Atypical conservation and pattern of small RNAs from mir-451. (A) mir-144/mir-451 region from the University of California Santa Cruz Genome Browser; mature miRNAs (green) are encoded by the bottom strand. mir-144 exhibits a typical evolutionary pattern with far greater divergence in the terminal loop (triangle) than in mature miRNA or star arm (yellow). mir-451 is more conserved in its terminal loop than its 3′ hairpin arm. (B) Vertebrate mir-451 alignments illustrate the constrained terminal loop; asterisk marks a variable position in the 3′ hairpin. (C) Dominant miR-451 reads are 23 nt (green highlighted box), but larger species up to 30 nt (light green triangle) are observed in multiple species; rare reads are derived from the 3p arm. Data were analyzed from GSM494811 (K562) and GSM433295 (mouse testis); see Dataset S1 for full analysis of these and other miR-451–containing libraries. (D) mir-144 generates typical pre-miRNA hairpin and mature miRNA. (E) mir-451 generates small RNAs ranging from 22 to 24 nt to >30 nt. Endogenous mir-144 and mir-451 were detected in K562 cells or MEL cells induced with HMBA; these miRNAs are not expressed by uninduced MEL or GFP-transduced MEL cells. Ectopic miRNAs were generated by transfection of HeLa cells with mir-144/451 plasmid or transduction of MEL cells with mir-144/451 lentivirus. (F) The two upper bands (**) in miR-451 blots exhibit differential mobility in different acrylamide gels from ∼32 to >40 nt. (G) Sensor assays in HeLa cells transfected with mir-144/451 show repression of miR-144-3p and miR-451 perfect targets and of miR-451 seed target. Sensor values were normalized against mir-1-2 construct; SDs from quadruplicate assays are shown.

Maturation of miR-451 requires Drosha complex but not Dicer. (A) siRNA-mediated knockdown of DGCR8 and Drosha showed their requirement for biogenesis of miR-144 and miR-451 produced by mir-144/mir-451 construct. A probe against the 3p arm of mir-451 hairpin defines pre-mir-451 hairpin bands (pre), which are substantially decreased on knockdown of DGCR8 and Drosha. The miR-451 blot was stripped and reprobed for U6 as a loading control. (B) Western blot verifies the absence of Dicer in a viable Dicer^−/− MEF cell line. Transfection of mir-144/mir-451 into these cells yielded mature miR-451 small RNAs. We used 16% gels, but blots in B were run at 500 V, whereas blots in A were run at 250 V, causing differential migration of the pre-miRNA hairpin (Fig. S1). (C) The mir-144/mir-451 construct was highly active in Dicer^−/− MEFs, both on perfect and bulged sensors. Quadruplicate assays were performed, and SDs were plotted; tests in HeLa were normalized to mir-1-2, whereas tests in Dicer^−/− were normalized to mir-144/451 construct in which miR-451 was reprogrammed with miR-23a-3p.

The mir-451 backbone confers Dicer-independent expression of other miRNAs. (A) mir-451 hairpins were reprogrammed with other miRNAs and tested against perfect sensors in HeLa (blue bars). Activities of canonical miRNA constructs are shown for comparison (orange bars); all tests were normalized to mir-1-2 as a noncognate miRNA (white bars). (B) Canonical miRNA constructs are essentially inactive in Dicer^−/− MEFs (orange bars), whereas reprogrammed mir-451 hairpins are highly active (blue bars). We also observed strong repression of bulged sensors (dark blue bars), indicating that the 5′ ends of miRNAs from reprogrammed constructs were defined accurately. Data were normalized to sensor activity in the presence of 451:miR-23a-3p as a noncognate functional control miRNA; 451:miR-199a-3p was used as a control for the reprogrammed miR-23a-3p sensor test. Quadruplicate assays were performed, and SDs were plotted. (C) Northern analysis confirms the failure of Dicer^−/− MEFs to process ectopic canonical miRNAs. In contrast, short RNAs were produced from reprogrammed mir-451 backbones in Dicer^−/− MEFs; analysis of untransfected cells reveals the accumulation of some endogenous pre-miRNA species (asterisks).

Structural requirements for miR-451 biogenesis. (A) Schematic of human mir-451 hairpin. Site mutants were generated in the context of a functional mir-144/451 plasmid. (B) Regulatory activity of mir-451 variants was assayed using a 2× perfect sensor normalized to its activity in the presence of mir-1-2 (Fig. 1G). Pairing at the putative cleavage site across from positions 10 and 11 from the 5′ end of mature miR-451 is essential for miR-451 activity as is the integrity of the lower stem. (C) Northern analysis reveals that the three cleavage site mutants accumulate >40-nt species; the lower stem mutant did not produce any short RNA species. (D) Stripping and reprobing reveal the accumulation of pre-mir-144 and mature miR-144 from the different structural variants, providing a loading control.

Ago2 Slicer activity mediates miR-451 maturation. (A) Microarray profiling of wild-type and Ago2^−/− bone marrow revealed that miR-451 is the highest-expressed miRNA in bone marrow and uniquely deficient in the absence of Ago2. (B) Northern blot verifies loss of processed miR-451 intermediates in Ago2^−/− marrow. (C Top) Western blot verification of Ago2^−/− MEFs reconstituted with control virus, Ago2-expressing virus, or Ago2[D669A] virus. (Bottom) Transfection of mir-144/mir-451 construct into this panel of cells shows that Ago2 Slicer function is strictly required for production of mature miR-451. (D Upper) Ago2 proteins were immunoprecipitated from the panel of reconstituted Ago2^−/− MEFs. (Lower) Associated small RNAs were analyzed by Northern blot and probed for miR-451; Ago2[D669A] cannot mature miR-451. (E) Model for mir-451 processing.