Dual Strategies for Argonaute2-Mediated Biogenesis of Erythroid miRNAs Underlie Conserved Requirements for Slicing in Mammals

Abstract

While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis. While it is Dicer dependent, it requires slicing to eliminate its star strand. Thus, in Ago2-CD conditions, miR-486-5p is functionally inactive due to duplex arrest. Genome-wide analyses reveal miR-486 and miR-451 as the major slicing-dependent miRNAs in the hematopoietic system. Moreover, mir-486-KO mice exhibit erythroid defects, and double knockout of mir-486/451 phenocopies the cell-autonomous effects of Ago2-CD in the hematopoietic system. Finally, we observe that Ago2 is the dominant-expressed Argonaute in maturing erythroblasts, reflecting a specialized environment for processing slicing-dependent miRNAs. Overall, the mammalian hematopoietic system has evolved multiple conserved requirements for Slicer-dependent miRNA biogenesis.

Argonaute Proteins: From Structure to Function in Development and Pathological Cell Fate Determination

“…This differentiation defect was rescued by the reintroduction of a wild-type AGO2 or a catalytic dead AGO2 in mESCs, but not by an RNA-bindingdeficient AGO2 (Ngondo et al, 2018). In line with these results, Ago2 catalytic dead mice were previously shown to be viable until a few hours after birth and subsequently died of anemia Jee et al, 2018). In these cases, the slicing activity of AGO2 is needed to process the pre-miRNA-451 and miRNA-486-3p, two miRNAs required in the development of the erythroblasts Papapetrou et al, 2010;Jee et al, 2018).…”

Depletion of erythropoietic miR-486-5p and miR-451a improves detectability of rare microRNAs in peripheral blood-derived small RNA sequencing libraries

“…In addition to whole blood, we observed that these miRNAs are also dominant in erythrocytes, serum and partly in exosomes. Interestingly, miR-486-5p and miR-451a were previously shown to be involved in erythroid development (6), which partly explains why these miRNAs are so abundant in erythrocytes. Since erythrocytes and reticulocytes together comprise more than 90% of blood cells, these miRNAs are even more abundant in whole blood.…”

“…For example, in addition to globin mRNAs, red blood cells also contain highly abundant miRNAs including miR-486-5p and miR-451a (4,5). These two conserved, non-canonical miRNA molecules represent dominant miRNAs, which are specifically upregulated in the erythroid lineage (6). Loss of mir-486 and mir-451a genes in mice leads to erythroid defects, showing their importance in erythrocyte development (6), and explaining their high abundance in erythroid cells as well as in whole blood.…”

“…These two conserved, non-canonical miRNA molecules represent dominant miRNAs, which are specifically upregulated in the erythroid lineage (6). Loss of mir-486 and mir-451a genes in mice leads to erythroid defects, showing their importance in erythrocyte development (6), and explaining their high abundance in erythroid cells as well as in whole blood. In RNA-Seq workflows, overabundance of transcripts reduces the complexity of PCR-amplified libraries and exhausts sequencing space, which leads to unwanted effects such as inaccurate detectability and quantification of low abundant transcripts.…”

A new member in the Argonaute crew: the mt-miRNAs

“…According to the original authors (Jee et al, 2018;Viljetic et al, 2017), the experiments on mice sourced from the Jackson laboratory were carried out in compliance with their institutional protocols, thus we believe that correct ethics for the experiments have been followed.…”