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

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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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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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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Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

Nature Communications ◽

10.1038/s41467-021-23702-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hauke S. Hillen ◽

Elena Lavdovskaia ◽

Franziska Nadler ◽

Elisa Hanitsch ◽

Andreas Linden ◽

...

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Structural Basis ◽

Peptidyl Transferase ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome ◽

In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

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GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8565-8574.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8565-8574 ◽

Cited By ~ 20

Author(s):

Anthony J. Greenberg ◽

Paul Schedl

Keyword(s):

Fusion Protein ◽

Transcriptional Activation ◽

Functional Difference ◽

Wild Type ◽

Gaga Factor ◽

Phenotypic Analysis ◽

Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

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gar2 is a nucleolar protein fromSchizosaccharomyces pomberequired for 18S rRNA and 40S ribosomal subunit accumulation

Nucleic Acids Research ◽

10.1093/nar/23.11.1912 ◽

1995 ◽

Vol 23 (11) ◽

pp. 1912-1918 ◽

Cited By ~ 41

Author(s):

Marie-Pierre Gulli ◽

Jean-Philippe Girard ◽

Dan Zabetakis ◽

Bruno Lapeyre ◽

Teri Melese ◽

...

Keyword(s):

18S Rrna ◽

Ribosomal Subunit ◽

Nucleolar Protein ◽

40S Ribosomal Subunit

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Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation

Nucleic Acids Research ◽

10.1093/nar/gkg139 ◽

2003 ◽

Vol 31 (2) ◽

pp. 734-742 ◽

Cited By ~ 30

Author(s):

B. Tallet-Lopez

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antisense Oligonucleotides ◽

Ribosomal Subunit ◽

In Vitro Translation ◽

40S Ribosomal Subunit

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The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

10.1101/2020.07.29.226936 ◽

2020 ◽

Author(s):

Laura Plassart ◽

Ramtin Shayan ◽

Christian Montellese ◽

Dana Rinaldi ◽

Natacha Larburu ◽

...

Keyword(s):

Ribosomal Subunit ◽

Ribosomal Subunits ◽

40S Ribosomal Subunit ◽

Inactive Form ◽

Final Maturation ◽

Translation Apparatus ◽

Translation Accuracy ◽

Functional Analyses

Preventing premature interaction of preribosomes with the translation apparatus is essential to translation accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATPloaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

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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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