Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA-protein interactions during small ribosomal subunit biogenesis

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

RNA ◽

10.1261/rna.078994.121 ◽

2022 ◽

pp. rna.078994.121

Author(s):

Haina Huang ◽

Melissa D Parker ◽

Katrin Karbstein

Keyword(s):

Quality Control ◽

Binding Site ◽

Ribosomal Rna ◽

Ribosomal Proteins ◽

Kinase Activity ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Yeast Strains

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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The complete structure of the small subunit processome

10.1101/175547 ◽

2017 ◽

Cited By ~ 2

Author(s):

Jonas Barandun ◽

Malik Chaker-Margot ◽

Mirjam Hunziker ◽

Kelly R. Molloy ◽

Brian T. Chait ◽

...

Keyword(s):

Ribosomal Proteins ◽

18S Rrna ◽

Molecular Mimicry ◽

Ribosomal Subunit ◽

Small Subunit ◽

Ribosome Assembly ◽

Complete Structure ◽

Small Ribosomal Subunit ◽

Rna Remodeling ◽

Rrna Cleavage

The small subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small subunit processome at an overall resolution of 3.8 Å, which provides an essentially complete atomic model of this assembly. In this nucleolar superstructure, 51 ribosome assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry, steric hindrance as well as protein-and RNA-mediated RNA remodeling are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.

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Interaction between Ribosome Assembly Factors Krr1 and Faf1 Is Essential for Formation of Small Ribosomal Subunit in Yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m114.584490 ◽

2014 ◽

Vol 289 (33) ◽

pp. 22692-22703 ◽

Cited By ~ 18

Author(s):

Sanduo Zheng ◽

Pengfei Lan ◽

Ximing Liu ◽

Keqiong Ye

Keyword(s):

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Assembly Factors

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Conformational switches control early maturation of the eukaryotic small ribosomal subunit

eLife ◽

10.7554/elife.45185 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 11

Author(s):

Mirjam Hunziker ◽

Jonas Barandun ◽

Olga Buzovetsky ◽

Caitlin Steckler ◽

Henrik Molina ◽

...

Keyword(s):

Ribosomal Rna ◽

Conformational Changes ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Small Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Early Maturation ◽

External Transcribed Spacer ◽

Assembly Factors

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

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METTL15 interacts with the assembly intermediate of murine mitochondrial small ribosomal subunit to form m4C840 12S rRNA residue

Nucleic Acids Research ◽

10.1093/nar/gkaa522 ◽

2020 ◽

Vol 48 (14) ◽

pp. 8022-8034 ◽

Cited By ~ 1

Author(s):

Ivan Laptev ◽

Ekaterina Shvetsova ◽

Sergey Levitskii ◽

Marina Serebryakova ◽

Maria Rubtsova ◽

...

Keyword(s):

Protein Biosynthesis ◽

Mitochondrial Protein ◽

Ribosomal Subunit ◽

12S Rrna ◽

Small Ribosomal Subunit ◽

Mitochondrial Ribosomes ◽

Murine Cell Line ◽

Assembly Factor ◽

Mitochondrial 12S Rrna ◽

Assembly Intermediate

Abstract Mammalian mitochondrial ribosomes contain a set of modified nucleotides, which is distinct from that of the cytosolic ribosomes. Nucleotide m4C840 of the murine mitochondrial 12S rRNA is equivalent to the dimethylated m4Cm1402 residue of Escherichia coli 16S rRNA. Here we demonstrate that mouse METTL15 protein is responsible for the formation of m4C residue of the 12S rRNA. Inactivation of Mettl15 gene in murine cell line perturbs the composition of mitochondrial protein biosynthesis machinery. Identification of METTL15 interaction partners revealed that the likely substrate for this RNA methyltransferase is an assembly intermediate of the mitochondrial small ribosomal subunit containing an assembly factor RBFA.

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Role of Era in Assembly and Homeostasis of the Ribosomal Small Subunit

10.1101/525360 ◽

2019 ◽

Cited By ~ 1

Author(s):

Aida Razi ◽

Joseph H. Davis ◽

Yumeng Hao ◽

Dushyant Jahagirdar ◽

Brett Thurlow ◽

...

Keyword(s):

Ribosomal Subunit ◽

Small Subunit ◽

Folding Pathway ◽

Ribosome Assembly ◽

Combined Approach ◽

Quantitative Mass Spectrometry ◽

Cryo Electron Microscopy ◽

30S Subunit ◽

Assembly Factor

SUMMARYTo reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains were able to fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era’s role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.

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