Identification of distinct maturation steps involved in human 40S ribosomal subunit biosynthesis

AbstractTechnical problems intrinsic to the purification of preribosome intermediates have limited our understanding of ribosome biosynthesis in humans. Addressing this issue is important given the implication of this biological process in human disease. Here we report a preribosome purification and tagging strategy that overcomes some of the existing technical difficulties. Using these tools, we find that the pre-40S precursors go through two distinct maturation phases inside the nucleolus and follow a regulatory step that precedes late maturation in the cytoplasm. This regulatory step entails the intertwined actions of both PARN (a metazoan-specific ribonuclease) and RRP12 (a phylogenetically conserved 40S biogenesis factor that has acquired additional functional features in higher eukaryotes). Together, these results demonstrate the usefulness of this purification method for the dissection of ribosome biogenesis in human cells. They also identify distinct maturation stages and metazoan-specific regulatory mechanisms involved in the generation of the human 40S ribosomal subunit.

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Recent Advances on the Structure and Function of RNA Acetyltransferase Kre33/NAT10

Cells ◽

10.3390/cells8091035 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1035 ◽

Cited By ~ 8

Author(s):

Sophie Sleiman ◽

Francois Dragon

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

40S Ribosomal Subunit ◽

Functional Features ◽

And Function ◽

Progeria Syndrome ◽

Hutchinson Gilford Progeria Syndrome ◽

Nucleolar Rna

Ribosome biogenesis is one of the most energy demanding processes in the cell. In eukaryotes, the main steps of this process occur in the nucleolus and include pre-ribosomal RNA (pre-rRNA) processing, post-transcriptional modifications, and assembly of many non-ribosomal factors and ribosomal proteins in order to form mature and functional ribosomes. In yeast and humans, the nucleolar RNA acetyltransferase Kre33/NAT10 participates in different maturation events, such as acetylation and processing of 18S rRNA, and assembly of the 40S ribosomal subunit. Here, we review the structural and functional features of Kre33/NAT10 RNA acetyltransferase, and we underscore the importance of this enzyme in ribosome biogenesis, as well as in acetylation of non-ribosomal targets. We also report on the role of human NAT10 in Hutchinson–Gilford progeria syndrome.

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Human PDCD2L Is an Export Substrate of CRM1 That Associates with 40S Ribosomal Subunit Precursors

Molecular and Cellular Biology ◽

10.1128/mcb.00303-16 ◽

2016 ◽

Vol 36 (24) ◽

pp. 3019-3032 ◽

Cited By ~ 9

Author(s):

Anne-Marie Landry-Voyer ◽

Sarah Bilodeau ◽

Danny Bergeron ◽

Kiersten L. Dionne ◽

Sarah A. Port ◽

...

Keyword(s):

Nuclear Export ◽

Ribosome Biogenesis ◽

Nuclear Export Signal ◽

Ribosomal Subunit ◽

Stable Complex ◽

Dependent Manner ◽

Reporter Protein ◽

Ribosomal Subunits ◽

40S Ribosomal Subunit ◽

Late Maturation

Protein arginine methyltransferase 3 (PRMT3) forms a stable complex with 40S ribosomal protein S2 (RPS2) and contributes to ribosome biogenesis. However, the molecular mechanism by which PRMT3 influences ribosome biogenesis and/or function still remains unclear. Using quantitative proteomics, we identified human programmed cell death 2-like (PDCD2L) as a novel PRMT3-associated protein. Our data suggest that RPS2 promotes the formation of a conserved extraribosomal complex with PRMT3 and PDCD2L. We also show that PDCD2L associates with 40S subunit precursors that contain a 3′-extended form of the 18S rRNA (18S-E pre-rRNA) and several pre-40S maturation factors. PDCD2L shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner using a leucine-rich nuclear export signal that is sufficient to direct the export of a reporter protein. Although PDCD2L is not required for the biogenesis and export of 40S ribosomal subunits, we found that PDCD2L -null cells accumulate free 60S ribosomal subunits, which is indicative of a deficiency in 40S subunit availability. Our data also indicate that PDCD2L and its paralog, PDCD2, function redundantly in 40S ribosomal subunit production. Our findings uncover the existence of an extraribosomal complex consisting of PDCD2L, RPS2, and PRMT3 and support a role for PDCD2L in the late maturation of 40S ribosomal subunits.

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Evolutionarily Conserved Function of RRP36 in Early Cleavages of the Pre-rRNA and Production of the 40S Ribosomal Subunit

Molecular and Cellular Biology ◽

10.1128/mcb.00999-09 ◽

2009 ◽

Vol 30 (5) ◽

pp. 1130-1144 ◽

Cited By ~ 17

Author(s):

Marie Gérus ◽

Chrystelle Bonnart ◽

Michèle Caizergues-Ferrer ◽

Yves Henry ◽

Anthony K. Henras

Keyword(s):

Ribosome Biogenesis ◽

18S Rrna ◽

Ribosomal Subunit ◽

Rapid Decrease ◽

Cellular Activity ◽

Uncharacterized Gene ◽

40S Ribosomal Subunit ◽

Evolutionarily Conserved ◽

Human Orthologue ◽

Ribosome Biosynthesis

ABSTRACT Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes.

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Chaperone-E3 Ligase Complex HSP70-CHIP Mediates Ubiquitination of Ribosomal Protein S3

International Journal of Molecular Sciences ◽

10.3390/ijms19092723 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2723 ◽

Cited By ~ 2

Author(s):

Inwoo Hwang ◽

Sung-Woo Cho ◽

Jee-Yin Ahn

Keyword(s):

Ribosomal Protein ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

E3 Ligase ◽

Interacting Protein ◽

Akt Activation ◽

Protein Levels ◽

40S Ribosomal Subunit ◽

Subsequent Degradation ◽

Ribosomal Protein S3

In addition to its role in ribosome biogenesis, ribosomal protein S3 (RPS3), a component of the 40S ribosomal subunit, has been suggested to possess several extraribosomal functions, including an apoptotic function. In this study, we demonstrated that in the mouse brain, the protein levels of RPS3 were altered by the degree of nutritional starvation and correlated with neuronal apoptosis. After endurable short-term starvation, the apoptotic function of RPS3 was suppressed by Akt activation and Akt-mediated T70 phosphorylation, whereas after prolonged starvation, the protein levels of RPS3 notably increased, and abundant neuronal death occurred. These events coincided with ubiquitination and subsequent degradation of RPS3, controlled by HSP70 and the cochaperone E3 ligase: carboxy terminus of heat shock protein 70-interacting protein (CHIP). Thus, our study points to an extraribosomal role of RPS3 in balancing neuronal survival or death depending on the degree of starvation through CHIP-mediated polyubiquitination and degradation.

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Novel Stress-responsive Genes EMG1 and NOP14 Encode Conserved, Interacting Proteins Required for 40S Ribosome Biogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3644 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3644-3657 ◽

Cited By ~ 59

Author(s):

Phillip C. C. Liu ◽

Dennis J. Thiele

Keyword(s):

Gene Expression ◽

Ribosome Biogenesis ◽

Expression Patterns ◽

Ribosomal Subunit ◽

Temperature Sensitive ◽

Physical Interaction ◽

Ribosomal Protein Genes ◽

Yeast Saccharomyces Cerevisiae ◽

40S Ribosomal Subunit ◽

Responsive Gene

Under stressful conditions organisms adjust the synthesis, processing, and trafficking of molecules to allow survival from and recovery after stress. In baker's yeast Saccharomyces cerevisiae, the cellular production of ribosomes is tightly matched with environmental conditions and nutrient availability through coordinate transcriptional regulation of genes involved in ribosome biogenesis. On the basis of stress-responsive gene expression and functional studies, we have identified a novel, evolutionarily conserved gene, EMG1, that has similar stress-responsive gene expression patterns as ribosomal protein genes and is required for the biogenesis of the 40S ribosomal subunit. The Emg1 protein is distributed throughout the cell; however, its nuclear localization depends on physical interaction with a newly characterized nucleolar protein, Nop14. Yeast depleted of Nop14 or harboring a temperature-sensitive allele of emg1 have selectively reduced levels of the 20S pre-rRNA and mature18S rRNA and diminished cellular levels of the 40S ribosomal subunit. Neither Emg1 nor Nop14 contain any characterized functional motifs; however, isolation and functional analyses of mammalian orthologues of Emg1 and Nop14 suggest that these proteins are functionally conserved among eukaryotes. We conclude that Emg1 and Nop14 are novel proteins whose interaction is required for the maturation of the 18S rRNA and for 40S ribosome production.

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Processing of the Ribosomal Ubiquitin-Like Fusion Protein FUBI-eS30/FAU is Required for 40S Maturation and Depends on USP36

10.1101/2021.05.21.445149 ◽

2021 ◽

Author(s):

Jasmin van den Heuvel ◽

Caroline Ashiono ◽

Ludovic Gillet ◽

Kerstin Doerner ◽

Emanuel Wyler ◽

...

Keyword(s):

Fusion Protein ◽

Ribosome Biogenesis ◽

18S Rrna ◽

Affinity Purification ◽

Ribosomal Subunit ◽

Wild Type ◽

Functional Importance ◽

40S Ribosomal Subunit ◽

Differential Affinity

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

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Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36

eLife ◽

10.7554/elife.70560 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jasmin van den Heuvel ◽

Caroline Ashiono ◽

Ludovic C Gillet ◽

Kerstin Dörner ◽

Emanuel Wyler ◽

...

Keyword(s):

Fusion Protein ◽

Ribosome Biogenesis ◽

18S Rrna ◽

Affinity Purification ◽

Ribosomal Subunit ◽

Wild Type ◽

Functional Importance ◽

40S Ribosomal Subunit ◽

Differential Affinity

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Citrus exocortis viroid causes ribosomal stress in tomato plants

Nucleic Acids Research ◽

10.1093/nar/gkz679 ◽

2019 ◽

Vol 47 (16) ◽

pp. 8649-8661 ◽

Cited By ~ 8

Author(s):

Patrick Cottilli ◽

Borja Belda-Palazón ◽

Charith Raj Adkar-Purushothama ◽

Jean-Pierre Perreault ◽

Enrico Schleiff ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Tomato Plants ◽

Translation Machinery ◽

Citrus Exocortis Viroid ◽

40S Ribosomal Subunit ◽

Infected Tomato ◽

Stress Mediator ◽

Ribosomal Stress

Abstract Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.

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A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0138 ◽

2002 ◽

Vol 13 (10) ◽

pp. 3683-3695 ◽

Cited By ~ 6

Author(s):

Petra Björk ◽

Göran Baurén ◽

ShaoBo Jin ◽

Yong-Guang Tong ◽

Thomas R. Bürglin ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Rna Binding ◽

Ribosomal Subunit ◽

Binding Domain ◽

Dependent Manner ◽

Binding Domains ◽

40S Ribosomal Subunit ◽

Rna Binding Domain

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

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USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit

eLife ◽

10.7554/elife.54435 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Christian Montellese ◽

Jasmin van den Heuvel ◽

Caroline Ashiono ◽

Kerstin Dörner ◽

André Melnik ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Synthesis ◽

Ribosome Biogenesis ◽

18S Rrna ◽

Ribosomal Subunit ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Novel Proteins ◽

40S Ribosomal Subunit ◽

Final Maturation

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.

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