Absolute Quantification of Noncoding RNA by Microscale Thermophoresis.

Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization-based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for example, single tRNA species with a turnaround time of about one hour. We developed MST to quantify the effect of tRNA toxins and of heat stress and RNA modification on single tRNA species. A comparative analysis also revealed significant differences to RNA-Seq-based quantification approaches, strongly suggesting a bias due to tRNA modifications in the latter. Further applications include the quantification of rRNA as well as of polyA levels in cellular RNA.

Transfer RNA instability as a stress response in Escherichia coli: Rapid dynamics of the tRNA pool as a function of demand.

Production of the translation apparatus of E. coli is carefully matched to the demand for protein synthesis posed by a given growth condition. For example, the fraction of RNA polymerases that transcribe rRNA and tRNA drops from 80% during rapid growth to 24% within minutes of a sudden amino acid starvation. We recently reported in Nucleic Acids Research that the tRNA pool is more dynamically regulated than previously thought. In addition to the regulation at the level of synthesis, we found that tRNAs are subject to demand-based regulation at the level of their degradation. In this point-of-view article we address the question of why this phenomenon has not previously been described. We also present data that expands on the mechanism of tRNA degradation, and we discuss the possible implications of tRNA instability for the ability of E. coli to cope with stresses that affect the translation process.

A robust method for measuring aminoacylation through tRNA-Seq.

Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge transfer RNA sequencing (tRNA-Seq) method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.

Beyond the Anticodon: tRNA Core Modifications and Their Impact on Structure, Translation and Stress Adaptation.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional chemical modifications. Approximately 100 different modifications have been identified in tRNAs, and each tRNA typically contains 5-15 modifications that are incorporated at specific sites along the tRNA sequence. These modifications may be classified into two groups according to their position in the three-dimensional tRNA structure, i.e., modifications in the tRNA core and modifications in the anticodon-loop (ACL) region. Since many modified nucleotides in the tRNA core are involved in the formation of tertiary interactions implicated in tRNA folding, these modifications are key to tRNA stability and resistance to RNA decay pathways. In comparison to the extensively studied ACL modifications, tRNA core modifications have generally received less attention, although they have been shown to play important roles beyond tRNA stability. Here, we review and place in perspective selected data on tRNA core modifications. We present their impact on tRNA structure and stability and report how these changes manifest themselves at the functional level in translation, fitness and stress adaptation.