The life span of a mammalian mRNA is determined, in part, by the binding of regulatory proteins and small RNA-guided complexes. The conserved endonuclease activity of Argonaute2 requires extensive complementarity between a small RNA and its target and is not used by animal microRNAs, which pair with their targets imperfectly. Here we investigate the endonucleolytic function of Ago2 and other nucleases by transcriptome-wide profiling of mRNA cleavage products retaining 5' phosphate groups in mouse embryonic stem cells (mESCs). We detect a prominent signature of Ago2-dependent cleavage events and validate several such targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in site-specific mRNA cleavage. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events that functionally regulate mRNA levels in mESCs, including one in the Dgcr8 mRNA. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals.
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