Drosophila Orb2 targets genes involved in neuronal growth, synapse formation, and protein turnover

Abstract

In the study of long-term memory, how memory persists is a fundamental and unresolved question. What are the molecular components of the long-lasting memory trace? Previous studies in Aplysia and Drosophila have found that a neuronal variant of a RNA-binding protein with a self-perpetuating prion-like property, cytoplasmic polyadenylation element binding protein, is required for the persistence of long-term synaptic facilitation in the snail and long-term memory in the fly. In this study, we have identified the mRNA targets of the Drosophila neuronal cytoplasmic polyadenylation element binding protein, Orb2. These Orb2 targets include genes involved in neuronal growth, synapse formation, and intriguingly, protein turnover. These targets suggest that the persistent form of the memory trace might be comprised of molecules that maintain a sustained, permissive environment for synaptic growth in an activated synapse.

Fig. 1.

Drosophila Orb2 binds to the 3′UTR of Oskar, Tequila, DaPKC, and Murashka. (A) A schematic diagram shows the experimental design for the mRNA protein pull-down assay. (B) Endogenous Orb2RB and (C) exogenously expressed Orb2RAHA (Elav-GAL4:: UAS-Orb2RAHA) specifically bind to the 3′UTR of Oskar, Murashka, Tequila, and DaPKC. Specificity of the assay was determined by using nonspecific RNA EF1α and Actin88F. The asterisk indicates an Orb2 immunoreactive polypeptide that is most likely derived from the degradation of Orb2RB, because it was not observed when pull down was performed using orb2 null fly heads (Fig. S1). (D) Orb2RAHA binds only to long-term memory-related genes. The endogenous Orb2RB binds to the intermediate-term memory gene Amnesiac but not the short-term memory genes Dunce, Rutabaga, and Nf1. The amount of Orb2RB bound to Nf1 is similar to control EF1α mRNA and thus, is considered as background binding. Orb2RAHA binds only to the control Oskar mRNA but none of the other genes. The same blot was probed with RNA-binding protein Hrp48 to ensure the presence of the probe RNA. (E) The conserved Zn-finger domain in the C-terminal end of Orb2RB is required for mRNA binding. (Upper) The RRMs and the Zn-finger domain are indicated. The numbers indicate amino acid numbers. (Lower) The pull down from head extracts of Orb2RB lacking the Zn-finger domain (Act-GAL4::UAS-Orb2RBΔZnHA) or wild-type Orb2RB (Act-GAL4:: UAS-Orb2RBHA) were probed with anti-HA antibodies. The lysates show the relative amounts of Orb2 proteins in head extracts used in the pull down. (F) The CPE element is not essential for Orb2 binding. Mutation of the putative CPE element in Tequila 3′UTR UUUUGU (TeqWT) to UUCAGU (TeqMT) reduces but does not abolish the endogenous Orb2RB binding.

Fig. 2.

The long-term memory-related genes Tequila, DaPKC, and Murashka are translationally repressed by Orb2 in S2 cells. When overexpressed, both Orb2RB (A) and Orb2RA (B) selectively suppress the translation of target mRNA in S2 cells. The relative activity of firefly luciferase over renilla luciferase activity in the presence (+) or absence (−) of Orb2RB (A) or Orb2RA (B) is plotted. (C) The translation of firefly luciferase-bearing control SV40 3′UTR or the 3UTR of Tequila RC isoform or Actin88F were not affected. The Tequila gene has five predicted isoforms of which Tequila RC has its own 3′UTR. Both forms of Orb2 bind to the common 3′UTR but not to the RC 3′UTR. The ratios were obtained from at least three independent experiments for each construct, and each experiment consists of three independent transfections. The significance was tested by Student t test, and a P value < 0.05 was considered significant and is indicated with an asterisk. The error bars indicate SEM. (D) The long-term memory gene Tequila is regulated by Orb2 in the adult fly brain. Western blot using anti-Tequila antibody shows that, in orb2 null mutant (orb2^−/−), Tequila protein level is up-regulated, whereas transgenic overexpression of Orb2RA (Orb2^+/+;ELAVGAL4:UAS-RAHA) and Orb2RB (Orb2^+/+; ELAVGAL4:UAS-RBHA) results in about 50% reduction of Tequila expression levels. Teq^f10792 is a hypomorphic allele of the Tequila gene and serves as antibody control. The asterisk indicates a nonspecific immunoreactive band, and coomassie staining of the same membrane serves as a loading control. (E) The steady-state level of Tequila and Murashka mRNA levels are altered on misregulation of Orb2. Tequila and Murashka mRNA levels are increased in orb2 null mutants and in Orb2 overexpressing flies. RT-PCR was used to analyze the Tequila, Murashka, and DaPKC mRNA on total RNA isolated from indicated genetic backgrounds. GAPDH served as PCR control. DaPKC mRNA level was not changed, implying that mRNA levels of all Orb2 targets are not susceptible to changes in Orb2 protein level.

Fig. 3.

Orb2 binds to the bona fide target mRNA. (A) A representative blot of binding of endogenous Orb2RB protein to the targets isolated from GeneChip analysis. The gene CG14528 did not bind to Orb2RB. (B) The common and distinct targets of Orb2RA and Orb2RB were obtained from GeneChip analysis. The asterisk indicates an Orb2 immunoreactive polypeptide, which is most likely derived from the degradation of Orb2RB. The genes marked with a box did not bind to either form of Orb2. Fig. S3 shows binding to additional targets. (C–E) Orb2RB represses the translation of the majority of targets in S2 cells. (C) Representative plots of Orb2RB-dependent translational suppression of Orb2 targets. Fig. S3 shows additional Orb2 targets. (D) The translation of the majority of the target genes was reduced except Synaptobrevin and CG6129, which showed modest but statistically insignificant suppression. P value < 0.05 was considered significant in a Student t test. (E) The translation of firefly luciferase with SV40 3′UTR or the 3′UTR from CG14528, which did not bind to Orb2RB, was not affected by Orb2RB.