August 6th

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, Song et al. present a comprehensive functional model of m6A regulation mediated by m6A reader proteins in plants. Kejiou et al. look at identified post-transcriptional regulators associated with the endoplasmic reticulum (ER), and Albers et al. look at nonsense suppression therapy via engineered tRN

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. and Jia, G.

The RNA N6-methyladenosine (m6A) modification plays a vital role in plant growth and crop yield. Proteins responsible for reading m6A modifications enable the effective functioning of m6A in gene regulation. Among these reader proteins are ECT2, ECT3, and ECT4, which redundantly regulate trichome branching and leaf growth in plants. Despite their significance, the precise molecular mechanism underlying the role these proteins play is yet to be fully understood.

In this study, it is demonstrated that ECT2, ECT3, and ECT4 exhibit direct interactions with each other within the cytoplasm, and they function redundantly in regulating the response to abscisic acid (ABA) during both seed germination and post-germination growth. It was shown that ECT2, ECT3, and ECT4 play a role in stabilizing their targeted m6A-modified mRNAs, but do not participate in alternative polyadenylation and translation processes.

Using mRNA stability profiling and FA-CLIP data analysis, the authors found that ECT2/ECT3/ECT4 work together to enhance the stability of m6A-modified mRNAs they bind to, influencing gene expression. Specifically, it is found that ECT2 interacts directly with two poly(A) binding proteins, PAB2 and PAB4, which enhances the stabilization of m6A-modified mRNAs by preventing their deadenylation. When ECT2, ECT3, and ECT4 are disrupted, the mRNAs of ABA signaling-related genes become destabilized, leading to an accumulation of ABI5 and subsequently causing ABA hypersensitivity.

In conclusion, this study presents a comprehensive functional model of m6A regulation mediated by m6A reader proteins in plants. According to this model, ECT2/ECT3/ECT4 play a crucial role in stabilizing their target mRNAs within the cytoplasm.

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., and Yee, B.A

In light of numerous studies that have identified post-transcriptional regulators associated with the endoplasmic reticulum (ER), the researchers embarked on an investigation to explore whether specific factors regulate compartment-specific mRNA translation in human cells. Utilising a proteomic survey of spatially regulated polysome interacting proteins, they successfully identified Pyruvate Kinase M (PKM), a glycolytic enzyme, as a polysome interactor localized in the cytosol (i.e., excluded from the ER).

The authors discovered that the interaction between PKM and polysomes is directly regulated by ADP levels, establishing a noteworthy connection between carbohydrate metabolism and mRNA translation. Employing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), the researchers found that PKM crosslinks to mRNA sequences located immediately downstream of regions encoding lysine- and glutamate-enriched tracts. Furthermore, ribosome profiling was used to show that PKM binding to ribosomes leads to translational stalling near sequences in which lysine and glutamate codons are overrepresented.

The researchers also noted that PKM’s recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation), indicating a potential reliance on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, their study has uncovered a novel role for PKM in post-transcriptional gene regulation, establishing a crucial link between cellular metabolism and mRNA translation in human cells.

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., Santos, L., Mukthavaram, R., Delgado-Toscano, M., Molina, B., Kuakini, K., Alayyoubi, M., Park, K., Acharya, G., Gonzalez, J., Sagi, A., Birket, S., Tearney, G., Rowe, S., Manfredi, C., Hong, J., Tachikawa, K., Karmali, P., Matsuda, D., Sorscher, E., Chivukula, P., & Ignatova, Z.

Nonsense mutations are responsible for about 11% of inherited genetic diseases. These mutations change a sense codon that normally encodes for an amino acid into a premature stop signal, causing the translation process to stop prematurely and resulting in non-functional proteins. One approach to tackle these mutations is to use altered natural tRNAs to pair with the premature stop signal and allow translation to continue. However, current tRNA-based therapies have not achieved the desired balance between effectiveness and safety, and there is still no treatment available for individuals with nonsense mutations.

n this study, a new strategy is introduced where native tRNAs are modified to become highly efficient suppressor tRNAs (sup-tRNAs). These sup-tRNAs are tailored to match the specific properties of the amino acids they carry. When administered to mice through lipid nanoparticles, the sup-tRNAs successfully restored the production of functional proteins affected by nonsense mutations. To assess potential off-target effects of sup-tRNA on native stop codons, the authors analysed the whole lung and liver organs of mice treated with tSA1T5 (a sup-tRNA variant that exhibited one of the highest readthrough activities) intratracheally or intravenously respectively, using ribosome profiling. This was also tested in cell systems and patient-derived nasal epithelia. It was determined that sup-tRNA uniformly restored translation, as exemplified by the effect of the arginine sup-tRNA variant tRT5 on the cystic fibrosis transmembrane conductance regulator gene (CFTR) PTC-containing variant CFTRR553X, which restored the translation level to 22% of the level of cells transfected with wild-type CFTR.

Overall, this research presents a promising approach for developing tRNA-based therapies with high molecular safety and targeted effectiveness in suppressing premature stop signals in genetic diseases.

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