Poly(A)\nTail Length of Messenger RNA Regulates Translational\nEfficiency of the Mitochondria-Targeting Delivery System

Mitochondria\nare essential for cellular functions, such\nas energy\nproduction. Human mitochondrial DNA (mtDNA), encoding 13 distinct\ngenes, two rRNA, and 22 tRNA, is crucial for maintaining vital functions,\nalong with nuclear-encoded mitochondrial proteins. However, mtDNA\nis prone to somatic mutations due to replication errors and reactive\noxygen species exposure. These mutations can accumulate, leading to\nheteroplasmic conditions associated with severe metabolic diseases.\nTherefore, developing methodologies to improve mitochondrial health\nis highly demanded. Introducing nucleic acids directly into mitochondria\nis a promising strategy to control mitochondrial gene expression.\nMessenger RNA (mRNA) delivery especially offers several advantages\nsuch as faster gene expression and reduced risk of genome integration\nif accidentally delivered to the cell nucleus. In this study, we investigated\nthe effect of the poly(A) tail length of mRNA on the mitochondrial\ntranslation to achieve efficient expression. We used a peptide-based\nmitochondrial targeting system, mitoNEET-(RH)<sub>9</sub>, comprising\na mitochondria-targeting sequence (MTS) and a cationic sequence, to\ndeliver mRNA with various poly(A) tails into the mitochondria. The\npoly(A) tail length significantly affected translational efficiency,\nwith a medium length of 60 nucleotides maximizing protein expression\nin various cell lines due to enhanced interaction with mitochondrial\nRNA-binding proteins. Our findings highlight the importance of optimizing\npoly(A) tail length for efficient mitochondrial mRNA translation,\nproviding a potential strategy for improving mitochondrial gene therapy.\nThese results pave the way for further exploration of the mechanisms\nand clinical applications of mitochondrial mRNA delivery systems.