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Chromosoma

, Volume 126, Issue 3, pp 375–388 | Cite as

Loss of Drosophila nucleostemin 2 (NS2) blocks nucleolar release of the 60S subunit leading to ribosome stress

  • Yubo Wang
  • Patrick DiMario
Original Article

Abstract

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.

Keywords

Nucleostemin Ngp1 Drosophila Ribosome stress Autophagy Apoptosis 

Abbreviations

NS

Nucleostemin

HNS

Human nucleostemin

UAS

Upstream activation sequences

TEM

Transmission electron microscopy

PBST

Phosphate-buffered saline with Triton X-100

TTBS

Tris-buffered saline with Tween 20

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

DAPI

4-6-Diamidino-2-phenylindole dihydrochloride

RNAi

RNA interference

FC

Fibrillar center

DFC

Dense fibrillar component

GC

Granular component

GFP

Green fluorescent protein

rER

Rough endoplasmic reticulum

Notes

Acknowledgments

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

Funding

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.

Supplementary material

412_2016_597_Fig9_ESM.gif (104 kb)
Fig. S1

Conventional fluorescence microscopy localized GFP-NS2 relative to endogenous fibrillarin and coilin in wild-type larval midgut cells. a–d GFP-NS2 located mostly to the peripheral regions of the one large nucleolus and to two unknown extra-nucleolar bodies (white arrowheads). Mab72B9 labeled fibrillarin. DAPI stained DNA foci within the nucleolus. We interpret these foci as FCs. e–f and g–h Midgut cells expressing GFP-NS2 but counterstained with rabbit anti-Drosophila coilin to show Cajal bodies (blue arrowheads). Extra-nucleolar bodies containing GFP-NS2 (white arrowheads) were well separated from the Cajal bodies. (GIF 103 kb)

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High resolution image (TIF 15,009 kb)
412_2016_597_Fig10_ESM.gif (59 kb)
Fig. S2

RT-PCR assays to test for off-target transcripts potentially affected by siRNAs expressed in da-GAL4 > UAS-RNAi-NS2.1 larvae. Actin5C and NS2 transcripts served as controls; Actin5C transcript remained steady in both control and NS2-depleted larvae, while NS2 transcripts dropped as expected. CG17259 and CG31140 transcripts consistently remained unchanged. D19A and jnj transcripts showed consistent depletions (see text) (GIF 59 kb)

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High resolution image (TIF 2700 kb)
412_2016_597_Fig11_ESM.gif (221 kb)
Fig. S3

Variable loss of ribosomes in the midgut of da-GAL4 > UAS-RNAi-NS2.1 larvae. a Toluidine blue uniformly stained a thick (0.5 μm) section of wild-type midgut. Panel a represented 18 images. b A toluidine blue stained thick (0.5 μm) section of midgut from a da-GAL4 > UAS-RNAi-NS2.1 larva. Unequal staining suggested variable ribosome contents in neighboring cells. Panel b represented 38 images. c TEM analysis of two neighboring cells from the midgut shown in panel b. The basophilic cell on the left contained large amounts of cytosolic and rER-associated ribosomes, but its nucleolus showed extended GRs (arrow). The non-basophilic cell on the right contained few cytosolic ribosomes but numerous vesicles with surface-attached ribosomes. d Higher magnification of a similar non-basophilic cell as shown in panel c. Clusters of apparent cytosolic ribosomes were likely tethered to vesicle membranes not included in the section. Panels c and d represented 151 images (GIF 221 kb)

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High resolution image (TIF 16,858 kb)
412_2016_597_MOESM4_ESM.docx (27 kb)
ESM 4 (DOCX 27 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Biological SciencesLouisiana State UniversityBaton RougeUSA

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