A study of Drosophila CPEB protein Orb2: expression, functions and regulatory mechanisms

Abstract

Translational control plays an essential role in regulating gene expression. Members of the CPEB (Cytoplasm Polyadenylation Element Binding Protein) family of proteins have been shown to bind to the 3' UTR of the target mRNAs and regulate their translation. While the classic CPEB (the CPEB1 subfamily) members have conserved roles in germline development, the newly discovered CPEB2 subfamily members are enriched in the nervous system. Earlier studies in Drosophila revealed that orb (a CPEB1 subfamily member) is required for the specification and also the establishment of proper axes of the oocyte. However, Orb is only found in the germline, with no roles in other tissue. My thesis has focused on the newly discovered CPEB2 subfamily member orb2. Our analysis on Orb2 expression and function has revealed that Orb2 is involved in multiple processes including embryogenesis, spermatogenesis, and adult locomotion control. We discovered that Orb2 is highly expressed in embryonic CNS and its presence is required for the proper asymmetric division of neural and some somatic precursor cells. In the neuroblasts (NB), loss-of-function mutations in orb2 result in the disruption of correct mitotic spindle orientation and also influence the apical cortical localization of aPKC, a critical factor in determining the asymmetric neuroblast (NB) division plane. Orb2 also functions in the germline during spermatogenesis. Specifically, Orb2 is required for meiosis progression in spermatocytes, and differentiation of spermatids (meiotic division product). The orb236 null allele displays an over-accumulation of Twine-lacZ (Twine is a meiotic CDC25), and the mutant spermatocytes arrest at the Meiosis I G2-M transition. Analyzing a hypomorphic allele of orb2 revealed another function of Orb2 during subsequent differentiation steps: the proper asymmetric elongation of the spermatids. Orb2 accumulates preferentially in the apical termini of elongating spermatids. Moreover, like Orb2, aPKC - a critical factor involved in defining apical-basal polarity in many other cell types - is also found concentrated in the apical termini of elongating spermatids. In orb2 mutants we find that the elongation of the spermatids is randomized with respect to the testes apical-terminal axis, correlated with the loss of localized aPKC in the orb2 mutants. As the same thing was observed in dividing neuroblasts, it suggests that orb2 has a conserved role in localizing aPKC, and neuroblast asymmetric division regulators may be conserved in regulating spermatids asymmetric elongation. To better understand the mechanism of Orb2 mediated translational control, we used immunoprecipitation to pull down Orb2 associated proteins in vivo and analyzed their identities by large-scale mass spectrometry. The results support the idea that Orb2 functions as a classic CPEB in an mRNP complex to regulate polyadenylation of its target mRNAs. Analysis of the data also suggests that Orb2 can physically bind to other asymmetric division regulators such as Bazooka. Further studies will help us understand how Orb2, a novel member of the CPEB2 subfamily proteins, executes its role during translational regulation of target mRNAs.