Avoidance of antisense, antiterminator tRNA anticodons in vertebrate mitochondria

Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the ‘antisense’ tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs.     

Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response

Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNA^Glu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics.     

Molecular Mimicry Between Protein and tRNA

Mimicry is a sophisticated development in animals, fish, and plants that allows them to fool others by imitating a shape or color for diverse purposes, such as to prey, evade, lure, pollinate, or threaten. This is not restricted to the macro-world, but extends to the micro-world as molecular mimicry. Recent advances in structural and molecular biology uncovered a set of translation factors that resembles a tRNA shape and, in one case, even mimics a tRNA function for deciphering the genetic code. Nature must have evolved this art of molecular mimicry between protein and ribonucleic acid by using different protein structures until the translation factors sat in the cockpit of a ribosome machine, on behalf of tRNA, and achieved diverse actions. Structural, functional, and evolutionary aspects of molecular mimicry will be discussed. Received: 9 January 2001 / Accepted: 22 March 2001     

The flexible evolutionary anchorage-dependent Pardee's restriction point of mammalian cells. How its deregulation may lead to cancer

Living cells oscillate between the two states of quiescence and division that stand poles apart in terms of energy requirements, macromolecular composition and structural organization and in which they fulfill dichotomous activities. Division is a highly dynamic and energy-consuming process that needs be carefully orchestrated to ensure the faithful transmission of the mother genotype to daughter cells. Quiescence is a low-energy state in which a cell may still have to struggle hard to maintain its homeostasis in the face of adversity while waiting sometimes for long periods before finding a propitious niche to reproduce. Thus, the perpetuation of single cells rests upon their ability to elaborate robust quiescent and dividing states. This led yeast and mammalian cells to evolve rigorous Start [L.H. Hartwell, J. Culotti, J. Pringle, B.J. Reid, Genetic control of the cell division cycle in yeast, Science 183 (1974) 46–51] and restriction (R) points [A.B. Pardee, A restriction point for control of normal animal cell proliferation, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 1286–1290], respectively, that reduce deadly interferences between the two states by enforcing their temporal insulation though still enabling a rapid transition from one to the other upon an unpredictable change in their environment. The constitutive cells of multicelled organisms are extremely sensitive in addition to the nature of their adhering support that fluctuates depending on developmental stage and tissue specificity. Metazoan evolution has entailed, therefore, the need for exceedingly flexible anchorage-dependent R points empowered to assist cells in switching between quiescence and division at various times, places and conditions in the same organism. Programmed cell death may have evolved concurrently in specific contexts unfit for the operation of a stringent R point that increase the risk of deadly interferences between the two states (as it happens notably during development). But, because of their innate flexibility, anchorage-dependent R points have also the ability to readily adjust to a changing structural context so as to give mutated cells a chance to reproduce, thereby encouraging tumor genesis. The Rb and p53 proteins, which are regulated by the two products of the Ink4a-Arf locus [C.J. Sherr, The INK4a/ARF network in tumor suppression, Nat. Rev., Mol. Cell Biol. 2 (2001) 731–737], govern separable though interconnected pathways that cooperate to restrain cyclin D- and cyclin E-dependent kinases from precipitating untimely R point transit. The expression levels of the Ink4a and Arf proteins are especially sensitive to changes in cellular shape and adhesion that entirely remodel at the time when cells shift between quiescence and division. The Arf proteins further display an extremely high translational sensitivity and can activate the p53 pathway to delay R point transit, but, only when released from the nucleolus, ‘an organelle formed by the act of building a ribosome’ [T. Mélèse, Z. Xue, The nucleolus: an organelle formed by the act of building a ribosome, Curr. Opin. Cell Biol. 7 (1995) 319–324]. In this way, the Ink4a/Rb and Arf/p53 pathways emerge as key regulators of anchorage-dependent R point transit in mammalian cells and their deregulation is, indeed, a rule in human cancers. Thus, by selecting the nucleolus to mitigate cell cycle control by the Arf proteins, mammalian cells succeeded in forging a highly flexible R point enabling them to match cell division with a growth rate imposed by factors controlling nucleolar assembling, such as nutrients and adhesion. It is noteworthy that nutrient control of critical size at Start in budding yeast has been shown recently to be governed by a nucleolar protein interaction network [P. Jorgensen, J.L. Nishikawa, B.-J. Breitkreutz, M. Tyers, Systematic identification of pathways that couple cell growth and division in yeast, Science 297 (2002) 395–400].     

The intron of tRNA-TrpCCA is dispensable for growth and translation of Saccharomyces cerevisiae

A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3' to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of (35)S-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.     

Nonsense and missense translational suppression in plant cells mediated by tRNALys

A nuclear tRNA^Lys gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA^Lys and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA _CUA ^Lys from non-replicative vectors promoted 10–40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA^Lys suppressor genes from a geminivirus vector capable of replication promoted 30–80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons.     

Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.     

第一类肽链释放因子研究进展

第一类释放因子是新生肽链释放所必需的因子,它能正确地识别终止信号,水解肽酰-tRNA酯键,释放出新合成的多肽链.它的高级结构与tRNA结构相似,从而解释了它们功能上的相似性,其氨基酸序列的高度保守区GGQ motif和三肽反密码子区在新生肽链的释放中分别执行重要的功能.     

血管生成素:RNA代谢过程中重要的核糖核酸酶

血管生成素是核糖核酸酶A超家族成员之一,具有较弱的核糖核酸酶活性.最新研究发现,血管生成素参与细胞内多种RNA的代谢过程.在生长条件下,血管生成素可以发生核转位聚集于细胞核中,促进rRNA转录,并可参与其剪切加工,同时它也调控一系列mRNA基因的转录,最终促进细胞的生长和增殖;在应激条件下,血管生成素能降解tRNA形成tiRNA,抑制细胞内整体蛋白质的翻译水平,并促进应激小体的形成,激活细胞内应激保护机制,从而促进细胞存活.此外,血管生成素还可参与非编码小RNA等RNA代谢过程.本文概述了血管生成素在RNA代谢中的作用与分子机制等方面的进展,并探讨了其在疾病发生和发展中的作用,以期开拓血管生成素的研究新思路.