Several interesting questions remain unsolved regarding the biogenesis of miR-451. First, how is the 30-nt, Ago2-cleaved pre-mir-451 (ac-pre-mir-451
) trimmed back to yield the mature product? Using in vitro processing assay with synthetic 42-nt pre-mir-451 and recombinant Ago2, the Giraldez group showed that the processing of mir-451
stopped at ac-pre-mir-451
, but that addition of RNAse I promoted the generation of shorter products.38
This is consistent with the view that a cellular nuclease(s) is required in the trimming step. It has been reported that pre-let-7
stability is regulated post-transcriptionally by terminal uridylyl transferase 4 (TUT4),65,66
which adds multiple uridine residues to the 3′ end of a pre-miRNA (tailing) that possibly recruit a nuclease to trigger miRNA degradation (trimming). Recently, mature miRNAs that encounter perfectly complementary targets were also found to be subject to a tailing and trimming mechanism resulting in their downregulation.67
Untemplated nucleotide additions were detected at the 3′ end of ac-pre-mir-451
suggesting that a possible tailing/trimming mechanism might be involved in maturation of miR-451.
Second, if the mir-451 backbone is going be applied effectively in the future, is it possible to increase the miRNA expression level and knockdown efficiency by manipulating the mir-451 hairpin to make it a better substrate for Ago2 and/or the resection pathway? Structure-function studies should be able to address what parameters of mir-451 sequence, hairpin length, and basepairing type are most critical for optimal maturation and activity. Such knowledge will permit the rational design of reprogrammed mir-451 hairpins towards arbitrary targets of choice.
Third, and perhaps most interestingly, we do not understand the underlying biology to the existence of the mir-451
pathway. Why hasn't this locus evolved as a conventional miRNA? Despite the facile capacity of reprogrammed mir-451
backbones to produce diverse functional miRNAs, the fact is that mir-451
currently stands as structurally unique amongst endogenous miRNA loci. No doubt, the discovery of the direct Ago2-loading of short hairpins has stimulated searches for additional loci of this class using the vast published catalogs of short RNA sequences. However, it should also be noted that mir-451
was itself annotated many years ago,26
prior to recognition of its exotic biogenesis mechanism. Many computational studies have focused on the recovery of conserved hairpins, and by this criterion one might have expected additional candidates to have emerged already.
One wonders then, what is the special utility of the mir-451
pathway that has not only led to its strict conservation amongst all vertebrates, but has apparently been coupled to a suppression of similar hairpins in vertebrate genomes. At present, the only possible clue comes from the cell specificity of miR-451, which is expressed at extraordinary levels in erythrocytes. Three studies of mir-451
mouse knockouts (some in combination with mir-144
) provide evidence for the endogenous influence of miR-451 on erythrocyte maturation.41,46,47
Rasmussen et al. showed that the phenotype of mir-451
-deficient mice recapitulated that of the mir-144/451
-deficient mice, which had a late erythroblast maturation defect.41
More dramatically, these mutant mice were strongly sensitive to oxidative stress induced by phenylhydrazine, which induces hemolytic anemia. Wildtype mice can recover their red blood cells in this situation, but half of the miRNA mutant mice expired within 6 days.41
Similarly, Patrick et al.46
and Yu et al.45
observed that mir-451
knockout and mir-144/451
knockout mice, respectively, had erythroid differentiation defects in both embryonic stage and adulthood, and were unable to sustain robust erythropoiesis following phenylhydrazine challenge. However, unlike knockin of catalytically dead Ago2, which is lethal,37
these three studies showed that mice deleted for miR-451 are viable and only have a mild anemia that was exacerbated when encountering oxidative stress. Thus, the Slicer activity of Ago2 may play other additional roles than miR-451 processing in erythroid differentiation.
At the stage of erythroblast differentiation, the expression level of hemoglobin starts to accumulate, which sensitizes the cells to oxidative damage. miR-451 may be important at this stage to protect erythroblast from oxidative stress by downregulating the level of 14-3-3ζ, a negative regulator of the transcriptional factor FoxO3 that mediates the expression of many anti-oxidant-encoding genes.45,46
It should also not escape notice that red blood cells are notable as the sole cell type that exists lacking its nucleus in mammals. Perhaps some aspect of this exceptional lifestyle is linked to the simplification of its miRNA profile to consist largely of miR-451, and perhaps this is in turn linked to the disposal of Dicer-dependent pathways. The level of Dicer in “elderly” red blood cells, which can circulate for 3–4 months, has not been specifically examined, nor have the properties of old erythrocytes that lack miR-451. The genetic tools exist to study this with clean knockouts, so their analysis may prove revealing.
In summary, the study of vertebrate miR-451 biogenesis has revealed a Dicer-independent, Ago2-mediated strategy for miRNA biogenesis, and provided a new platform for gene suppression. It is interesting to note that archaebacterial genomes encode Argonaute proteins,68–70
but not apparently Drosha or Dicer enzymes. It has been reported that Dicer-independent “primal” short RNAs are directly incorporated to Ago1 in Schizosaccharomyces pombe
, and serve to initiate Dicer-dependent siRNA amplification during heterochromatin formation.71
It has also been previously reported that the miRNA-like RNA-2 (milR-2) in Neurospora can be produced by direct loading of the pre-miRNA-like hairpin to Argonaute protein QDE-2, whose Slicer activity is responsible for the processing of pre-milR-2
Such studies may suggest that the direct loading and processing of small RNA by Argonaute proteins is an ancient strategy, and conceivably may have preceded the incorporation of RNAse III enzymes into small RNA pathways.