Mapping the Functional Domains of Yeast NMD3, the Nuclear Export Adapter for the 60 S Ribosomal Subunit

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

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New Bone Marrow Failure Genes: DNAJC21 and ERCC6L2

Blood ◽

10.1182/blood.v130.suppl_1.sci-22.sci-22 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. SCI-22-SCI-22

Author(s):

Inderjeet Dokal

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Nuclear Export ◽

Conflicts Of Interest ◽

Excision Repair ◽

Bone Marrow Failure ◽

Retinal Dystrophy ◽

Ribosomal Subunit ◽

Genotoxic Stress ◽

Biallelic Mutations

A significant number of cases with bone marrow failure present with one or more extra-hematopoietic abnormality. This suggests a constitutional or genetic basis, and yet many of them remain uncharacterized. Through exome sequencing, we have recently identified two sub groups of these cases, one defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21) and the other in ERCC6L2 (excision repair cross complementing 6 like-2). Patients with DNAJC21 mutations are characterized by global bone marrow failure in early childhood. They can also have a variable number of extra-hematopoietic abnormalities such as short stature and retinal dystrophy. The encoded protein associates with ribosomal RNA (rRNA) and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid patient cells exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced levels of rRNA. Characterisation of mutations has revealed impairment in interactions with cofactors (PA2G4, HSPA8 and ZNF622) involved in 60S maturation. DNAJC21 deficiency results in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles and increased cell death. Collectively these findings demonstrate that biallelic mutations in DNAJC21 cause disease due to defects in early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit. Patients harbouring biallelic ERCC6L 2 mutations are characterized by bone marrow failure (in childhood or early adulthood) and one or more extra-hematopoietic abnormality such as microcephaly. Knockdown of ERCC6L2 in human cells significantly reduces their viability upon exposure to the DNA damaging agent irofulven but not etoposide and camptothecin suggesting a role in nucleotide excision repair. ERCC6L2 knockdown cells and patient cells harbouring biallelic ERCC6L2 mutations also display H2AX phosphorylation that significantly increases upon genotoxic stress, suggesting an early DNA damage response. ERCC6L2 is seen to translocate to mitochondria as well as the nucleus in response to DNA damage and its knockdown induces intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger, N-acetyl-cysteine, attenuates the irofulven-induced cytotoxicity in ERCC6L2 knockdown cells and abolishes its traffic to mitochondria and nucleus in response to this DNA damaging agent. Collectively, these observations suggest that ERCC6L2has a pivotal rolein DNA repair and mitochondrial function. In conclusion, ERCC6L2 and DNAJC21 have an important role in maintaining genomic stability and ribosome biogenesis, respectively. They bring into focus new biological connections/pathways whose constitutional disruption is associated with defective hematopoiesis since patients harbouring germline biallelic mutations in these genes uniformly have bone marrow failure. Disclosures No relevant conflicts of interest to declare.

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Requirements for the nuclear export of the small ribosomal subunit

Journal of Cell Science ◽

10.1242/jcs.115.14.2985 ◽

2002 ◽

Vol 115 (14) ◽

pp. 2985-2995 ◽

Cited By ~ 4

Author(s):

Terence I. Moy ◽

Pamela A. Silver

Keyword(s):

Nuclear Export ◽

Ribosome Biogenesis ◽

Large Scale ◽

Ribosomal Subunit ◽

Small Subunit ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Small Ribosomal Subunit ◽

Factors Affecting ◽

The Stability

Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants,we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.

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Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel

Nature Structural & Molecular Biology ◽

10.1038/nsmb.2438 ◽

2012 ◽

Vol 19 (12) ◽

pp. 1234-1241 ◽

Cited By ~ 80

Author(s):

Bettina Bradatsch ◽

Christoph Leidig ◽

Sander Granneman ◽

Marén Gnädig ◽

David Tollervey ◽

...

Keyword(s):

Nuclear Export ◽

Ribosomal Subunit ◽

Exit Tunnel

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Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.11.11.3777 ◽

2000 ◽

Vol 11 (11) ◽

pp. 3777-3789 ◽

Cited By ~ 91

Author(s):

Tracy Stage-Zimmermann ◽

Ute Schmidt ◽

Pamela A. Silver

Keyword(s):

Ribosomal Protein ◽

Nuclear Export ◽

Ribosomal Proteins ◽

Protein Import ◽

Fluorescent Protein ◽

Ribosomal Subunit ◽

Rrna Processing ◽

Factors Affecting ◽

Processing Factors

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit inS. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ∼45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b–GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b–GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b–GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b–GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120.

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Faculty Opinions recommendation of Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.4242979.15781062 ◽

2012 ◽

Author(s):

Susan Gerbi

Keyword(s):

Nuclear Export ◽

Ribosomal Subunit ◽

Adaptor Protein

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Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

Nature Communications ◽

10.1038/s41467-021-26323-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jan Andreas Ruland ◽

Annika Marie Krüger ◽

Kerstin Dörner ◽

Rohan Bhatia ◽

Sabine Wirths ◽

...

Keyword(s):

Single Molecule ◽

Nuclear Export ◽

Ribosomal Subunit ◽

Super Resolution ◽

Stable Cell Line ◽

Electron Microscopic ◽

Rate Limiting Step ◽

Biochemical Genetic ◽

Maximum Particle

AbstractRibosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of ∼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s−1 in vivo.

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CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev

10.1101/710137 ◽

2019 ◽

Cited By ~ 1

Author(s):

Francisco García-de-Gracia ◽

Daniela Toro-Ascuy ◽

Sebastián Riquelme-Barrios ◽

Camila Pereira-Montecinos ◽

Bárbara Rojas-Araya ◽

...

Keyword(s):

Translation Initiation ◽

Viral Protein ◽

Nuclear Export ◽

Ribosomal Subunit ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Viral Transcript ◽

40S Ribosomal Subunit ◽

Cellular Mrnas ◽

Hiv 1

ABSTRACTTranslation initiation of the human immunodeficiency virus type-1 (HIV-1) unspliced mRNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the unspliced mRNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF restricts HIV-1 replication by interfering with Gag synthesis from the unspliced mRNA. Our results indicate that CTIF associates with Rev through its N-terminal domain and is recruited onto the unspliced mRNA ribonucleoprotein complex in order to block translation. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that CTIF restricts HIV-2 but not MLV Gag synthesis indicating an inhibitory mechanism conserved in Rev-expressing human lentiviruses.

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Bud23 Methylates G1575 of 18S rRNA and Is Required for Efficient Nuclear Export of Pre-40S Subunits

Molecular and Cellular Biology ◽

10.1128/mcb.01674-07 ◽

2008 ◽

Vol 28 (10) ◽

pp. 3151-3161 ◽

Cited By ~ 62

Author(s):

Joshua White ◽

Zhihua Li ◽

Richa Sardana ◽

Janusz M. Bujnicki ◽

Edward M. Marcotte ◽

...

Keyword(s):

Nuclear Export ◽

Ribosome Biogenesis ◽

18S Rrna ◽

Fluorescent Protein ◽

Ribosomal Subunit ◽

Small Subunit ◽

Nuclear Accumulation ◽

Methyltransferase Activity ◽

Late Step ◽

Green Fluorescent

ABSTRACT BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5′ internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Δ mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.

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