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RNAs are central to protein synthesis, with ribosomal RNA, transfer RNAs and messenger RNAs comprising the core components of the translation machinery. In addition to the four canonical bases (uracil, cytosine, adenine, and guanine) these RNAs contain an array of enzymatically incorporated chemical modifications. Transfer RNAs (tRNAs) are responsible for ferrying amino acids to the ribosome, and are among the most abundant and highly modified RNAs in the cell across all domains of life. On average, tRNA molecules contain 13 post-transcriptionally modified nucleosides that stabilize their structure and enhance function. There is an extensive chemical diversity of tRNA modifications, with over 90 distinct varieties of modifications reported within tRNA sequences. Some modifications are crucial for tRNAs to adopt their L-shaped tertiary structure, while others promote tRNA interactions with components of the protein synthesis machinery. In particular, modifications in the anticodon stem-loop (ASL), located near the site of tRNA:mRNA interaction, can play key roles in ensuring protein homeostasis and accurate translation. There is an abundance of evidence indicating the importance of ASL modifications for cellular health, and in vitro biochemical and biophysical studies suggest that individual ASL modifications can differentially influence discrete steps in the translation pathway. This review examines the molecular level consequences of tRNA ASL modifications in mRNA codon recognition and reading frame maintenance to ensure the rapid and accurate translation of proteins., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2023. Published by Elsevier Ltd.)

There is growing recognition that viral RNA genomes possess enzymatically incorporated modified nucleosides. These small chemical changes are analogous to epigenomic modifications in DNA and have the potential to be similarly important modulators of viral transcription and evolution. However, the molecular level consequences of individual sites of modification remain to be broadly explored. Here we describe an in vitro assay to examine the impact of nucleoside modifications on the rate and fidelity of SARS-CoV-2 RNA transcription. Establishing the role of modified nucleotides in SARS-CoV-2 is of interest both for advancing fundamental knowledge of RNA modifications in viruses, and because modulating the modification-landscape of SARS-CoV-2 may represent a therapeutic strategy to interfere with viral RNA replication. Our approach can be used to assess the influence both of modifications present in a template RNA, as well nucleotide analog inhibitors. These methods provide a reproducible guide for generating active SARS-CoV-2 replication/transcription complexes capable of establishing how RNA modifications influence the pre-steady state rate constants of nucleotide addition by RNA-dependent RNA polymerases., (Copyright © 2024. Published by Elsevier Inc.)

The ribosome translates the information stored in the genetic code into functional proteins. In this process messenger RNAs (mRNAs) serve as templates for the ribosome, ensuring that amino acids are linked together in the correct order. Chemical modifications to mRNA nucleosides have the potential to influence the rate and accuracy of protein synthesis. Here, we present an in vitro Escherichia coli translation system utilizing highly purified components to directly investigate the impact of mRNA modifications on the speed and accuracy of the ribosome. This system can be used to gain insights into how individual chemical modifications influence translation on the molecular level. While the fully reconstituted system described in this chapter requires a lengthy time investment to prepare experimental materials, it is highly verstaile and enables the systematic assessment of how single variables influence protein synthesis by the ribosome., (Copyright © 2021 Elsevier Inc. All rights reserved.)