Recent applications of translational control in Arabidopsis (Arabidopsis thaliana) highlight the potential power of manipulating mRNA translation for crop improvement. However, to what extent translational regulation is conserved between Arabidopsis and other species is largely unknown, and the translatome of most crops remains poorly studied. Here, we combined de novo transcriptome assembly and ribosome profiling to study global mRNA translation in tomato (Solanum lycopersicum) roots. Exploiting features corresponding to active translation, we discovered widespread unannotated translation events, including 1,329 upstream open reading frames (uORFs) within the 59 untranslated regions of annotated coding genes and 354 small ORFs (sORFs) among unannotated transcripts. uORFs may repress translation of their downstream main ORFs, whereas sORFs may encode signaling peptides. Besides evolutionarily conserved sORFs, we uncovered 96 Solanaceae-specific sORFs, revealing the importance of studying translatomes directly in crops. Proteomic analysis confirmed that some of the unannotated ORFs generate stable proteins in planta. In addition to defining the translatome, our results reveal the global regulation by uORFs and microRNAs. Despite diverging over 100 million years ago, many translational features are well conserved between Arabidopsis and tomato. Thus, our approach provides a high-throughput method to discover unannotated ORFs, elucidates evolutionarily conserved and unique translational features, and identifies regulatory mechanisms hidden in a crop genome.
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...
Upstream ORFs (uORFs) are widespread cis-regulatory elements in the 5 prime untranslated regions of eukaryotic genes. Translation of uORFs could negatively regulate protein synthesis by repressing main ORF (mORF) translation and by reducing mRNA stability presumably through nonsense-mediated decay (NMD). While the above expectations were supported in animals, they have not been extensively tested in plants. Using ribosome profiling, we systematically identified 2093 Actively Translated uORFs (ATuORFs) in Arabidopsis seedlings and examined their roles in gene expression regulation by integrating multiple genome-wide datasets. Compared with genes without uORFs, we found ATuORFs result in 38%, 14%, and 43% reductions in translation efficiency, mRNA stability, and protein levels, respectively. The effects of predicted but not actively translated uORFs are much weaker than those of ATuORFs. Interestingly, ATuORF-containing genes are also expressed at higher levels and encode longer proteins with conserved domains, features that are common in evolutionarily older genes. Moreover, we provide evidence that uORF translation in plants, unlike in vertebrates, generally does not trigger NMD. We found ATuORF-containing transcripts are degraded through 5 prime to 3 prime decay, while NMD targets are degraded through both 5 prime to 3 prime and 3 prime to 5 prime decay, suggesting uORF-associated mRNA decay and NMD have distinct genetic requirements. Furthermore, we showed ATuORFs and NMD repress translation through separate mechanisms. Our results reveal that the potent inhibition of uORFs on mORF translation and mRNA stability in plants are independent of NMD, highlighting a fundamental difference in gene expression regulation by uORFs in the plant and animal kingdoms.
Aims To determine the role of sialylation on α5β1 and α2β1 integrins in the regulation of adhesion between breast cancer cells and extracellular matrix (ECM). Main methods Static cell adhesion assays were performed to quantify avidity of breast cancer cells to ECM. The effects of sialidases on α2,6 sialylation was assessed by flow cytometry using biotin conjugated sambucus nigra lectin. Lectin affinity assays were used to determine expression of α2,6 sialylated integrins. Cell migration and invasion were investigated by wound healing and transwell invasion assays. Key findings α2, α5 and β1 integrins had considerable α2,6 sialylation on MDA-MB-231 cells, whereas signals from MCF-7 cells were undetectable. Cleavage of α2,6 sialylation increased adhesion of MDA-MB-231 cells to ECM, while adhesion of MCF-7 cells was unaffected, consistent with the latter’s lack of endogenous α2,6 sialylated surface integrins. Neither surface expression of α2β1 and α5β1 integrins, nor activated β1 integrin, changed in MDA-MB-231 cells after sialidase treatment. However, sialidase treatment did not have significant impact on migration or invasion of MDA-MB-231 cells. Significance Cell adhesion is an important early step of cancer metastasis, yet the roles of sialylation in regulating integrin-mediated breast cancer cell adhesion in comparison to migration and invasion are not well-understood. Our data suggest desialylation of α2,6-sialylated integrins increases adhesion, but not migration or invasion, of MDA-MB-231 cells to ECM without altering integrin expression. It should be considered that α2,6 sialylation may play different roles in regulating cell adhesion of different cancer cells when developing potential therapeutics targeting α2,6 sialylation.
Long intergenic noncoding RNAs (lincRNAs) are a large yet enigmatic class of eukaryotic transcripts that can have critical biological functions. The wealth of RNA-sequencing (RNA-seq) data available for plants provides the opportunity to implement a harmonized identification and annotation effort for lincRNAs that enables cross-species functional and genomic comparisons as well as prioritization of functional candidates. In this study, we processed >24 Tbp of RNA-seq data from >16,000 experiments to identify ∼130,000 lincRNAs in four Brassicaceae: Arabidopsis thaliana, Camelina sativa, Brassica rapa, and Eutrema salsugineum. We used Nanopore RNA-seq, transcriptome-wide structural information, peptide data, and epigenomic data to characterize these lincRNAs and identify conserved motifs. We then used comparative genomic and transcriptomic approaches to highlight lincRNAs in our dataset with sequence or transcriptional conservation. Finally, we used guilt-by-association analyses to assign putative functions to lincRNAs within our dataset. We tested this approach on a subset of lincRNAs associated with germination and seed development, observing germination defects for Arabidopsis lines harboring T-DNA insertions at these loci. LincRNAs with Brassicaceae-conserved putative miRNA binding motifs, small open reading frames, or abiotic-stress modulated expression are a few of the annotations that will guide functional analyses into this cryptic portion of the transcriptome.
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