April 7th, 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, Ramachandra et al. uncover the importance of BCL6 in governing transcriptional regulation of skeletal muscle differentiation and translation, while Shi et al. delve deep into the world of tRNA pseudouridylation, and the impact it has on protein synthesis in erythrocytes. Finally, Li et al. work to discover the mechanism of action of carrimycin, a novel anti-viral compound with high broad-spectrum potential.
Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis
Nature Metabolism, 2024
Ramachandran, K., Futtner, C.R., Sommars, M.A., Quattrocelli, M., Omura, Y., Fruzyna, E., Wang, J.C., Waldeck, N.J., Senagolage, M.D., Telles, C.G. and Demonbreun, A.R.
Skeletal muscle proteostasis is crucial for metabolic and motor functions, although its mechanisms remain incompletely understood. IGF-1 is one of the key players in muscle growth, activating the AKT and mTORC1 pathways, simultaneously suppressing autophagy and promoting protein synthesis. Additionally, a host of other molecules are also involved, such as FOXO transcription factors, myostatin, and certain androgens. B cell lymphoma 6 (BCL6) is a transcriptional repressor, known mainly for its role in the functionality of germinal centres. It is known to be present in high levels in skeletal muscle, and its expression is inversely correlated with VO2 max. These observations, along with others, suggest that BCL6 may have an important role as a transcriptional regulator in skeletal muscle. Here, the authors sought to investigate this role further.
Inducible BCL6 knockouts revealed rapid muscle loss, suggesting a role in muscle maintenance. mRNA sequencing in these knockouts vs controls indicated that translation, ribosome assembly, and muscle development are key functionalities influenced by this protein. More specifically, translational inhibitors such as eIF4EBP1 and ZFP36L1 were induced in knockout, and stimulators such as CPEB1, AMD1 and SMOX were ablated. Furthermore, puromycin incorporation analysis revealed a 54% decrease in protein synthesis after a week of BCL6 knockout. Interestingly, ribosome profiling analysis uncovered that BCL6 knockout influenced 3 times as many genes at the translational level as at the transcriptional level. This was determined to be due to the action of BCL6 on eIF4EBP1, which influences cap-dependent translation initiation.
PUS1 regulates erythropoiesis via tRNA pseudouridylation and cytoplasmic translation
iScience, 2024
Shi, D., Wang, B., Li, H., Lian, Y., Ma, Q., Liu, T., Cao, M., Ma, Y., Shi, L., Yuan, W. and Shi, J.
Pseudouridine is the most common RNA modification, and was first uncovered in tRNA. tRNA pseudouridylation itself is thought to promote tRNA stability, and may play a role in determining cell fate. This modification can be catalysed by snoRNAs, or by pseudouridine synthetases, such as PUS1. Mutations in such synthetases have been associated with a range of conditions, including mitochondrial myopathy, lactic acidosis and sideroblastic anemia (MLASA). Erythropoiesis, on the other hand, and the subsequent synthesis of hemoglobin, is known to be a highly translationally regulated process, due to the lack of transcription in these cells. Here, the authors aim to investigate if there is a link between tRNA pseudouridylation and erythropoiesis.
They demonstrate that a R110W PUS1 mutation leads to anemia within 4 weeks in mice, which also exhibited significantly lower red blood cell count and hemoglobin levels compared to controls. Interestingly, this mutation also led to impaired oxidative phosphorylation in bone marrow erythroblasts, appearing to specifically impact complex I and complex III activity. Indeed, NDUFS1 and NDUFS2 (complex I subunits) were significantly upregulated. tRNA profiles suggested that PUS1 predominantly impacts on cytoplasmic tRNAs as opposed to their mitochondrial counterparts, with 16 being significantly upregulated. Ribosome profiling analysis revealed a slight decrease in translational efficiency generally. This appeared to be mediated by a translational decrease in the expression of ribosomal proteins. PUS1 knockout appeared to have little effect on erythropoiesis differentiation but did have a striking impact on hemoglobin production, as revealed by Western blot, with no impact on hemoglobin mRNA levels, emphasising the impact of the PUS1 mutation on translation, and subsequent anemia.
Carrimycin inhibits coronavirus replication by decreasing the efficiency of programmed –1 ribosomal frameshifting through directly binding to the RNA pseudoknot of viral frameshift-stimulatory element
Acta Pharmaceutica Sinica B, 2024
The pandemic propagated by the spread of SARS-CoV-2 emphasised the need for effective antiviral treatments outside of the prophylactics of mRNA vaccines. Many drugs have been given emergency approval in the face of this pandemic, many of which target viral entry and replicases. However, the strong variability of the spike protein, and poor efficacy of anti-replicate compounds, constitute a need for broader spectrum medicines. Many viruses, such as coronaviruses, utilise -1 programmed ribosome frameshifting (PRF) in order to express their entire repertoire of encoded proteins. As such, this mechanism constitutes an ideal target for novel therapies. Here, the authors investigate the mechanism of action of carrimycin, currently undergoing clinical trials for treatment of SARS-CoV-2 infection.
Immunofluorescent staining of viral RNA revealed that carrimycin was dramatically more effective compared to other similar macrolide antibiotics in reducing viral load. Subsequent assays showed that it does not inhibit any SARS-CoV-2 replicase activity, suggesting an active target prior to these processes. Ribosome profiling analysis later suggested that carrimycin reduced the efficiency of -1 PRF, with a decreased in ORF1b/ORF1a translatome ratios (indicative of post/pre frameshift expression). Adding to this, a dual luciferase reporter was able to demonstrate a reduction in -1 PRF efficiency of up to 50% in Huh7 cells, at a concentration of 2umol/L. Using surface plasmon resonance spectroscopy, it was determined that carrimycin was able to directly bind to the frameshift stimulatory element (FSE) of SARS-CoV-2. It was also able to bind to other coronavirus FSE, with the exception of hCoV-NL63, which was subsequently found to be resistant to this drug, increasing the confidence of this hypothesised mechanism of action.