Diversity and roles of (t)RNA ligases

The discovery of discontiguous tRNA genes triggered studies dissecting the process of tRNA splicing. As a result, we have gained detailed mechanistic knowledge on enzymatic removal of tRNA introns catalyzed by endonuclease and ligase proteins. In addition to the elucidation of tRNA processing, these studies facilitated the discovery of additional functions of RNA ligases such as RNA repair and non-conventional mRNA splicing events. Recently, the identification of a new type of RNA ligases in bacteria, archaea, and humans closed a long-standing gap in the field of tRNA processing. This review summarizes past and recent findings in the field of tRNA splicing with a focus on RNA ligation as it preferentially occurs in archaea and humans. In addition to providing an integrated view of the types and phyletic distribution of RNA ligase proteins known to date, this survey also aims at highlighting known and potential accessory biological functions of RNA ligases.     

The accuracy of aminoacylation--ensuring the fidelity of the genetic code

The fidelity of protein biosynthesis rests not only on the proper interaction of the messenger RNA codon with the anticodon of the tRNA, but also on the correct attachment of amino acids to their corresponding (cognate) transfer RNA (tRNA) species. This process is catalyzed by the aminoacyl-tRNA synthetases which discriminate with remarkable selectivity amongst many structurally similar tRNAs. The basis for this highly specific recognition of tRNA by these enzymes (also referred to as 'tRNA identity') is currently being elucidated by genetic, biochemical and biophysical techniques. At least two factors are important in determining the accuracy of aminoacylation: a) 'identity elements' in tRNA denote nucleotides in certain positions crucial for protein interactions determining specificity, and b) the occurrence in vivo of competition between synthetases for a particular tRNA which may have ambiguous identity.     

Misacylation of tRNA in prokaryotes: a re-evaluation

Misacylation of tRNA by a non-cognate amino acid is a natural phenomenon and occurs with a frequency of approximately 1 in 10,000 due to occasional mistakes in aminoacyl transfer RNA (tRNA) synthesis. In a number of prokaryotic organisms, misacylation of selenocysteinyl tRNA, glutaminyl tRNA and aspartyl tRNAs has particular physiological meaning. Recently, misacylation has emerged as a powerful tool for studying specific interactions between aa-tRNAs and associated protein factors. The present review provides an overview of the application of misacylated tRNA in research. Received 27 April 2005; received after revision 2 November 2005; accepted 5 December 2005     

Suppression and the code: beyond codons and anticodons

Specificity and accuracy in the decoding of genetic information during mRNA-programmed, ribosome-dependent polypeptide synthesis (translation) involves more than just hydrogen bonding between two anti-parallel trinucleotides, the mRNA codon and the tRNA anticodon. Other macromolecules are also involved, and translational suppression has been and continues to be an appropriate and effective way to identify them, as well as other parts of mRNA and tRNA, and to elucidate the structural determinants of their functions and interactions. Experimental results are presented that bear upon codon context effects, the role of tRNA structural features in aminoacyl-tRNA selection and in codon selection (reading-frame maintenance), determinants of tRNA identity, elongation factor suppressor mutants, and termination codon recognition by the ribosomal RNA of the small subunit. The examples presented illustrate the complexity of the decoding process and the interconnectedness of translational macromolecules in achieving specificity and accuracy in polypeptide synthesis.     

Altered or increased transfer-RNA methylation in the course of Interferon action on cells in culture?

The induction of the antiviral state by Interferon might reflect the decrease of the rate of biosynthesis, the degradation or the alteration of one or several tRNAs. This could result in rate-limiting concentrations for codons common in viral RNA but rare in host mRNA. Altered methylation of tRNA could be the basis of such a phenomenon. However, we could not find an altered extent of methylation of total tRNA or an altered pattern of methylation, if mixed tRNAs were chromatographed on MAK- or BD-cellulose columns, despite a large range of conditions of pretreatment of chick embryo fibroblast cultures with interferon.     

The E-site story: the importance of maintaining two tRNAs on the ribosome during protein synthesis

In the sixties James Watson suggested a twosite model for the ribosome comprising the P site for the peptidyl transfer RNA (tRNA) before peptide-bond formation and the A site, where decoding takes place according to the codon exposed there. In the eighties a third tRNA binding site was detected, the E site, which was specific for deacylated tRNA and turned out to be a universal feature of ribosomes. However, despite having three tRNA binding sites, only two tRNAs occupy the ribosome at a time during protein synthesis: at the A and P sites before translocation (PRE state) and at the P and E sites after translocation (POST state). The importance of having two tRNAs in the POST state has been revealed during the last 25 years, showing that the E site contributes two fundamental features: (i) the fact that incorporation of a wrong amino acid is not harmful for the cell (only 1 in about 400 misincorporations destroys the function of a protein) stems from the presence of an E-tRNA; (ii) maintenance of the reading frame is one of the most remarkable achievements of the ribosome, essential for faithful translation of the genetic information. The presence of the POST state E-tRNA prevents loss of the reading frame. Received 14 March 2006; received after revision 8 June 2006; accepted 4 August 2006     

Altered or increased transfer-RNA methylation in the course of Interferon action on cells in culture?

Summary The induction of the antiviral state by Interferon might reflect the decrease of the rate of biosynthesis, the degradation or the alteration of one or several tRNAs. This could result in rate-limiting concentrations for codons common in viral RNA but rare in host mRNA. Altered methylation of tRNA could be the basis of such a phenomenon. However, we could not find an altered extent of methylation of total tRNA or an altered pattern of methylation, if mixed tRNAs were chromatographed on MAK- or BD-cellulose columns, despite a large range of conditions of pretreatment of chick embryo fibroblast cultures with interferon.Work supported by the Swiss National Science Foundation, grants 3.1050 and 3.540.     

4-Thiouridine and photoprotection in Escherichia coli K 12]

A high level of protection is observed in the Escherichia coli K 12 strain AB 1157 rec A 1 nuv+ whose transfer RNA contains 4-thiouridine. In contrast, the photoprotection level is low and observed at higher doses in a strain which differs from the former by a single mutation, nuv-, (lack of 4-thiouridine). This nucleoside is therefore an important chromophore leading to photoprotection. This conclusion is corroborated by the similarity of the action spectra for 8-13 link formation in tRNA and for photoprotection.     

Dictyostelium mobile elements: strategies to amplify in a compact genome

Dictyostelium discoideum is a eukaryotic microorganism that is attractive for the study of fundamental biological phenomena such as cell-cell communication, formation of multicellularity, cell differentiation and morphogenesis. Large-scale sequencing of the D. discoideum genome has provided new insights into evolutionary strategies evolved by transposable elements (TEs) to settle in compact microbial genomes and to maintain active populations over evolutionary time. The high gene density (about 1 gene/2.6 kb) of the D. discoideum genome leaves limited space for selfish molecular invaders to move and amplify without causing deleterious mutations that eradicate their host. Targeting of transfer RNA (tRNA) gene loci appears to be a generally successful strategy for TEs residing in compact genomes to insert away from coding regions. In D. discoideum, tRNA gene-targeted retrotransposition has evolved independently at least three times by both non-long termina l repeat (LTR) retrotransposons and retrovirus-like LTR retrotransposons. Unlike the nonspecifically inserting D. discoideum TEs, which have a strong tendency to insert into preexisting TE copies and form large and complex clusters near the ends of chromosomes, the tRNA gene-targeted retrotransposons have managed to occupy 75% of the tRNA gene loci spread on chromosome 2 and represent 80% of the TEs recognized on the assembled central 6.5-Mb part of chromosome 2. In this review we update the available information about D. discoideum TEs which emerges both from previous work and current large-scale genome sequencing, with special emphasis on the fact that tRNA genes are principal determinants of retrotransposon insertions into the D. discoideum genome. Received 10 May 2002; received after revision 10 June 2002; accepted 12 June 2002 RID="*" ID="*"Corresponding author.     

RNA-splicing endonuclease structure and function

The RNA-splicing endonuclease is an evolutionarily conserved enzyme responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs. Since its first identification from yeast in the late 1970s, significant progress has been made toward understanding the biochemical mechanisms of this enzyme. Four families of the splicing endonucleases possessing the same active sites and overall architecture but with different subunit compositions have been identified. Two related consensus structures of the precursor RNA splice sites and the critical elements required for intron excision have been established. More recently, a glimpse was obtained of the structural mechanism by which the endonuclease recognizes the consensus RNA structures and cleaves at the splice sites. This review summarizes these findings and discusses their implications in the evolution of intron removal processes. Received 24 August 2007; received after revision 24 November 2007; accepted 27 November 2007     

tRNA nucleotidyltransferases: ancient catalysts with an unusual mechanism of polymerization

RNA polymerases are important enzymes involved in the realization of the genetic information encoded in the genome. Thereby, DNA sequences are used as templates to synthesize all types of RNA. Besides these classical polymerases, there exists another group of RNA polymerizing enzymes that do not depend on nucleic acid templates. Among those, tRNA nucleotidyltransferases show remarkable and unique features. These enzymes add the nucleotide triplet C–C–A to the 3′-end of tRNAs at an astonishing fidelity and are described as “CCA-adding enzymes”. During this incorporation of exactly three nucleotides, the enzymes have to switch from CTP to ATP specificity. How these tasks are fulfilled by rather simple and small enzymes without the help of a nucleic acid template is a fascinating research area. Surprising results of biochemical and structural studies allow scientists to understand at least some of the mechanistic principles of the unique polymerization mode of these highly unusual enzymes.     

Oxidative damage to RNA: mechanisms, consequences, and diseases

Overproduction of free radicals can damage cellular components resulting in progressive physiological dysfunction, which has been implicated in many human diseases. Oxidative damage to RNA received little attention until the past decade. Recent studies indicate that RNA, such as messenger RNA and ribosomal RNA, is very vulnerable to oxidative damage. RNA oxidation is not a consequence of dying cells but an early event involved in pathogenesis. Oxidative modification to RNA results in disturbance of the translational process and impairment of protein synthesis, which can cause cell deterioration or even cell death. In this review, we discuss the mechanisms of oxidative damage to RNA and the possible biological consequences of damaged RNA. Furthermore, we review recent evidence suggesting that oxidative damage to RNA may contribute to progression of many human diseases.     

Novel features in the tRNA-like world of plant viral RNAs

tRNA-like domains are found at the 3' end of genomic RNAs of several genera of plant viral RNAs. Three groups of tRNA mimics have been characterized on the basis of their aminoacylation identity (valine, histidine and tyrosine) for aminoacyl-tRNA synthetases. Folding of these domains deviates from the canonical tRNA cloverleaf. The closest sequence similarities with tRNA are those found in valine accepting structures from tymoviruses (e.g. TYMV). All the viral tRNA mimics present a pseudoknotted amino acid accepting stem, which confers special structural and functional characteristics. In this review emphasis is given to newly discovered tRNA-like structures (e.g. in furoviruses) and to recent advances in the understanding of their three-dimensional architecture, which mimics L-shaped tRNA. Identity determinants in tRNA-like domains for aminoacylation are described, and evidence for their functional expression, as in tRNAs, is given. Properties of engineered tRNA-like domains are discussed, and other functional mimicries with tRNA are described (e.g. interaction with elongation factors and tRNA maturation enzymes). A final section reviews the biological role of the tRNA-like domains in amplification of viral genomes. In this process, in which the mechanisms can vary in specificity and efficiency according to the viral genus, function can be dependent on the aminoacylation properties of the tRNA-like domains and/or on structural properties within or outside these domains.     

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.     

RNA recognition by double-stranded RNA binding domains: a matter of shape and sequence

The double-stranded RNA binding domain (dsRBD) is a small protein domain of 65–70 amino acids adopting an αβββα fold, whose central property is to bind to double-stranded RNA (dsRNA). This domain is present in proteins implicated in many aspects of cellular life, including antiviral response, RNA editing, RNA processing, RNA transport and, last but not least, RNA silencing. Even though proteins containing dsRBDs can bind to very specific dsRNA targets in vivo, the binding of dsRBDs to dsRNA is commonly believed to be shape-dependent rather than sequence-specific. Interestingly, recent structural information on dsRNA recognition by dsRBDs opens the possibility that this domain performs a direct readout of RNA sequence in the minor groove, allowing a global reconsideration of the principles describing dsRNA recognition by dsRBDs. We review in this article the current structural and molecular knowledge on dsRBDs, emphasizing the intricate relationship between the amino acid sequence, the structure of the domain and its RNA recognition capacity. We especially focus on the molecular determinants of dsRNA recognition and describe how sequence discrimination can be achieved by this type of domain.     

The structure and function of initiation factors in eukaryotic protein synthesis

Protein synthesis is one of the most complex cellular processes, involving numerous translation components that interact in multiple sequential steps. The most complex stage in protein synthesis is the initiation process. It involves initiation factor-mediated assembly of a 40S ribosomal subunit and initiator tRNA into a 48S initiation complex at the initiation codon of an mRNA and subsequent joining of a 60S ribosomal subunit to form a translationally active 80S ribosome. The basal set of factors required for translation initiation has been determined, and biochemical, genetic, and structural studies are now beginning to reveal details of their individual functions in this process. The mechanism of translation initiation has also been found to be influenced significantly by structural properties of the 5' and 3' termini of individual mRNAs. This review describes some of the major developments in elucidating molecular details of the mechanism of initiation that have occurred over the last decade.