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At least 703 records · Page 39Linked to original sources
PubMed1997

Identification of rapid turnover transcripts overexpressed in thyroid tumors and thyroid cancer cell lines: use of a targeted differential RNA display method to select for mRNA subsets.

The mRNAs of transiently expressed proteins such as cytokines and proto-oncogenes are commonly subject to rapid transcriptional activation and degradation. Transcript turnover is determined in part by association of certain proteins with consensus AU-rich motifs (AUUUA) in the 3'-untranslated region of the transcripts. Here we report a modification of differential RNA display (DRD) to detect differentially expressed rapid turnover mRNAs containing AU-rich motifs from thyroid cancer tissues and cell lines. RNA of normal and thyroid cancer tissues was differentially displayed using a 3'anchor primer to the poly(A) tail and an arbitrary 5'primer incorporating an AUUUA sequence. The appropriateness of the strategy was established by its ability to display known early response genes, such as c- fos, using partially degenerate primers. To test whether the novel cDNAs isolated coded for transcripts subject to rapid turnover, they were used as probes for Northern blots of RNA from clonal human thyroid carcinoma cell lines treated for varying periods with either cycloheximide or actinomycin D. A number of novel differentially expressed cDNA fragments were isolated from human papillary thyroid carcinoma tissues, among them a cDNA with zinc finger motifs and homology to other zinc finger proteins. Using this fragment to probe a cDNA library, a full-length cDNA (ZnF20) was isolated that was 4333 bp in length and contained an open reading frame of 1029 amino acids. The ZnF20 cDNA hybridized to multiple transcripts in a thyroid cancer cell line (8.0, 4.5 and 2 kb) that increased after cycloheximide treatment and decayed <2 h after addition of actinomycin D. The ZnF20 mRNA was overexpressed in three of six thyroid papillary carcinomas as compared with paired normal thyroid tissue controls. The data presented here support the use of a targeted DRD approach for the isolation of rapid turnover mRNAs, many of which may be interesting candidate oncogenes.

Amino Acid Sequence↗
PubMed2006

The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing.

RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification.

Adenosine Deaminase↗
PubMed2006

Amount changes of tRNA modification enzymes in Thermus thermophilus HB8 cells according to culture temperatures.

Thermus thermophilus HB8 is an extreme thermophilic eubacrium, which grows at 50-80 degrees C. Transfer RNA molecules in T. thermophilus HB8 contains modified nucleosides such as Gm18, m(7)G46, m(5)s(2)U54, Psi55, and m(1)A58. Recently, all responsible genes for these modifications have been identified. To clarify the relationship of the amounts of the RNA modification enzymes (proteins) and culture conditions, we investigated the activities and quantities of the proteins. In this meeting, we report the amount changes of tRNA (Gm18) methyltransferase [TrmH] in T. thermophilus HB8 cells cultured at 52, 67, 75, and 79 degrees C. 60 mug of total proteins in the crude extract from cells cultured at 67 degrees C contained about 10-30 ng of the TrmH protein. Thus, the content of the TrmH protein was estimated to be around 1/5000 of the total proteins. Through the log phase, the content of the TrmH protein was not changed obviously. However, in the stationary phase, the content of the TrmH protein was slowly reduced. The TrmH contents in the cells cultured at 75 or 79 degrees C were similar to that at 67 degrees C. However, the content of the TrmH protein in the extract from 52 degrees C cultured cells slightly decreased as compared with that in the 67 degrees C cultured cells.

Bacterial Proteins↗
PubMed1990

Guanosine modifications in runoff transcripts of synthetic transfer RNA-Phe genes microinjected into Xenopus oocytes.

We have investigated whether unmodified yeast phenylalanine transfer RNA as well as one of its precursors containing an intron of nineteen nucleotides in the anticodon (pre-tRNA-Phe) can become substrates for selected tRNA modification enzymes present in a eukaryotic cell. This study was done by microinjecting into the cytoplasm of Xenopus laevis oocytes transcripts completely deprived of the naturally occurring modified nucleotides; these were obtained in vitro from appropriate synthetic genes under the control of bacteriophage T7 promoter. During the in vitro transcription, 32P labels were introduced with the guanosine triphosphate thus allowing easy detection of guanosine modifications in tRNA by two-dimensional chromatography after complete digestion into 5'-mononucleotides by nuclease P1. Results indicate that modifications occur on five guanosines (at positions 10, 26, 34, 37 and 46) in yeast tRNA-Phe and only on three guanosines (at 10, 26 and 46) in yeast precursor tRNA-Phe. These are the modifications expected from the known nucleotide sequences of naturally occurring Xenopus and yeast tRNA-Phe, i.e. N2-methyl-G10, N2,N2-dimethyl-G26, 2'-O-methyl-G34, N1-methyl-G37 or Y nucleoside-37 and N7-methyl-G46. The rates of modifications occurring in the two kinds of tRNA-Phe are faster in the intron-less tRNA-Phe than in the intron-containing tRNA-Phe. However quantitative modifications are only observed after as long as 75 h incubation in the oocytes.

Animals↗
PubMed2000

Structural genomics of RNA.

A detailed understanding of the functions and interactions of biological macromolecules requires knowledge of their molecular structures. Structural genomics, the systematic determination of all macromolecular structures represented in a genome, is focused at present exclusively on proteins. It is clear, however, that RNA molecules play a variety of significant roles in cells, including protein synthesis and targeting, many forms of RNA processing and splicing, RNA editing and modification, and chromosome end maintenance. To comprehensively understand the biology of a cell, it will ultimately be necessary to know the identity of all encoded RNAs, the molecules with which they interact and the molecular structures of these complexes. This report focuses on the feasibility of structural genomics of RNA, approaches to determining RNA structures and the potential usefulness of an RNA structural database for both predicting folds and deciphering biological functions of RNA molecules.

Computational Biology↗
PubMed1975

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.

Adenoviridae↗
PubMed2002

[A RAPD method acceptable for the analysis of mitochondrial gene expression].

Several techniques are available in detecting variations in gene expression between different samples, such as SSH, RACE, etc. However, they can not be applied to analyze mitochondrial gene expression due to the specific characteristics of mitochondrial RNA. So some modifications were made to the conventional techniques. Here we reported a demonstration of this modified technique, taking rice mitochondria as materials. In this technique, using random hexamers to prime the RT, the resultant cDNA likely included coding regions because it was not locked to the poly(A) tail of the messenger RNA.

English Abstract↗
PubMed1981

Post-transcriptional modifications of mRNA. Purification and characterization of cap I and cap II RNA (nucleoside-2'-)-methyltransferases from HeLa cells.

The existence in HeLa cell extracts of two separate RNA (nucleoside-2')-methyltransferases involved in the modification of mRNA was established using assays that specifically measure the conversion of cap O [m7G(5')pppNpN-] to cap I [m7G(5')pppNmpN-] and cap I to cap II [m7G(5')pppNmpNm-]. Cap II methyltransferase activity was found almost exclusively in cytoplasmic fractions while cap I methyltransferase activity was also found in the nucleus, its apparent biological site of action. The two enzymes were purified by DEAE-cellulose and phosphocellulose chromatography and their optimal reaction conditions were determined. The substrate specificity of cap I methyltransferase was examined with particular regard to information that would help elucidate the natural order of capping and methylation was drawn from data presented here. Both purine and pyrimidine nucleosides in the N position of M7G(5')pppN- were methylated by purified cap I methyltransferase.

Cell Nucleus↗
PubMed1999

Chlorella viruses as a source of novel enzymes.

A special advantage has been conferred upon Chlorella cells as tools in biotechnology when viruses (Phycodnaviridae) infecting Chlorella cells were discovered and isolated. The viruses are large icosahedral particles (150-200 nm in diameter), containing a giant, 330-380 kbp long, linear dsDNA genome. Recently, the nucleotide sequence of the 330,740-bp genome of PBCV-1, the prototype virus of Phycodnaviridae, was determined, and up to 702 open reading frames (ORFs) were identified along the genome. The possible genes present include those encoding a variety of enzymes involved in the modification of DNA, RNA, protein and polysaccharides as well as those involved in the metabolism of sugars, amino acids, lipids, nucleotides and nucleosides. Many of these genes are actually expressed during viral infection, with functional enzymes detected in the host cytoplasm or incorporated into the virion. The successful utilization of these viral enzymes as various DNA restriction and modification enzymes (Cvi enzymes) that are now commercially available is well documented. Also noteworthy are virion-associated chitinase and chitosanase activities that have potentially important applications in the recycling of natural resources. The virions of Chlorella viruses contain more than 50 different structural proteins, ranging in size from 10 to 200 kDa. Some of these proteins may be replaced with useful foreign proteins using recombinant DNA technology. The proteins of interest can be recovered easily from the viral particles, and collected by centrifugation after complete lysis of the host Chlorella cells.

Journal Article↗
PubMed2004

The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific.

In cells infected with herpes simplex virus 1, the RNA encoded by the stress-inducible immediate early response gene IEX-1 was up-regulated immediately after infection. However, the accumulated RNA was degraded 3'-5', and the protein was detectable only at very early times after infection. The degradation was dependent on the U(L)41 gene encoding the virion host shutoff (vhs) protein and resulted in the accumulation of truncated RNA containing the 5'-end portion of the transcript. IEX-1 contains an AU-rich element (ARE) in its 3'-untranslated domains known to regulate negatively the RNA lifespan. To examine the role of ARE in signaling the degradation, we compared the stability of several RNAs up-regulated during infection to WT virus. These were ARE-containing RNAs encoding IEX-1, c-fos, and IkappaBalpha and the non-ARE-containing RNAs GADD45beta and tristetraprolin. We report that the ARE-containing RNAs exemplified by IEX-1 RNA are deadenylated and cleaved in the ARE within the 3' UTR in a U(L)41-dependent manner. In contrast, Northern blot hybridizations and analyses of poly(A) tails revealed no evidence of degradation of GADD45beta RNA. GADD45beta protein was detected in WT virus-infected cells. These results indicate that the degradation of RNAs and the mechanism by which cellular RNAs are degraded are selective and may be sequence specific. The persistence of partially degraded ARE-containing RNAs may reflect specific targeting of the vhs proteins to the ARE and the modification of the RNA degradation machinery of the cell induced by the presence of the vhs protein.

3' Untranslated Regions↗