The translatome, a profile of the translational status of genetic information within cells, provides a new perspective on gene expression. Although many plant genomes have been sequenced, comprehensive translatomic annotations are not available for plants due to a lack of efficient translatome profiling techniques. Here, we developed a new technique termed 3′ ribosome-profiling sequencing (3′Ribo-seq) for reliable, robust translatomic profiling. 3′Ribo-seq combines polysome profiling and 3′ selection with a barcoding and pooling strategy. Systematic translatome profiling of different tissues of
Arabidopsis
, rice, and maize using conventional ribosome profiling (Ribo-seq) and 3′Ribo-seq revealed many novel translational genomic loci, thereby complementing functional genome annotation in plants. Using the low-cost, efficient 3′Ribo-seq technique and genome-wide association mapping of translatome expression (eGWAS), we performed a population-level dissection of the translatomes of 159 diverse maize inbred lines and identified 1,777 translational expression quantitative trait loci (eQTLs). Notably, local eQTLs are significantly enriched in the 3′ untranslated regions of genes. Detailed eQTL analysis suggested that sequence variation around the polyadenylation (polyA) signal motif plays a key role in translatomic variation. Our study provides a comprehensive translatome annotation of plant functional genomes and introduces 3′Ribo-seq, which paves the way for deep translatomic analysis at the population level.
Small peptides (sPeptides), <100 amino acids (aa) long, are encoded by small open reading frames (sORFs) often found in the 5′ and 3′ untranslated regions (or other parts) of mRNAs, in long non-coding RNAs, or transcripts from introns and intergenic regions; various sPeptides play important roles in multiple biological processes. In this study, we conducted a comprehensive study of maize (Zea mays) sPeptides using mRNA sequencing, ribosome profiling (Ribo-seq), and mass spectrometry (MS) on six tissues (each with at least two replicates). To identify maize sORFs and sPeptides from these data, we set up a robust bioinformatics pipeline and performed a genome-wide scan. This scan uncovered 9,388 sORFs encoding peptides of 2–100 aa. These sORFs showed distinct genomic features, such as different Kozak region sequences, higher specificity of translation, and high translational efficiency, compared with the canonical protein-coding genes. Furthermore, the MS data verified 2,695 sPeptides. These sPeptides perfectly discriminated all the tissues and were highly associated with their parental genes. Interestingly, the parental genes of sPeptides were significantly enriched in multiple functional gene ontology terms related to abiotic stress and development, suggesting the potential roles of sPeptides in the regulation of their parental genes. Overall, this study lays out the guidelines for genome-wide scans of sORFs and sPeptides in plants by integrating Ribo-seq and MS data and provides a more comprehensive resource of functional sPeptides in maize and gives a new perspective on the complex biological systems of plants.
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