Overexpression of initiator methionine tRNA leads to global reprogramming of tRNA expression and increased proliferation in human epithelial cells

Transfer RNAs (tRNAs) are typically considered housekeeping products with little regulatory function. However, several studies over the past 10 years have linked tRNA misregulation to cancer. We have previously reported that tRNA levels are significantly elevated in breast cancer and multiple myeloma cells. To further investigate the cellular and physiological effects of tRNA overexpression, we overexpressed tRNA_i^Met in two human breast epithelial cell lines. We then determined tRNA abundance changes and performed phenotypic characterization. Overexpression of tRNA_i^Met significantly altered the global tRNA expression profile and resulted in increased cell metabolic activity and cell proliferation. Our results extend the relevance of tRNA overexpression in human cells and underscore the complexity of cellular regulation of tRNA expression.     

第18届国际tRNA学术研讨会介绍

第 18届国际ｔＲＮＡ学术研讨会于 2 0 0 0年 4月8— 12日在英国剑桥大学皇后学院举行。来自各国的 2 4 0名科学家参加了会议。提交会议的论文共2 0 0篇。会议由新当选的国际生物化学与分子生物学联合会 (ＩＵＢＭＢ)主席Ｂ .Ｃｌａｒｋ主持 ,每天从上午 8点 4 5分开始 ,进行到晚上 11点 ,实际会议时间达10小时。共有 80位科学家作了学术报告。其余论文以墙报形式交流。这次会议原安排在去年 ,以纪念国际ｔＲＮＡ学术研讨会 30周年。故会议的第一部分 ,放了很多旧照片 ,回忆ｔＲＮＡ学术研讨会和ｔＲＮＡ研究的历史。在Ｈｏｌｌｙ因测定了第…     

细菌的毒力岛

毒力岛是指细菌染色体上一段具有典型结构特征的基因簇,主要编码与细菌的毒力及代谢等功能相关的产物,已在致病菌中发现了30几个毒力岛。由于毒力岛具有可移动性,使其在细菌的进化,毒力的获得,以及新病原的出现中均具有重要的意义。     

The length of the 5' leader of Escherichia coli tRNA precursors influences bacterial growth

Based on a computational analysis of the 5' regions of tRNA-encoding genes, the average length of the 5' leaders in tRNA precursors in Escherichia coli appears to be 17-18 residues long. An in vivo assay based on tRNA nonsense suppression was developed and used to investigate the function of the 5' leader of the tRNA precursors on tRNA processing and bacterial growth. Our data indicate that the 5' leader influences bacterial growth but is surprisingly not absolutely necessary for growth. These findings are consistent with previous in vitro data where it was demonstrated that the 5' leader plays a role in the interaction with RNase P, the endoribonuclease responsible for removing the 5' leader in the cell. We discuss the plausible role of the 5' leader in processing and tRNA gene expression.     

镁离子稳定tRNA高级结构及其重要意义--生化所tRNA研究的一段历史回顾

镁离子稳定tRNA高级结构并具有重要的生理功能已经写入生物化学教科书。上世纪60年代初,中国科学院上海生物化学研究所的研究者与国外同行同时发现了这一现象。镁离子稳定tRNA的高级结构在tRNA的序列分析中可以得到较大的片段,甚至tRNA半分子。将这一事实运用于“片段重叠”法测定tRNA序列工作中,极大地推动了tRNA的序列测定。     

线粒体tRNAThr G15927A突变可能影响耳聋相关的12S rRNA A1555G突变的表型表达

摘要: 对1个中国汉族耳聋家系进行了临床和分子遗传学特征分析。家系中听力下降的母系成员表现为程度不等、听力图形态不同的听力损害, 但同为双侧对称的感觉神经性耳聋。该家系耳聋外显率很高, 包括药物致聋的耳聋外显率为75%, 而非药物致聋的外显率为41.7%。对母系成员进行线粒体DNA(mtDNA)全序列扩增分析, 发现了耳聋相关12S rRNA A1555G同质性突变位点和多态性位点, 属于东亚人群B5b单体型。在这些变异位点中, mtDNA 15927位点的G-A碱基变化破坏tRNA^Thr反密码子结构上十分保守的C-G碱基对, 这可能加重由A1555G突变造成的线粒体功能缺陷。这表明tRNAThrG15927A突变可能增强携带12S rRNA A1555G的中国汉族耳聋家系的外显率和表现度。     

人线粒体tRNA基因突变与疾病

线粒体是体内重要的细胞器,存在于胞质内。许多生物化学反应都在线粒体内进行,其中尤为重要的是通过氧化磷酸化作用产生ＡＴＰ,供生命活动需要。线粒体内ｔＲＮＡ的总数只有２２个（核编码的ｔＲＮＡ总数至少为３０多个）。这样,线粒体ｔＲＮＡ与编码氨基酸之比基本是...     

tRNA个性研究进展

ｔＲＮＡ参与蛋白质生物合成中至关重要的两个功能上相连的生物化学事件。一种是由氨基酰－ｔＲＮＡ合成酶（ａａＲＳ）催化的ｔＲ－ＮＡ的氨基酰化（ｔＲＮＡ的个性）,通过ａａＲＳ对ｔＲＮＡ的序列和结构元件的专一识别和氨基酰化,使ｔＲＮＡ转化为氨基酰ｔＲＮＡ。另...     

tRNA体外抑制L929细胞生长的作用观察

目的:探讨tRNA对不同哺乳动物细胞生长的影响.方法:L929细胞、NIH3T3细胞、MCF-7细胞和PC12细胞接种96孔板,37℃,5% CO2细胞培养箱中培养4 h后,直接加入酵母tRNA,继续培养一定时间后采用MTT法检测细胞生长情况;将200 μg/ml酵母tRNA加入至L929细胞中,在不同时间点观察细胞形态并收集细胞进行细胞流式分析.结果:tRNA对L929细胞有特异的抑制作用,并且表现出一定的剂量依赖性;tRNA处理后L929细胞与正常细胞相比,形态明显变大,而且突起增长;流式细胞仪分析进一步发现tRNA使细胞阻滞于S期.结论:tRNA的这种细胞生长的抑制效应可能是通过其一些小的降解片段发挥的,提示tRNA或者其降解片段可能具有调控L929细胞增殖的重要作用.     

RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

Queuine is incorporated into brain transfer RNA

Pig brain tRNA was assayed for the presence of queuosine in the first position of the anticodon for each of the Q-family of tRNAs (aspartyl, asparaginyl, histidyl and tyrosyl). The brain tRNA was aminoacylated with each of the four amino acids and the aminoacylated tRNA's analyzed by RPC-5 chromatography. The results of this study show that for all four tRNAs of the family, queuine is substituted for guanine in virtually 100% of the anticodons. Therefore, it can be concluded that queuine is able to cross the blood-brain barrier and that brain contains quanine-queuine tRNA transglycosylase, the enzyme responsible for the excision of guanine from the orginal transcipts of these tRNAs and insertion of queuine. The determination of whether the tRNA contained queuine was made from the elution profile of the RPC-5 chromatrograms and the results confirmed by a change in the RPC-5 elution profile when the tRNAs were reacted with BrCN or NaIO₄.     

Mature methyl-deficient tRNA isolated from a mammary adenocarcinoma

A transplantable rat tumor, mammary adenocarcinoma 13762, accumulates tRNA which can be methylated in vitro by mammalian tRNA (adenine-1) methyltransferase. This unusual ability of the tumor RNA to serve as substrate for a homologous tRNA methylating enzyme is correlated with unusually low levels of the A₅₈-specific adenine-1 methyltransferase. The nature of the methyl-accepting RNA has been examined by separating tumor tRNA on two-dimensional polyacrylamide gels. Comparisons of ethidium bromide-stained gels of tumor vs. liver tRNA show no significant quantitative differences and no accumulation of novel tRNAs or precursor tRNAs in adenocarcinoma RNA. Two-dimensional separations of tumor RNA after in vitro [¹⁴C]methylation using purified adenine-1 methyltransferase indicate that about 25% of the tRNA species are strongly methyl-accepting RNAs. Identification of six of the tRNAs separated on two-dimensional gels has been carried out by hybridization of cloned tRNA genes to Northern blots. Three of these, tRNA^Lys3, tRNA^Gln and tRNA^Met_i, are among the adenocarcinoma methyl-accepting RNAs. The other three RNAs, all of which are leucine-specific tRNAs, show no methyl-accepting properties. Our results suggest that low levels of a tRNA methyltransferase in the adenocarcinoma cause selected species of tRNA to escape the normal A₅₈ methylation, resulting in the appearance of several mature tRNAs which are deficient in 1-methyladenine. The methyl-accepting tRNAs from the tumor appear as ethidium bromide-stained spots of similar intensity to those seen for RNA from rat liver; therefore, methyladenine deficiency does not seem to impair processing of these tRNAs.     

Reduced Amino Acid Specificity of Mammalian Tyrosyl-tRNA Synthetase Is Associated with Elevated Mistranslation of Tyr Codons

Quality control operates at different steps in translation to limit errors to approximately one mistranslated codon per 10,000 codons during mRNA-directed protein synthesis. Recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. Here we examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were previously found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Despite this comparatively high mistranslation rate, no significant change in cellular viability was observed. Monitoring of Phe and Tyr levels revealed that changes in error rates correlated with changes in amino acid pools, suggesting that mischarging of tRNA^Tyr with noncognate Phe by tyrosyl-tRNA synthetase was responsible for mistranslation. Steady-state kinetic analyses of CHO cytoplasmic tyrosyl-tRNA synthetase revealed a 25-fold lower specificity for Tyr over Phe as compared with previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. Functional comparisons of mammalian and bacterial tyrosyl-tRNA synthetase revealed key differences at residues responsible for amino acid recognition, highlighting differences in evolutionary constraints for translation quality control.     

Genes, enzymes and coenzymes of queuosine biosynthesis in procaryotes

In almost all known tRNAs that are specific for Asp, Asn, His or Tyr the wobble position of the anticodon is occupied by the hypermodified tRNA nucleoside queuosine. This unusual deazaguanine derivative is synthesised only in eubacteria. The biosynthesis, as investigated in Escherichia coli, is accomplished in four steps involving many unprecedented enzymatic reactions.     

析遗传密码子多态性之谜

建立了1个由16个“3读2”原始密码子组成的系统。它们分为“语义确切”的,和“双义的”,两大类。后者,通过不同的分化方式,进一步分化为语义确切的“3读3”现代密码子;前者则无需再分化,仍保留着“3读2”原始形态,成为孑遗密码子。首次解释了氨基酸具有不同数目密码子,以及线粒体内存在反常密码子的多态性现象,初步建立了密码子进化树,并提出了原始氨酰基－tRNA合成酶可能在密码子进一步分化中起关键作用的观点。     

tRNA转录后修饰的功能及其与人类疾病的关系

转移核糖核酸(tRNA)的转录后修饰对tRNA正常行使生物学功能具有重要意义,这些功能包括tRNA的正确折叠和维持其稳定性、在核糖体上正确解码。虽然tRNA转录后大部分核苷酸修饰形式在20世纪70年代已被鉴定出,但最近才在大肠杆菌及酵母中鉴定出催化这些tRNA核苷酸修饰的酶的绝大部分基因。这些修饰酶基因的鉴定为研究tRNA转录后修饰的生物功能开启了新的大门。人胞质tRNA和线粒体tRNA(mt tRNA)都存在大量核苷酸修饰,这些修饰的缺陷常常与多种人类疾病相关。因此,研究tRNA核苷酸修饰有助于我们了解相关疾病的发病机理。