Distinct genome protective vs. ribosome synthetic functions of the paralogous nucleolar proteins nucleostemin and GNL3L

Abstract

The mammalian nucleolar proteins nucleostemin and GNL3-like (GNL3L) are encoded by paralogous genes that arose from an ancestral invertebrate gene, GNL3. Invertebrate GNL3 has been implicated in ribosome biosynthesis, as has its mammalian descendent, GNL3L. The paralogous mammalian nucleostemin protein has, instead, been implicated in cell renewal. Here, we found that depletion of nucleostemin in a human breast carcinoma cell line triggers prompt and significant DNA damage in S-phase cells without perturbing the initial step of ribosomal (r)RNA synthesis and only mildly affects the total ribosome production. By contrast, GNL3L depletion markedly impairs ribosome production without inducing appreciable DNA damage. These results indicate that, during vertebrate evolution, GNL3L retained the role of the ancestral gene in ribosome biosynthesis, whereas the paralogous nucleostemin acquired a novel genome-protective function. Our results provide a coherent explanation for what had seemed to be contradictory findings about the functions of the invertebrate versus vertebrate genes and are suggestive of how the nucleolus was fine-tuned for a role in genome protection and cell-cycle control as the vertebrates evolved.

Turning a new page on nucleostemin and self-renewal

“…In a recent study from my laboratory, we observed a cell cycle arrest upon nucleostemin knockdown in human breast cancer cells (MDA-MB-231) after 12 hours, and found that, at this early time point compared with other related studies, nucleostemin depletion causes an increase in cells in S phase and a decrease of those in G2/ M, indicative of an intra-S-phase arrest (Lin et al, 2014). Most importantly, our dose-dependent study revealed that the apparent discrepancy with regard to the cell cycle effects of nucleostemin depletion among different studies might be due to the level of protein knockdown.…”

“…It is apparent that DNA damage arises at the earliest time point of measurement, that is 12 hours after the initiation of nucleostemin depletion, whereas no significant effect on rRNA synthesis or nucleolar structure can be noted up to 48 hours (Lin et al, 2014). This finding establishes that DNA damage occurs considerably before ribosomal perturbation in response to nucleostemin depletion.…”

“…Two studies have reported a perturbed ribosomal effect following the knockdown of vertebrate nucleostemin in zebrafish and in human cancer cells (Essers et al, 2014;Romanova et al, 2009). Although these findings might appear to be consistent with a role of nucleostemin in ribosomal synthesis, there have been multiple lines of evidence that contradict a direct involvement of mammalian nucleostemin in ribosomal synthesis, including the failure of human nucleostemin to rescue the ribosomal phenotype of yeast null for GNL3 (the ortholog of nucleostemin), the non-congruent localization of nucleostemin and nascent 28S rRNA inside the human nucleolus, and the late occurrence of ribosomal defects following nucleostemin knockdown (Du et al, 2006;Kudron and Reinke, 2008;Lin et al, 2014;Politz et al, 2005). Whether nucleostemin has a ribosomal or non-ribosomal role is not the only issue under debate.…”

“…Although the failure to rescue GNL3 deficiency with mammalian nucleostemin alone might simply reflect the inability of the latter to bind to the invertebrate rRNA or ribosome, the fact that human GNL3L is able to reverse the ribosomal phenotype of GNL3-depleted S. pombe poses the interesting possibility that mammalian nucleostemin might have functionally diverged from mammalian GNL3L and invertebrate GNL3 (Du et al, 2006). Indeed, recent work has shown that nucleostemin-knockdown triggers DNA damage without causing a significant disturbance of ribosomal synthesis, whereas depletion of GNL3L perturbs the processing of pre-rRNA without causing DNA damage in human breast cancer cells (Lin et al, 2014). On the basis of these studies, I propose the idea that during the evolution of vertebrate species, GNL3L might have retained the role of its ancestral gene in ribosome biosynthesis, whereas, nucleostemin, the paralog that arose, acquired a new function in genome protection.…”

“…Most importantly, our dose-dependent study revealed that the apparent discrepancy with regard to the cell cycle effects of nucleostemin depletion among different studies might be due to the level of protein knockdown. A more efficient loss of nucleostemin causes an early S-phase block with fewer cells in G2/M, whereas a less efficient loss of nucleostemin results in arrest in late S and G2/M with an increased number of G2/M cells (Lin et al, 2014). This idea resonates with the finding of an earlier study showing that HeLa cells with nucleostemin knockdown are unable to complete DNA synthesis to pass through S phase, resulting in an increase in the percentage of cells in G0/G1 and a concomitant decrease of S phase cells (Sijin et al, 2004).…”