The Homologous Putative GTPases Grn1p from Fission Yeast and the Human GNL3L Are Required for Growth and Play a Role in Processing of Nucleolar Pre-rRNA

Abstract

Grn1p from fission yeast and GNL3L from human cells, two putative GTPases from the novel HSR1_MMR1 GTP-binding protein subfamily with circularly permuted G-motifs play a critical role in maintaining normal cell growth. Deletion of Grn1 resulted in a severe growth defect, a marked reduction in mature rRNA species with a concomitant accumulation of the 35S pre-rRNA transcript, and failure to export the ribosomal protein Rpl25a from the nucleolus. Deleting any of the Grn1p G-domain motifs resulted in a null phenotype and nuclear/nucleolar localization consistent with the lack of nucleolar export of preribosomes accompanied by a distortion of nucleolar structure. Heterologous expression of GNL3L in a ⌬grn1 mutant restored processing of 35S pre-rRNA, nuclear export of Rpl25a and cell growth to wild-type levels. Genetic complementation in yeast and siRNA knockdown in HeLa cells confirmed the homologous proteins Grn1p and GNL3L are required for growth. Failure of two similar HSR1_MMR1 putative nucleolar GTPases, Nucleostemin (NS), or the dose-dependent response of breast tumor autoantigen NGP-1, to rescue ⌬grn1 implied the highly specific roles of Grn1p or GNL3L in nucleolar events. Our analysis uncovers an important role for Grn1p/GNL3L within this unique group of nucleolar GTPases.

Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

“…Certain nucleolar proteins such as guanine nucleotide-binding proteinlike 3-like (GNL3L), nucleostemin (NS), 2 nucleolin, and nucleophosmin modulate ribosomal as well as nonribosomal pathways (3)(4)(5)(6)(7)(8)(9)(10). GNL3L and NS belong to the HSR1-MMR1 family of GTPases and are up-regulated in various cancers (8,(11)(12)(13)(14).…”

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

“…The present findings can aid in the interpretation of earlier studies in which invertebrate GNL3 and mammalian nucleostemin have been investigated. Thus, because genetic deletion of the invertebrate ortholog, GNL3, was observed to impair ribosome biosynthesis (Du et al, 2006;Kudron and Reinke, 2008;Rosby et al, 2009), reports of similar effects of mammalian nucleostemin knockdown naturally led some investigators to regard nucleostemin as the mammalian equivalent of invertebrate GNL3. Here, we show that a significant impairment of ribosome biosynthesis occurs only after prolonged nucleostemin knockdown, and that it is in fact GNL3L that plays a major role in rRNA biosynthesis.…”

“…Nucleostemin plays an indispensable role in early embryogenesis and the maintenance of the self-renewal of stem or progenitor cells (Qu and Bishop, 2012;Tsai, 2011;Zhu et al, 2006). GNL3L was discovered in a screen for nucleostemin-related genes (Du et al, 2006;Yasumoto et al, 2007). Later studies revealed that nucleostemin and GNL3L exist as separate genes only in vertebrate species ).…”

“…Grn1, NST-1 and NS1). It has been reported that deletion of Grn1 in S. pombe perturbs 35S preribosomal (pre-r)RNA processing and nucleolar export of the Rpl25a (60S) complex (Du et al, 2006). In C. elegans, loss of NST-1 decreases the levels of 18S and 26S rRNAs (Kudron and Reinke, 2008), and in Drosophila depletion of NS1 protein results in nucleolar accumulation of the large ribosomal subunit proteins L11 and L26 (Rosby et al, 2009).…”

“…Although these studies indicate that the loss of nucleostemin might eventually lead to the perturbation of ribosomes, they fail to establish a coherent mechanism or a direct target of nucleostemin action in the ribosomal-synthetic pathway. Indeed, a direct role of mammalian nucleostemin in pre-rRNA processing is contradicted by a study showing that the impaired 35S pre-rRNA processing and Rpl25a nucleolar export phenotypes of Grn1-null yeast can be restored by human GNL3L, but not by human nucleostemin (Du et al, 2006). Moreover, mammalian nucleostemin fails to rescue the growth phenotype in NST-1-deficient C. elegans, and NST-1 is absent from nucleolar regions where rRNA transcription and processing occur (Kudron and Reinke, 2008).…”