October 29th
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, Volegova et al. investigate the specific role of the helix-loop-helix motif in the eIF3A subunit, and its impact on selective translation. Later, we take a dive into the C9ORF72 repeat expansion, with two papers detailing its impact on translation. First, Latallo et al. reveal in the workings of this expansion on translation at a single-molecule level not seen before in this area. Finally, Malnar Črnigoj et al. uncover an aminoacyl tRNA synthetase specific dysregulation seen in the presence of these expansions.
The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs
PLOS One, 2023
Volegova, M.P., Hermosillo, C. and Cate, J.H
eIF3 is possibly the most complex initiation factor in mammals, consisting of up to 13 subunits. Typically thought to act as a structural backbone to the initiation complex, it also has roles in translation under stress conditions, specific cellular mRNA translation, and IRES-based translation initiation. A key element conducive to such IRES-based translation is the N-terminal helix-loop-helix (HLH) motif of eIF3A, crucial for the translation of HCV mRNA. However, the exact mechanisms underlying the specificity of the cellular mRNA translation seen with this subunit still remain elusive. Here, the authors aim to uncover such mechanisms, utilising the technique of ribosome profiling, among others.
Using a HLH mutated version of eIF3A (eIF3A HLH*) in conjunction with ribosome profiling revealed differential translation efficiency on nearly 170 transcripts, with an enrichment of genes relating to proliferation, such as MYC and MET. Later, using a fractionated in vitro translation system incorporating the eIF3A HLH* mutant, it was observed that the eIF3A and eIF5B distribution was disturbed compared to controls, suggesting the HLH structure is crucial for both these factors associated with the scanning ribosome. Furthermore, MYC mRNA-enhanced lysates displayed abnormal eIF4A incorporation, suggesting the HLH structure is also important for stable integration of this factor with regard to eIF4A-dependent transcripts.
Single-molecule imaging reveals distinct elongation and frameshifting dynamics between frames of expanded RNA repeats in C9ORF72-ALS/FTD
Nature Communications, 2023
Latallo, M.J., Wang, S., Dong, D., Nelson, B., Livingston, N.M., Wu, R., Zhao, N., Stasevich, T.J., Bassik, M.C., Sun, S. and Wu, B.
The most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide (GGGGCC) repeat expansion within the C9ORF72 gene, resulting in the expression of toxic dipeptide repeat (DPR) proteins, such as poly-GR, poly-PR, poly-GA, and poly-GP. Overall DPR synthesis is typically determined through the dynamics of initiation and elongation. Individual initiation factors (such as eIF2A, eIF2D, and eIF4B) have been shown to influence RAN (repeat-associated non-AUG) translation. Other elements, such as strong secondary structure, optimal codon usage, and aberrant ribosomal frameshifting, are suggested to play a role. Here, the authors examine much of these factors at a single-molecule level, allowing insights into the heterogeneity of DPR translation that would not be available with more traditional ensemble methodologies.
Using the SunTag imaging system, it was demonstrated that frameshifting is comparatively rare, occurring only in 15% of individual mRNAs observed. Frameshifting from the GR-encoding to the GA-encoding reading frame was the most common of the conditions tested, at nearly 11% efficiency. Elongation in the GR-encoding reading frame was also found to be the slowest, followed by the GP-associated frame. These slower speeds were suggested to encourage the higher GR-to-GA frameshifting efficiency. Utilising CRISPR-Cas9 knockout screenings, ZNF598 and Pelota were identified as genes which influenced GR DPR levels. Both are involved in ribosome-associated quality control.
Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA
Nature Communications, 2023
Malnar Črnigoj, M., Čerček, U., Yin, X., Ho, M.T., Repic Lampret, B., Neumann, M., Hermann, A., Rouleau, G., Suter, B., Mayr, M. and Rogelj, B.,
Expanded hexanucleotide repeats (GGGGCC) in the C9ORF72 gene are known to be associated with ALS and FTD. Transcriptionally, these repeats are found in both sense and antisense RNA, which both interact and can sequester a number of RNA-binding proteins, possibly resulting in an overall loss of function. Phenylalanine-tRNA aminoacylation is carried out by the tRNA synthetase FARS, and recent evidence increasingly implicates a range of aminoacyl tRNA synthetases (ARSs) in a range of neurodegenerative diseases. In preliminary experiments done by this group, FARS was identified as one of the major proteins binding to the above repeats, and was thus investigated further.
The same experiments by this group revealed that interactions with other ARSs was lacking, indicating a specificity of GGGGCC repeat-FARS binding. Correspondingly, the presence of such repeats was associated with lower phenylalanine tRNA aminoacylation. Later analysis using click chemistry showed that when HEK293T cells were transfected with C9ORF72 repeats, there was a 20% reduction in the levels of phenylalanine incorporation into the proteome. Furthermore, Western blots of C9ORF72 patient-derived cells revealed a lower expression of phenylalanine-rich proteins, while those of control proteins remained unchanged.