Eur. J. Biochem. 181, 363-370]. It also gives a very low level of 5'-OH-U ends. These results are consistent with generalizations derived from our previous studies [Cannistraro, V. J., Subbaro, M. N. & Kennell, D. (1986) J. Mol. Biol. 192, 257-274] and suggest that RNase M is a primary endoribonuclease for mRNA degradation in E. coli. The results also indicate that RNase I* contributes a smaller fraction of cleavages to larger RNA oligonucleotides and accounts for most of the degradation of the very small oligonucleotides and almost all degradation of dinucleotide to mononucleotide.

Endoribonucleases↗
PubMed1995

Use of monoclonal antibodies in the functional characterization of the Saccharomyces cerevisiae Sep1 protein.

The Saccharomyces cerevisiae strand-exchange protein 1 (Sep1 also known as Xrn1, Kem1, Rar5, Stp beta/DST2) has been demonstrated to mediate the formation of hybrid DNA from model substrates of linear double-stranded and circular single-stranded DNA in vitro. To delineate the mechanism by which Sep1 acts in the strand-exchange reaction, we analyzed mouse anti-Sep1 monoclonal antibodies for inhibition of the Sep1 in vitro activity. Of 12 class-G immunoglobulins tested, four were found to consistently inhibit the Sep1-mediated strand-exchange reaction. The inhibiting antibodies were tested for inhibition of a variety of Sep1-catalyzed DNA reactions including exonuclease activity on double-stranded and single-stranded DNA, renaturation of complementary single-stranded DNA and condensation of DNA into large aggregates. All four inhibiting antibodies had no effect on the exonuclease activity of Sep1. Three antibodies specifically blocked DNA aggregation. In addition, one antibody inhibited renaturation of complementary single-stranded DNA. This inhibition pattern underlines the importance of condensation of DNA into large aggregates in conjunction with double-stranded DNA exonuclease activity for the in vitro homologous pairing activity of Sep1. The implications of these data for the interpretation of proteins which promote homologous pairing of DNA are discussed, in particular in light of the reannealing activity of the p53 human tumor-suppressor protein.

Antibodies, Monoclonal↗
PubMed1996

Pre-tRNA 3'-processing in Saccharomyces cerevisiae. Purification and characterization of exo- and endoribonucleases.

We investigated ribonucleases from Saccharomyces cerevisiae which are active in pre-tRNA 3'-processing in vitro. Two pre-tRNA 3'-exonucleases with molecular masses of 33 and 60 kDa, two pre-tRNA 3'-endonucleases with molecular masses of 45 kDa/60 kDa and 55 kDa and 70-kDa 3'-pre-tRNase were purified from yeast whole cell extracts by several successive chromatographic purification steps. The purified exonucleases are non-processive 3'-exonucleases that catalyze the exonucleolytic processing of 3'-trailer sequences of pre-tRNAs to produce mature tRNAs. The 45-kDa/60-kDa 3'-endonuclease is tRNA-specific and catalyzes the processing of pre-tRNAs in a single endonucleolytic step. Two isoenzymes of this activity (p45 and p60) were identified by chromatography. The second endonuclease, p55, is dependent on monovalent ions and cleaves about three nucleotides downstream the mature 3'-end. All of the purified 3'-pre-tRNases accept homologous as well as heterologous pre-tRNA substrates. Pre-tRNAs carrying a 5'-leader are processed with almost the same efficiency as those lacking this 5'-leader. Mature tRNAs carrying the CCA 3'-sequence and tRNA pseudogene products carrying mutations in the mature domain are processed by the 3'-exonucleases, not by the 3'-endonucleases. The specific endonuclease p45/p60 discriminates between UUUOH as a 3'-flank, which is cleaved, and the CCA 3'-end of mature tRNAs, which is not cleaved. This study suggests that several 3'-pre-tRNases are active on tRNA precursors in vitro and might therefore in pre-tRNA 3'-processing in yeast, partly in a cooperative manner.

Base Sequence↗
PubMed2004

Immunohistochemical analysis of 14-3-3 sigma and related proteins in hyperplastic and neoplastic breast lesions, with particular reference to early carcinogenesis.

In order to confirm the role of 14-3-3 sigma (sigma) as a tumor suppressor in breast carcinogenesis, we have studied the expression of 14-3-3sigma immunohistochemically in usual ductal hyperplasia (UDH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) breast lesions. Immunostaining for estrogen receptor alpha (ERalpha), p53 and estrogen-responsive RING finger protein (Efp) was also carried out. Immunohistochemically, expression of 14-3-3sigma was seen in 92% UDH lesions and gradually decreased from 65% in DCIS to 23% in IDC. The expression of ERalpha decreased gradually from UDH to DCIS to IDC, while p53 showed an inverse staining pattern to that of ERalpha. The expression of Efp showed no significant difference among the three breast lesions. Hence, the present immunohistochemical study confirmed 14-3-3sigma as a tumor suppressor in breast carcinogenesis. A similar immunohistochemical analysis was then carried out on columnar cell hyperplasia with atypia (CCHA), in which the expression pattern of tumor suppressor 14-3-3sigma, ERalpha and p53 suggested that it might be possible that CCHA is a precancerous lesion.

14-3-3 Proteins↗
PubMed2004

Immunohistochemical expression of 14-3-3 sigma protein in various histological subtypes of uterine cervical cancers.

14-3-3 sigma (sigma) has been a major G2/M checkpoint control gene and has demonstrated that its inactivation in various cancers occurs mostly by epigenetic hypermethylation, not by genetic change. In order to confirm 14-3-3sigma protein expression together with p16 and p53 in cervical cancers, immunohistochemistry was performed using various histological subtypes of cervical cancers and dysplasia. Strong and diffuse immunoreactivity for 14-3-3sigma was uniformly observed in all the cervical dysplasia (17/17) and squamous cell carcinomas (29/29) including human papillomavirus (HPV)-negative cases. Even in adenosquamous carcinomas and adenocarcinomas of the cervix, immunohistochemical expression of 14-3-3sigma was shown with relatively high frequency (13/15, 87% and 22/27, 81%). In the in situ hybridization study, mRNA of 14-3-3sigma was expressed in six of eight immunohistochemical-negative cases. Therefore, the undetectable expression of 14-3-3sigma protein in cervical cancers might, at least in part, be due to a proteolysis not epigenetic hypermethylation. It is of interest that cancers without 14-3-3sigma expression were predominantly those lacking HPV DNA, and that there were no cases with concomitant inactivation of 14-3-3sigma and p16 in the present study. These observations are consistent with the hypothesis that inactivation of either 14-3-3sigma or p16 has an effect equivalent to the expression of E6 and E7 oncoproteins of HPV.

14-3-3 Proteins↗
PubMed2005

Immunohistochemical expression of 14-3-3 sigma protein in human urological and gynecological tumors using a multi-tumor microarray analysis.

14-3-3 sigma is an exclusive epithelial marker and data on its expression in different malignancies are very scarce. The aims of the present study are to screen its expression in the most common neoplasms occurring in the urological and gynecological tract and to evaluate its use as a diagnostic marker. A tissue microarray was constructed using 350 samples from 13 different neoplasms. Immunohistochemical analysis using a polyclonal 14-3-3 sigma antibody was performed. Overall, this protein was positive in 141 and negative in 209 tumors. The most frequent expression was seen in squamous cell carcinoma of the cervix and urothelial bladder carcinoma, followed by prostatic and endometrial adenocarcinoma. 14-3-3 sigma was able to distinguish prostate adenocarcinoma from urothelial bladder carcinoma, with an odds ratio of 0.028 (P = 0.001; 95% CI, 0.0003-0.222), and distinguish seminoma from embryonal carcinoma of the testis, with an odds ratio of 0.061 (P = 0.009; 95% CI, 0.007-0.5014). It also has a good value in differentiating renal clear cell carcinoma from papillary carcinoma, with an odds ratio 0.470 (P < 0.001; 95% CI, 0.008-0.261). 14-3-3 sigma seems to have good potential use as an epithelial marker, after confirmation with further targeted studies. Finally, as with all immunohistochemical markers, we can optimize the utility of this protein to distinguish tumor mimics by including it in an appropriate immunohistochemical panel.

14-3-3 Proteins↗
PubMed2005

Increasing 14-3-3 sigma expression with declining estrogen receptor alpha and estrogen-responsive finger protein expression defines malignant progression of endometrial carcinoma.

14-3-3 sigma (sigma) is a negative regulator of the cell cycle and contributes to G2 arrest. Lack of its expression due to hypermethylation of CpG islands has been reported in some carcinomas. A recent study showed that 14-3-3 sigma was down-regulated through proteolysis by estrogen-responsive finger protein (Efp). Here, we investigated the expression of 14-3-3 sigma, hormone receptors, Efp and p53 in 86 cases of endometrial adenocarcinoma and 46 cases of normal or non-neoplastic endometria by means of immunohistochemistry and methylation-specific polymerase chain reaction. In normal endometrium, 14-3-3 sigma was overexpressed in the mid- to late-secretory phase due to hypomethylation. In endometrial adenocarcinoma, 14-3-3 sigma expression was low in low grade endometrioid adenocarcinoma due to hypermethylation, and increased significantly with increasing histological grade due to hypomethylation. 14-3-3 sigma expression inversely correlated with estrogen receptor alpha, progesterone receptor and Efp, and positively correlated with myometrial invasion and lymph node metastasis. These results suggest that 14-3-3 sigma was one of the menstrual cycle-related proteins regulated by epigenetic methylation, and its expression was influenced by epigenetic methylation or hormone receptors in progression of endometrial adenocarcinoma, and therefore was more than just a cell-cycle regulator.

14-3-3 Proteins↗
PubMed1995

Construction and characterization of an absolute deletion mutant of Escherichia coli ribonuclease II.

The degradation of mRNA plays a central role in the control of protein synthesis. In Escherichia coli, the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay. We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tetr cassette. This is the first rnb absolute deletion mutant that shows the complete absence of rnb-specific mRNA. This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism.

Cloning, Molecular↗
PubMed1996

A new role for RNase II in mRNA decay: striking differences between RNase II mutants and similarities with a strain deficient in RNase E.

The effect of Escherichia coli ribonuclease II and polynucleotide phosphorylase was analysed on the degradation of Desulfovibrio vulgaris cytochrome c3 (cyc) mRNA. In the absence of these exoribonucleolytic activities, cyc mRNA was stabilised but the two enzymes had a different role in its decay. Surprisingly, a temperature-sensitive mutation in ribonuclease II gave a degradation pattern similar to what had been observed in the absence of endoribonuclease E activity. In an RNase II deletion mutant this was not observed. We propose and verify a model in which the temperature-sensitive ribonuclease II interferes with the action of ribonuclease E.

Antibiotics, Antitubercular↗
PubMed1998

RNase III deficient Salmonella typhimurium LT2 contains intervening sequences (IVSs) in its 23S rRNA.

Salmonella typhimurium LT2 contains intervening sequences (IVSs) of 90-110 nt within all its 23S rRNA that are cleaved out by RNase III, resulting in rRNA fragmentation. In order to determine the functionality of 23S rRNA that contains unexcised IVSs, we constructed an S. typhimurium RNase III (rnc) deficient strain by transducing a mini-Tn10 (rnc-14::Tn10) from Escherichia coli K-12. The resulting strain of S. typhimurium was viable, contained IVSs within all of its 23S rRNA, and showed a growth reduction similar to that observed for the RNase III deficient strain of E. coli. These results indicate that ribosomes containing 23S rRNA in which IVSs are not excised are functional in translation, and make it unlikely that RNase III excision of IVSs from strain LT2 23S rRNA is dictated by a selective pressure to uphold the functional integrity of ribosomes.

Base Sequence↗