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Streamlined identification of permissive non-canonical amino acid incorporation sites in mammalian cell lines with an expanded genetic code

ABSTRACT: The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efﬁciencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identiﬁcation of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efﬁciencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-ﬂuorescence reporter for high-throughput ﬂow-cytometry analysis that reproducibly yields context-speciﬁc ncAA incorporation efﬁciencies. We show that nucleotides up- and downstream of UAG synergistically inﬂuence ncAA incorporation efﬁciency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons ﬂanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simpliﬁes identiﬁcation as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Enes Ugur

LAB HEAD: Michael Wierer

PROVIDER: PXD019815 | Pride | 2021-02-23

REPOSITORIES: Pride

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			Action	DRS
	20200207_QX6_EnUg_SA_ST00_MB_2-4_1.raw	Raw
	20200207_QX6_EnUg_SA_ST00_MB_2-4_10.raw	Raw
	20200207_QX6_EnUg_SA_ST00_MB_2-4_11.raw	Raw
	20200207_QX6_EnUg_SA_ST00_MB_2-4_12.raw	Raw
	20200207_QX6_EnUg_SA_ST00_MB_2-4_13.raw	Raw

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Project description:Readthrough of a translation termination codon is regulated by ribosomal A site recognition and insertion of near-cognate tRNAs. Small molecules exist that mediate the incorporation of amino acids at the stop codon and the production of full-length, often functional protein but defining the actual amino acid that is incorporated remains a challenging area. We report on the development of a human cell model that can be used to determine whether rules can be developed using mass spectrometry that defines the type of amino acid that is placed at a premature termination codon during readthrough mediated by an aminoglycoside. The first premature termination codon we analysed contained the relatively common cancer-associated termination signal at codon 213 in the p53 gene. Although we could detect a tryptic peptide with the incorporation of an R at codon 213 in the presence of the aminoglycoside, there were no other tryptic peptides detected across codon 213 that could be recovered so we needed to create a more robust artificial premature stop codon model. P53 expression plasmids were developed that incorporate a string of single synthetic UGA (opal) stop codons at S127P128A129 within the relatively abundant tryptic p53 peptide 122-SVTCTYSPALNK-132. The treatment of cells stably expressing the p53-UGA129 mutation, treated with Gentamycin, followed by immunoprecipitation and trypsinization of p53, resulted in the identification R, W, or C within the tryptic peptide at codon-UGA129; as expected based on the two base pairing of the respective anticodons to UGA with R being the most abundant using all three codons. By contrast, incorporating the amber or ochre premature stop codons, UAA129 or UAG129 resulted in the incorporation of a Y or Q amino acid, again as expected based on the two base pairings to the anticodons, with Q being the most abundant. The incorporation of these amino acids at codons 127, 128, or 129 generally results in a p53 protein that is predicted to be ‘unfolded’ or inactive as defined by Molecular Dynamic Simulations. As such, the data also highlight the need in the future to not only produce novel small molecules that can read through premature termination codons but also the need to design methods to insert the required amino acid at the position that could result in a ‘wild-type’ functional protein.

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Streamlined identification of permissive non-canonical amino acid incorporation sites in mammalian cell lines with an expanded genetic code

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