Replacement of Hydroxylated His39 in Ribosomal Protein uL15 with Ala or Thr Impairs the Translational Activity of Human Ribosomes

Yanshina, Darya D.; Gopanenko, Alexander V.; Карпова, Г. Г.; Malygin, Alexey A.

doi:10.1134/s0026893320030206

Cited by 8 publications

(9 citation statements)

References 27 publications

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“…However, the polysome/monosome ratio (P/M) in the polysome profiles with mutant protein was markedly lower than that in the profiles with the wild-type protein, indicating inefficient translation in cells where mut-uL15 3×FLAG replaces endogenous uL15 in ribosomes. This is consistent with the early data [ 22 ] and points to the importance of uL15 hydroxylation for the correct functioning of the ribosome.…”

Section: Resultssupporting

confidence: 93%

“…The plasmids design for wt-uL15 3×FLAG and mut-uL15 3×FLAG , cloning of the minigene of C-terminal 3×FLAG-tagged human ribosomal protein uL15 in vector pcDNA3.1 and its mutagenesis for the H39A codon substitution, were described in [ 22 ]. The HEK293T cells (CVCL_0063) were cultured in the 10 cm Petri dishes containing DMEM, 10% FBS, and 100 U/mL of penicillin–streptomycin, in a CO 2 incubator (5% CO 2 ) at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…We have previously shown that exogenous FLAG-tagged uL15 carrying the His39Ala substitution, and therefore being incapable of hydroxylation, can successfully incorporate into 60S ribosomal subunits in HEK293 cells [ 22 ]. Nevertheless, the level of polysomes in cells producing the mutant form of uL15 was significantly reduced compared to cells containing the wild-type form of exogenous uL15 [ 22 ]. Thus, one can propose that specific disturbances in mRNA translation occur in cells with non-hydroxylated form of uL15 in the ribosomes.…”

Section: Introductionmentioning

confidence: 99%

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Mutation at the Site of Hydroxylation in the Ribosomal Protein uL15 (RPL27a) Causes Specific Changes in the Repertoire of mRNAs Translated in Mammalian Cells

Zolotenkova

Gopanenko

Tupikin

et al. 2023

IJMS

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Ribosomal protein uL15 (RPL27a) carries a specific modification, hydroxylation, at the His39 residue, which neighbors the CCA terminus of the E-site-bound tRNA at the mammalian ribosome. Under hypoxia, the level of hydroxylation of this protein decreases. We transiently transfected HEK293T cells with constructs expressing wild-type uL15 or mutated uL15 (His39Ala) incapable of hydroxylation, and demonstrated that ribosomes containing both proteins are competent in translation. By applying RNA-seq to the total cellular and polysome-associated mRNAs, we identified differentially expressed genes (DEGs) in cells containing exogenous uL15 or its mutant form. Analyzing mRNA features of up- and down-regulated DEGs, we found an increase in the level of more abundant mRNAs and shorter CDSs in cells with uL15 mutant for both translated and total cellular mRNAs. The level of longer and rarer mRNAs, on the contrary, decreased. Our data show how ribosome heterogeneity can change the composition of the translatome and transcriptome, depending on the properties of the translated mRNAs.

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Section: Resultssupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mutation at the Site of Hydroxylation in the Ribosomal Protein uL15 (RPL27a) Causes Specific Changes in the Repertoire of mRNAs Translated in Mammalian Cells

Zolotenkova

Gopanenko

Tupikin

et al. 2023

IJMS

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“…The minigene of the human ribosomal protein uS10 with the N-terminal 3×FLAG insertion was amplified using HEK293T cDNA and specific primers ( Table S6 ). The plasmid pcDNA-uS10 wt was generated by inserting the above minigene into the pcDNA3.1 vector at the Bam HI site as described [ 68 ]. This plasmid was a template for preparing pcDNA-uS10 mut1 , pcDNA-uS10 mut2 and pcDNA-uS10 mut3 plasmids bearing targeted mutations in the CDS of the minigene (insertion, substitution and deletion, respectively) by the quick-change mutagenesis method, using specific primers ( Table S6 ).…”

Section: Methodsmentioning

confidence: 99%

Changes in the Transcriptome Caused by Mutations in the Ribosomal Protein uS10 Associated with a Predisposition to Colorectal Cancer

Tian

Babaylova

Gopanenko

et al. 2022

IJMS

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A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g., PPM1D and PIGN. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer.

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“…It is interesting to note that uL15 contains a 36GGQ38 motif in the peptide 30GGRGMAGGQHHHR42 involved in the binding of CHX, and a Q38E transition was found to be responsible for the resistance [30]. Interestingly, replacement in rp uL15 of the His-39 residue (adjacent to Gln-38) by Ala or Thr impairs the activity of human ribosomes [31]. Since rp uL15 is universally conserved in all kingdoms of life, these data strongly agree with early biochemical studies that had demonstrated that peptidyl transferase is an enzyme with an ionizable functional group of pKa 7.2, which was supposed to be a catalytic His residue [32,33].…”

Section: Gank or Ggqtkpmentioning

confidence: 99%

COVID-19: Mechanisms of the Antiviral Activities of Selective Antibiotics Targeting the Human 80S Ribosome

Hountondji¹,

Poupaert²,

Aguida³

et al. 2021

TOBIOCJ

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Background: The majority of scientists, physicians, and healthcare professionals were trained with the paradigm: “antibiotics are for bacteria only !”, because they misunderstood the definition of the ribosome targeting antibiotics. In the context of the current worldwide COVID-19 pandemic, it might be useful to recall as precisely as possible the definition of the word antibiotic and provide evidence that some classes of antibiotics could offer excellent means to counteract viral infections via specific mechanisms. Methods: Molecular modeling and docking studies were used, as well as the tRNAox labeling reaction of the ribosomal protein eL42 in situ on human 80S ribosomes to demonstrate that cycloheximide and its thiosemicarbazone analogues bind to the catalytic Lys-53 residue of the human large subunit ribosomal protein eL42. Results: Comparison of the binding sites for Cycloheximide (CHX) and Sparsomycin (SPS) on the evolutionarily conserved E. coli bL12 and S. cerevisiae eL42 by means of molecular modeling and docking studies showed that: (i) SPS binds in proximity to the catalytic Lys-65 residue of the GANK motif of rp bL12 and to the catalytic Lys-55 residue of the GGQTKP motif of rp eL42; (ii) CHX failed to bind to the GANK motif, while the glutarimide moiety of SPS and CHX was found to make contact with Lys-55 of the GGQTKP motif of rp eL42. Conclusion: In this report, we demonstrate that cycloheximide and its thiosemicarbazone analogues are capable of inhibiting the human 80S ribosomes selectively through their binding to the ε-amino group of the side chain of Lys-53. As a consequence, these small-molecule inhibitors of translation are susceptible to exhibit antiviral activities by preventing the human ribosomes of the SARS-CoV-2 infected cells from synthesizing the viral proteins and enzymes.

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Replacement of Hydroxylated His39 in Ribosomal Protein uL15 with Ala or Thr Impairs the Translational Activity of Human Ribosomes

Cited by 8 publications

References 27 publications

Mutation at the Site of Hydroxylation in the Ribosomal Protein uL15 (RPL27a) Causes Specific Changes in the Repertoire of mRNAs Translated in Mammalian Cells

Mutation at the Site of Hydroxylation in the Ribosomal Protein uL15 (RPL27a) Causes Specific Changes in the Repertoire of mRNAs Translated in Mammalian Cells

Changes in the Transcriptome Caused by Mutations in the Ribosomal Protein uS10 Associated with a Predisposition to Colorectal Cancer

COVID-19: Mechanisms of the Antiviral Activities of Selective Antibiotics Targeting the Human 80S Ribosome

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