Modifications in the T arm of tRNA globally determine tRNA maturation, function, and cellular fitness.

Almost all elongator tRNAs (Transfer RNAs) harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, generated by the enzymes TrmA and TruB, respectively, in Escherichia coli. TrmA and TruB both act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wild type. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modifications such as acp3U47. While overall protein synthesis is not affected in ΔtrmA and ΔtruB strains, the translation of a subset of codons is significantly impaired. As a consequence, we observe translationally reduced expression of many specific proteins, that are either encoded with a high frequency of these codons or that are large proteins. The resulting proteome changes are not related to a specific growth phenotype, but overall cellular fitness is impaired upon deleting trmA and truB in accordance with a general protein synthesis impact. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness irrespective of the growth conditions which explains the conservation of trmA and truB.

Profiling Selective Packaging of Host RNA and Viral RNA Modification in SARS-CoV-2 Viral Preparations.

Viruses package host RNAs in their virions which are associated with a range of functions in the viral life cycle. Previous transcriptomic profiling of host RNA packaging mostly focused on retroviruses. Which host RNAs are packaged in other viruses at the transcriptome level has not been thoroughly examined. Here we perform proof-of-concept studies using both small RNA and large RNA sequencing of six different SARS-CoV-2 viral isolates grown on VeroE6 cells to profile host RNAs present in cell free viral preparations and to explore SARS-CoV-2 genomic RNA modifications. We find selective enrichment of specific host transfer RNAs (tRNAs), tRNA fragments and signal recognition particle (SRP) RNA in SARS-CoV-2 viral preparations. Different viral preparations contain the same set of host RNAs, suggesting a common mechanism of packaging. We estimate that a single SARS-CoV-2 particle likely contains up to one SRP RNA and four tRNA molecules. We identify tRNA modification differences between the tRNAs present in viral preparations and those in the uninfected VeroE6 host cells. Furthermore, we find uncharacterized candidate modifications in the SARS-CoV-2 genomic RNA. Our results reveal an under-studied aspect of viral-host interactions that may be explored for viral therapeutics.