You'd Prefer An Argonaute

A dicer-independent miRNA biogenesis pathway that requires Ago catalysis

Sihem Cheloufi, Camila O. Dos Santos, Mark M. W. Chong  & Gregory J. Hannon

Nature, 465: 584–589, 3 June 2010.
Nature AOP, 27 April 2010.
doi:10.1038/nature09092

This week’s summary and ruminative analysis by Vikram Agarwal. It’s Vikram’s second contribution to the blog:

In this article, Cheloufi and colleagues demonstrate an alternative biogenesis pathway for the maturation of a microRNA. A key question that this study seeks to address is why a member of the Argonaute family of proteins has retained its catalytic activity throughout millions of years of mammalian evolution. Though a handful of examples are known of miRNA-mediated cleavage events in animals, none have been shown to be crucial for target gene regulation and cell viability. The evidence thus suggests the possibility that the catalytically active residues of Argonaute have been conserved for purposes that are distinct from target cleavage.

The authors initially explore the consequences of losing catalytic activity of Argonaute during early mouse embryogenesis. They find that Ago2 is expressed ubiquitously in the early mouse embryo and in nearby placental tissues. Mice with a mutated catalytic residue of Ago2 develop normally through embryogenesis, but exhibit an anemic phenotype and die shortly after birth. Further cell sorting experiments confirm a problem in the maturation of erythrocytes, leading to an accumulation of pro-erythroblasts prior to birth.

During a check of global miRNA expression patterns in wild type and mutant mice, the authors discover that a single miRNA, miR-451, is aberrantly expressed in catalytically deficient mice. This miRNA is already known to have important roles in erythrocyte differentiation. More interestingly, small RNAs mapping to its precursor do not correspond to a canonical pattern one would expect if the miRNA were processed by Dicer. The precursor lacks a detectable miR* fragment, and the 3′ end of the mature miRNA is derived from the loop of the hairpin rather than the stem. The processing of the precursor is biochemically shown to be Drosha, but not Dicer, dependent, when compared to canonical miRNAs such as miR-294 and miR-16. The concluding experiments of the work investigate the role of Argonaute in the processing of pre-miR-451. In vitro purified Ago2 is demonstrated to be sufficient to produce a mature miR-451 cut, although the cleavage product is nearly 8-10 bases longer than expected. Detection of shorter products in in vivo samples, along with untemplated U addition in the 3′ end, implicate the role of an unknown exonuclease in the ultimate maturation of miR-451 into its shorter, functional form.

Overall, the biochemical evidence presented in this study is compelling, and strongly supports a model in which the Piwi domain of Ago2 catalytically cleaves miR-451. A conclusion that is less clear, though presented as an underlying motivation for the study, is that this non-canonical processing pathway explains the retention of Ago2 catalytic activity. An experiment that would provide support to this hypothesis would be to see whether a miR-451 knock-out recapitulates a similar phenotype as a catalytically-deficient Ago2 mutant. At present, it is possible that the target cleavage ability of Argonaute, or a yet unknown mechanism requiring its catalytic potential, could explain the evolutionary pressure for its conservation. Intuitively, it is difficult to comprehend why this non-canonical pathway has been conserved for only a single miRNA out of the hundreds encoded in the genome. The time spans involved during the evolution of the precursor are so vast that there must have been at least some opportunity for small insertions to have converted the miRNA into one that is processed canonically, or produced a novel miRNA convergently with the same seed sequence. Only further work will help elucidate why such strong evolutionary pressure exists to preserve this non-canonical processing mechanism.

Citation for researchblogging.org:

Cheloufi S, Dos Santos CO, Chong MM, & Hannon GJ (2010). A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature PMID: 20424607