Global investigation of the 3' extremity of mRNA (3'-terminome), despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences and relative paucity of mRNA. We here develop a method, TAIL-seq, to sequence the very end of mRNA molecules. TAIL-seq allows us to measure poly(A) tail length at the genomic scale. Median poly(A) length is 50-100 nt in HeLa and NIH 3T3 cells. Poly(A) length correlates with mRNA half-life, but not with translational efficiency. Surprisingly, we discover widespread uridylation and guanylation at the downstream of poly(A) tail. The U tails are generally attached to short poly(A) tails (<25 nt), while the G tails are found mainly on longer poly(A) tails (>40 nt), implicating their generic roles in mRNA stability control. TAIL-seq is a potent tool to dissect dynamic control of mRNA turnover and translational control, and to discover unforeseen features of RNA cleavage and tailing.
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