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

Abstract

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.

Translational landscape in tomato revealed by transcriptome assembly and ribosome profiling

“…Several studies have illustrated the power of altering mRNA translation via uORFs to improve agriculture (1–3). For example, engineering rice that specifically induces defense proteins when a uORF is repressed by pathogen attack enables immediate plant resistance without compromising plant growth in the absence of pathogens (2).…”

“…Besides being an essential step in gene expression, mRNA translation directly shapes the proteome, which contributes to cellular structure, function, and activity in all organisms. The characterization of translational regulation has enabled crop improvement, including increasing tomato sweetness, rice immunity and lettuce resistance to oxidative stress (1–3). However, due to limited genomic resources and methods, most crop translatomes remain understudied.…”

Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing

“…Since its demonstration in plants in late 2013 (Li et al ., ; Nekrasov et al ., ; Shan et al ., ), CRISPR‐Cas9 has been widely applied in many plant species (Malzahn et al ., ; Yin et al ., ). Most studies have relied on non‐homologous end joining (NHEJ) DNA repair pathway to introduce insertion or deletion (InDel) mutations, achieving editing outcomes such as gene knockout (Malzahn et al ., ), mutagenesis of microRNAs (Zhou et al ., ) or cis ‐regulatory sequences (Rodriguez‐Leal et al ., ; Zhang et al ., ), as well as large chromosomal deletions (Zhou et al ., ) and gene replacement (Li et al ., ). In some cases, homology directed repair (HDR) was used for targeted gene replacement (Endo et al ., 2016b; Gil‐Humanes et al ., ; Li et al ., ; Miki et al ., ; Schiml et al ., ; Svitashev et al ., ).…”

Transcription factor AP‐4 (TFAP4)‐upstream ORF coding 66 aa inhibits the malignant behaviors of glioma cells by suppressing the TFAP4/long noncoding RNA 00520/microRNA‐520f‐3p feedback loop

“…Upstream ORF could inhibit the translation initiation rate of downstream CDS regions by retaining or dissociating ribosomes from mRNA. A short ribosome‐encoded peptide interacts with the ribosome to induce the arrest of the ribosome located on the initiation codons of uORFs to the terminator, which affects the translation of downstream CDS . Two studies reported that the presence of uORFs of 25 codons in the 5′UTR region of CPA1 mRNA, which encodes a fungal AAP that regulates ribosome function.…”

“…A short ribosome-encoded peptide interacts with the ribosome to induce the arrest of the ribosome located on the initiation codons of uORFs to the terminator, which affects the translation of downstream CDS. 33 Two studies reported that the presence of uORFs of 25 codons in the 5′UTR region of CPA1 mRNA, which encodes a fungal AAP that regulates ribosome function. Ribosomal retention of AAP at the stop codon, which is induced by increasing arginine, inhibits the translation of CPA1.…”