The Drosophila CPEB Protein Orb2 Has a Novel Expression Pattern and Is Important for Asymmetric Cell Division and Nervous System Function

Abstract

Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. Here we characterize the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. We also show that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm. C YTOPLASMIC polyadenylation element binding (CPEB) family proteins bind to target sequences in the 39-UTR of mRNAs and control their localization and translation. CPEB proteins function in many different biologocial contexts including oogenesis in Xenopus and Drosophila (Lantz et al. 1992(Lantz et al. , 1994Christerson and McKearin 1994;Hake and Richter 1994), synaptic plasticity in the rat hippocampus (Wu et al. 1998), and long-term memory in Aplysia . In Xenopus oocytes, CPEB regulates oocyte maturation. Prior to progesterone stimulation, it functions to repress target mRNAs in conjunction with proteins such as Maskin. After hormone stimulation, CPEB is phosphorylated, and the phosphorylated isoform recruits factors that stimulate poly(A) addition and translational activation of mRNAs that mediate the maturation process. In Drosophila, the CPEB protein Orb is required for oocyte determination and the establishment of the anterior-posterior and dorsal-ventral axes of the developing egg chamber. It functions by promoting the on-site translation of mRNAs that are localized in the developing oocyte, and like the Xenopus oocyte CPEB, its activity is regulated by phosphorylation.Most animals have two or more CPEB genes. Completed genome sequences reveal that humans, mice, and Caenorhabditis elegans have four CPEB genes, but only two CPEBs in Drosophila. The homology between the CPEB proteins is limited to the C-terminal half of the protein, which has two RNA-recognition motif (RRM) domains and a zinc finger domain, while the N-terminal half is highly divergent. Phylogenetic trees indicate that the CPEB genes fall into two differen...

RNA-binding profiles of Drosophila CPEB proteins Orb and Orb2

“…Neurogenesis is one of the top-ranking GO terms associated with Orb2 CLIP targets, supporting Orb2's crucial role in this process (Fig. 5A) (12,13,20). Moreover, Orb2-bound transcripts are generally associated with the development of epithelial tissues and germline, suggesting that Orb2 may regulate a wide array of tissue-patterning and polarization processes (Dataset S5).…”

“…Orb2 has been best studied for its role in LTM formation and nervous system development, and some of its previously identified targets were suggested to contribute to these processes (12,13,19,20,26). To determine what other biological processes may be regulated by Orb2 targets,we turned to Gene Ontology (GO) analysis (50) performed with the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (51,52).…”

“…The molecular machinery controlling local protein synthesis has been best studied in vertebrate oocyte development during which members of the cytoplasmic polyadenylation element binding (CPEB) protein family, in concert with other RNA-binding proteins, control the temporal expression of maternal RNAs (8,9). More recently, CPEB proteins also have been found to be expressed in adult nervous systems (10)(11)(12)(13) where they are thought to be involved in synaptic plasticity and LTM (12,(14)(15)(16)(17), suggesting evolutionary conservation of the mechanism of local translation across species, tissues, and developmental stages.…”

“…Although both proteins play critical and nonredundant roles in germline formation (18,19), Orb2 is also essential for nervous system development (13) and is acutely required for LTM (12,20), as is Orb (15). Both proteins contain an unstructured N-terminal poly-glutamine (poly-Q) stretch and a well-conserved C-terminal RNA-binding domain (RBD) consisting of two RNA recognition motifs (RRMs) and a zinc finger (Znf) region (12).…”

“…Orb2 has two variants, Orb2A and Orb2B (12), which differ in the composition of the N terminus preceding the poly-Q and share a common RBD (12). The poly-Q is required exclusively for LTM, whereas the RBD is required for both development and LTM (20), and its mutations are lethal (12,13). Moreover, the RBD of Orb2 can be functionally replaced by the RBD of mouse CPEB2 (mCPEB2) but not by that of Orb or mCPEB1, suggesting the conservation of target specificity within but not between the CPEB subfamilies, at least in regard to their developmental function (20).…”