Fig. 1: Ribosome profiling revealed defects in the transition from translation initiation to elongation in hot3-2.
a A metagene analysis was conducted to determine normalized P-site counts that were mapped to regions close to the translation initiation site (TIS) and stop codon of coding sequence (CDS). A 41-nt region located near the TIS and stop codon is displayed. A diagram representing the A-site (entry point for aminoacyl-tRNA), P-site (where peptide bond formation occurs), and E-site (exit site for uncharged tRNA) within ribosomes is provided below. The P-site counts for the initiating ribosome (P-site position on TIS) and terminating ribosome (A-site position on the stop codon) are shown. Three biological replicates were performed to generate the average value and standard deviation shown for each nucleotide. The relative Pausing at Start codon of CDS (rPS) represents the inability in translational I-E transition by calculating the ratio of P-site counts mapped to the TIS vs. the average P-site counts on each codon within the E region (CDS downstream of the TIS). Transcripts with at least 10 P-site counts mapped to the E region were included for further analysis. b A scatter plot showing the global increase in rPS values in inflorescence (IF) and seedling (S) tissues of hot3-2 mutants. A total of 21,425 and 20,805 transcripts with valid p values were utilized to calculate the fold change in rPS in hot3-2 mutants as compared to wild type. The median log2-transformed fold-change in rPS values was 0.48 and 0.33 for inflorescence and seedling samples, respectively. The Wilcoxon signed-rank test was employed to assess significance, resulting in two-tailed p value presented. c A Venn diagram showing the overlap between transcripts in IF_Stalled and S_Stalled, which were defined as those with significantly increased rPS values in hot3-2 vs. WT in inflorescences and seedlings, respectively. The 1584 transcripts in common are designated as IF&S_Stalled. d A Gene Ontology (GO) term enrichment plot for IF&S_Stalled. The terms “BP”, “CC”, and “MF” stand for biological process, cellular component, and molecular function, respectively. The GO term analysis was conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID)76,77. The enrichment plot was generated at https://www.bioinformatics.com.cn, an online platform for data analysis and visualization.
