A dicer-independent miRNA biogenesis pathway that requires Ago catalysis

Abstract

The nucleolytic activity of animal Argonaute proteins is deeply conserved, despite its having no obvious role in microRNA-directed gene regulation. In mice, Ago2 (also known as Eif2c2) is uniquely required for viability, and only this family member retains catalytic competence. To investigate the evolutionary pressure to conserve Argonaute enzymatic activity, we engineered a mouse with catalytically inactive Ago2 alleles. Homozygous mutants died shortly after birth with an obvious anaemia. Examination of microRNAs and their potential targets revealed a loss of miR-451, a small RNA important for erythropoiesis. Though this microRNA is processed by Drosha (also known as Rnasen), its maturation does not require Dicer. Instead, the pre-miRNA becomes loaded into Ago and is cleaved by the Ago catalytic centre to generate an intermediate 3′ end, which is then further trimmed. Our findings link the conservation of Argonaute catalysis to a conserved mechanism of microRNA biogenesis that is important for vertebrate development.Argonaute proteins are the key effectors of small RNA-mediated regulatory pathways that modulate gene expression, regulate chromosome structure and function, and provide an innate immune defence against viruses and transposons 1 . The structure of Ago proteins is well conserved, consisting of an amino-terminal domain, the Mid domain, and their signature PAZ and Piwi domains. Structure-function relationships in this family are becoming increasingly well understood 2 . The PAZ and Mid domains help to anchor the small RNA guide, with PAZ binding the 3′ end using a series of conserved aromatic residues and the Mid domain providing a binding pocket for the 5′ end. The Piwi domain contains an RNase H motif that was cryptic in the primary sequence but easily recognizable in the tertiary structure. Loading of a highly complementary target into an Ago brings the scissile ©2010 Macmillan Publishers Limited. All rights reservedCorrespondence and requests for materials should be addressed to G.J.H. (hannon@cshl.edu). Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions S.C. and G.J.H. planned experiments, interpreted data and wrote the paper. S.C. and C.O.D.S. performed studies, and M.M.W.C. provided critical unpublished reagents and discussion.Author Information Sequencing data has been deposited in GEO and assigned accession number GSE21370. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 December 1. Animal microRNAs function differently from their plant counterparts, with nearly all microRNA-target interactions providing insufficient complementarity to orient the ...

Regulation of erythroid differentiation by miR-376a and its targets

“…Moreover, over-expression of miR-376a, via its ability to negatively regulate Ago2 expression, may suppress erythroid differentiation through inhibition of Ago2, which is required for miRNA biogenesis. This is consistent with the report that Ago2 catalytic deficiency results in a strong reduction in miR-451, whose expression is indispensable to erythrocyte maturation [26,27].…”

“…Ago2 demonstrates nucleolytic activity in siRNA and miRNA biogenesis [25,26]. To date, two labs have reported a dicer-independent miRNA biogenesis pathway that requires Ago2 catalysis [27,28]. Therefore, the observation that Ago2 modulates erythroid differentiation can be partly explained by its involvement in miRNA activity, especially of those miRNAs involved in erythropoiesis.…”

“…Furthermore, the erythroid-related miRNA (such as miR-451 and miR-144) levels were also increase when Ago2 was rescued (Supplementary information, Figure S7). Since the catalytic activity of Ago2 is known to be essential to mature miR-451 expression [27], we also constructed a catalytically dead Ago2 (pcDNA-Ago2_Mut) with a substitution of alanine for the aspartate residue in the catalytic DDH motif. We performed a similar ''rescue'' assay with the inclusion of pcDNA-Ago2_Mut, and miR-451 showed the most significant reduction in transfection groups containing pcDNA-Ago2_Mut when compared to pcDNAAgo2_WT, suggesting the requirement of Ago2 catalysis for miR-451 biogenesis (Supplementary information, Immunoblotting of CDK2, phosphorylated RB, total RB, and GAPDH in extracts from K562 cells induced by hemin for 3, 6, 12, and 24 h after cell transfection with pre-miR-376a or the negative control.…”

“…A well-known characteristic of Ago2 is its catalytic activity in the miRNA-directed cleavage of target mRNAs and in miRNA biogenesis [26,[34][35][36]. Homozygous Ago2 mutant mice demonstrate obvious anaemia and severe defects in erythropoiesis, and die shortly after birth [27]. The arrest in the maturation of erythroid precursors caused by Ago2 deficiency can be explained by either its catalysis-independent or cleavagedependent roles in the transition of cells from progenitors to mature erythroblasts.…”

From guide to target: molecular insights into eukaryotic RNA-interference machinery

“…The remaining guide strand directs Ago to its target via complementary base pairing. Some specific miRNAs are processed in a Dicerindependent pathway [128,129]. (b) siRNAs typically derive from exogenous sources (i.e.…”