Loss of Drosophila nucleostemin 2 (NS2) blocks nucleolar release of the 60S subunit leading to ribosome stress
Four nucleostemin-like proteins (nucleostemin (NS) 1–4) were identified previously in Drosophila melanogaster. NS1 and NS2 are nucleolar proteins, while NS3 and NS4 are cytoplasmic proteins. We showed earlier that NS1 (homologous to human GNL3) enriches within the granular components (GCs) of Drosophila nucleoli and is required for efficient maturation or nucleolar release of the 60S subunit. Here, we show that NS2 is homologous to the human nucleostemin-like protein, Ngp1 (GNL2), and that endogenous NS2 is expressed in both progenitor and terminally differentiated cell types. Exogenous GFP-NS2 enriched within nucleolar GCs versus endogenous fibrillarin that marked the dense fibrillar components (DFCs). Like NS1, depletion of NS2 in midgut cells blocked the release of the 60S subunit as detected by the accumulation of GFP-RpL11 within nucleoli, and this likely led to the general loss of 60S subunits as shown by immunoblot analyses of RpL23a and RpL34. At the ultrastructural level, nucleoli in midgut cells depleted of NS2 displayed enlarged GCs not only on the nucleolar periphery but interspersed within the DFCs. Depletion of NS2 caused ribosome stress: larval midgut cells displayed prominent autophagy marked by the appearance of autolysosomes containing mCherry-ATG8a and the appearance of rough endoplasmic reticulum (rER)-derived isolation membranes. Larval imaginal wing disc cells depleted of NS2 induced apoptosis as marked by anti-caspase 3 labeling; loss of these progenitor cells resulted in defective adult wings. We conclude that nucleolar proteins NS1 and NS2 have similar but non-overlapping roles in the final maturation or nucleolar release of 60S ribosomal subunits.
KeywordsNucleostemin Ngp1 Drosophila Ribosome stress Autophagy Apoptosis
Upstream activation sequences
Transmission electron microscopy
Phosphate-buffered saline with Triton X-100
Tris-buffered saline with Tween 20
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Dense fibrillar component
Green fluorescent protein
Rough endoplasmic reticulum
We thank Ying Xiao of LSU’s Socolofsky Microscopy Center for sectioning embedded Drosophila tissues. We thank Joe Gall and Ji-Long Liu for the rabbit anti-Drosophila coilin antiserum and Thomas Neufeld for the UAS-mCherry-ATG8a fly line.
Compliance with ethical standards
This study was funded by the National Science Foundation, award MCB0919709.
Conflict of interest
The authors declare that they have no conflict of interest.
Humans and animals
This article does not contain any studies with human participants performed by any of the authors.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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