Identification of host tRNAs preferentially recognized by the Plasmodium surface protein tRip.

Malaria is a life-threatening and devastating parasitic disease. Our previous work showed that parasite development requires the import of exogenous transfer RNAs (tRNAs), which represents a novel and unique form of host-pathogen interaction, as well as a potentially druggable target. This import is mediated by tRip (tRNA import protein), a membrane protein located on the parasite surface.

The nature of the purine at position 34 in tRNAs of 4-codon boxes is correlated with nucleotides at positions 32 and 38 to maintain decoding fidelity.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix.

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions.

Pleiotropic Roles of Non-Coding RNAs in TGF-β-Mediated Epithelial-Mesenchymal Transition and Their Functions in Tumor Progression.

Epithelial-mesenchymal transition (EMT) is a spatially- and temporally-regulated process involved in physiological and pathological transformations, such as embryonic development and tumor progression. While the role of TGF-β as an EMT-inducer has been extensively documented, the molecular mechanisms regulating this transition and their implications in tumor metastasis are still subjects of intensive debates and investigations. TGF-β regulates EMT through both transcriptional and post-transcriptional mechanisms, and recent advances underline the critical roles of non-coding RNAs in these processes.

SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels.

Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E.

The Enzymatic Paradox of Yeast Arginyl-tRNA Synthetase: Exclusive Arginine Transfer Controlled by a Flexible Mechanism of tRNA Recognition.

Identity determinants are essential for the accurate recognition of transfer RNAs by aminoacyl-tRNA synthetases. To date, arginine determinants in the yeast Saccharomyces cerevisiae have been identified exclusively in vitro and only on a limited number of tRNA Arginine isoacceptors. In the current study, we favor a full cellular approach and expand the investigation of arginine determinants to all four tRNA Arg isoacceptors.

A dual fluorescent reporter for the investigation of methionine mistranslation in live cells.

In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells.

MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification.

Regulation of translation dynamic and neoplastic conversion by tRNA and their pieces.

Research on transfer RNA (tRNA) has gone a long way since the existence of this essential adapter of the genetic code was first hypothesized five decades ago. With the new and fascinating discovering of connections between tRNAs and cellular pathways beyond genetic translation, the field of tRNA research has reached a new era. Here, we review some aspects of the emerging variety of tasks performed by full length tRNAs as well as their fragments generated by specific nuclease cleavage.

Distinct functions of erythropoietin and stem cell factor are linked to activation of mTOR kinase signaling pathway in human erythroid progenitors.

Erythropoietin (EPO) and Stem Cell Factor (SCF) have partially distinct functions in erythroid cell development. The primary functions of EPO are to prevent apoptosis and promote differentiation, with a minor role as a mitogen. On the other hand SCF acts primarily as a mitogenic factor promoting erythroid cell proliferation with a minor role in inhibition of apoptosis.

Anti-tumor effects of an engineered "killer" transfer RNA.

A hallmark of cancer cells is their ability to continuously divide; and rapid proliferation requires increased protein translation. Elevating levels of misfolded proteins can elicit growth arrest due to ER stress and decreased global translation. Failure to correct prolonged ER stress eventually results in cell death via apoptosis.

Functional analysis of human tRNA isodecoders.

tRNA isodecoders share the same anticodon but have differences in their body sequence. An unexpected result from genome sequencing projects is the identification of a large number of tRNA isodecoder genes in mammalian genomes. In the reference human genome, more than 270 isodecoder genes are present among the approximately 450 tRNA genes distributed among 49 isoacceptor families.

Chimeric tRNAs as tools to induce proteome damage and identify components of stress responses.

Misfolded proteins are caused by genomic mutations, aberrant splicing events, translation errors or environmental factors. The accumulation of misfolded proteins is a phenomenon connected to several human disorders, and is managed by stress responses specific to the cellular compartments being affected. In wild-type cells these mechanisms of stress response can be experimentally induced by expressing recombinant misfolded proteins or by incubating cells with large concentrations of amino acid analogues.

tRNA over-expression in breast cancer and functional consequences.

Increased proliferation and elevated levels of protein synthesis are characteristics of transformed and tumor cells. Though components of the translation machinery are often misregulated in cancers, what role tRNA plays in cancer cells has not been explored. We compare genome-wide tRNA expression in cancer-derived versus non-cancer-derived breast cell lines, as well as tRNA expression in breast tumors versus normal breast tissues.

Solid-phase combinatorial synthesis of a lysyl-tRNA synthetase (LysRS) inhibitory library.

The solid-phase combinatorial synthesis of a new library with potential inhibitory activity against the cytoplasmic lysyl-tRNA synthetase (LysRS) isoform of Trypanosoma brucei is described. The library has been specifically designed to mimic the lysyl adenylate complex. The design was carried out by dividing the complex into four modular parts.

Not just because it is there: aminoacyl-tRNA synthetases gain control of the cell.

In a recent issue of Molecular Cell, Jia et al. (2008) demonstrate that time-controlled repression of interferon-induced transcripts depends upon the interaction between an RNA structure in these transcripts and protein domains harbored by a mammalian aminoacyl-tRNA synthetase.

An operational RNA code for faithful assignment of AUG triplets to methionine.

The assignment of AUG codons to methionine remains a central question of the evolution of the genetic code. We have unveiled a strategy for the discrimination among tRNAs containing CAU (AUG-decoding) anticodons. Mycoplasma penetrans methionyl-tRNA synthetase can directly differentiate between tRNA(Ile)(CAU) and tRNA(Met)(CAU) transcripts (a recognition normally achieved through the modification of anticodon bases).

Trypanosoma seryl-tRNA synthetase is a metazoan-like enzyme with high affinity for tRNASec.

Trypanosomatids are important human pathogens that form a basal branch of eukaryotes. Their evolutionary history is still unclear as are many aspects of their molecular biology. Here we characterize essential components required for the incorporation of serine and selenocysteine into the proteome of Trypanosoma.

Regulation of RNA function by aminoacylation and editing?

The chemical modification of nucleic acids is a ubiquitous phenomenon. Aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a reaction essentially devoted to protein synthesis but it is used also as an emergency mechanism to recycle stalled ribosomes, and it is required for genome replication in some RNA viruses. In several aminoacyl-tRNA synthetases a correction mechanism known as editing is present to prevent aminoacylation errors.

A yeast arginine specific tRNA is a remnant aspartate acceptor.

High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems.

Mutation and evolution of the magnesium-binding site of a class II aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases contain one or three Mg(2+) ions in their catalytic sites. In addition to their role in ATP binding, these ions are presumed to play a role in catalysis by increasing the electropositivity of the alpha-phosphate and stabilizing the pentavalent transition state. In the class II aaRS, two highly conserved carboxylate residues have been shown to participate with Mg(2+) ions in binding and coordination.

Limited set of amino acid residues in a class Ia aminoacyl-tRNA synthetase is crucial for tRNA binding.

The aim of this work was to characterize crucial amino acids for the aminoacylation of tRNA(Arg) by yeast arginyl-tRNA synthetase. Alanine mutagenesis was used to probe all the side chain mediated interactions that occur between tRNA(Arg2)(ICG) and ArgRS. The effects of the substitutions were analyzed in vivo in an ArgRS-knockout strain and in vitro by measuring the aminoacylation efficiencies for two distinct tRNA(Arg) isoacceptors.

A yeast knockout strain to discriminate between active and inactive tRNA molecules.

Here we report the construction of a yeast genetic screen designed to identify essential residues in tRNA(Arg). The system consists of a tRNA(Arg) knockout strain and a set of vectors designed to rescue and select for variants of tRNA(Arg). By plasmid shuffling we selected inactive tRNA mutants that were further analyzed by northern blotting.