metabolic-labeling-and-profiling-of-transfer-rnas-using-macroarrays

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 ...

College of Charleston

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. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.

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

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. More precisely, this work scrutinizes the relevance of the tRNA nucleotides at position 20, 35 and 36 in the yeast arginylation reaction. We built 21 mutants by site-directed mutagenesis and tested their functionality in YAL5, a previously engineered yeast knockout deficient for the expression of tRNA Arg CCG. Arginylation levels were also monitored using Northern blot. Our data collected in vivo correlate with previous observations. C35 is the prominent arginine determinant followed by G36 or U36 (G/U36). In addition, although there is no major arginine determinant in the D loop, the recognition of tRNA Arg ICG relies to some extent on the nucleotide at position 20. This work refines the existing model for tRNA Arg recognition. Our observations indicate that yeast Arginyl-tRNA synthetase (yArgRS) relies on distinct mechanisms to aminoacylate the four isoacceptors. Finally, according to our refined model, yArgRS is able to accommodate tRNA Arg scaffolds presenting N34, C/G35 and G/A/U36 anticodons while maintaining specificity. We discuss the mechanistic and potential physiological implications of these findings.

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

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. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.

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

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. Although microRNAs and lncRNAs have been clearly identified as effectors of TGF-β-mediated EMT, the contributions of other atypical non-coding RNA species, such as piRNAs, snRNAs, snoRNAs, circRNAs, and even housekeeping tRNAs, have only been suggested and remain largely elusive. This review discusses the current literature including the most recent reports emphasizing the regulatory functions of non-coding RNA in TGF-β-mediated EMT, provides original experimental evidence, and advocates in general for a broader approach in the quest of new regulatory RNAs.

Article	Year
MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases. PloS one\| PubMed ID: 26067673	2015
The Enzymatic Paradox of Yeast Arginyl-tRNA Synthetase: Exclusive Arginine Transfer Controlled by a Flexible Mechanism of tRNA Recognition. PloS one\| PubMed ID: 26844776	2016
SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels. PloS one\| PubMed ID: 28545122	2017
Pleiotropic Roles of Non-Coding RNAs in TGF-β-Mediated Epithelial-Mesenchymal Transition and Their Functions in Tumor Progression. Cancers\| PubMed ID: 28671581	2017

Renaud Geslain

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