June 30th, 2024

Recent Publications Harnessing the Power of Translatomics.

Every week we provide a digest of a small number of recent interesting papers in the field of translatomics.

In this week’s Sunday papers, Awad S et al. find that longer genes in S. cerevisiae have lower ribosome drop-off rates, while Rocha AL et al. characterise the function of the uORF-encoded microprotein of SLC35A4 and Krueger J et al. show that GidA-dependent tRNA modifications in P. aeruginosa drive translation of transcripts containing rare codons and modulates expression of genes encoding virulence regulators.

A data-driven estimation of the ribosome drop-off rate in S. cerevisiae reveals a correlation with the genes length

NAR Genomics and Bioinformatics, 2024

Awad S, Valleriani A and Chiarugi D.

Several factors influence protein synthesis efficiency, including ribosome drop-off – the premature separation of ribosomes from the mRNA template. In this study the ‘Ribofolio’ tool was used to provide a data-driven estimation of ribosome drop-off rates in S. cerevisiae, analysing ribosome profiling data under both standard and stress conditions.

The author’s analysis revealed that under normal conditions, the average density of the ribosomes along the CDS declines exponentially with the rate per codon being in a range of 4 × 10−4 events. This estimate is consistent with values obtained for E. coli, however, the exponential model in S. cerevisiae is a less optimal fit, suggesting a greater complexity of the dynamics of ribosome trafficking in yeast. The study suggests that global drop-off rates might be influenced by gene-specific factors, with a consistent rate observed across each gene subset. Notably, lower drop-off rates were associated with longer CDSs, hinting at potential compensatory mechanisms in sequences that reduce drop-off rates.

Further comparisons between standard and non-standard conditions, like methionine and glucose restriction, revealed significant differences in drop-off rates, suggesting environmental impacts on protein synthesis.

These insights offer valuable understanding into protein synthesis regulation, potentially aiding the optimization of protein expression vectors for gene therapy and the production of recombinant proteins.

An inner mitochondrial membrane microprotein from the SLC35A4 upstream ORF regulates cellular metabolism

Journal of Molecular Biology2024

Rocha AL, Pai V, Perkins G, Chang T, Ma J, De Souza EV, Chu Q, Vaughan JM, Diedrich JK, Ellisman MH and Saghatelian A.

uORFs are well-recognized as regulators of translation for downstream open reading frames. There has been significant interest among researchers in exploring other potential roles for uORFs beyond regulation, such as encoding functional proteins. Several instances have already been identified where microproteins produced from uORFs play critical roles, including the microprotein from MIEF1 uORF, which regulates the mitochondrial ribosome.

In their study, Rocha AL et al. focus on a microprotein encoded by the uORF of the SLC35A4 mRNA (SLC35A4-MP), employing a combination of biochemical fractionation, cell biology, and cellular imaging techniques. Previously known for its regulatory impact on SLC35A4 translation, this uORF has now been shown to have additional functional roles.

The research team used immunofluorescence and biochemical fractionation, complemented by confocal microscopy and Western blots, to pinpoint the localization of SLC35A4-MP predominantly within the mitochondrial fraction, specifically within the inner mitochondrial membrane (IMM). Intriguingly, SLC35A4-MP and SLC35A4 localise to different cellular compartments—the former in the mitochondria and the latter in the Golgi-ER compartments.

Further investigations into the functional implications of SLC35A4-MP utilised CRISPR-Cas9 knockout cell lines targeting the SLC35A4 uORF. Findings revealed that loss of SLC35A4-MP significantly reduced ATP-linked oxygen consumption rates, whereas its overexpression slightly but significantly enhanced maximal capacity rates. No consistent changes were observed in mitochondrial structure upon loss of SLC35A4-MP.

This study highlights the broader potential of hundreds of uORFs predicted to encode conserved microproteins, suggesting that further characterization of these elements could significantly advance our understanding of microprotein biology.

tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen Pseudomonas aeruginosa

Proc Natl Acad Sci USA, 2024

Krueger J, Preusse M, Oswaldo Gomez N, Frommeyer YN, Doberenz S, Lorenz A, Kordes A, Grobe S, Müsken M, Depledge DP, Svensson SL, Weiss S, Kaever V, Pich A, Sharma CM, Ignatova Z and Häussler S.


In this study, the authors combined transcriptomic, ribosome profiling, proteomic data, and virulence datasets from over 400 clinical isolates to investigate GidA-dependent carboxymethylaminomethyl modifications on specific tRNAs in Pseudomonas aeruginosa. The research highlighted that GidA enzyme activity leads to the addition of a carboxymethylaminomethyl group to the wobble uridine base of the tRNA’s anticodon.

To explore the impact of lacking this modification, the researchers utilized Nano-tRNA-seq to measure tRNA levels in both control and gidA-deficient (tngidA) strains, finding no significant overall change. However, specific tests in GFP reporters on codons that involve U-A pairing, like Arg AGA and Leu UUA, showed markedly lower fluorescence in the tngidA strain, indicating decoding issues.

Using ribosome profiling, the study assessed the broader effects of the GidA modification across all translated transcripts in wild-type and tngidA strains. It was observed that genes with one or more UUA or AGA codons had reduced translational efficiency in the tngidA strain. Despite this, variations in ribosome occupancy at these specific codons were minimal, likely due to their rare usage. However, ribosome occupancy notably decreased downstream of AGA codons.

Furthermore, employing the stable isotope labelling by amino acids in cell culture (SILAC) method, the researchers found that proteins rich in GidA-dependent codons, particularly AGA and UUA, were significantly underrepresented in the proteome of gidA-deficient cells. Many of these proteins are associated with virulence and motility, underscoring the biological significance of GidA-mediated tRNA modifications.

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