Translational landscape in tomato revealed by transcriptome assembly and ribosome profiling

Abstract

25mRNA translation is a critical step in gene expression, but our understanding of the 26 landscape and control of translation in diverse crops remains lacking. Here, we combined de 27 novo transcriptome assembly and ribosome profiling to study global mRNA translation in tomato 28 roots. Taking advantage of the 3-nucleotide periodicity displayed by translating ribosomes, we 29 identified 354 novel small ORFs (sORFs) translated from previously unannotated transcripts, as 30 well as 1329 upstream ORFs (uORFs) translated within the 5' UTRs of annotated protein-coding 31 genes. Proteomic analysis confirmed that some of these novel uORFs and sORFs generate 32 stable proteins in planta. Compared with the annotated ORFs, the uORFs use more flexible 33 Kozak sequences around translation start sites. Interestingly, uORF-containing genes are 34 enriched for protein phosphorylation/dephosphorylation and signaling transduction pathways, 35 suggesting a regulatory role for uORFs in these processes. We also demonstrated that 36 ribosome profiling is useful to facilitate the annotation of translated ORFs and noncanonical 37 translation initiation sites. In addition to defining the translatome, our results revealed the global 38 control of mRNA translation by uORFs and microRNAs in tomato. In summary, our approach 39 provides a high-throughput method to discover unannotated ORFs, elucidates evolutionarily 40 conserved translational features, and identifies new regulatory mechanisms hidden in a crop 41 genome. 42 43 48 transcriptome assembly and ribosome profiling, we mapped and quantified translating 49 ribosomes across the entire transcriptome in tomato roots. This is the first experiment-based 50 survey to systematically identify actively translated ORFs in a crop. Our results reveal numerous 51 unannotated translation events and uncover new regulatory mechanisms of gene expression in 52 tomato. Our approach not only facilitates our understanding of the tomato translational 53 landscape but also provides a practical strategy to study the translatomes of other species. 54 55 56 Introduction 57Besides being an essential step in gene expression, mRNA translation directly shapes 58 the proteome, which contributes to cellular structure, function, and activity in all organisms. The 59 characterization of translational regulation has enabled crop improvement, including increasing 60 tomato sweetness, rice immunity and lettuce resistance to oxidative stress (1-3). However, due 61 to limited genomic resources and methods, most crop translatomes remain understudied. 62Ribosome profiling, or Ribo-seq, has emerged as a high-throughput technique to study 63 global translation (4-6). In a Ribo-seq experiment, ribosomes in the sample of interest are 64 immobilized, and the lysate is treated with nucleases to obtain ribosome-protected mRNA 65 fragments (i.e. ribosome footprints). Finally, sequencing of the ribosome footprints reveals the 66 quantity and positions of ribosomes on a given transcript. Features corresponding to active 67 translat...

Construction of High-Quality Rice Ribosome Footprint Library

“…Recently, a genome-wide methodology named as ribosome profiling or ribo-seq has been first proposed by Ingolia et al (2009) to evaluate in vivo RNA translation dynamics in yeast via high-throughput sequencing of ribosome footprints, and subsequently been rapidly extended to related research in bacteria ( Li et al, 2012 ; Mohammad et al, 2016 ; Mohammad et al, 2019 ), animals ( Ingolia et al, 2011 ; Gonzalez et al, 2014 ; Chen and Dickman, 2017 ; Sugiyama et al, 2017 ; Zhang et al, 2018a ), human being ( Ingolia et al, 2014 ; Raj et al, 2016 ; Blair et al, 2017 ; Holmes et al, 2019 ; Chen et al, 2020 ) and higher plants ( Liu et al, 2013 ; Juntawong et al, 2014 ; Lei et al, 2015 ; Merchante et al, 2015 ; Hsu et al, 2016 ; Bazin et al, 2017 ; Lorenzo-Orts et al, 2019 ; Wu H. L. et al, 2019 ). To perform ribosome profiling, total polysomes are first isolated from tissues of interest such as rice seedling shoots, then the monosomes that are derived from the nuclease-treated total polysomes are collected for isolation of ribosome footprints, which are subjected to rRNA trimming and library construction, and the final product is obtained for high-throughput sequencing followed by data analysis after PCR enrichment and PAGE purification of ribosome footprint library ( Figure 1 ).…”

“…In comparison to its widespread application in bacteria, yeast, animals and human being, only a limited number of studies on ribosome profiling have been conducted in higher plants ( Liu et al, 2013 ; Juntawong et al, 2014 ; Lei et al, 2015 ; Merchante et al, 2015 ; Hsu et al, 2016 ; Bazin et al, 2017 ; Lorenzo-Orts et al, 2019 ; Wu H. L. et al, 2019 ), and most of them are restricted to the model plant Arabidopsis . Up to date, no available protocol has been proposed for construction of high-quality ribosome footprint library in rice, which feeds most of world population.…”

Translational regulation contributes to the elevated CO2 response in two Solanum species

“…GO terms related to transcriptional regulation and transcription factor activity were uniquely enriched in Pattern 2, a marked contrast with the translational regulatory pathways observed in the transcriptional profiling. Such enrichment for entire categories related to transcriptional regulation have not previously been identified in response to abiotic stresses (Mustroph et al , ; Ribeiro et al , ; Juntawong and Bailey‐Serres, ; Yángüez et al , ; Aubry et al , ; Lin et al , ; Cheng et al , ; Vragović et al , ; Basbouss‐Serhal et al , ; Zoschke et al , ; Meteignier et al , ; Xu et al , ; Li et al , ; Chen et al , ; Wu et al , ; Tian et al , ). Other patterns had few and general terms associated with them (patterns 3, 6, 9, 10, and 11; Figure S6; Table S4).…”

Translational gene regulation in plants: A green new deal

“…Ribosome footprinting has yielded direct evidence that many uORFs are actively translated (Bazin et al, 2017;Hsu et al, 2016;Juntawong et al, 2014;Kurihara et al, 2020;M. J. Liu et al, 2013;H. L. Wu, Song, Walley, & Hsu, 2019).…”