Noncanonical function of Capicua as a growth termination signal in Drosophila oogenesis.

Capicua (Cic) proteins are conserved HMG-box transcriptional repressors that control receptor tyrosine kinase (RTK) signaling responses and are implicated in human neurological syndromes and cancer. While Cic is known to exist as short (Cic-S) and long (Cic-L) isoforms with identical HMG-box and associated core regions but distinct N termini, most previous studies have focused on Cic-S, leaving the function of Cic-L unexplored. Here we show that Cic-L acts in two capacities during Drosophila oogenesis: 1) as a canonical sensor of RTK signaling in somatic follicle cells, and 2) as a regulator of postmitotic growth in germline nurse cells. In these latter cells, Cic-L behaves as a temporal signal that terminates endoreplicative growth before they dump their contents into the oocyte. We show that Cic-L is necessary and sufficient for nurse cell endoreplication arrest and induces both stabilization of CycE and down-regulation of Myc. Surprisingly, this function depends mainly on the Cic-L-specific N-terminal module, which is capable of acting independently of the Cic HMG-box-containing core. Mirroring these observations, basal metazoans possess truncated Cic-like proteins composed only of Cic-L N-terminal sequences, suggesting that this module plays unique, ancient roles unrelated to the canonical function of Cic.

Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs.

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. Here, we have analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, we show that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1. Overall, our results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.

Using CRISPR-Cas9 to Study ERK Signaling in Drosophila.

Genome engineering using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated nuclease 9 (Cas9) technology is revolutionizing biomedical research. CRISPR-Cas9 enables precise editing of genes in a wide variety of cells and organisms, thereby accelerating molecular studies via targeted mutagenesis, epitope tagging, and other custom genetic modifications. Here, we illustrate the CRISPR-Cas9 methodology by focusing on Capicua (Cic), a nuclear transcriptional repressor directly phosphorylated and inactivated by ERK/MAPK. Specifically, we use CRISPR-Cas9 for targeting an ERK docking site of Drosophila Cic, thus generating ERK-insensitive mutants of this important signaling sensor.