Abstract Non-templated poly(A) tails are added to the 3′-end of most mRNAs, which have important roles in post-transcriptional regulation 1–3 . Recent studies have revealed that poly(A) tails are not composed purely of A residues, but also contain U, C and G residues internally and at their 3′-ends 4–6 , revealing new levels of complexity. However, no method is able to analyze these internal and terminal non-A residues simultaneously. Here, we developed a new method called unbiased capture of 3-terminal followed by full-length RNA sequencing (UCTF-seq) which captures RNA 3′-ends by direct 3′ adaptor ligation and rRNA removal by CRISPR/Cas9. This method allows simultaneous evaluation of the poly(A) tail length and 5′-end, internal, and 3′-end non- A residues together with the full-length cDNA for a transcript. Applying this method, we achieved the first complete transcriptome-wide 3′ tail map of mRNA within the nuclear and cytoplasmic compartments of mammalian cells, uncovering differences in poly(A) tail length and non-A residues between these two mRNA populations. A survey of diverse eukaryotic species revealed the conservation of a subset of poly(A) tails containing consecutive U residues in the internal positions, whereas those with consecutive C or G residues were of much lower abundance. Together, we established the first method to be able to comprehensively analyze poly(A) tail 5′-end, internal and 3′-end non-A residues in addition to the length simultaneously, and reveal the first complete mRNA 3′ tail map, providing rich insights into the regulatory roles of poly(A) tails.

Comprehensive analysis of mRNA poly(A) tail reveals complex and conserved regulation