Use of tRNA-Mediated Suppression to Assess RNA Chaperone Function

Porat, Jennifer; Bayfield, Mark A.

doi:10.1007/978-1-0716-0231-7_6

Cited by 12 publications

(13 citation statements)

References 24 publications

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“…Yeast strains were grown, after counterselection of a cover plasmid where applicable, at 30°C or at 37°C in synthetic selective medium and harvested at mid-log phase. Total RNA was isolated with hot acid phenol and northern blot analysis was performed as described 36,65 using separation on 8% TBE-urea polyacrylamide gels. Band intensities were quantified with ImageQuant TL v2005 software.…”

Section: Methodsmentioning

confidence: 99%

A substrate binding model for the KEOPS tRNA modifying complex

et al. 2020

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The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway–Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3’ CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

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Section: Methodsmentioning

confidence: 99%

A substrate binding model for the KEOPS tRNA modifying complex

et al. 2020

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“…Bulk tRNA were isolated according to the reported method (39) using hot acid-saturated phenol with slight modification. In particular, the cells were grown in 25 ml medium at 75°C to OD 600 of 0.8 before collection.…”

Section: Methodsmentioning

confidence: 99%

The archaeal KEOPS complex possesses a functional Gon7 homolog and has an essential function independent of cellular t⁶A modification level

Gan

Zhang

et al. 2022

Preprint

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Double-stranded DNA break (DSB) repair is a fundamental process for all cellular life. Recently, KEOPS, a multiple-subunit protein complex that is conserved in eukaryotes and archaea and primarily involved in N6- threonylcarbamoyl adenosine (t6A) modification of tRNAs is reported to participate in homologous recombination in yeast. To functionally characterize archaeal KEOPS (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus. We show that aKEOPS possesses five subunits, Pcc1, Pcc1-like (or Gon7-like), Kae1, Bud32 and Cgi121, just as eukaryotic KEOPS. Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1-Pcc1-Pcc1-Kae1), suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1-like, can be deleted in the wild type and in a t6A modification complementary strain constructed, indicating that aKEOPS complex is essential in multiple cellular processes in this archaeon. Moreover, knock-down of the subunit genes leads to increase or decrease in the sensitivity of the cells to hydroxyurea and ultraviolet radiation both of which are DNA damage agents. These results indicated that aKEOPS plays an important role in DNA repair. In vitro, archaeal Cgi121 possesses dsDNA-binding activity which relies on its tRNA 3' CCA tail binding module. Our study indicates that DNA repair is an original intrinsic function of the evolutionarily conserved complex and reveals a possible link between two functions of the complex, t6A modification and DSB repair.

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“…Total RNA was isolated from 1% of native protein extracts by incubation with 0.5% SDS, 0.2 mg/mL proteinase K (Sigma, P2308), 20 mM Tris HCl pH 7.4, and 10 mM EDTA pH 8.0 for 15 minutes at 50°C, followed by phenol: chloroform extraction and ethanol precipitation. Northern blot analysis was performed as described using 8% TBE-urea polyacrylamide gels 58 . Briefly, electrophoresed RNA was transferred to positively charged nylon membranes (Perkin Elmer, NEF988001) with the iBlot 2 system (Thermo, IB21001) and probed with 32 P γ -ATP-labeled DNA probes (probe sequences provided in Table S6).…”

Section: Methodsmentioning

confidence: 99%

The methyl phosphate capping enzyme Bin3 is a stable component of the fission yeast telomerase holoenzyme

Porat

Grigull

et al. 2021

Preprint

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The telomerase holoenzyme is critical for maintaining eukaryotic genome integrity. In addition to a reverse transcriptase and an RNA template, telomerase contains additional proteins that protect the telomerase RNA and promote holoenzyme assembly. Here we report that the methyl phosphate capping enzyme (MePCE) Bin3 is a stable component of the S. pombe telomerase holoenzyme. Bin3 associates with the telomerase holoenzyme and the U6 snRNA through an interaction with the recently described LARP7 family member Pof8, and we demonstrate that these two factors are evolutionarily linked in fungi. Our data suggest that the association of Bin3 with telomerase is independent of its methyltransferase activity, but rather that Bin3 negatively regulates TER1 levels and telomere length. Taken together, this work yields new insight into the composition, assembly, and regulation of the telomerase holoenzyme in fission yeast as well as the breadth of its evolutionary conservation.

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Use of tRNA-Mediated Suppression to Assess RNA Chaperone Function

Cited by 12 publications

References 24 publications

A substrate binding model for the KEOPS tRNA modifying complex

A substrate binding model for the KEOPS tRNA modifying complex

The archaeal KEOPS complex possesses a functional Gon7 homolog and has an essential function independent of cellular t⁶A modification level

The methyl phosphate capping enzyme Bin3 is a stable component of the fission yeast telomerase holoenzyme

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Use of tRNA-Mediated Suppression to Assess RNA Chaperone Function

Cited by 12 publications

References 24 publications

A substrate binding model for the KEOPS tRNA modifying complex

A substrate binding model for the KEOPS tRNA modifying complex

The archaeal KEOPS complex possesses a functional Gon7 homolog and has an essential function independent of cellular t6A modification level

The methyl phosphate capping enzyme Bin3 is a stable component of the fission yeast telomerase holoenzyme

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The archaeal KEOPS complex possesses a functional Gon7 homolog and has an essential function independent of cellular t⁶A modification level