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Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains
Published on: November 6, 2017
Two splice variants of Nopp140 in Drosophila melanogaster
John M Waggener1, Patrick J DiMario
1Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803-1715, USA.
Abstract:
The Nopp140 gene of Drosophila maps within 79A5 of chromosome 3. Alternative splicing yields two variants. DmNopp140 (654 residues) is the sequence homolog of vertebrate Nopp140. Its carboxy terminus is 64% identical to that of the prototypical rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains a glycine/arginine-rich domain that is often found in RNA-binding proteins such as vertebrate nucleolin. Both Drosophila variants localize to nucleoli in Drosophila Schneider II cells and Xenopus oocytes, specifically within the dense fibrillar components. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, whereas DmNopp140 partitions HeLa nucleoli into phase-light and phase-dark regions. The phase-light regions contain DmNopp140 and endogenous fibrillarin, whereas the phase-dark regions contain endogenous nucleolin. When coexpressed, both Drosophila variants colocalize to HeLa cell nucleoli. Both variants fail to localize to endogenous Cajal bodies in Xenopus oocyte nuclei and in HeLa cell nuclei. Endogenous HeLa coilin, however, accumulates around the periphery of phase-light regions in cells expressing DmNopp140. The carboxy truncation (DmNopp140DeltaRGG) also fails to localize to Cajal bodies, but it forms similar phase-light regions that peripherally accumulate endogenous coilin. Conversely, we see no unusual accumulation of coilin in cells expressing DmNopp140-RGG.
Insights
The Drosophila Nopp140 gene produces two variants that localize to nucleoli. One variant, DmNopp140, alters nucleolar structure and recruits coilin, while the other, DmNopp140-RGG, does not.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The Nopp140 gene in Drosophila melanogaster is crucial for nucleolar function.
- Alternative splicing generates distinct DmNopp140 variants with varying functional domains.
Purpose of the Study:
- To investigate the localization and function of Drosophila Nopp140 variants in nucleoli.
- To determine the impact of these variants on nucleolar structure and protein recruitment.
Main Methods:
- Gene mapping and sequence analysis of Drosophila Nopp140 variants.
- Subcellular localization studies in Drosophila, Xenopus, and HeLa cells.
- Immunofluorescence microscopy to observe protein distribution and nucleolar organization.
Main Results:
- Both DmNopp140 and DmNopp140-RGG variants localize to the dense fibrillar components of nucleoli.
- DmNopp140 induces nucleolar partitioning and recruits endogenous coilin, unlike DmNopp140-RGG.
- Neither variant localizes to Cajal bodies, but DmNopp140 and its truncation affect coilin localization.
Conclusions:
- Drosophila Nopp140 variants exhibit differential effects on nucleolar organization and protein interactions.
- The glycine/arginine-rich domain in DmNopp140-RGG may influence its distinct localization and functional properties.
- These findings provide insights into nucleolar dynamics and the roles of Nopp140 homologs in different organisms.
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