Mouse GNL3 peptide

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.

A quintessential trait of stem cells is embedded in their ability to self-renew without incurring DNA damage as a result of genome replication. One key self-renewal factor is the nucleolar GTP-binding protein nucleostemin (also known as guanine-nucleotide-binding protein-like 3, GNL3, in invertebrate species). Several studies have recently pointed to an unexpected role of nucleostemin in safeguarding the genome integrity of stem and cancer cells. Since its discovery, the predominant presence of nucleostemin in the nucleolus has led to the notion that it might function in the card-carrying event of the nucleolus - the biogenesis of ribosomes. As tantalizing as this might be, a ribosomal role of nucleostemin is refuted by evidence from recent studies, which argues that nucleostemin depletion triggers a primary event of DNA damage in S phase cells that then leads to ribosomal perturbation. Furthermore, there have been conflicting reports regarding the p53 dependency of nucleostemin activity and the cell cycle arrest profile of nucleostemin-depleted cells. In this Commentary, I propose a model that explains how the many contradictory observations surrounding nucleostemin can be reconciled and suggest that this protein might not be as multi-tasking as has been previously perceived. The story of nucleostemin highlights the complexity of the underlying molecular events associated with the appearance of any cell biological phenotype and also signifies a new understanding of the genome maintenance program in stem cells.