Direct profiling of non-adenosines in poly(A) tails of endogenous and therapeutic mRNAs with Ninetails

Stability and translation of mRNAs, both endogenous and therapeutic, is determined by poly(A) tail. Direct RNA sequencing enables single-molecule measurements of poly(A) lengths, avoiding amplification bias. It also holds potential for observation of non-adenosines within poly(A), known to influence mRNA fate. However, there is no computational method to detect composite tails in Direct Sequencing data. To address this gap, we introduce the Ninetails, a neural network-based tool that accurately identifies and quantifies non-adenosines in poly(A) tails. Examination of different biological contexts revealed widespread non-adenosine decorations, with frequencies influenced by the origin of poly(A) tails differing by mRNA class, cell type, and species. Notably, substrates of cytoplasmic TENT5-polymerases and mitochondrially encoded mRNAs are enriched in composite tails. For mRNA therapeutics, we show that the composition of poly(A) tails in mRNA vaccines is dynamic during its cellular lifetime and that the manufacturing protocol of synthetic mRNAs affects the purity of poly(A) tails.