September 10th
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, Albers, S. et al developed strategies to overcome limitations in restoring full-length protein production in genetic disorders caused by nonsense mutations. A study conducted by Kejiou, N.S. et al delved into the distribution of post-transcriptional regulatory elements across cellular sub compartments, specifically the cytosol and endoplasmic reticulum (ER), where protein synthesis takes place. Lastly, Song, P. et al. investigated how cytoplasmic m6A reader proteins (ECT2, ECT3, and ECT4) interact with each other to enhance their m6A-binding ability.
Engineered tRNAs suppress nonsense mutations in cells and in vivo
Nature, 2023
Albers, S., Allen, E. C., Bharti, N., Davyt, M., Joshi, D., Perez-Garcia, C. G., Santos, L., Mukthavaram, R., Delgado-Toscano, M. A., Molina, B., Kuakini, K., Alayyoubi, M., Park, K.-J. J., Acharya, G., Gonzalez, J. A., Sagi, A., Birket, S. E., Tearney, G. J., Rowe, S. M., Manfredi, C., Hong, J. S., Tachikawa, K., Karmali, P., Matsuda, D., Sorscher, E. J., Chivukula, P., & Ignatova, Z.
Efforts to treat nonsense mutations involve using pharmacological compounds or synthetic suppressor transfer RNAs (sup-tRNAs) to enable translation through premature stop codons. Pharmacological approaches face challenges due to off-target effects and safety concerns. Natural tRNA-based therapies have struggled with efficacy and mRNA decay. To overcome these issues, in this study researchers have modified bacterial tRNAs to suppress stop codons and optimized human tRNA segments for better performance. Encapsulation of sup-tRNAs in lipid nanoparticles enhances in vivo effectiveness while maintaining safety. These strategies aim to overcome limitations in restoring full-length protein production in genetic disorders caused by nonsense mutations.
This study demonstrates the modification of native tRNAs for efficient decoding of clinically significant nonsense mutations. The process involves altering tRNA sequences to improve decoding accuracy and binding affinity to elongation factors. Ribosome profiling was used to assess unintended effects of sup-tRNA on natural stop codons. In mice treated with tSA1T5 (tRNAser variant), ribosome profiling unveiled that readthrough at targeted UGA stop codons was minimal. Comparable to readthrough levels in untreated mice and other non-targeted stop codons (UAA and UAG). This suggests tSA1T5 did not significantly enhance readthrough beyond baseline levels, even at sites with naturally high readthrough rates. These findings highlight effective readthrough suppression and underscore the potential of sup-tRNA in treating PTC-linked liver and respiratory diseases.
Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADP ribosylation dependent manner to induce translational stalling
Nucleic Acids Research, 2023
Kejiou, N. S., Ilan, L., Aigner, S., Luo, E., Tonn, T., Ozadam, H., Lee, M., Cole, G. B., Rabano, I., Rajakulendran, N., Yee, B. A., Najafabadi, H. S., Moraes, T. F., Angers, S., Yeo, G. W., Cenik, C., & Palazzo, A. F
The often-overlooked spatial organisation of post-transcriptional regulators within cells plays a crucial role in mRNA translation control. This arrangement has significance in specific cellular compartments, like the endoplasmic reticulum (ER), where responses to unique needs and stressors such as unfolded protein response occur. This study delved into the distribution of post-transcriptional regulatory elements across cellular sub compartments, specifically the cytosol and endoplasmic reticulum (ER), where protein synthesis takes place.
Polysomes were isolated from both these fractions using human osteosarcoma (U2OS) cells. A total of 496 polysome interactors were identified, spanning various categories, including metabolic enzymes. Notably, eukaryotic translation initiation factors were slightly more concentrated in the ER, while cytoskeletal components and carbohydrate metabolic enzymes were enriched in the cytosol. Pyruvate Kinase M (PKM), a glycolytic enzyme, was identified as a cytoplasmic polysome interactor. This investigation focused on understanding PKM’s impact on mRNA translation. It was revealed that PKM-polysome interaction is influenced by ADP levels, linking carbohydrate metabolism and mRNA translation. Ribosome profiling showed that PKM binding to ribosomes slows down translation near lysine and glutamate encoding sequences. Furthermore, their observations indicated that PKM’s recruitment to polysomes hinges on the activity of poly-ADP ribosylation (PARylation). This process may require co-translational PARylation of lysine and glutamate residues within emerging polypeptide chains. Additionally, PKM’s depletion suggested a role in DNA repair, replication, and mitosis. PKM-ribosome interaction decreases during mitosis, aligned with decreased PARylation, indicating enhanced mitotic transcript elongation.
Overall, this study reveals a novel role for PKM in post-transcriptional genetic regulation, establishing a connection between cellular metabolism and mRNA translation.
m6A readers ECT2/ECT3/ECT4 enhance mRNA stability through direct recruitment of the poly(A) binding proteins in Arabidopsis
Genome Biology, 2023
Song, P., Wei, L., Chen, Z., Cai, Z., Lu, Q., Wang, C., Tian, E., & Jia, G.
Chemical modifications in RNA, particularly N6-methyladenosine (m6A), play a significant role in regulating gene expression, thereby impacting various biological processes. In plants, such as Arabidopsis thaliana, the m6A modification is controlled by a complex of proteins including mRNA adenosine methylase (MTA), methyltransferase B (MTB), and others. This modification is crucial in regulating processes like stem cell proliferation, stress responses, and signalling. In Arabidopsis, the m6A modification has been shown to impact diverse processes like stem cell growth, stress responses, and flowering. However, the precise mechanisms underlying its regulatory functions are not well understood. In this study, researchers investigated how cytoplasmic m6A reader proteins (ECT2, ECT3, and ECT4) interact with each other to enhance their m6A-binding ability.
Ribosome profiling was employed to assess the translation efficiency of specific mRNA molecules. The analysis conducted on targeted mRNAs, including those associated with ECT2 or ECT2 & m6A, as well as non-targets, was performed in different plant genetic backgrounds (ect2-1, ect2/3/4, and WT). The results showed that there were no significant differences in translation efficiency between these groups. This implies that the proteins ECT2, ECT3, and ECT4 do not play a role in modulating protein translation. They found that these proteins work together to stabilize m6A-modified mRNAs, thereby affecting gene expression. This complex was found to specifically impact the plant response to abscisic acid (ABA) during seed germination and growth after germination. The study also identified specific mRNA stabilizers (PAB2 and PAB4) that bind to ECT2, further enhancing the stability of targeted mRNAs.
In conclusion, this study demonstrated that multiple m6A reader proteins in plants collaborate to regulate mRNA stability and gene expression. This has important implications for understanding how plants respond to environmental cues and stresses through precise regulation of gene expression via RNA modifications.