Non-templated poly(A) tails are added to the 3'-end of most mRNAs, which have important roles in post-transcriptional regulation. 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, 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 PAIso-seq2 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.
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