Functioning of theDrosophila orbgene ingurkenmRNA localization and translation

Abstract

The orb gene encodes an RNA recognition motif (RRM)-type RNA-binding protein that is a member of the cytoplasmic polyadenylation element binding protein (CPEB) family of translational regulators. Early in oogenesis, orb is required for the formation and initial differentiation of the egg chamber, while later in oogenesis it functions in the determination of the dorsoventral (DV) and anteroposterior axes of egg and embryo. In the studies reported here, we have examined the role of theorb gene in the gurken (grk)-Drosophila epidermal growth factor receptor (DER) signaling pathway. During the previtellogenic stages of oogenesis, the grk-DER signaling pathway defines the posterior pole of the oocyte by specifying posterior follicle cell identity. This is accomplished through the localized expression of Grk at the very posterior of the oocyte. Later in oogenesis, thegrk-DER pathway is used to establish the DV axis. Grk protein synthesized at the dorsal anterior corner of the oocyte signals dorsal fate to the overlying follicle cell epithelium. We show that orb functions in both the early and late grk-DER signaling pathways, and in each case is required for the localized expression of Grk protein. We have found thatorb is also required to promote the synthesis of a key component of the DV polarity pathway, K(10). Finally, we present evidence that Orb protein expression during the mid- to late stages of oogenesis is, in turn, negatively regulated by K(10).

Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly

“…Interestingly, expression of the PLP-GFP transgene partially restored embryonic viability (53% hatched, p<0.0001, orb vs. PLP-GFP; orb by chi-squared test), supporting a genetic interaction between orb and plp required for viability (Figure 6D). Orb likely further supports embryonic viability through regulating other mRNA targets or protein partners (Chang et al, 2001;Mansfield et al, 2002;Norvell et al, 2015). Nonetheless, our data argue that plp is a critical downstream target subject to Orb regulation.…”

“…Embryonic lethality in orb mutants is largely attributed to the requirement of Orb in establishing the embryonic patterning axes via its role in mediating localization and translation of osk and grk mRNAs (Christerson and McKearin, 1994;Chang et al, 1999;Chang et al, 2001;Castagnetti and Ephrussi, 2003). Interestingly, expression of the PLP-GFP transgene partially restored embryonic viability (53% hatched, p<0.0001, orb vs. PLP-GFP; orb by chi-squared test), supporting a genetic interaction between orb and plp required for viability (Figure 6D).…”

“…Drosophila encodes two CPEB proteins, oo18 RNA-binding protein (Orb) and Orb2 (Lantz et al, 1992;Hafer et al, 2011). While Orb2 functions are best defined in the testis and central nervous system (Keleman et al, 2007;Mastushita-Sakai et al, 2010;Hafer et al, 2011;Xu et al, 2012), Orb regulates RNA localization and translation during oogenesis and early embryogenesis (Lantz et al, 1992;Christerson and McKearin, 1994;Chang et al, 2001;Castagnetti and Ephrussi, 2003;Rojas-Ríos et al, 2015). Orb is orthologous to mammalian CPEB1 and similarly promotes polyadenylation to regulate the stability and translation of its target mRNAs, including oskar (osk), gurken (grk), and Autophagy-related 12 (Atg12) mRNAs (Christerson and McKearin, 1994;Chang et al, 1999;Chang et al, 2001;Castagnetti and Ephrussi, 2003;Norvell et al, 2015;Rojas-Ríos et al, 2015;Davidson et al, 2016).…”

Tudor domain containing protein 5-like(Tdrd5l) identifies a novel germline body and regulates maternal RNAs during oogenesis

“…Work by other labs has shown that Grk translation in the oocyte is activated by Oo18 RNA binding protein (Orb) (43–45). Orb is a cytoplasmic polyadenylation element binding protein (CPEB) that is highly expressed in the oocyte where it recruits Wispy to lengthen the grk poly(A) tail to activate its translation (44).…”

“…In the most extreme cases we observed diffuse Kin-LacZ localization in the center of the oocyte (Fig S7C white arrow and Fig S7D yellow arrow). These data indicate that the defects in osk posterior localization may be caused by loss of grk function rather than defects in the direct regulation of osk itself.Tdrd5l represses Orb protein expression in nurse cellsWork by other labs has shown that Grk translation in the oocyte is activated by Oo18RNA binding protein (Orb)(43)(44)(45). Orb is a cytoplasmic polyadenylation element binding protein (CPEB) that is highly expressed in the oocyte where it recruits Wispy to lengthen the grk poly(A) tail to activate its translation(44).…”