Functional parameters of Dicer-independent microRNA biogenesis

Abstract

Until recently, a Dicer-class RNase III enzyme was believed to be essential for microRNA (miRNA) biogenesis in all animals. The conserved vertebrate locus mir-451 defies this expectation and instead matures by direct cleavage of its pre-miRNA hairpin via the Slicer activity of Argonaute2 (Ago2). In this study, we used structure-function analysis to define the functional parameters of Ago2-mediated miRNA biogenesis. These include (1) the requirement for base-pairing at most, but not all, positions within the pre-mir-451 stem; (2) surprisingly little influence of the 5'-nucleotide on Ago sorting; (3) substantial influence of Ago protein stoichiometry on mir-451 maturation; (4) strong influence of G:C content in the distal stem on 3' resection of cleaved mir-451 substrates; and (5) the influence of hairpin length on substrate utilization by Ago2 and Dicer. Unexpectedly, we find that certain hairpin lengths confer competence to mature via both Dicer-mediated and Ago2-mediated pathways, and we show, in fact, that a conventional shRNA can traverse the Dicer-independent pathway. Altogether, these data inform the design of effective Dicer-independent substrates for gene silencing and reveal novel aspects of substrate handling by Ago proteins.

FIGURE 1.

Systematic mutational analysis of pre-mir-451. (A) Schematic of the human pri-mir-451 hairpin used for mutagenesis within a previously described mir-451-mir-144 operon construct (Yang et al. 2010). The pre-mir-451 hairpin is shaded to denote the Ago2-cleaved byproduct (yellow), the resected region following Ago2 cleavage (light green), and the mature miR-451 product (dark green). The mutants are designated by their positions within the pre-mir-451 hairpin and the nucleotides to which they were changed. (B) Activity of mutant mir-451 constructs in HeLa cells, assayed on a Renilla luciferase construct bearing two antisense matches to mature miR-451. Sensor values were normalized to the level observed in cells transfected with a functional mir-451 variant reprogrammed with the miR-23a sequence (Yang et al. 2010). (C) Northern blot analysis of variant mir-451 constructs. The blot was probed for mature miR-451, which detects the hairpin (42 nt), Ago2-cleaved (30 nt), and resected species (23–30 nt). The blot was stripped and reprobed for miR-144 produced from the same construct. The relative levels of total ≤30 nt miR-451 species were normalized to mature miR-144-3p. Note that mispairing caused by the 35G mutation was compatible with the biogenesis and function of miR-451, with levels slightly higher than the corresponding wild-type construct.

FIGURE 2.

Mutational analysis of the mir-451 hypervariable stem position. (A) Sequence alignment of selected mir-451 orthologs. These emphasize strict conservation of mature miR-451 and the 3′ resected region, from man to fish, as well as divergence in the pri-miRNA lower stem (highlighted by red boxes). Note also that one position on the 3′ hairpin arm is highly variable during evolution, creating both paired and unpaired configurations; the specific nucleotide in human is not even preserved in rhesus. (B) Hypervariable site mutants that change the 35U found in wild-type human to 35G or 35A. (C) Processing of hypervariable site mutants in HeLa cells and Dicer^−/− MEFs. The 35G and 35A mutants accumulated slightly less ac-pre-miRNA (arrow) and slightly more mature miR-451 (arrowhead) than the wild-type construct. The banding patterns were not as substantially altered in the absence of Dicer; the lack of mature miR-144 confirms the genotype. (D) When tested on sensors bearing two perfect miR-451 sites, the hypervariable site mutants were detectably more active than normal human mir-451. All errors represent SEM; a Student's two-tailed t-test was performed.

FIGURE 3.

Influence of 5′-nucleotide and Ago protein levels on miR-451 biogenesis. (A) 5′-Nucleotide variants. The pre-mir-451 hairpin normally begins with A, which was altered to 5′-U/C/G. Mutation to 5′-G alone generates a base pair with the terminal C (G–C); therefore, we also made a 5′-G variant that is unpaired (G–A). (B) Maturation of the 5′ variants in HeLa cells. All of the 5′ variants that were unpaired at the +1 position were efficiently matured, although the 5′-C and 5′-G variants were processed less efficiently. Pairing at +1 (5′-G, G–C) strongly diminished accumulation of cleaved and resected forms. Quantification is the level of total ≤30-nt miR-451 species normalized to mature miR-144-3p. (C) Activity of 5′ variants on perfect (“2x-si”) and bulged (“4x-mi”) sensors showed that their activities paralleled the maturation efficiency judged from Northern blotting. (D) Analysis of mir-451 5′ variants in HeLa cells cotransfected with myc-Ago1/2/3. Ago-IP analysis indicates that Ago2 associates exclusively with cleaved and 3′ resected species from all four 5′ variants, whereas Ago1 and Ago3 associate exclusively with the pre-mir-451 hairpin of all four 5′ variants. All Ago proteins associated equally with mature miR-144. (E) Analysis of companion total RNA samples indicates distinct effects of ectopic Ago proteins on miR-451 and miR-144. Maturation of all 5′ variants was enhanced by elevated Ago2, but uniformly inhibited by elevation of either Ago1 or Ago3. Expression of Ago1/2/3 increased the levels of mature miR-144 in all cases. (F) Functional consequences of Ago competition on miR-451 function. Repression of a miR-451 perfect sensor was enhanced by coexpression of Ago2, and inhibited by coexpression of Ago1 or Ago3.

FIGURE 4.

Influence of distal hairpin G:C content of 3′ resection efficiency. (A) mir-451 constructs reprogrammed with pre-mir-199a (mir-199a-RP) or pre-mir-375 (mir-375-RP). These were compared with chimeric constructs in which the hairpin stem distal to the Ago2 cleavage site (marked by the horizontal line) was exchanged. GC pairs in the resection region are marked with blue asterisks. (B,C) Northern analysis of the wild-type and chimeric constructs in HeLa cell or Dicer^−/− MEFs. Similar patterns were observed in these cell lines except that expression of the mir-451-type constructs is higher in the absence of Dicer, whereas expression of the linked mir-144 is arrested at the pre-miRNA stage. The blots show that the 3′ resection pattern is correlated with the identity of the hairpin region distal to the Ago2 cleavage site; i.e., 5′-375:199a-3′ resembles miR-199a, while 5′-199a:375-3′ resembles miR-375. (D) Direct manipulation of GC content in the human pre-mir-451 backbone. It normally contains two GCs in the 3′ resected region, and variant constructs sampled 3/4/5/8 GC pairs. (E) Northern analysis of the GC variant panel of mir-451. Increase of distal GC content impairs generation of 3′ resected forms of miR-451 in HeLa cells. The levels of Ago-cleaved and 3′ resected forms of miR-451 are severely reduced with high GC content. (*) The pre-mir-451, which appears to decrease with higher GC content. However, control experiments using oligonucleotide standards show that these high-GC-content hairpins are inefficiently detected; in contrast, the linear Ago-cleaved forms of these hairpins are detected with equal efficiency (Supplemental Fig. 4). Expression of miR-144 validates equal transfection and loading. (F) Functional consequences of increased GC content on miR-451 function. These were tested in Dicer^−/− cells against a luciferase sensor bearing four seed matches for miR-451. The variants with the highest GC content in the 3′ resected region were severely impaired in their repression activity.

FIGURE 5.

Influence of hairpin length on entry into the Ago2-dependent miRNA pathway. (A) The pre-mir-451 hairpin was extended by adding base pairs distal to the mature 23-nt miR-451 product (dark green box); thus, the sequence of mature miR-451 is not expected to change across this panel of length variants. Also highlighted are the inferred 12-nt 3′ passenger strand resulting from Ago2 cleavage (yellow box) and the 3′ resected region (light green box) following Ago2 cleavage. The solid reference line depicts the location of Ago2-mediated passenger strand cleavage; the dotted reference line depicts the putative location of 21-nt Dicer cleavage. (B) Northern blot analysis of the depicted constructs in Ago^−/− MEFs reconstituted with wild-type Ago2 (Ago2-WT), with catalytic dead Ago2 (Ago2-CD), or in Dicer^−/− MEFs, using a mature miR-451-5p probe in the common sequence of the panel of length variants. The blots were stripped and reprobed with U6 as a loading control. The absence of matured species in Ago2-CD cells for WT, +1, +2, and +3 constructs indicates that they are processed predominantly by the Ago2-dependent pathway; this is substantiated by their processing in Dicer^−/− cells. With the +4 variant, a modest amount of Ago2-independent product appears and is enhanced in the +5, +6, and +7 constructs. This product is shorter than the predominant species detected in companion Dicer^−/− cells, indicating that they are generated by distinct nucleases. The +5 variant is capable of maturing via both Ago2-dependent and Dicer-dependent pathways. The +7 variant is strictly Dicer-dependent. (C) Sensor assays of the length variant panel in HeLa cells and Dicer^−/− MEFs against a miR-451 luciferase sensor bearing four seed matches. The repression capacity of Ago2-dependent and Dicer-independent hairpins is relatively equivalent in HeLa cells, except for the +3 and +4 variants (which correlates with their lower efficiency of biogenesis, panel B). A subset of these hairpins is also active in Dicer^−/− MEFs, with shorter hairpin lengths exhibiting enhanced activity.

FIGURE 6.

Maturation of a conventional shRNA through the Dicer-independent pathway. (A) Sequence of a conventional Pol III shRNA bearing a 21-bp stem and a 5-nt loop. The position of Ago2-mediated cleavage, if it occurs, is predicted to be at +37 from the 5′ end. (B,C) Expression of the shRNA in Ago^−/− MEFs reconstituted with wild-type Ago2 (Ago2-WT) or with catalytic dead Ago2 (Ago2-CD), or in Dicer^−/− MEFs. Small RNA products from both hairpin arms are detected in Ago2-WT and Ago2-CD cells; the shRNA hairpin is also detected at a low level. The hairpin accumulates to a high level in Dicer^−/− cells, which also accumulate a heterogeneous set of <30-nt products detected with a 5p arm probe, but not the 3p arm probe. (D,E) Sorting and processing of shRNA-derived small RNAs in Dicer^−/− cells cotransfected with tagged hAgo1 or hAgo2. In total RNA from the input samples, the ∼37-nt Ago-cleaved hairpin and ∼23-nt to 27-nt 3′ resected species are detected with the 5p probe (D), but the 3p probe detects only the full-length shRNA hairpin (E). The hAgo1-IP samples contain only the full-length shRNA hairpin that is codetected by 5p and 3p probes, but hAgo2-IP samples contain the Ago-cleaved and 3′ resected mature species.