Ribosomal mutation in helix 32 of 18S rRNA alters fidelity of eukaryotic translation start site selection

A, Charles Antony; Ram, Anup Kumar; Dutta, Kalloly; Alone, Pankaj V.

doi:10.1002/1873-3468.13369

Cited by 4 publications

(4 citation statements)

References 49 publications

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“…The eIF2 S264Y mutation was extensively used to study the eIF2 function [3,5,17,[23][24][25][26][27][28][29][30][31][32].…”

Section: Resultsmentioning

confidence: 99%

eIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding

Gayen,

Alone

2024

Bioscience Reports

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The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2β subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2β-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2βS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2βS264Y mutation has eIF2β-γ interaction defect. Consistently, the eIF2βT238A intragenic suppressor mutation restored the eIF2β-γ and Met-tRNAiMet binding. The eIF2β-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2βT238A mutation, suggesting the eIF2β-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2βF217A/Q221A double mutation in the HTH domain. The eIF2βT238A suppressor mutation could not rescue the eIF2β binding defect of the eIF2γV281K mutation; however, combining the eIF2βS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2β with the eIF2γ subunit via its α1-helix, the eIF2β-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2β-γ interacts via two distinct binding sites.

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“…The eIF2 S264Y mutation was extensively used to study the eIF2 function [3,5,17,[23][24][25][26][27][28][29][30][31][32].…”

Section: Resultsmentioning

confidence: 99%

eIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding

Gayen,

Alone

2024

Bioscience Reports

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“…Translation from these out-of-frame upORFs may preclude the translation of main ORF, resulting in the altered proteome in Sui − mutant cells. The eIF5 G31R mutation is one of the strongest Sui − mutants that preferentially recognize UUG as a start codon, unlike other Sui − mutants that can also utilize CUG or GUG as a start codon 16 , 18 . The eIF5 G31R mutation in the recessive condition is lethal, whereas the dominant eIF5 G31R mutation represses GCN4 translation (Gcn − phenotype) and shows 3-amino-1,2,4-triazole (3AT) sensitivity due to utilization of upUUG codons from the 5′ regulatory region of GCN4 transcript and show severe slow growth phenotype 19 , 20 .…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we reported that the eIF5 G31R mutation recognizes UUG codon but does not initiate translation from CUG or GUG codon 18 . In this study, we decipher the underpinning effect of the eIF5 G31R mutation on UUG codon recognition that alters cellular proteome in dominant conditions.…”

Section: Discussionmentioning

confidence: 96%

“…The Sui − mutants can utilize the in-frame or the out-of-frame UUG, CUG, or GUG codons as a translation start site at the 5′ UTR of the mRNA 16 . The dominant-negative eIF5 G31R hyper GAP mutant is the strongest Sui − mutation identified that preferentially recognizes UUG as a start codon and shows severe slow growth, whereas in the recessive conditions the eIF5 G31R mutation is lethal 18 . This preferential recognition of UUG start from the 5′ regulatory region of the GCN4 transcript causes repression of GCN4 expression (general control non-derepressed: Gcn − phenotype) 19 .…”

Section: Introductionmentioning

confidence: 99%

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Altered proteome in translation initiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage

Ram

Mallik

Reddy

et al. 2022

Sci Rep

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The recognition of the AUG start codon and selection of an open reading frame (ORF) is fundamental to protein biosynthesis. Defect in the fidelity of start codon selection adversely affect proteome and have a pleiotropic effect on cellular function. Using proteomic techniques, we identified differential protein abundance in the translation initiation fidelity defective eIF5G31R mutant that initiates translation using UUG codon in addition to the AUG start codon. Consistently, the eIF5G31R mutant altered proteome involved in protein catabolism, nucleotide biosynthesis, lipid biosynthesis, carbohydrate metabolism, oxidation–reduction pathway, autophagy and re-programs the cellular pathways. The utilization of the upstream UUG codons by the eIF5G31R mutation caused downregulation of uridylate kinase expression, sensitivity to hydroxyurea, and DNA damage. The eIF5G31R mutant cells showed lower glutathione levels, high ROS activity, and sensitivity to H2O2.

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Sequence-context dependency shown by eIF5^G31Rmutant for UUG start codon selection inSaccharomyces cerevisiae

Ram

2023

Preprint

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In higher eukaryotes, the efficiency of start codon selection depends upon its sequence context A/GxxAUGG, especially at the -3 and +4 position. However, S. cerevisiae prefers AAA/UAUGU sequence context surrounding the AUG start codon. Furthermore, it was demonstrated that the first AUG codon on the mRNA serves as a signal for ribosomal recognition of the initiation site selection. In yeast, the G31R mutation in eIF5 shows Suppressor of initiation codon (Sui) phenotype enabling 40S ribosome to initiate at UUG start codon. To understand whether the UUG codon recognition by the eIF5G31R mutant is sensitive to the sequence context, we made different HIS4-LacZ reporter constructs with UUG as start codon and changed the sequence context at -3 and -2 positions. The HIS4UUG-LacZ transcripts that carry purine (A/G) at the -3-position showed better β-Galactosidase activity than pyrimidine (U/C). Furthermore, on changing the -1 position for the AAA context, we discovered purines are more preferred at the -1 position for the UUG start codon selection.

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Ribosomal mutation in helix 32 of 18S rRNA alters fidelity of eukaryotic translation start site selection

Cited by 4 publications

References 49 publications

eIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding

eIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding

Altered proteome in translation initiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage

Sequence-context dependency shown by eIF5^G31Rmutant for UUG start codon selection inSaccharomyces cerevisiae

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Ribosomal mutation in helix 32 of 18S rRNA alters fidelity of eukaryotic translation start site selection

Cited by 4 publications

References 49 publications

eIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding

eIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding

Altered proteome in translation initiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage

Sequence-context dependency shown by eIF5G31Rmutant for UUG start codon selection inSaccharomyces cerevisiae

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Sequence-context dependency shown by eIF5^G31Rmutant for UUG start codon selection inSaccharomyces cerevisiae