Stepwise Splitting of Ribosomal Proteins from Yeast Ribosomes by LiCl

Piir, Kerli; Tamm, Tiina; Kisly, Ivan; Tammsalu, Triin; Rèmme, Jaanus

doi:10.1371/journal.pone.0101561

Cited by 9 publications

(11 citation statements)

References 25 publications

(30 reference statements)

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“…Yeast Saccharomyces cerevisiae ribosome (80S) was purified according to Chakraborty et al 49 . Yeast 80S rRNA was extracted as mentioned in Piir et al 50 . Tau K18 and Ht40 were purified using a two-step purification method: direct-boiling method 51,52 and cation-exchange purification method 22 .…”

Section: Methodsmentioning

confidence: 99%

“…Seeding assay for tau induced 80S aggregation. 50 µM K18 was reduced in Buffer A for 2 hours at 37 °C, after which 0.1 µM 80S was added to it. 1 µl aliquots were drawn from this reaction mixture after 0, 1.5, 3, 4 hours of incubation (for K18 induced 80S aggregation) and 0, 2, 5, 8 hours of incubation (for Ht40 induced aggregation) added to 999 µl of fresh 0.1 µM 80S in Buffer A.…”

Section: Human In Vitro Transcription-translation Assay the In Vitromentioning

confidence: 99%

“…Light scattering study. 50 µM of K18 or Ht40 was reduced in Buffer A with 1 mM DTT for 2 hours at 37 °C after which 1 µM of LiCl extracted 80SrRNA was added to the reaction mixture (t = 0 h) and further incubated for 48 hours at 37 °C. The light scattering of the solutions was measured at t = 0 h and t = 48 h and the increase was plotted in the form of bar diagrams.…”

Section: Human In Vitro Transcription-translation Assay the In Vitromentioning

confidence: 99%

“…Delayed heparin/tRNA addition assay for tau induced 80S aggregation. 50 µM K18 was reduced in Buffer A for 2 hours at 37 °C in presence of 1 mM DTT, after which 0.1 µM 80S was added to it. 1 µM heparin or tRNA was added after 0, 1.5, 3 and 4 hours of addition of the ribosome in case of K18 and after 0, 2, 5 and 8 hours of addition of the ribosome in case of Ht40.…”

Section: Human In Vitro Transcription-translation Assay the In Vitromentioning

confidence: 99%

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Tau protein- induced sequestration of the eukaryotic ribosome: Implications in neurodegenerative disease

Banerjee

Ferdosh

Ghosh

et al. 2020

Sci Rep

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the human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer's disease, ribosomal dysfunction might be a crucial event in the disease pathology. our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRnA-ribosomal protein aggregates. incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. the incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRnA, thereby indicating the electrostatic nature of the aggregation process.

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Section: Methodsmentioning

confidence: 99%

Section: Human In Vitro Transcription-translation Assay the In Vitromentioning

confidence: 99%

Section: Human In Vitro Transcription-translation Assay the In Vitromentioning

confidence: 99%

Section: Human In Vitro Transcription-translation Assay the In Vitromentioning

confidence: 99%

See 2 more Smart Citations

Tau protein- induced sequestration of the eukaryotic ribosome: Implications in neurodegenerative disease

Banerjee

Ferdosh

Ghosh

et al. 2020

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Ribosome-polysome profile analysis was performed as described previously 51; 52 with modifications. Briefly, 100 ml of yeast cells was grown in YPD at 30°C or 20°C to mid-exponential phase.…”

Section: Methodsmentioning

confidence: 99%

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome

Kisly

Gulay

Mäeorg

et al. 2016

Journal of Molecular Biology

Self Cite

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During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote-specific. These are mainly localized to the peripheral regions and are believed to stabilise the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 which is composed of an N-terminal globular domain, a middle region and a long C-terminal α-helix. Analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilising interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg2+ concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasising the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality.

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Amyloid protein‐induced sequestration of the eukaryotic ribosome: effect of stoichiometry and polyphenolic inhibitors

et al. 2022

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Alzheimer's disease (AD) is characterized by the appearance of neurofibrillary tangles comprising of the Tau protein and aggregation of amyloid‐β peptides (Aβ 1–40 and Aβ 1–42). A concomitant loss of the ribosomal population is also observed in AD‐affected neurons. Our studies demonstrate that, similarly to Tau protein aggregation, in vitro aggregation of Aβ peptides in the vicinity of the yeast 80S ribosome can induce co‐aggregation of ribosomal components. The RNA‐stimulated aggregation of Aβ peptides and the Tau‐K18 variant is dependent on the RNA:protein stoichiometric ratio. A similar effect of stoichiometry is also observed on the ribosome–protein co‐aggregation process. Polyphenolic inhibitors of amyloid aggregation, such as rosmarinic acid and myricetin, inhibit RNA‐stimulated Aβ and Tau‐K18 aggregation and can mitigate the co‐aggregation of ribosomal components.

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Stepwise Splitting of Ribosomal Proteins from Yeast Ribosomes by LiCl

Cited by 9 publications

References 25 publications

Tau protein- induced sequestration of the eukaryotic ribosome: Implications in neurodegenerative disease

Tau protein- induced sequestration of the eukaryotic ribosome: Implications in neurodegenerative disease

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome

Amyloid protein‐induced sequestration of the eukaryotic ribosome: effect of stoichiometry and polyphenolic inhibitors

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