Genome editing of upstream open reading frames enables translational control in plants

Translational regulation by upstream open reading frames (uORFs) is becoming established as a general mechanism for controlling the amount of protein that is synthesized from downstream primary ORFs (pORFs). We found that genome editing of endogenous uORFs in plants enabled the modulation of translation of mRNAs from four pORFs that are involved in either development or antioxidant biosynthesis. A single-guide RNA that targeted the region harboring a uORF initiation codon can produce multiple mutations. Following uORF editing, we observed varying amounts of mRNA translation in four pORFs. Notably, editing the uORF of LsGGP2, which encodes a key enzyme in vitamin C biosynthesis in lettuce, not only increased oxidation stress tolerance, but also increased ascorbate content by ∼150%. These data indicate that editing plant uORFs provides a generalizable, efficient method for manipulating translation of mRNA that could be applied to dissect biological mechanisms and improve crops.

[1]  Silvia Zucchelli,et al.  Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs , 2016, Computational and structural biotechnology journal.

[2]  W. Miller,et al.  3' cap-independent translation enhancers of plant viruses. , 2013, Annual review of microbiology.

[3]  J. Sheen,et al.  Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis , 2007, Nature Protocols.

[4]  Hui-Li Xing,et al.  A CRISPR/Cas9 toolkit for multiplex genome editing in plants , 2014, BMC Plant Biology.

[5]  N. Sonenberg,et al.  Targeting the eIF4F translation initiation complex: a critical nexus for cancer development. , 2015, Cancer research.

[6]  M. van Zanten,et al.  An element within the 5' untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation. , 2001, European journal of biochemistry.

[7]  Q. Xie,et al.  High-Efficiency Genome Editing in Arabidopsis Using YAO Promoter-Driven CRISPR/Cas9 System. , 2015, Molecular plant.

[8]  J. Qiu,et al.  Progress and prospects in plant genome editing , 2017, Nature Plants.

[9]  Soon Il Kwon,et al.  DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins , 2015, Nature Biotechnology.

[10]  R. Hellens,et al.  Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants , 2005, Plant Methods.

[11]  Russell L. Ault,et al.  A comprehensive catalog of predicted functional upstream open reading frames in humans , 2018, Nucleic acids research.

[12]  J. Chory,et al.  BRI1 is a critical component of a plasma-membrane receptor for plant steroids , 2001, Nature.

[13]  Gerald M Rubin,et al.  Using translational enhancers to increase transgene expression in Drosophila , 2012, Proceedings of the National Academy of Sciences.

[14]  V. Valpuesta,et al.  Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase , 2003, Nature Biotechnology.

[15]  M. Selbach,et al.  Global quantification of mammalian gene expression control , 2011, Nature.

[16]  Yanpeng Wang,et al.  Genome editing in rice and wheat using the CRISPR/Cas system , 2014, Nature Protocols.

[17]  Qingzhen Zhao,et al.  Arabidopsis Ubiquitin Conjugase UBC32 Is an ERAD Component That Functions in Brassinosteroid-Mediated Salt Stress Tolerance[W][OA] , 2012, Plant Cell.

[18]  Q. Xie,et al.  The RING Finger Ubiquitin E3 Ligase SDIR1 Targets SDIR1-INTERACTING PROTEIN1 for Degradation to Modulate the Salt Stress Response and ABA Signaling in Arabidopsis , 2015, Plant Cell.

[19]  Piero Carninci,et al.  Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat , 2012, Nature.

[20]  Daowen Wang,et al.  Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana. , 2007, The Plant journal : for cell and molecular biology.

[21]  A. Allan,et al.  Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. , 2012, Plant biotechnology journal.

[22]  David R. Liu,et al.  Evolved Cas9 variants with broad PAM compatibility and high DNA specificity , 2018, Nature.

[23]  David R. Liu,et al.  Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage , 2016, Nature.

[24]  Meng Yuan,et al.  uORF-mediated translation allows engineered plant disease resistance without fitness costs , 2017, Nature.

[25]  Ha-il Jung,et al.  Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana. , 2009, Journal of visualized experiments : JoVE.

[26]  R. Hellens,et al.  An Upstream Open Reading Frame Is Essential for Feedback Regulation of Ascorbate Biosynthesis in Arabidopsis , 2015, Plant Cell.

[27]  Justin N. Vaughn,et al.  Regulation of plant translation by upstream open reading frames. , 2014, Plant science : an international journal of experimental plant biology.

[28]  S. Crooke,et al.  Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames , 2016, Nature Biotechnology.

[29]  Xun Xu,et al.  Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce , 2017, Nature Communications.

[30]  L. Kovács,et al.  Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC , 2016 .

[31]  Jin-Soo Kim,et al.  Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases , 2014, Bioinform..

[32]  V. Mootha,et al.  Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans , 2009, Proceedings of the National Academy of Sciences.