Our results indicated that miRNA-directed target cleavage might prove important for erythrocyte maturation. As a step towards identifying such a target, we profiled miRNAs expressed in the liver, one of the fetal haematopoietic sites. Deep sequencing from wild-type animals and Ago2ADH homozygotes showed that virtually all microRNAs were present at nearly identical levels. One miRNA, miR-451, represented 11% of all miRNA reads in normal fetal liver but was markedly reduced in the mutants ().
Mature miR-451 expression depends on Ago2 catalysis
Previous studies have demonstrated a strong dependency of the development of pro-E into basophilic erythroblasts on the expression of miR-451 (ref. 37
). Together, miR-451 and miR-144 form a microRNA cluster with robust expression in erythroid cells. This pattern can be explained in part on the basis of the presence of regulatory sites for the GATA-1 zinc finger transcription factor, which acts as a master regulator of erythroid differentiation38
. The regulatory circuit seems to be intact in Ago2ADH
animals, because we observe no changes in the levels of precursor pre-miR-144-451 in homozygous mutants (). This strongly pointed to an impact of catalysis on miR-451 maturation rather than miR-451 expression.
MicroRNA biogenesis occurs via a two-step processing pathway wherein Drosha initially cleaves the primary microRNA transcript to liberate a hairpin pre-miRNA39
. This is exported to the cytoplasm and recognized and cleaved by Dicer to yield the mature duplex, which is loaded into Ago. The passenger strand is removed through unknown mechanisms to yield a complex ready for target recognition.
An examination of the miR-451 precursor and its mature strand revealed an unusual feature. As annotated, the six terminal nucleotides of the 23-nucleotide-long mature miR-451 span the loop region and extend into the complementary strand of the hairpin precursor. This arrangement seems incompatible with the well-studied enzymatic activities of Drosha and Dicer, which would normally liberate the mature microRNA mapping to the stem only (). We therefore explored the possibility that miR-451 might adopt an unusual mode of biogenesis.
We began by assessing the dependency of miR-451 on Drosha. We created a construct that drives the expression of the miR-144/451 precursor from a strong viral promoter and introduced this into mouse embryonic fibroblasts (MEFs) homozygous for a conditional Drosha allele. After activation of Cre-ER and Drosha loss of function, we noted a 20-fold reduction in levels of mature miR-451. This was even more marked than the effect on a miRNA, let-7c, with a well-established dependency on canonical processing factors ().We also assessed the ability of Drosha to liberate pre-miR-451 in vitro. Drosha complexes were affinity-purified from human 293T cells and mixed with in vitro-synthesized fragments of primary pri-miR-451 or pri-miR-144. In both cases, bands of the appropriate size for the pre-miRNA were observed (). In the case of pri-miR-451 processing the 5′ flank of the transcript folds into an additional hairpin, which may be released by Drosha to give additional fragments. As a result, only one flank was observed. The identities of pre-miRNA bands were confirmed by northern blotting with oligonucleotide probes corresponding to the predicted species (, not shown). Considered together, these experiments provide both genetic and biochemical support for Drosha catalysing the excision of pre-miR-451 from its primary transcript.
Non-canonical biogenesis of miR-451
Pre-miR-451 has an unusually short, 17-nucleotide stem region. Previous studies indicate that this is too short to be efficiently recognized and processed by Dicer40
. We therefore examined the role of Dicer in miR-451 maturation. We introduced the pri-miR-451 expression vector into ES cells that are homozygous for Dicer conditional alleles and express tamoxifen-inducible oestrogen receptor–Cre fusion protein (Cre-ER). Whereas acute Dicer loss caused a roughly 80-fold reduction in a control ES cell microRNA (miR-294), miR-451 levels did not change (). A pure population of continuous Dicer-null ES cells showed a more than 500-fold reduction in conventional microRNA, whereas levels of miR-451 were unaffected (). We also confirmed these results using northern blot analysis of Dicer-null ES cells transiently expressing the miR-451 precursor (). Finally, we incubated synthetic miR-451 pre-miRNA with recombinant Dicer and observed no mature cleavage products, although pre-let-7c was efficiently processed (not shown). Thus, conversion of pre-miR-451 into a mature miRNA proceeds independently of Dicer. We therefore strove to identify an alternative maturation pathway.
We examined miR-451 species in wild-type and Ago2ADH mutant livers by northern blotting. This confirmed loss of the mature miRNA in the mutant animals. However, we noted the appearance of a ~40- nucleotide band that co-migrated with a synthetic pre-miR-451 and hybridized to probes to its 5′ and 3′ arms (). This indicated accumulation of the Drosha cleavage product in mutant animals. Notably, the same bands seen in total RNA were also detected in Ago2 immunoprecipitates (). This demonstrated the direct loading of the pre-miRNA-451 into Ago2 and raised the possibility that the Ago2 catalytic centre might help to catalyse the maturation of this microRNA.
Ago2 catalysis is required for miR-451 biogenesis
The well-established biochemical properties of Ago2 predict that it would cleave a loaded pre-miR-451 after its thirtieth base. We searched for evidence of such an intermediate in fetal liver small RNA libraries encompassing an expanded size range. Plotting a size distribution of reads corresponding to a conventional miRNA, miR-144, gave the expected pattern, a sharp peak at ~20 nucleotides. In contrast, miR-451 showed a heterogeneous size distribution, exclusively because of variation at its 3′ end. One abundant species corresponded precisely to the predicted Ago cleavage product ().
We confirmed the capacity of Ago2 to load and cleave pre-miR-451 using in vitro assays (). Wild-type or catalytically inactive Ago2 complexes () were affinity-purified from 293T cells and mixed with 5′ end-labelled pre-miR-451. Only wild-type Ago2 produced the expected product, and this depended on the presence of Mg2+ (not shown). No product was produced if we provided a mutant version of the precursor in which a single point mutation disrupted pairing at the cleavage site. Beta elimination and ligation reactions confirmed that cleavage left a free 3′ OH terminus as expected of Argonaute proteins. These data strongly support a role for the Ago2 catalytic centre in miR-451 maturation. This is perhaps akin to the proposed role of passenger strand cleavage in the maturation of RISC (RNA-induced silencing complex). Ago1 could load pre-miR-451 but was unable to process it to its mature form ()
To investigate sequence versus structural requirements for entry into the alternative miRNA biogenesis pathway, we created a structural mimic of miR-451 that might instead produce let-7c. At the concentration tested, this was as potent as the native pre-let-7c in suppressing a green fluorescent protein (GFP) or luciferase reporter containing perfect let-7c complementary sites ( and Supplementary Fig. 5a
). The miR-451 precursor could also be remodelled to express a short hairpin RNA that efficiently represses p53 (also known as Trp53; Supplementary Fig. 5b
). Given the unique ability of Ago2 to productively process miR-451, these data could have practical implications in the experimental use of shRNAs.
Considered together, our results suggest a model (Supplementary Fig. 6
) in which miR-451 enters RISC through an alternative biogenesis pathway. Although Drosha cleavage proceeds normally, the Dicer step is skipped and the pre-miRNA is loaded directly into Argonaute. This is surprising, considering earlier studies indicating a coupling of Dicer cleavage and RISC loading41,42
. Such a complex would also lack interactions between the PAZ domain and the 3′ end of a conventional Dicer product43,44
. A previous report indicated the ability of RISC to accommodate such species and posited a potential for Ago cleavage in the maturation of canonical microRNAs45
. However, no physiological role for such an activity was demonstrated, and we detect no measurable defects in the processing of canonical miRNAs in Ago2ADH
mutants. miR-451 maturation proceeds with Ago-mediated cleavage, producing an intermediate that is further trimmed. Although this could occur via either endo- or exonucleolytic digestion, the observed distribution of 3′ ends, many bearing single non-templated U residues (Supplementary Figs 6, 7
), seems more consistent with the latter model. Although the precise enzymology of this step remains obscure, preliminary studies fail to support roles for Eri-1 (also known as Eri1) or the exosome complex (not shown).
A previous report noted severe defects in erythropoiesis in Ago2-null cells46
. In that case, the phenotype could be rescued by transplantation of adult haematopoietic stem cells expressing a catalytically inactive Ago2 allele. Although the two studies may seem at odds, we feel that the differences simply reflect the quantitative nature of our respective phenotypes and the fact that the strong reduction in miR-451 that we observe results in only a partial loss of RBCs. Anaemia in the Ago2 mutants could be a complex phenotype, of which miR-451 loss is only one component. Finally, in the earlier study, a different inactive Ago2 allele was expressed in adult haematopoietic stem cells from a strong viral promoter, whereas in the work presented here, a knock-in allele had its impact on the haematopoietic niche as a whole during fetal development.
Although the anaemia of the Ago2ADH
animals is profound, it is unclear whether this alone is sufficient to cause the perinatal death of mutant animals. Although no other well-defined pathologies were observed, we did find haemorrhages in the lungs and intra-abdominal bleeding in some animals. Moreover, miR-451 is expressed in the gastric epithelium, where its loss could have impacts that we have as yet failed to detect47
. We leave open the possibility that additional microRNAs might rely on the alternative biogenesis pathway for maturation, although none presently annotated in miRBase share the usual structure or 3′ heterogeneity of miR-451. Additional defects could also arise from a role of Ago2-mediated target mRNA cleavage. Thus far, miR-196 and microRNAs from the imprinted Rtl1
cluster have been ascribed this property; however, none of the phenotypes that we observe can be explained by defects in these particular species16–18
The structure of the miR-451 precursor and the extension of its mature sequence around the loop and into the complementary strand of the precursor are present throughout vertebrate evolution (Supplementary Fig. 8
). Thus, a conserved pathway of miR-451 maturation may provide at least some of the evolutionary pressure to maintain a catalytic Argonaute protein in animals.