Ribosome assembly coming into focus

Klinge, Sebastian; Woolford, John L.

doi:10.1038/s41580-018-0078-y

Cited by 404 publications

(461 citation statements)

References 204 publications

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“…The substoichiometric presence of eS25 and other RPs on cellular ribosomes tempts the possibility that such compositional alterations are selected for translational control and participate in cellular functions such as tissue diversification (Shi et al, 2017). Yet it is still unclear how and why a cell would produce such heterogeneity given the complex and energy expensive nature of ribosome biogenesis (Klinge and Woolford, 2019;Peña et al, 2017). Potentially supporting this interpretation, a recent study found that maternal ribosomes are sufficient for embryogenesis in C. elegans (Cenik et al, 2019), meaning that functional ribosome heterogeneity in embryonic cell types must occur after assembly and transport.…”

Section: Discussionmentioning

confidence: 99%

“…RPs do much more than chaperone rRNA maturation concordant with their assembly, and several biogenesis checkpoints exist, providing opportunities for feedback loops to sense proper assembly (Klinge and Woolford, 2019;Peña et al, 2017). The complexity of ribosome biogenesis in eukaryotes is amplified by the existence of the nucleolus-a subnuclear, membraneless organelle that is a key hub for stress sensing (Boulon et al, 2010;Yang et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

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A memory of RPS25 loss drives resistance phenotypes

Johnson

Flynn

Lapointe

et al. 2019

Preprint

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In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand the genetic disruption of numerous ribosomal protein genes, indicating that select ribosome variants can sustain cellular life. Genetic alterations of ribosomal protein genes have been further linked to diverse cellular phenotypes and human disease, yet the direct and indirect consequences of sustained alterations in ribosomal protein levels are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the small subunit ribosomal proteins RPS25 or RACK1 in a human haploid cell line. To our surprise, we found that multiple cellular phenotypes previously assumed to result from a direct effect on translation were instead caused by indirect effects. During characterization of ribosomes isolated from the RPS25 knockout cell line, we discovered a partial remodeling of the large subunit via the ribosomal protein paralog eL22L1. Upon further examination, we found that RPS25 knockout clones display irreversible rewiring of viral-and toxin-resistance phenotypes, suggesting that the cells had undergone a stable transition to a new cell state. This new state appears to drive pleiotropic phenotypes and is characterized by a dramatically altered transcriptome and membrane proteome. By homology-directed repair of a RPS25 knockout cell line, we demonstrate that even when RPS25 expression is rescued at the native genomic locus, cells fail to correct for the phenotypic hysteresis. Our results illustrate how the elasticity of cells to a ribosome perturbation can manifest as specific but indirect phenotypic outcomes. The eukaryotic ribosome is comprised of four strands of rRNA and ~80 ribosomal proteins (RPs)

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A memory of RPS25 loss drives resistance phenotypes

Johnson

Flynn

Lapointe

et al. 2019

Preprint

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“…Las1 plays a fundamental role in processing precursor ribosomal RNA (pre-rRNA) (6,(14)(15)(16)(17). Ribosome production involves a complex and highly coordinated pre-rRNA processing cascade tasked with removing four spacer sequences from the pre-rRNA (14,(18)(19)(20)(21). Removal of the ITS2 (internal transcribed spacer 2), which lies between the 5.8S and 25S rRNAs, is initiated by Las1 pre-rRNA cleavage at the defined C2 site ( Fig.…”

Section: Textmentioning

confidence: 99%

It takes two (Las1 HEPN Endoribonuclease Motifs) to cut the RNA right

Pillon¹,

Goslen²,

Williams³

et al. 2019

Preprint

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Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the HEPN (higher eukaryotes and prokaryotes nucleotide-binding) nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif important for RNA cleavage. While most HEPN nucleases participate in stress activated RNA cleavage pathways, Las1 plays a fundamental role in processing the pre-ribosomal RNA. Underscoring the significance of Las1 function, mutations to the LAS1L gene have been associated with neurological dysfunction. Two juxtaposed Las1 HEPN nuclease motifs create its composite nuclease active site, however the roles of the individual HEPN residues are poorly defined. Here we show through a combination of in vivo and in vitro studies that both Las1 HEPN nuclease motifs are required for nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we define the consensus Las1 HEPN nuclease motif and uncover its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN¢ chimeras, we define the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif are necessary for nuclease specificity revealing that both HEPN motifs participate in coordinating the RNA within the active site. Taken together, our work reveals critical information about HEPN nuclease function and establishes that HEPN nucleases can be re-wired to cleave alternative RNA substrates. 185 aa (H134A) + LCT: 469-502 aa

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“…Unlike yeast and other eukaryotes, ribosome biogenesis in bacteria occurs in the same cell compartment as protein synthesis (Klinge and Woolford, 2019;Shajani et al, 2011). Although bacteria lack membrane compartments, they must nevertheless encode robust biochemical checkpoints that gate the transition from ribosome biogenesis to translation.…”

Section: Introductionmentioning

confidence: 99%

IF3 licenses newly made 30S subunits for translation during stress

Sharma

Woodson

2019

Preprint

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Bacterial ribosome biogenesis and translation occur in the same cellular compartment. Therefore, a biochemical gate-keeping step is required to prevent immature ribosomes from engaging in protein synthesis. Here, we show that the abundant ribosome assembly factor, RbfA, creates this checkpoint by suppressing protein synthesis by immature E. coli 30S subunits. After 30S maturation, RbfA is released by initiation factor 3 (IF3), which remains bound to 30S subunits to promote translation initiation. Genetic interactions between RbfA and IF3 show that IF3 is important for RbfA release during logarithmic growth. Moreover, IF3 is the main pathway for RbfA release in stationary phase when the activity of a less abundant RbfA-release factor, RsgA GTPase, is inhibited by the alarmone (p)ppGpp. By gating the transition from 30S biogenesis to translation initiation, RbfA and IF3 maintain the integrity of bacterial protein synthesis under a range of growth conditions and especially under stress.

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Ribosome assembly coming into focus

Cited by 404 publications

References 204 publications

A memory of RPS25 loss drives resistance phenotypes

A memory of RPS25 loss drives resistance phenotypes

It takes two (Las1 HEPN Endoribonuclease Motifs) to cut the RNA right

IF3 licenses newly made 30S subunits for translation during stress

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