March 10th, 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, Xu et al. uncover a novel functionality of rRNA modifications in selective mRNA translation in colorectal cancer, while Ren et al. hypothesise a greater role for ribosomal frameshifting in mammalian gene expression. Finally, Misra et al. delve into great detail in their exploration of the multiple pathways responsible for GCN2 activation, known for its important role in the integrated stress response.
SNORA56-mediated pseudouridylation of 28 S rRNA inhibits ferroptosis and promotes colorectal cancer proliferation by enhancing GCLC translation
Journal of Experimental & Clinical Cancer Research, 2023
Xu, C., Bian, Z., Wang, X., Niu, N., Liu, L., Xiao, Y., Zhu, J., Huang, N., Zhang, Y., Chen, Y. and Wu, Q.
Colorectal cancer (CRC) is a major health concern, with rising incidence and mortality, partially attributed to the lack of early-stage biomarkers. Small nucleolar RNAs (snoRNAs) have been previously implicated in CRC, guiding 2’-O-methylation of rRNA, thereby ultimately influencing translation. The stability of these snoRNAs gives arise to their potential as early biomarkers for this disease. In addition to this, metabolic reprogramming, particularly so with regard to ferroptosis, has been uncovered as a feature of some CRCs. Further recent studies have brought to light the involvement of long non-coding RNAs in ferroptosis, although the relationship between snoRNAs and this process is unclear. Here, the authors investigate snoRA56, a CRC-overexpressed snoRNA, and its role in ferroptosis.
They first uncover a relationship between higher snoRA56 expression and increased mortality in CRC patients, demonstrating its relevance as a target. Indeed, stable upregulation of snoRA56 significantly increased proliferative capacity. In investigating snoRA56’s impact on ribosome maturation, they found that its silencing led to increased levels of immature 28S rRNA, but no impact was noted for 18S rRNA. Using polysome profiling, the authors also showed that snoRA56 overexpression also upregulated ribosomal translational capacity. Proteomic analysis of snoRA56 knockdown cells revealed ~250 impacted proteins. KEGG functional enrichment analysis revealed that the downregulated proteins were enriched for the ribosome and proteins involved in metabolic processes associated with ferroptosis. The protein GCLC, crucial for the synthesis of glutathione, needed for ferroptosis inhibition, was specifically translationally regulated by snoRA56, with transcriptional regulation ruled out. When analysing ferroptosis with relation to snoRA56 expression, higher snoRA56 levels repeatedly improved several known ferroptosis markers, resulting in increased CRC proliferation.
Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human
Nucleic Acids Research, 2024
Ren, G., Gu, X., Zhang, L., Gong, S., Song, S., Chen, S., Chen, Z., Wang, X., Li, Z., Zhou, Y. and Li, L.
Ribosomal translation is a strictly regulated process. However, there are exceptions to the typical conventions of this form of gene expression, coming under the umbrella term of recoding. Frameshifting (FS) is probably one of the most studied forms of recoding in this regard, wherein a ribosome may slip into the +1 or -1 frame during the elongation process, resulting in novel chimeric proteins. It is more common in viral and bacterial genomes, with known cases of programmed ribosome frameshifting (PRF) in eukaryotes being comparatively rare. A number of cis- and trans-acting factors can contribute to FS, such as RNA secondary structure and particular slippery sequences. Here, the authors aim to investigate the impact of codon repeats on FS efficiency.
Initially, the phenylalanine codon UUU was investigated, given its association with slippery sites. UUU repeats of up to 4 and 5 appeared to induce significant FS in dual luciferase assays, with evidence also seen via Western blots. Extending this out to all 61 codons, the authors found 46 5-codon repeats gave statistically significant FS over control, with 13 of these giving comparatively robust FS. Later, by comparing a known proteomic database with predicted unique protein sequences downstream of possible codon repeated-associated FS sites (termed CRFS), they theorised the existence of ~450 CRFS loci. Narrowing these down to loci predicted to be widespread across multiple tissues, they focussed on HDAC1, a histone deacetylase, as a CRFS candidate. A number of assays used suggested +1 FS occurred just downstream of the canonical start codon, at a poly-tyrosine stretch. Interestingly, when this HDAC1 FS product was overexpressed, it was noted as having multiple binding partners known to interact with HDAC1. Indeed, overexpression of this product led to increased acetylation of specific histones, suggesting this novel ORF may function to regulate the process of HDAC itself.
Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions
Nucleic Acids Research, 2024
GCN2 is a kinase responsible for the phosphorylation of eIF2α, thus inducing the integrated stress response (ISR), greatly limiting generalised translational output, while inducing select stress responsive mRNAs. GCN2 is also known to be induced by a number of environmental conditions, such as UV radiation, nutrient starvation, and oxidative stress. In a more granular view, an increase of uncharged tRNAs, as well as ribosome collisions, have been found to activate GCN2. Furthermore, the protein kinase ZAK is thought to mediate at least some of these mechanisms leading to GCN2 activation. However, the precise relationship and pathways between these myriad of influences, all converging on GCN2, remains not fully elucidated. Here, the authors aim to clarify such influences.
Using wild type and ZAK knockout cell lines, it was found that ZAK itself does not mediate GCN2 activation resulting from the accumulation of uncharged tRNAs, as seen with halofuginone treatment. Using puromycin treatment (which is known to reduce ribosome collisions via release of bound mRNA), researchers were able to determine that such collisions are not responsible for GCN2 activation as a result of the accumulation of uncharged tRNAs, suggesting these tRNAs themselves are enough for robust activation of GCN2. It was determined, however, that the HARS-like domain of GCN2 is needed for its response to ribosome collisions. This domain was also shown to bind uncharged tRNAPro. When investigating the impact of UV radiation on GCN2, it was found that, contrary to previous reports, that ZAK, nor ribosome collisions, are required for GCN2 activation in these conditions. Instead, UV radiation lowered levels of cellular amino acids and TrnaSer charging, later attributed to the need of these amino acids to feed into antioxidative response pathways, with this having a knock on effect on GCN2 activation.