Scheme for the generation of the pasha^KO deletion allele. (A) FLP-mediated recombination was used to delete genomic sequence between FRT-bearing piggyBac elements that flank the pasha locus. (B) The progenitor chromosomes bear piggyBac insertion in trans, which were brought in cis as a hybrid element after recombination. (C) PCR analysis verifies that the pasha^KO allele juxtaposes the left (L) and right (R) arms of the progenitor piggyBacs, while presence of the novel hybrid (H) product reflects the deletion of the intervening pasha locus. (D) RT-PCR tests demonstrate that pasha transcripts are absent from pasha^KO larvae, while expression of the neighboring locus CG1792 is maintained. Quantitative tests indicate 1.65-fold increase in CG1792 mRNA in this mutant (see Fig. S1 in the supplemental material).

Pasha^KO larvae strongly accumulate pri-miRNA species. (A) qRT-PCR) was used to assess pri-miRNA levels in Canton S and homozygous pasha^KO larvae, as normalized to rp49. Three independent RNA samples were reverse transcribed for each genotype, and six qPCR measurements were made for each cDNA preparation. The y axis depicts the pri-miRNA ratio and standard error determined for each biological replicate. (B) Northern analysis of pri-mir-1 in Canton S and homozygous pasha^KO larvae revealed the accumulation of an ∼8-kb transcript in the pasha mutant. Hybridization with an ago2 probe and ethidium staining of rRNA served as loading controls.

Phenotypes of dcr-1 and pasha^KO adult mutant clones. Compared to wild-type, clonal loss of either Dcr-1 or Pasha results in small rough eyes (A to C) and loss of external mechanosensory bristle structures (D to F). Eye clones were made using the EGUF system (49), which generates eyes that are nearly composed entirely of mutant tissue.

Canonical miRNA and mirtron pathways in Drosophila. Key protein families include RNase III endonucleases (Drosha and Dicer-1), double-stranded RNA-binding domain proteins (Pasha and Loqs) and Argonaute effectors (AGO1 and AGO2). Canonical miRNA precursors are cleaved by the Drosha/Pasha complex in the nucleus, cleaved again by the Dicer-1/Loqs complex in the cytoplasm, and predominantly loaded into AGO1. Mirtrons are short hairpin introns that use the splicing and debranching machinery to bypass the requirement for Drosha/Pasha but are subsequently processed by Dicer-1 to generate miRNA-class regulatory RNAs.

Small RNA expression in mutants for canonical miRNA and RNAi factors. Total RNAs were extracted from homozygous larvae of the following genotypes: Canton S (CS) = wild type, ago2 = ago2⁴¹⁴; dcr2 = dcr-2^L811fs; loqs = loqs^KO; pasha = pasha^KO. Blots were stripped and probed for 2S rRNA as a loading and transferring control (shown beneath each miRNA blot). Some blots were probed with multiple miRNAs and therefore have the same 2S control. (A) Canonical miRNAs that are strongly dependent on Loqs; i.e., for which the mature species is reduced (arrowhead) and there is accumulation of pre-miRNA hairpins (bracket). All of these show reduced miRNA and pre-miRNA levels in pasha^KO. (B) miRNAs that are mildly dependent on Loqs; i.e., for which there is pre-miRNA accumulation but mature species are relatively unaffected. All of these still show reduced miRNA and pre-miRNA in pasha. (C) Mirtron-derived miRNAs. These are strongly affected in loqs but mostly unaffected in pasha. There is a slight reduction in pre-mir-1010 and miR-1010 in pasha, although this is potentially due to an effect on the expression of its host gene CG31163.

pasha distinguishes the function of canonical miRNAs and mirtrons. Shown are eye imaginal discs stained for DNA and a GFP sensor, with DsRed fluorescence marking active small RNA transgenes; the right panels depict the merged GFP/DsRed channels. (A to C) MARCM clones that are homozygous for a given chromosome 3R and ectopically express a hybrid DsRed:mir-7 transgene were tested for their ability to repress a miRNA sensor, a ubiquitously transcribed GFP target bearing two miR-7 binding sites. miRNA-mediated target repression was detected in control clones (A), but not in dcr-1 clones (B) or pasha^KO clones (C). (D to F) MARCM clones that are homozygous for a given chromosome 3R and ectopically express a hybrid DsRed:mir-1004 (mirtron) transgene were tested for their ability to repress a mirtron sensor, a ubiquitously transcribed GFP transcript bearing two miR-1004 binding sites. Mirtron-mediated target repression was detected in control clones (D) but not dcr-1 clones (E); pasha^KO homozygous cells generated active mirtrons (F).

Distinct growth defects observed in mutant clones compared to homozygous pasha^KO animals. (A to C) Small retinal clones in third instar eye imaginal discs stained with β-galactosidase antibody. Homozygous mutant cells have no β-galactosidase (−/−), their twin-spots have a high level of β-galactosidase (+/+), and unrecombined heterozygous cells have an intermediate level of β-galactosidase (+/−). Compared to control clones, dcr-1 and pasha^KO clones are much smaller than their respective twin-spots. (D to F) Large clones in third-instar eye imaginal discs generated with the Minute technique and stained with GFP antibody; the magnification is lower than in panels A to C. Minute homozygosity is cell lethal, and Minute heterozygous cells have a severe growth disadvantage, thus permitting the recovery of large, GFP^−/− mutant clones. (D) Control clones occupy nearly the entire disc; the arrow and arrowhead point to small patches of heterozygous cells in the antenna and retina, respectively. Homozygous dcr-1 cells (E) and pasha^KO (F) cells compete poorly even against Minute cells, so large GFP⁺ sectors remain. (G) Wild-type brain/imaginal disc complex stained for the neural marker Elav; the olfactory lobe (OL), ventral ganglion (VG), eye-antennal disc (E), and leg disc (L) are indicated. Elav is highly expressed by the brain and the developing retina of the eye disc (arrows, G′). (H) Brain/imaginal disc complex from a homozygous pasha^KO larva is nearly devoid of imaginal discs and differentiate only small patches of Elav⁺ retina. The pasha^KO brain exhibits rudimentary optic lobes, but the ventral ganglion is of fairly normal size.