Precursor Of tRNA Research Articles

Circulating serum exosomes i-tRF-AspGTC and tRF-1-SerCGA as diagnostic indicators for non-small cell lung cancer.

tRF-RNA-a representative of non-coding RNA (ncRNA)-is a precursor or fragment of mature tRNA and plays a crucial regulatory role in the occurrence and development of cancer. There is currently little research on tRF-RNA as a diagnostic marker in cancer, especially for NSCLC from serum exosomes. Serum exosomes were successfully extracted from serum; their physical morphology was captured by transmission electron microscopy (TEM); appropriate particle size detection was performed using qNano; surface labeling was verified through western blotting. Serum exosomes i-tRF-AspGTC and tRF-1-SerCGA were selected through gene microarray, and qPCR was used to validate their significance in 242 patients and 201 healthy individuals. The area under the curve (AUC) was used to evaluate the diagnostic indicators of non-small cell lung cancer (NSCLC). Compared with 201 healthy individuals, i-tRF-AspGTC and tRF-1-SerCGA were significantly downregulated in 242 NSCLC patients and 95 early-stage patients. For tRF-AspGTC and tRF-1-SerCGA, the predictive diagnostic efficiency rates of AUC were 0.690 and 0.680, respectively, whereas the early diagnostic efficiency rates were 0.656 and 0.688, respectively. The result of combined diagnosis with CEA and CYFRA21-1 was 0.928, and the early diagnostic efficiency was 0.843, which is a very high biological predictive factor for NSCLC. The expression of serum exosomes i-tRF-AspGTC and tRF-1-SerCGA was significantly downregulated in NSCLC patients. These exosomes could be used as predictive indicators for diagnosis or early diagnosis of NSCLC.

Non-enzymatic aminoacylation of an RNA minihelix with an aminoacyl phosphate oligonucleotide

Efficient aminoacylation of an RNA minihelix (a proposed precursor of tRNA) was achieved without any ribozymes or protein enzymes. The reaction was promoted with a template-like bridging Oligonucleotide and a 5'-aminoacyl-phosphate-oligonucleotide. Aminoacylation was template-dependent and showed high preference for a particular ribose hydroxyl. The 5'-aminoacyl-phosphate-oligonucleotides have the same adenylate linkage as in aminoacyl-AMP--the universal intermediate used in modern aminoacylation systems with aminoacyl-tRNA synthetases. Thus, the results raise the possibility that this kind of system could have played a role in the transition from the RNA world to the theatre of proteins.

RNA editing in the acceptor stem of squid mitochondrial tRNA(Tyr).

In squid (Loligo bleekeri) mitochondria, the two 3'-terminal nucleotides (G72-G73) of the tRNA(Tyr) gene overlap with the two 5'-terminal nucleotides (G1-G2) of the downstream tRNA(Cys) gene. To elucidate the processing mechanism(s) of the tRNA molecules derived from this region, tRNAs were analyzed by sequencing cDNAs synthesized from circularized tRNAs. Nucleotides G1-G2 in tRNA(Cys) appeared to be without post-transcriptional conversion, whereas CCA was post-transcriptionally added to the 3'-terminus. In contrast, in the majority of tRNAs(Tyr), G72-G73 were found to be converted to A72-A73, accompanied by the CCA addition. These results indicate that a precursor of tRNA(Tyr) is processed at U71 and two adenosines are attached prior to the CCA addition. Thus, we suggest that 5' processing of the precursor tRNA dominates 3' processing and maturation of the tRNA is mediated by a polyadenylylation enzyme in the mitochondria, a scenario which is consistent with the editing process proposed in land snail mitochondria. We also obtained intermediates, such as a premature tRNA lacking CCA that terminated at U71 and one with a single adenosine attached at position 72, which support the suggested maturation process. However, although we failed to detect a tRNA(Cys) lacking G1-G2 at the 5'-terminus, we obtained cDNAs for tRNA(Tyr) with G72-G73 and the CCA terminus. This inconsistent result suggests the co-existence of another process(es) in the maturation of these tRNA molecules in squid mitochondria.

Relationship of the queuine content of transfer ribonucleic acids to histopathological grading and survival in human lung cancer : Huang B-S, Wu R-T, Chien K-Y. Graduate Clinical Medicine Institute, National Yang-Ming Medical College, Taipei. Cancer Res 1992;52:4696–4700

To elucidate the significance of tRNA hypomodified with queuine to the grade of malignancies in human solid tumors, the amount of tRNA having guanosine in place of queuosine was determined in human lung cancer and normal lung tissue, by exchanging the unmodified guanosine residue for [3H]guanine. The reaction is catalyzed by guanine:queuine tRNA transglycosylase. Total tRNA was extracted from 23 different lung cancer specimens and the precursor of isoacceptor tRNA that contains guanine instead of queuine in the first or wobble position of the anticodon [(Q-)tRNA] content was determined. In 12 cases the (Q-)-tRNA was determined in normal lung tissues as well. In each individual, the (Q-)tRNA content in lung cancer tissue was higher than that of the normal lung tissue. The (Q-)tRNA content was not correlated to the surgicopathological staging of the patients but was highly correlated to the histopathological classification of the tumors. The amounts of (Q-)-tRNA were 1.75 +/- 0.67 (SD), 2.36 +/- 0.89, 3.77 +/- 1.39, 5.18 +/- 2.32, and 7.65 +/- 1.34 pmol/A260 in normal, well, moderately, moderately to poorly, and poorly differentiated tumors. The difference from normal to moderately differentiated or less differentiated groups was significant (P less than 0.05). In 10 patients with (Q-)tRNA higher than 3.5 pmol/A260, their cancers relapsed and only 2 were alive after 4 years. In 11 patients with (Q-)tRNA less than 3.5 pmol/A260 in their lung cancer tissues, 7 patients were still alive without any evidence of disease, 3 were dead, and 1 had recurrence of disease. These results, taken together with other previous studies, suggest that a decreased queuosine content of tRNA may be a general feature of neoplasms and may be useful for grading malignancy and perhaps also for the prediction of survival in human lung cancer.

Origin of glutaminyl-tRNA synthetase: An example of palimpsest?

Sequence data and evolutionary arguments suggest that a similarity may exist between the C-terminal end of glutaminyl-tRNA synthetase (GlnRS) and the catalytic domain of glutamine amidotransferases (GATs). If true, this would seem to imply that the amidation reaction of the Glut-tRNA(Gln) complex was the evolutionary precursor of the direct tRNA(Gln) aminoacylation pathway. Since the C-terminal end of GlnRS does not now have an important functional role, it can be concluded that this sequence contains vestiges that lead us to believe that it represents a palimpsest. This sequence still conserves the remains of the evolutionary transition: amidation reaction-->aminoacylation reaction. This may be important in deciding which mechanism gave origin to the genetic code organization. These observations, together with results obtained by Gatti and Tzagoloff [J. Mol. Biol. (1991) 218:557-568], lead to the hypothesis that the class I aminoacyl-tRNA synthetases (ARSs) may be homologous to the GATs of the trpG subfamily, while the class II ARSs may be homologous to the GATs of the purF subfamily. Overall, this seems to point to the existence of an intimate evolutionary link between the proteins involved in the primitive metabolism and aminoacyl-tRNA synthetases.

Direct evidence for selective modulation of psbA, rpoA, rbcL and 16S RNA stability during barley chloroplast development.

The turnover of RNAs encoded by seven different barley chloroplast genes was analyzed after treatment of barley shoots with tagetitoxin, a selective inhibitor of chloroplast transcription. Changes in RNA stability were examined during chloroplast development using basal and apical leaf sections of 4.5-day-old dark-grown seedlings and apical leaf sections of 4.0-day-old dark-grown seedlings which had been illuminated for 12 h. Of the RNAs examined, a 2.6 kb unspliced precursor of tRNA(lys) exhibited the shortest half-life, which was estimated to be 3 h. The 16S rRNA and psbA mRNA had the longest estimated half-lives, which were greater than 40 h. Among mRNAs, half-lives were estimated to range from 6 h for psaA mRNA, to over 40 h for psbA mRNA. Therefore, barley chloroplast mRNAs have long half-lives relative to bacterial mRNAs. The stability of atpB mRNA and the unspliced precursor of tRNA-lys was not altered during chloroplast development, while the stability of psaA mRNA decreased 2-fold. In contrast, the stability of the 16S rRNA and mRNAs for rpoA, psbA and rbcL increased during chloroplast development. The stability of 16S rRNA increased markedly during chloroplast development in the dark and this increase was maintained in illuminated seedlings. The stability of rbcL mRNA increased 2.5-fold during chloroplast development in the dark, and then decreased 2-fold in chloroplasts of light-grown plants. The initial increase in rpoA and psbA mRNA stability was also light-independent, with total increases in stability of at least 5-fold. In the case of rpoA, the stability of 2 of the 13 polycistronic rpoA transcripts that were detected in dark-grown plants was selectively increased during chloroplast development. In conclusion, the stability of some transcripts is selectively increased and further modulated during chloroplast development in barley. We propose that the selective stabilization of chloroplast mRNA, which occurred independent of light, is an indication that non-light regulated developmental signals are involved in barley chloroplast mRNA stability.

The action of alpha-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids.

alpha-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of alpha-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of alpha-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10-20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by alpha-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.

Nucleotide Sequence of 4.5 S Ribonucleic Acidi of Novikoff Hepatoma Cell Nuclei

Abstract The primary sequence of nucleotides has been defined for 4.5 S RNAi, which is one type of low molecular weight nuclear RNA of Novikoff hepatoma ascites cells. This RNA is the first unique nuclear species of mammalian RNA to be sequenced. It is specifically localized to the extranucleolar portion of the nucleus and is not a precursor of tRNA. Highly labeled 4.5 S RNAi was obtained from cells incubated with [32P]orthophosphate in vitro. The RNA was purified by successive gel electrophoresis and chromatography on DEAE-Sephadex. The primary sequence of nucleotides of this RNA shows that it has a purine-rich 5' end and a pyrimidine-rich 3' end: [see PDF for sequence].

Rapidly labeled cytoplasmic RNA in normal and phytohaemagglutinin-stimulated human lymphocytes

1. 1. Phytohaemagglutinin caused a significant stimulation of the incorporation of [ 3H]uridine into RNA within 30 min of its addition to lymphocytes. While more than 90% of the radioactive RNA labeled in this period was in the nucleus, the stimulation of [ 3H]uridine incorporation into RNA located in the cytoplasm was greater than the stimulation of incorporation into whole cell RNA. This rapid stimulation of the labeling of cytoplasmic RNA by phytohaemagglutinin might be important for the changes in lymphocyte protein synthesis that began at this time. 2. 2. Analysis by sucrose gradient centrifugation of the cytoplasmic RNA labeled in a 30-min pulse with [ 3H]uridine showed that 4-S RNA was the predominant labeled species. However, gel filtration on Sephadex G-100 showed that this labeled RNA contained several components and that little of it was tRNA. Phytohaemagglutinin did not alter the elution pattern of the radioactive cytoplasmic RNA from Sephadex G-100 but stimulated the labeling of all its components to about the same extent. 3. 3. The predominant rapidly labeled cytoplasmic RNA species was retarded by Sephadex G-100 but eluted in a polydisperse fashion ahead of tRNA. This species was unstable to nuclease digestion and was not methylated. Its disappearance during an actinomycin chase was accompanied by the appearance of labeled tRNA. It did not seem to be attached to cytoplasmic particles, and it hybridized to lymphocyte DNA less readily than did nuclear polydisperse RNA. These properties are compatible with it being an unmethylated cytoplasmic precursor of tRNA. 4. 4. There was no evidence for an increase in the labeling of mature tRNA 30 min after the addition of phytohaemagglutinin, but such an increase was apparent after 90 min. After incubation of lymphocytes with phytohaemagglutinin for 20–24 h, the rate of tRNA synthesis was greatly stimulated. If the material discussed above is indeed a precursor to tRNA, the rate of its conversion to mature tRNA was also accelerated at this time.