GSE21975
Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases
Messenger RNAs are regulated by a variety of degradation mechanisms in mammalian cells.  In the canonical animal microRNA pathway, microRNAs in complex with Argonaute proteins bind to many mRNA targets with imperfect complementarity, leading to degradation of the mRNA through the regular decay machinery.  The ancestral “slicer” endonuclease activity of Argonaute2 itself, which requires more extensive complementarity with the target RNA, is not used in this pathway, and has only been observed in two microRNA-guided cases.  Nevertheless, the cleavage capacity of mammalian Ago2 is conserved and essential for viability.  Here, we assess the endonucleolytic function of Ago2 and other nucleases by identifying cleavage products retaining 5`-phosphate groups in mouse ES cells on a transcriptome-wide scale.  We detect a significant signature of Ago2-dependent cleavage events and validate several targets.  Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in this mode of mRNA regulation.  Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events, including one in the Dgcr8 mRNA, that functionally regulate mRNA levels in mES cells.  Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals. Overall design: Global 5`-phosphate-dependent RACE in WT, Ago2-KO and Drosha-excised mouse ES cells and human 293S cells