Adamczyk, M. | bioRxiv Science

bioRxiv2024

Direct RNA Oxford Nanopore sequencing distinguishes between modifications in tRNA at the U34 position

The measurement of tRNA modifications with single transcript resolution has been feasible for only a few modifications due to the lack of available methods. This limitation does not allow to advance basic research studies on the dynamic nature of tRNA modification and its cellular function in time and space, neither to develop modern diagnostic tools for several already known tRNA-dependent human diseases. Nanopore is a well-established sequencing method that has proven to be efficient for the study of RNA. The analysis of tRNA modifications by Nanopore is still under development. We have investigated the efficacy of nanopore technology to discriminate between complex modifications of uridine 34 in tRNA, which affect the base-calling properties of neighbouring bases and are therefore difficult to accurately predict. We have developed new methods to chemically and enzymatically synthesise single modified tRNA molecules with modifications at the anticodon loop. Nanopore technology captures the features produced by uridine with and without a thiol group when present on synthetic tRNA molecules. Thus, Oxford Nanopore Technology (ONT) has great potential for developing strategies to accurately identify the modification status of the tRNA anticodon loop (ACL), which encompasses the most complex modifications on uridine-containing RNA motifs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/630739v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@4317baorg.highwire.dtl.DTLVardef@caf204org.highwire.dtl.DTLVardef@1d7249dorg.highwire.dtl.DTLVardef@1a4f8c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2018

Glycolytic flux in Saccharomyces cerevisiae is dependent on RNA polymerase III and its negative regulator Maf1

Protein biosynthesis is energetically costly, is tightly regulated and is coupled to stress conditions including glucose deprivation. RNA polymerase III (RNAP III) driven transcription of tDNA genes for production of tRNAs is a key element in efficient protein biosynthesis. Here we present an analysis of the effects of altered RNAP III activity on the Saccharomyces cerevisiae proteome and metabolism under glucose rich conditions. We show for the first time that RNAP III is tightly coupled to the glycolytic system at the molecular systems level. Decreased RNAP III activity or the absence of the RNAP III negative regulator, Maf1 elicit broad changes in the abundance profiles of enzymes engaged in fundamental metabolism in S. cerevisiae. In a mutant compromised in RNAP III activity there is a repartitioning towards amino acids synthesis de novo at the expense of glycolytic throughput. Conversely, cells lacking Maf1 protein have greater potential for glycolytic flux.

cell biology↗