Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps

Saveanu, Cosmin

doi:10.1093/emboj/20.22.6475

Cited by 174 publications

(219 citation statements)

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“…Disruption of Nog1 function through RNA interference led to a dramatic decrease in the levels of free 60 S particles and the appearance of an atypical rRNA intermediate in which ITS2 was not cleaved (40). Likewise, depletion of Nug2 resulted in a dramatic decrease in 5.8 S rRNA levels and accumulation of the 7 S precursor as well as nucleolar accumulation of ITS2-containing precursors (6). These GTPases may act in concert with the Rea1 ATPase to coordinate the structural reorganization of late pre-60 S ribosomes, prior to the irreversible step of nuclear export.…”

Section: Discussionmentioning

confidence: 99%

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Rea1, a Dynein-related Nuclear AAA-ATPase, Is Involved in Late rRNA Processing and Nuclear Export of 60 S Subunits

Galani

Nissan

Petfalski

et al. 2004

Journal of Biological Chemistry

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Rea1, the largest predicted protein in the yeast genome, is a member of the AAA ؉ family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperaturesensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.The synthesis of ribosomes is one of the major and most energy-consuming processes in the cell. In Saccharomyces cerevisiae, ribosome biogenesis begins in the nucleolus with the transcription of two rRNA precursors, the 35 S and the pre-5 S RNA, by RNA polymerases I and III, respectively. The 35 S pre-rRNA contains the sequences for the mature 18 S, 5.8 S, and 25 S rRNAs, two external transcribed spacers (ETS) 1 and two internal transcribed spacers (ITS). During the maturation process, the pre-rRNA has to undergo a number of modifications and is subjected to cleavages and trimming events. At least 170 accessory proteins including putative RNA helicases, endo-and exonucleases, and putative GTPases and AAAATPases as well as small nucleolar ribonucleoprotein particles are involved in the maturation of rRNA and its assembly into ribosomal subunits (1, 2).Concomitant with rRNA processing, ribosomal and non-ribosomal proteins are assembled on the pre-35 S rRNA, giving rise to a large 90 S pre-ribosomal particle (see Fig. 6B) (3, 4). The initial cleavages at sites A 0 -A 2 separate the two subunits. The pre-40 S subunit is exported relatively rapidly to the cytoplasm, where it undergoes further processing. In contrast, the maturation of the large subunit continues in the nucleoplasm with recruitment of 60 S-specific biogenesis factors and further processing of the 27 S pre-rRNA. This includes the late cleavage and processing in the ITS2 region, which generates mature 5.8 S and 25 S rRNA.In the last few years, the maturation of 40 S and 60 S pre-ribosomes has been extensively analyzed by purification of pre-ribosomal particles (5-10). Interestingly, a large number of non-ribosomal proteins were identified in pre-60 S particles without an assigned function in RNA metabolism. In contrast to the pre-40 S particles, the nascent 60 S particles contain several putative GTPases and AAA-type ATPases (2, 11).To understand the events of ribosome biogenesis, w...

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Section: Discussionmentioning

confidence: 99%

“…In the last few years, the maturation of 40 S and 60 S pre-ribosomes has been extensively analyzed by purification of pre-ribosomal particles (5)(6)(7)(8)(9)(10). Interestingly, a large number of non-ribosomal proteins were identified in pre-60 S particles without an assigned function in RNA metabolism.…”

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confidence: 99%

Rea1, a Dynein-related Nuclear AAA-ATPase, Is Involved in Late rRNA Processing and Nuclear Export of 60 S Subunits

Galani

Nissan

Petfalski

et al. 2004

Journal of Biological Chemistry

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“…Yeast GTPases Nug1p and Nog2p/Nug2p are required for the maturation and export of the large ribosomal subunit (Bassler et al, 2001;Saveanu et al, 2001). To test whether Drosophila NS1 is required for large ribosomal subunit maturation/transport, we first examined transgenic larvae that expressed GFP-tagged RpL11 (GFP-RpL11/ϩ; da-GAL4/ϩ).…”

Section: Loss Of Ns1 Blocks Nucleolar Release Of the Large Ribosomal mentioning

confidence: 99%

Knockdown of theDrosophilaGTPase Nucleostemin 1 Impairs Large Ribosomal Subunit Biogenesis, Cell Growth, and Midgut Precursor Cell Maintenance

Rosby

Cui

Rogers

et al. 2009

MBoC

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Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response. INTRODUCTIONMammalian nucleostemin (NS) is a nucleolar guanosine triphosphate (GTP)-binding protein first characterized in embryonic and neuronal stem cells and in certain cancer cells where it probably plays regulatory roles in cell cycle progression and ribosome biogenesis McKay, 2002, 2005; reviewed by Ma and Pederson, 2008b). Steadystate concentrations of NS drop to undetectable levels just before rat cortical stem cell differentiation (Tsai and McKay, 2002), suggesting that reduced expression of NS regulates stem cell proliferation and differentiation by triggering exit from the cell cycle (Normile, 2002;Tsai and McKay, 2002;Beekman et al., 2006). Mammalian NS rapidly cycles between the nucleolus and the nucleoplasm. Retention of NS within the nucleolus is prolonged when it binds GTP (Misteli, 2005;Tsai and McKay, 2005;Meng et al., 2006Meng et al., , 2007. GTP bound to the central region of NS is thought to stabilize interactions between its amino-terminal basic domain and other nucleolar components, whereas NS redistributes to the nucleoplasm when GTP binding is impaired by mutation (Tsai and McKay, 2005). Coimmunoprecipitation, yeast twohybrid, and bimolecular fluorescence complementation assays demonstrated an interaction between NS and nucleophosmin (B23) (Ma and Pederson, 2008a), a multifunctional chaperone that probably participates in the later stages of ribosome assembly within the granular component of nucleoli. Similar to NS, nucleophosmin probably plays a role in cell proliferation (Szebeni et al., 2003;Grisendi et al., 2006). Recent studies suggest that NS ...

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“…A favored approach has been to purify these complexes from cell lysates [1][2][3][4] . But the limitations of presently available purification techniques pose a major hurdle to the study of RNA processing and export.…”

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confidence: 99%

Comprehensive analysis of diverse ribonucleoprotein complexes

et al. 2007

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The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.During and after eukaryotic transcription, RNA is packaged into RNP complexes, then modified, spliced, folded and exported into the cytoplasm through nuclear pore complexes (NPCs). There are two main forms of RNPs. Each contains a different type of RNA: mRNPs contain mRNA, and rRNPs contain ribosomal RNA (rRNA). Each also uses discrete processing factors, which associate with RNPs in a dynamic fashion to define the order of transcript maturation. The function and composition of many of these RNP complexes, however, is poorly understood.In recent years, there have been considerable efforts to study the composition of mRNPs and rRNPs in greater detail. A favored approach has been to purify these complexes from cell lysates [1][2][3][4] . But the limitations of presently available purification techniques pose a major hurdle to the study of RNA processing and export. Here we describe a method that aims to overcome these limitations and capture the dynamic interactome of maturing RNP complexes.We used this method to analyze complexes along both the ribosome and mRNA biogenesis pathways. RESULTSA strategy for isolation of RNP complexes Our strategy comprises seven steps that we optimized for efficient isolation of intact RNP complexes ( Fig. 1 and Supplementary Methods online). Collected cells are rapidly frozen in liquid nitrogen and broken open in the solid phase by milling 5 , using a planetary ball mill that produces particles of B1-2 mm in diameter (small enough for rapid and efficient RNP extraction). Because cell breakage occurs in the frozen state, we avoid damage to RNP complexes by released nucleases and proteases, as well as redistribution of proteins within the extract. The resulting frozen grindate is rapidly thawed into an RNP-compatible buffer (see Supplementary Methods), to produce an extract that is then immediately clarified by filtration. We isolate the tagged complexes using antibody-conjugated magnetic beads, rather than more commonly used antibody-conjugated resins such as Sepharose. The magnetic beads are small (2.8 mm) and so have a large surface area-to-volume ratio that increases the speed of binding. Moreover, the beads are impermeable, such that the antibodies are densely conjugated to their exposed surface; hence, there is no theoretical upper limit to the size of isolated complexes (unlike with permeable resins such as Sepharose, which are limited by their pore size). Incubation times...

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Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps

Cited by 174 publications

References 41 publications

Rea1, a Dynein-related Nuclear AAA-ATPase, Is Involved in Late rRNA Processing and Nuclear Export of 60 S Subunits

Rea1, a Dynein-related Nuclear AAA-ATPase, Is Involved in Late rRNA Processing and Nuclear Export of 60 S Subunits

Knockdown of theDrosophilaGTPase Nucleostemin 1 Impairs Large Ribosomal Subunit Biogenesis, Cell Growth, and Midgut Precursor Cell Maintenance

Comprehensive analysis of diverse ribonucleoprotein complexes

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