September 24th

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, Müller et al. investigate the mechanisms of translational quality control within the context of stop codon readthrough, while later on in the piece, Pandit et al. similarly investigate such readthrough, describing a novel case seen in the NNAT gene. Sandwiched between these two articles, we take a look at elongation, with Cirzi et al. delving into the role of the queuosine modification in altering elongation speed, with knock-on effects for learning and memory.

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Cell, 2023

Müller, M.B., Kasturi, P., Jayaraj, G.G. and Hartl, F.U.

Eukaryotic cells possess complex proteostasis mechanisms to ensure against the build-up of aberrant proteins. A reduction in translational fidelity is seen in older age groups, as well as increased levels of stop codon readthrough, particularly in neurons. Failure of efficient stop codon termination results in C-terminal extended (CTE) proteins. While readthrough into poly A tails is known to activate the ribosome quality control pathway, most readthrough products are unlikely to reach this mRNA region, instead having a much higher chance of encountering an additional termination codon. Thus, other mechanisms are likely to be involved in the degradation of abberant CTE proteins. Here, the authors aim to investigate such mechanisms.

Using fluorescence-based methods, they replicated a CTE-mediated reduction in expression, and linked it to proteosome degradation. The CTE interactome revealed the BAG6 chaperone complex as being highly enriched, as compared to non-readthrough controls, and that such a complex displays preference for hydrophobic CTEs. Furthermore, in addition to protein degradation, this study found that the mRNAs, once translated in their 3’ UTR regions, also displayed increased decay. This was linked to GCN1, a factor which detects ribosome collisions at non-optimal codons in 3’UTRs as revealed by selective ribosome profiling of monosomes and disomes. GCN1 itself recruits the CCR4/NOT mRNA decay complex to reduce the accumulation of undesirable readthrough products.

Queuosine-tRNA promotes sex-dependent learning and memory formation by maintaining codon-biased translation elongation speed

The EMBO Journal, 2023

Cirzi, C., Dyckow, J., Legrand, C., Schott, J., Guo, W., Hernandez, D. P., Hisaoka, M., Parlato, R., Pitzer, C., van der Hoeven, F., Dittmar, G., Helm, M., Stoecklin, G., Schirmer, L., Lyko, F., and Tuorto, F.

Translation elongation proceeds via tRNA-mRNA interactions, according to codon-anticodon base pairing. While this is typically a process of high fidelity, tRNA anticodons can sometimes match with more than one codon, due to the geometry of the “wobble” position (i.e., the position corresponding to the match between the third nucleotide of the codon and first nucleotide of the anticodon). This position 34 in the tRNA is frequently modified, one of which is the insertion of queuosine (Q), a derivative of queuine, only found in bacteria. Such a modification has previously been shown to impact on elongation speed, and Q deficiency has also been linked to a raft of neurological disorders, including Parkinson’s and schizophrenia. Here, the authors aim to investigate its role in translation further.

They demonstrate high levels of Q modification in the heart, brain, and skeletal muscle. However, the distribution of QTRT1, the enzyme responsible for this modification, only partially correlates with such a modification profile, suggesting other factors may be involved. In a QTRT1 knockout murine model, female mice exhibited comparatively increased levels of hyperactivity and learning/memory impairment vs male mice, as compared to their wild-type counterparts. Translationally, QTRT1 knockout mice increased ribosome occupancy on classically Q-decoded codons, such as AAU, AAC, GAU, GAC, and CAU. AU-rich codons also broadly seemed to be decoded at a slower rate in the absence of QTRT1. Furthermore, female mice displayed significantly more differentially regulated genes compared to their male counterparts (327 vs 92). Gene ontology analysis revealed an enrichment of these genes in the categories of protein synthesis, ribosome metabolism, and nonsense-mediated mRNA decay.

Termination codon readthrough of NNAT mRNA regulates calcium-mediated neuronal differentiation

Journal of Biological Chemistry, 2023

Pandit, M., Akhtar, M.N., Sundaram, S., Sahoo, S., Manjunath, L.E. and Eswarappa, S.M

NNAT is a maternally imprinted gene, with expression in endocrine, adipose, and neural tissues, particularly so in fetal brains. It functions to induce the neural lineage in embryonic stem cells, by inhibiting the calcium pump SERCA2, thus increasing cytoplasmic calcium concentrations. However, it also functions in glucose metabolism, being implicated in insulin secretion, as well as obesity. The gene itself is known to have at least two isoforms, formed as a result of alternative splicing. However, here, the authors describe a novel isoform, produced through the process of termination codon readthrough (TCR), resulting in a C-terminally extended isoform (termed NNATx), and investigated its function.

Evolutionary conservation at the nucleotide and amino acid level for the readthrough region in the 3’ UTR of the NNAT canonical mRNA suggested that the readthrough region of NNAT may be utilised translationally. Using fluorescence-based reporters, it was demonstrated that TCR does indeed occur, and requires at least the first 90 nucleotides of the 3’ UTR to take place. Additionally, a custom antibody localising to the C-terminally extended region confirmed the endogenous existence of this isoform. Later, using RBPsuite, a web tool designed to identify binding proteins to a particular RNA sequence, NONO was suggested as a binding factor to the first 100 nucleotides of the 3’ UTR of NNAT, and later demonstrated to reduce levels of TCR, and subsequently, expression levels of NNATx. NNATx, unlike NNAT, does not increase cytoplasmic calcium levels, nor induce neuronal differentiation, suggestive that TCR in this instance negatively regulates the function of the normal length isoform.

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