Reduced Amino Acid Specificity of Mammalian Tyrosyl-tRNA Synthetase Is Associated with Elevated Mistranslation of Tyr Codons

Raina, Medha; Moghal, Adil; Kano, Amanda; Jerums, Mathew; Schnier, Paul D.; Luo, Shun; Deshpande, Rohini; Bondarenko, Pavel V.; Lin, Henry; Ibba, Michael

doi:10.1074/jbc.m114.564609

Cited by 25 publications

(24 citation statements)

References 38 publications

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“…LC–MS is still the method of choice for the detection and quantification of translational misincorporations into the biotherapeutic protein, which can be a consequence of suboptimal media and cell culture parameters inducing cellular stress conditions . While cell culture conditions can be changed and optimized to reduce mistranslations later in development, it is critical to screen and differentiate for DNA mutations early, during CHO cell line development to minimize the negative impacts on development timelines and costs.…”

Section: Discussionmentioning

confidence: 99%

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

Aeschlimann

Graf

Mayilo

et al. 2019

Biotechnology Journal

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Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next‐generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single‐nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high‐level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.

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Section: Discussionmentioning

confidence: 99%

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

Aeschlimann

Graf

Mayilo

et al. 2019

Biotechnology Journal

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“…The vast majority of Val-tRNA Ile can be eliminated by the editing activity of the IleRS (Hendrickson et al , 2002), but the IleRS does inherently charge Val to tRNA Ile in appreciable levels. Similarly, mammalian tyrosine-tRNA synthetases (TyrRS) have been shown to charge Phe to tRNA Tyr at relatively high levels and this low specificity is further exacerbated during Tyr limitation (Raina et al , 2014). Additionally, the human alanine-tRNA (AlaRS) synthetase readily accepts noncognate tRNAs that have a G4:U69 base pair (e.g.…”

Section: Inherent Mistranslationmentioning

confidence: 99%

“…tRNA Cys ) in the acceptor stem (Sun et al , 2016). Despite the low specificity recently discovered for the mammalian TyrRS and AlaRS, the bacterial TyrRS and AlaRS still have high specificity for cognate amino acids and tRNAs, respectively, indicating that the inexplicable low specificity of these mammalian synthetases is not inherent to all biological systems (Raina et al , 2014, Sun et al , 2016). …”

Section: Inherent Mistranslationmentioning

confidence: 99%

Function and origin of mistranslation in distinct cellular contexts

Schwartz

Pan

2017

Critical Reviews in Biochemistry and Molecular Biology

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Mistranslation describes errors during protein synthesis that prevent the amino acid sequences specified in the genetic code from being reflected within proteins. For a long time, mistranslation has largely been considered an aberrant cellular process that cells actively avoid at all times. However, recent evidence has demonstrated that cells from all three domains of life not only tolerate certain levels and forms of mistranslation, but actively induce mistranslation under certain circumstances. To this end, dedicated biological mechanisms have recently been found to reduce translational fidelity, which indicates that mistranslation is not exclusively an erroneous process and can even benefit cells in particular cellular contexts. There currently exists a spectrum of mistranslational processes that differ not only in their origins, but also in their molecular and cellular effects. These findings suggest that the optimal degree of translational fidelity largely depends on a specific cellular context. This review aims to conceptualize the basis and functional consequence of the diverse types of mistranslation that have been described so far.

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“…In particular, a shift in the abundance of an aaRS cognate or non-cognate substrate may result in unusually high misacylation of tRNAs. Illustrative of this effect is the observation that Chinese hamster ovary cells starved for tyrosine exhibit a high degree of mistranslation of Tyr codons as Phe (9). Rather than relying on editing activity, tyrosyl-tRNA synthetase exhibits high kinetic discrimination between Tyr and Phe at the amino acid activation step.…”

mentioning

confidence: 97%

Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase

Moghal

Hwang

Faull

et al. 2016

Journal of Biological Chemistry

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Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA Phe . We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA Phe with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain noncognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA Phe species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.

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Reduced Amino Acid Specificity of Mammalian Tyrosyl-tRNA Synthetase Is Associated with Elevated Mistranslation of Tyr Codons

Cited by 25 publications

References 38 publications

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

Function and origin of mistranslation in distinct cellular contexts

Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase

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