The study investigates a Dicer-independent miRNA biogenesis pathway that requires Ago2 catalytic activity. Despite the lack of a clear role in microRNA-directed gene regulation, the nucleolytic activity of animal Argonaute proteins is deeply conserved. In mice, Ago2 is uniquely essential for viability, and only this family member retains catalytic competence. The authors engineered a mouse with catalytically inactive Ago2 alleles, which resulted in homozygous mutants that died shortly after birth with anemia. Examination revealed a loss of miR-451, a small RNA important for erythropoiesis. Although Drosha processes miR-451, its maturation does not require Dicer. Instead, the pre-miRNA is loaded into Ago2 and cleaved by the Ago2 catalytic center to generate an intermediate 3' end, which is then further trimmed. This finding links the conservation of Argonaute catalysis to a conserved mechanism of miRNA biogenesis crucial for vertebrate development. The study also highlights the importance of Ago2 in extra-embryonic development and postnatal development, particularly in erythroid maturation. The alternative biogenesis pathway for miR-451 involves Drosha cleavage followed by Ago2-mediated cleavage, producing an intermediate that is further trimmed. This pathway may provide evolutionary pressure to maintain a catalytic Argonaute protein in animals.The study investigates a Dicer-independent miRNA biogenesis pathway that requires Ago2 catalytic activity. Despite the lack of a clear role in microRNA-directed gene regulation, the nucleolytic activity of animal Argonaute proteins is deeply conserved. In mice, Ago2 is uniquely essential for viability, and only this family member retains catalytic competence. The authors engineered a mouse with catalytically inactive Ago2 alleles, which resulted in homozygous mutants that died shortly after birth with anemia. Examination revealed a loss of miR-451, a small RNA important for erythropoiesis. Although Drosha processes miR-451, its maturation does not require Dicer. Instead, the pre-miRNA is loaded into Ago2 and cleaved by the Ago2 catalytic center to generate an intermediate 3' end, which is then further trimmed. This finding links the conservation of Argonaute catalysis to a conserved mechanism of miRNA biogenesis crucial for vertebrate development. The study also highlights the importance of Ago2 in extra-embryonic development and postnatal development, particularly in erythroid maturation. The alternative biogenesis pathway for miR-451 involves Drosha cleavage followed by Ago2-mediated cleavage, producing an intermediate that is further trimmed. This pathway may provide evolutionary pressure to maintain a catalytic Argonaute protein in animals.