Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half assembled intermediate

Zhou, Danni; Zhu, Xing; Zheng, Sanduo; Tan, Dan; Dong, Meng‐Qiu; Ye, Keqiong

doi:10.1101/242446

Cited by 12 publications

(21 citation statements)

References 68 publications

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“…The role of this family of helicase proteins is to unwind RNA structures and/or RNA-protein complexes, thus they are highly involved in ribosome maturation. Mt-LAF1 shows similarity with Has1, previously described in yeast pre-60S maturation and with Mss116, involved in yeast mitoribosome maturation 13,14 (Extended Data Fig. 7).…”

Section: Intersubunit Sidementioning

confidence: 53%

Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

Soufari

Waltz

Parrot

et al. 2020

Preprint

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AbstractKinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis1. They possess a single large mitochondrion, essential for the parasite survival2. In kinetoplastids mitochondrion, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes3. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes4,5. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit3,6. However, several important aspects mainly related to the large subunit remain elusive, such as the structure and maturation of its ribosomal RNA3. Here, we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the large subunit as well as previously unidentified ribosomal proteins. Most importantly, we introduce the structure of an LSU assembly intermediate in presence of 16 identified maturation factors. These maturation factors act both on the intersubunit and solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

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Section: Intersubunit Sidementioning

confidence: 53%

Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

Soufari

Waltz

Parrot

et al. 2020

Preprint

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“…Further transcription does not lead to the addition of many additional factors, since domains III-V are flexible in the early stages of pre-60S maturation (Supplemental Fig. 2), as seen in the cryo-EM structure of nucleolar pre-60S particles (Kater et al 2017;Sanghai et al 2018;Zhou et al 2018). Completion of domain VI induces the incorporation of almost 20 ribosome assembly factors, the 5S rRNA and association of snR10.…”

Section: Resultsmentioning

confidence: 96%

“…Recent structures of nucleolar pre-60S particles have revealed some of the folding pathways that large subunit pre-rRNA follows (Kater et al 2017;Sanghai et al 2018;Zhou et al 2018) showing that domains I, II, and VI of the 25S rRNA, along with the 5.8S rRNA, fold first into a near-mature conformation. Domains III-V remain unfolded in the early stages of large subunit assembly.…”

Section: Introductionmentioning

confidence: 99%

Assembly and early maturation of large subunit precursors

Chaker-Margot

Klinge

2019

RNA

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The eukaryotic ribosome is assembled through a complex process involving more than 200 factors. As preribosomal RNA is transcribed, assembly factors bind the nascent pre-rRNA and guide its correct folding, modification, and cleavage. While these early events in the assembly of the small ribosomal subunit have been relatively well characterized, assembly of the large subunit precursors, or pre-60S, is less well understood. Recent structures of nucleolar intermediates of large subunit assembly have shed light on the role of many early large subunit assembly factors, but how these particles emerge is still unknown. Here, we use the expression and purification of truncated pre-rRNAs to examine the initial assembly of pre-60S particles. Using this approach, we can recapitulate the early recruitment of large subunit assembly factors mainly to the domains I, II, and VI of the assembling 25S rRNA.

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“…No overall particle structure is available, although a recent high throughput pre-rRNA probing has provided important insights into the folding of the 35S and 27SA2 pre-rRNAs [ 7 ]. Moreover, very recently, structures of nucleolar pre-60S particles have been reported that seem to lack the Npa1p complex and probably correspond to particles closely downstream from the first pre-60S particle in the maturation pathway [ 11 – 13 ]. Of the approximately 30 AFs present in the first pre-60S particle [ 6 ], a third are required for its production and/or stability.…”

Section: Discussionmentioning

confidence: 99%

“…It has been proposed that formation and clustering of the root helices, as well as folding of domain I that forms part of the solvent exposed surface of the large subunit, are key events that take place very early on during large subunit biogenesis and are prerequisites for all downstream maturation steps [ 10 ]. The recently published cryo-EM structures of yeast nucleolar pre-60S particles [ 11 – 13 ] support the idea that assembly of the large ribosomal subunit starts with the folding and stabilization of domain I followed by those of domains II and VI.…”

Section: Introductionmentioning

confidence: 88%

The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor

et al. 2018

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The early steps of the production of the large ribosomal subunit are probably the least understood stages of eukaryotic ribosome biogenesis. The first specific precursor to the yeast large ribosomal subunit, the first pre-60S particle, contains 30 assembly factors (AFs), including 8 RNA helicases. These helicases, presumed to drive conformational rearrangements, usually lack substrate specificity in vitro. The mechanisms by which they are targeted to their correct substrate within pre-ribosomal particles and their precise molecular roles remain largely unknown. We demonstrate that the Dbp6p helicase, essential for the normal accumulation of the first pre-60S pre-ribosomal particle in S. cerevisiae, associates with a complex of four AFs, namely Npa1p, Npa2p, Nop8p and Rsa3p, prior to their incorporation into the 90S pre-ribosomal particles. By tandem affinity purifications using yeast extracts depleted of one component of the complex, we show that Npa1p forms the backbone of the complex. We provide evidence that Npa1p and Npa2p directly bind Dbp6p and we demonstrate that Npa1p is essential for the insertion of the Dbp6p helicase within 90S pre-ribosomal particles. In addition, by an in vivo cross-linking analysis (CRAC), we map Npa1p rRNA binding sites on 25S rRNA adjacent to the root helices of the first and last secondary structure domains of 25S rRNA. This finding supports the notion that Npa1p and Dbp6p function in the formation and/or clustering of root helices of large subunit rRNAs which creates the core of the large ribosomal subunit RNA structure. Npa1p also crosslinks to snoRNAs involved in decoding center and peptidyl transferase center modifications and in the immediate vicinity of the binding sites of these snoRNAs on 25S rRNA. Our data suggest that the Dbp6p helicase and the Npa1p complex play key roles in the compaction of the central core of 25S rRNA and the control of snoRNA-pre-rRNA interactions.

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Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half assembled intermediate

Cited by 12 publications

References 68 publications

Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

Assembly and early maturation of large subunit precursors

The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor

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