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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
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
References
Attrill H, Falls K, Goodman JL, Millburn GH, Antonazzo G, Rey AJ, Marygold SJ, the FlyBase Consortium (2016) FlyBase: establishing a Gene Group resource for Drosophila melanogaster. Nucleic Acids Res 44(D1):D786–D792. doi:10.1093/nar/gkv1046
Baβler J, Grandi P, Gadal O, Leβmann T, Petfalski E, Tollervey D, Lechner J, Hurt E (2001) Identification of a 60S preribosomal particle that is closely linked to nuclear export. Mol Cell 8:517–529. doi:10.1016/S1097-2765(01)00342-2
Baβler J, Kallas M, Hurt E (2006) The Nug1 GTPase reveals an N-terminal RNA binding domain that is essential for association with 60S pre-ribosomal particles. J Biol Chem 281:24737–24744. doi:10.1074/jbc.M604261200
Bodenstein D (1950) The postembryonic development of Drosophila. In: Demerec M (ed) Biology of Drosophila. Wiley, New York, pp 275–367
Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2):401–415
Brand AH, Manoukian AS, Perrimon N (1994) Ectopic expression in Drosophila. Method Cell Biol 44:635–654. doi:10.1016/S0091-679X(08)60936-X
Chang YY, Neufeld TP (2009) An Atg1/Atg13 complex with multiple roles in TOR-mediated autophagy regulation. Mol Biol Cell 20:2004–2019. doi:10.1091/mbc.E08-12-1250
Cormier O, Mohseni N, Voytyuk I, Reed BH (2012) Autophagy can promote but is not required for epithelial cell extrusion in the amnioserosa of the Drosophila embryo. Autophagy 8(2):252–264. doi:10.4161/auto.8.2.18618
Cui Z, DiMario PJ (2007) RNAi knockdown of Nopp140 induces Minute-like phenotypes in Drosophila. Mol Biol Cell 18(6):2179–2191. doi:10.1091/mbc.E07-01-0074
Daigle DM, Rossi L, Berguis AM, Aravind L, Koonin EV, Brown ED (2002) YjeQ, an essential, conserved, uncharacterized protein from Escherichia coli, is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics. Biochemistry 41:11109–11117. doi:10.1021/bi020355q
de Cuevas M, Lee JK, Spradling AC (1996) alpha-spectrin is required for germline cell division and differentiation in the Drosophila ovary. Development 122(12):3959–3968
Denton D, Shravage B, Simin R, Baehrecke EH, Kumar S (2010) Larval midgut destruction in Drosophila: not dependent on caspases but suppressed by the loss of autophagy. Autophagy 6(1):163–165. doi:10.4161/auto.6.1.10601
Easton LE, Shibata Y, Lukavsky PJ (2010) Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography. RNA 16:647–653. doi:10.1261/rna.1862210
Fan Y, Bergmann A (2010) The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila. Cell Death Differ 17(3):534–539. doi:10.1038/cdd.2009.185
Feder N, Wolf MK (1965) Studies on nucleic acid metachromasy II. Metachromatic and orthochromatic staining by toluidine blue of nucleic acids in tissue sections. J Cell Biol 27:327–336. doi:10.1083/jcb.27.2.327
Filshie BK, Poulson DF, Waterhouse DF (1971) Ultrastructure of the copper-accumulating region of the Drosophila larval midgut. Tissue Cell 3(1):77–102. doi:10.1016/S0040-8166(71)80033-2
Finnegan DJ (1985) Transposable elements in eukaryotes. Int Rev Cytol 19:281–326. doi:10.1016/S0074-7696(08)61376-5
Florentin A, Arama E (2012) Caspase levels and execution efficiencies determine the apoptotic potential of the cell. J Cell Biol 196(4):513–527. doi:10.1083/jcb.201107133
Haerry TE, Khalsa O, O’Connor MB, Wharton KA (1998) Synergistic signaling by two BMP ligands through the SAX and TKV receptors controls wing growth and patterning in Drosophila. Development 125(20):3977–3987
Hartl TA, Ni J, Cao J, Suyama KL, Patchett S, Bussiere C et al (2013) Regulation of ribosome biogenesis by nucleostemin 3 promotes local and systemic growth in Drosophila. Genetics 194:101–115. doi:10.1534/genetics.112.149104
He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93. doi:10.1146/annurev-genet-102808-114910
He F, James A, Raje H, Ghaffari H, DiMario P (2015) Deletion of Drosophila Nopp140 induces subcellular ribosomopathies. Chromosoma 124(2):191–208. doi:10.1007/s00412-014-0490-9
Hedges J, West M, Johnson AW (2005) Release of the exporter adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsgp1. EMBO J 24:567–579. doi:10.1038/sj.emboj.7600547
Helminen HJ, Ericsson JLE (1971) Ultrastructural studies on prostatic involution in the rat. Mechanism of autophagy in epithelial cells, with special reference to the rough-surfaced endoplasmic reticulum. J Ultrastruct Res 36:708–724. doi:10.1016/S0022-5320(71)90025-6
Hernandez-Verdun D (2011) Structural organization of the nucleolus as a consequence of the dynamics of ribosome biogenesis. In: Olson MOJ (ed) The nucleolus, protein reviews 15. Springer, New York, pp 3–28. doi:10.1007/978-1-4614-0514-6_1
Hovhanyan A, Herter EK, Pfannsteil J, Gallant P, Raabe T (2014) Drosophila Mbm is a nucleolar Myc and casein kinase 2 target required for ribosome biogenesis and cell growth of central brain neuroblasts. Mol Cell Biol 34:1878–1891. doi:10.1128/MCB.00658-13
James A, Cindass R Jr, Mayer D, Terhoeve S, Mumphrey C, DiMario P (2013) Nucleolar stress in Drosophila melanogaster: RNAi-mediated depletion of Nopp140. Nucleus 4(2):123–133. doi:10.4161/nucl.23944
Jiang H, Edgar BA (2009) EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors. Development 136:483–493. doi:10.1242/dev.026955
Kallstrom G, Hedges J, Johnson A (2003) The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively. Mol Cell Biol 23:4344–4355. doi:10.1128/MCB.23.12.4344-4355.2003
Kaplan DD, Zimmermann G, Suyama K, Meyer T, Scott MP (2008) A nucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulin signaling and control body size. Genes Dev 22:1877–1893. doi:10.1101/gad.1670508
Leipe DD, Wolf YI, Koonin EV, Aravind L (2002) Classification and evolution of P-loop GTPases and related ATPases. J Mol Biol 317:41–72. doi:10.1006/jmbi.2001.5378
Li X, Zhuo R, Tiong S, Di Cara F, King-Jones K, Hughes SC, Campbell SD, Wevrick R (2013) The Smc5/Smc6/MAGE complex confers resistance to caffeine and genotoxic stress in Drosophila melanogaster. PLoS One 8(3):e59866. doi:10.1371/journal.pone.0059866
Liu J-L, Wu Z, Nizami Z, Deryusheva S, Rajendra TK, Beumer KJ, Gao H, Matera AG, Carroll D, Gall JG (2009) Coilin is essential for Cajal body organization in Drosophila melanogaster. Mol Biol Cell 20:1661–1670. doi:10.1091/mbc.E08-05-0525
Martoja R, Ballan-Dufrançais C (1984) The ultrastructure of the digestive and excretory organs. In: King RC, Akai H (eds) Insect ultrastructure vol 2. Plenum Press, New York, pp 199–268. doi:10.1007/978-1-4613-2715-8_6
Matsuo E, Kanno S, Matsumoto S, Tsuneizumi K (2010) Drosophila nucleostemin 2 proved essential for early eye development and cell survival. Biosci Biotechnol Biochem 74(10):2120–2123. doi:10.1271/bbb.100386
Matsuo E, Nagamine T, Matsumoto S, Tsuneizumi K (2011) Drosophila GTPase nucleostemin 2 changes cellular distribution during larval development and the GTP-binding motif is essential to nucleoplasmic localization. Biosci Biotechnol Biochem 75(8):1511–1515. doi:10.1271/bbb.110212
Matsuo Y, Granneman S, Thoms M, Manikas R-G, Tollervey D, Hurt E (2014) Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export. Nature 505:112–116. doi:10.1038/nature12731
Meng L, Yasumoto H, Tsai RYL (2006) Multiple controls regulate nucleoplasm partitioning between nucleolus and nucleoplasm. J Cell Sci 119:5124–5136. doi:10.1242/jcs.03292
Meng L, Zhu Q, Tsai RYL (2007) Nucleolar trafficking of nucleostemin family proteins: common versus protein-specific mechanisms. Mol Cell Biol 27(24):8670–8682. doi:10.1128/MCB.00635-07
Pieri L, Sassoli C, Romagnoli P, Domenici L (2002) Use of periodate-lysine-paraformaldehyde for the fixation of multiple antigens in human skin biopsies. Eur J Histochem 46:365–375. doi:10.4081/1749
Reimer G, Pollard KM, Penning CA, Ochs RL, Lischwe MA, Busch H, Tan EM (1987) Monoclonal autoantibody from a (New Zealand black x New Zealand white) F1 mouse and some scleroderma sera target an Mr 34,000 nucleolar protein of the U3 RNP particle. Arthritis Rheum 30(7):793–800. doi:10.1002/art.1780300709
Reynaud EG, Andrade MA, Bonneau F, Bach T, Ly N, Knop M, Scheffzek K, Pepperkok R (2005) Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization. BMC Biol 3:21. doi:10.1186/1741-7007-3-21
Rosby R, Cui Z, Rogers E, deLivron MA, Robinson VL, DiMario PJ (2009) Knockdown of the Drosophila GTPase nucleostemin 1 impairs large ribosomal subunit biogenesis, cell growth, and midgut precursor cell maintenance. Mol Biol Cell 20:4424–4434. doi:10.1091/mbc.E08-06-0592
Rubin GM (1983) Dispersed repetitive DNAs in Drosophila. In: Shapiro JA (ed) Mobile genetic elements. Academic, New York, pp 329–361. doi:10.1016/B978-0-12-638680-6.50012-0
Saveanu C, Bienvenu D, Namane A, Gleizes P-E, Gas N, Jacquier A, Fromont-Racine M (2001) Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps. EMBO J 20:6475–6484. doi:10.1093/emboj/20.22.6475
Schneider I (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27:353–365
Strand DJ, McDonald JF (1985) Copia is transcriptionally responsive to environmental stress. Nucleic Acids Res 13:4401–4410. doi:10.1093/nar/13.12.4401
Sun X, Artavanis-Tsakonas S (1997) Secreted forms of DELTA and SERRATE define antagonists of Notch signaling in Drosophila. Development 124:3439–3448
Tsai RYL (2011) New frontiers in nucleolar research: nucleostemin and related proteins. In: Olson MOJ (ed) The nucleolus, protein reviews 15. Springer, New York, pp 301–320. doi:10.1007/978-1-4614-0514-6_13
Tsai RYL (2014) Turning a new page on nucleostemin and self-renewal. J Cell Sci 127:3885–3891. doi:10.1242/jcs.154054
Tsai RYL, McKay RD (2002) A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev 16:2991–3003. doi:10.1101/gad.55671
Tsai RYL, McKay RD (2005) A multistep, GTP-driven mechanism controlling the dynamic cycling of nucleostemin. J Cell Biol 168:179–184. doi:10.1083/jcb.200409053
Yoshioka K, Honma H, Zushi M, Kondo S, Togashi S, Miyake T, Shiba T (1990) Virus-like particle formation of Drosophila copia through autocatalytic processing. EMBO J 9(2):535–541
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
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.
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
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)
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)
ESM 4
(DOCX 27 kb)
Rights and permissions
About this article
Cite this article
Wang, Y., DiMario, P. Loss of Drosophila nucleostemin 2 (NS2) blocks nucleolar release of the 60S subunit leading to ribosome stress. Chromosoma 126, 375–388 (2017). https://doi.org/10.1007/s00412-016-0597-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00412-016-0597-2