Drosha-independent DGCR8/Pasha pathway regulates neuronal morphogenesis

Abstract

Cleavage of microRNAs and mRNAs by Drosha and its cofactor Pasha/DGCR8 is required for animal development, but whether these proteins also have independent roles in development has been unclear. Known phenotypes associated with loss of either one of these two proteins are very similar and consistent with their joint function, even though both cofactors are involved with additional distinct RNA biogenesis pathways. Here, we report clear phenotypic differences between drosha and pasha/dgcr8 null alleles in two postembryonic lineages in the Drosophila brain: elimination of pasha/dgcr8 leads to defects that are not shared by drosha null mutations in the morphology of gamma neurons in the mushroom body lineage, as well as many neurons in the anterodorsal projection neuron lineage. These morphological defects are not detected in neurons that are genetically depleted of two additional microRNA pathway components, dicer-1 and argonaute1, indicating that they are not due to loss of microRNA activity. They are, however, phenocopied by a newly identified recessive gain-of-function allele in drosha that probably interferes with the microRNA independent functions of Pasha/DGCR8. These data therefore identify a general Drosha-independent DGCR8/Pasha pathway that promotes proper morphology in multiple neuronal lineages. Given that reduction of human DGCR8/Pasha may contribute to the cognitive and behavioral characteristics of DiGeorge syndrome patients, disruption of this newly described pathway could underlie human neurological disease.

Keywords: RNA metabolism; miRNA; neurodevelopment.

Fig. 1.

Genetic screen yields two unique drosha alleles that are defective in miRNA biogenesis. (A) Schematic of Drosha protein indicates two RNase III domains (RIII) and double-stranded RNA binding domain (gray square), as well as locations of mutations. The drosha^∆,E859K allele deletes 147 amino acids (∆) and contains a missense mutation at residue 859. (B–E) Eye discs from third-instar larvae containing WT (B), drosha^R662X (C), drosha^R1113X (D), and drosha^∆,E859K (E) clones stained with anti-Drosha antibodies (magenta). Clones are indicated by the absence of GFP. (Scale bar, 10 μm.) (F) Northern blots of total RNA from third-instar larvae of indicated genotypes probed for miR-2b, let-7, miR-125, and U6 snRNA (loading control). (G) In vitro processing of pri-let-7 by WT, E859K, ∆, E859K+∆, or dominant negative (DN) Drosha proteins. Length of incubation is indicated in minutes.

Fig. 2.

Core microRNA pathway components are required for NB maintenance in the mushroom body lineages. OK107-Gal4, UAS-mCD8::GFP-labeled WT (A), drosha^R662X (B), drosha^R1113X (C), drosha^∆,E859K (D), dcr-1^Q1147X (E), ago1^Q127X (F), and pasha^KO (G) adult MB neuroblast clones generated in newly hatched larvae and stained with anti-FasII antibodies. Arrowheads indicate α′/β′ neuron mistargeting and arrows indicate ɣ neuron overextension. (H) Average number of cells in adult MB clones of indicated genotypes. Differences between WT and all mutants are statistically significant (P < 0.0001). (Scale bar for A–G, 25 μm.)

Fig. 3.

Distinct roles for miRNA processing components in the adPN lineage. (A) Average number of cells in adult Acj6-labeled adPN clones of indicated genotypes. Numbers of clones analyzed are indicated in parentheses. Acj6-Gal4, UAS-mCD8::GFP-labeled adult WT (B), dcr-1^Q1147X (C), ago1^Q127X (D), pasha^KO (E), drosha^R662X (F), drosha^∆,E859K (G), and rescued drosha^∆,E859K (H) adPN NB clones generated in newly hatched larvae and stained with anti-GFP (green) and anti-nc82 (magenta) antibodies. Glomeruli names are labeled in white. Areas exhibiting targeting defects are circled and arrowheads point to dendritic mistargeting. (Scale bar for B–H, 10 μm.)

Fig. 4.

DL1 drosha^∆,E859K mutant neurons display phenotypes that are rescued by WT drosha. Acj6-Gal4, UAS-mCD8::GFP-labeled WT (A and B), drosha^∆,E859K (C and D), and rescued drosha^∆,E859K (E and F) adult DL1 clones generated in newly hatched larvae and stained with anti-GFP (green) and anti-nc82 (blue) antibodies. (Scale bar: A, C, and E, 10 μm; B, D, and F, 20 μm.)

Fig. 5.

Forced expression of Drosha^∆,E859K leads to neuronal overgrowth. Drosha^R662X (A, C, E, G, and I) or WT (B, D, F, H, and J) OK107-Gal4, UAS-mCD8::GFP-labeled adult MB clones expressing UAS-Drosha^WT (A and B), UAS-Drosha^∆+E859K (C and D) UAS-Drosha^∆ (E and F), UAS-Drosha^E859K (G and H), or UAS-Drosha^DN (I and J) and stained with anti-FasII antibody (magenta) and anti-Flag antibody (magenta; Insets in C, E, and I). Arrows point to mistargeted neurons. (Scale bar, 25 μm.)