Two Splice Variants of Nopp140 in Drosophila Melanogaster.

Abstract

The activities of non-ribosomal nucleolar proteins are now understood to be important for the normal functions of both nucleoli and C&barbelow;ajal B&barbelow;odies (CBs). Although these proteins have been studied extensively in other eukaryotes, knowledge of non-ribosomal nucleolar proteins in Drosophila melanogaster lags far behind. The n&barbelow;ucleo&barbelow;lar p&barbelow;hosphop&barbelow;rotein of 140 kDa (Nopp140) may function to shuttle box C/D and box H/ACA s&barbelow;mall n&barbelow;ucleo&barbelow;lar (sno)RNAs from the nucleus to the nucleolus, where they function in the 2 '-O-methylation and pseudouridylation of rRNA, respectively. Nopp140 homologues have been described in rat, human, Xenopus laevis , and yeast. This dissertation describes the cloning of cDNAs that encode two splice variants of Nopp140 in D. melanogaster. In addition, this dissertation addresses the localization patterns of the D. melanogaster Nopp140 splice variants in various cell types with respect to endogenous nucleolar proteins and CBs. The D. melanogaster Nopp140 gene maps within 79A5 of chromosome 3. Alternative mRNA splicing yields two variants. DmNopp140 (654 residues) is the true D. melanogaster homologue of vertebrate Nopp140 in that its carboxy terminus is 58% identical to the carboxy terminus of rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains an extensive ar&barbelow;rginine-g&barbelow;lycine-g&barbelow;lycine (RGG) domain that is found in many RNA-binding proteins such as vertebrate nucleolin. Both D. melanogaster Nopp140 variants localize to the dense fibrillar component (DFC) of D. melanogaster Schneider II cells and X. laevis oocytes. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, while DmNopp140 segregates nucleoli into phase-light and phase-dark regions. The phase light regions contain DmNopp140 and endogenous fibrillarin, while the phase-dark regions contain endogenous nucleolin. Both D. melanogaster variants co-localize to nucleoli when co-expressed in HeLa cells. Both proteins also co-localize with exogenously expressed X. laevis coilin to enlarged C&barbelow;ajal b&barbelow;odies (CBs) within HeLa cell nucleoli, but only DmNopp140 localizes to CBs in Schneider II cells. Both variants fail to localize to CBs in X. laevis oocyte nuclei. A carboxy terminal truncation, DmNopp140-DeltaRGG, fails to localize to nucleoli in HeLa cells, but like DmNopp140, it localizes with exogenously expressed coilin in HeLa cell CBs.