Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes

Hernández, Greco; Osnaya, Vincent G.; Pérez-Martı́nez, Xochitl

doi:10.1016/j.tibs.2019.07.001

Cited by 89 publications

(67 citation statements)

References 93 publications

(233 reference statements)

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“…Kozak sequences surrounding an ATG start codon can direct translation from this ATG with varying degrees of strength [25]. The most important factor is the presence of a purine (A or G) 3 nucleotides upstream of the ATG start codon, and of a G (or less favourably an U) immediately after the ATG.…”

Section: Analysis Of Kozak Consensus Sequences Of Potential Atg Startmentioning

confidence: 99%

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Sequence Properties of the MAAP Protein and of the VP1 Capsid Protein of Adeno-Associated Viruses

Karlin¹

2020

Preprint

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Adeno-associated viruses (AAVs, genus dependoparvovirus) are promising gene therapy vectors. In strains AAV1-12, the capsid gene VP1 encodes a recently discovered protein, MAAP, in an overlapping frame.MAAP binds the cell membrane by an unknown mechanism. We discovered that MAAP is also encoded in bovine AAV and in porcine AAVs (which have shown promise for gene transfer into muscle tissues), in which it is probably translated from a non-canonical start codon. MAAP is predicted to be mostly disordered except for a predicted C-terminal, membrane-binding amphipathic α-helix. MAAP has a highly unusual composition. In particular, it lacks internal methionines, and is devoid of tyrosines in most strains.Unexpectedly, we discovered that the N-terminus of VP1 also lacks several amino acids. In all AAVs that encode MAAP, the first 200 aas of VP1 are devoid of internal methionines, probably owing to a selection against ATG codons that could prevent translation of MAAP and of capsid isoforms (VP2, VP3). The Nterminus of VP1 also lacks cysteines, likely to avoid the formation of disulfide bridges when it becomes exposed outside of the capsid during post-endocytic trafficking. Finally, the region common to VP1 and VP2 lacks tyrosine in the vast majority of AAVs that encode MAAP. Avoiding these "forbidden" aas in MAAP and VP1 when creating recombinant AAV capsids might increase the efficiency of capsid design. Conversely, the presence of "forbidden" aas in some rare strains probably indicates that they have unusual properties that could help us understand the viral cycle.Preprints (www.preprints.org) | NOT PEER-REVIEWED |

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Section: Analysis Of Kozak Consensus Sequences Of Potential Atg Startmentioning

confidence: 99%

“…3) "moderate" ones match the consensus (A/G)NNATG(A/C/U) or (C/U)NNATGG; 4) "weak" Kozak sequences do not match any of these consensus sequences [25].…”

Section: Analysis Of Kozak Consensus Sequences Of Potential Atg Startmentioning

confidence: 99%

Sequence Properties of the MAAP Protein and of the VP1 Capsid Protein of Adeno-Associated Viruses

Karlin¹

2020

Preprint

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“…Kozak sequences surrounding an AUG start codon can direct translation from this AUG with varying degrees of strength [42]. The most important factor is the presence of a purine (A or G) 3 nucleotides upstream of the AUG start codon, and of a G (or less favourably an U) immediately after the AUG. For the ORFs considered here, we classified Kozak sequences of potential AUG start codons in 4 categories, as in a recent exhaustive analysis in vertebrates [42]: 1) "optimal" Kozak sequences match the consensus (A/G)CCAUGG. 2) "strong" ones match the consensus (A/G)NNAUGG, where N is any nucleotide; 3) "moderate" match the consensus (A/G)(A/C)(A/C)AUG(G/U); finally 4) "weak" Kozak sequences do not match any of these consensus sequences [42].…”

Section: Methodsmentioning

confidence: 99%

“…In vertebrates, two main factors influence canonical translation from an AUG codon: 1) the strength of the "Kozak sequence" surrounding it [42] (we present Kozak sequences and their degrees of strength in the Methods); and 2) the position of the AUG codon in the mRNA. In general, the first AUG with an optimal Kozak sequence is used to initiate translation, but many exceptions are known.…”

Section: Second Hypothesis: the X Protein Is Translated From The Vp1 mentioning

confidence: 99%

Parvovirus B19 and Human Parvovirus 4 Encode a Homologous "X Protein" in a Reading Frame Overlapping the VP1 Capsid Gene

Karlin¹

2020

Preprint

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30 years ago, researchers noticed that the capsid (VP1) gene of B19 parvovirus might encode a second protein, called "X", in an overlapping reading frame. Since then, experimental approaches failed to detect it. In contrast, sequence analyses can reliably predict whether a protein is expressed from an overlapping frame, provided that it is beneficial to the virus and thus under selection pressure. We used a dedicated software, Synplot2, to identify regions of VP1 likely to encode functional proteins in overlapping frames. Synplot2 detected the X open reading frame and confirmed it is under highly significant selection pressure. We discovered that the X protein is homologous to the ARF1 protein of human parvovirus 4, another suspected protein encoded in a frame overlapping VP1. These findings provide compelling evidence that the X protein must be expressed and functional. We predict that it contains a predicted transmembrane region. We found that the X frame contains a potential AUG start codon in parvovirus B19 and in all related species. Yet no currently known viral transcript has the potential to encode the X protein in a monocistronic fashion. Therefore, the X protein is probably expressed either from an unmapped monocistronic mRNA, or translated by a non-canonical mechanism from the VP1 mRNA or from a short transcript, R3, which has no currently known function. Finally, Synplot2 also detected proteins likely to be expressed from a frame overlapping VP1 in species distantly related to parvovirus B19: porcine parvovirus 2 and bovine parvovirus 3.

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“…Kozak has reported CCRCCATGG (R = A or G; start codon underlined) to be a highly efficient mammalian TIS (Kozak, 1981; Noderer et al, 2014). Within that sequence, the purine in position −3 (three nucleotides upstream of the ATG codon) is most highly conserved in vertebrate messenger RNA (Hernandez, Osnaya, & Perez‐Martinez, 2019; Kozak, 1987a). Point mutational studies provide evidence of the importance of A or G in position −3, and G in position +4 (immediately following the ATG codon) as crucial for optimal translational efficiency (Kozak, 1997).…”

Section: Introductionmentioning

confidence: 99%

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product‐related impurities in CHO cells

Blánco¹,

Williams²,

Tang³

et al. 2020

Biotech & Bioengineering

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Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co‐expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3‐fold compared with the wild‐type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild‐type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product‐related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.

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Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes

Cited by 89 publications

References 93 publications

Sequence Properties of the MAAP Protein and of the VP1 Capsid Protein of Adeno-Associated Viruses

Sequence Properties of the MAAP Protein and of the VP1 Capsid Protein of Adeno-Associated Viruses

Parvovirus B19 and Human Parvovirus 4 Encode a Homologous "X Protein" in a Reading Frame Overlapping the VP1 Capsid Gene

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product‐related impurities in CHO cells

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