The Drosophila CPEB Homolog, Orb, Is Required for Oskar Protein Expression in Oocytes

Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix–fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3′UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3′UTRs, and provide structural insights into a novel protein–RNA interaction motif involving a hydrolase-related domain.

mentioning

confidence: 99%

Structure of Drosophila Oskar reveals a novel RNA binding protein

Yang

et al. 2015

“…1 C and D) for Orb2RA. In the absence of known Orb2 targets, we used the 3′UTR of Oskar gene, a target of the other CPEB family member in Drosophila (Orb1) as a possible positive control (16). The Oskar gene is required for stable long-term memory in Drosophila (14).…”

Section: Resultsmentioning

confidence: 99%

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

Mastushita-Sakai

White-Grindley²,

Samuelson³

et al. 2010

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.protein synthesis | synaptic plasticity | memory

“…Although a small number of fly Orb2 mRNA targets was described (26), the authors could not identify the bound sequence unambiguously. Thus, the full spectrum of fly CPEB targets is largely unknown, with previous studies characterizing only a limited number of interacting mRNAs (26)(27)(28). Elucidation of the RNA-binding specificity of Orb2 and the identification of its mRNA targets is essential to understand the role of downstream effectors in local translation-dependent processes, including LTM formation.…”

Section: Significancementioning

confidence: 99%

RNA-binding profiles of Drosophila CPEB proteins Orb and Orb2

Stepien

Oppitz

Gerlach

et al. 2016

Localized protein translation is critical in many biological contexts, particularly in highly polarized cells, such as neurons, to regulate gene expression in a spatiotemporal manner. The cytoplasmic polyadenylation element-binding (CPEB) family of RNA-binding proteins has emerged as a key regulator of mRNA transport and local translation required for early embryonic development, synaptic plasticity, and long-term memory (LTM). Drosophila Orb and Orb2 are single members of the CPEB1 and CPEB2 subfamilies of the CPEB proteins, respectively. At present, the identity of the mRNA targets they regulate is not fully known, and the binding specificity of the CPEB2 subfamily is a matter of debate. Using transcriptome-wide UV cross-linking and immunoprecipitation, we define the mRNA-binding sites and targets of Drosophila CPEBs. Both Orb and Orb2 bind linear cytoplasmic polyadenylation element-like sequences in the 3′ UTRs of largely overlapping target mRNAs, with Orb2 potentially having a broader specificity. Both proteins use their RNA-recognition motifs but not the Zinc-finger region for RNA binding. A subset of Orb2 targets is translationally regulated in cultured S2 cells and fly head extracts. Moreover, pan-neuronal RNAi knockdown of these targets suggests that a number of these targets are involved in LTM. Our results provide a comprehensive list of mRNA targets of the two CPEB proteins in Drosophila, thus providing insights into local protein synthesis involved in various biological processes, including LTM.CPEB | CLIP | Orb2 | translation | long-term memory