Identification and functional characterization of mRNAs that exhibit stop codon readthrough in Arabidopsis thaliana

Figures

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  Figure 1. Selection and analysis of ribosome profiling datasets.A, heat map showing the three-nucleotide periodicity profile of the dataset SRP074840. Ribosomal footprints on all coding sequences were analyzed to get this profile for read lengths 24, 25, and 26. Reads were assigned to three frames based on which frame the first nucleotide aligns with on an mRNA sequence. The start codon ATG is indicated by the position 0 on the X-axis. B, distribution of ribo-seq read lengths. The graph shown is from the dataset SRP074840. C, flow chart showing the four-level screening method to identify mRNAs that show SCR in A. thaliana. Another flow chart with more details is shown in Fig. S2. CDS, coding sequence; ISR, interstop codon region; SCR, stop codon readthrough; UTR, untranslated region.

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  Figure 2. Ribosomal density in the ISR of five SCR-positive mRNAs—RPS15AD, CURT1B, CAM1, MUB6, and DXR. Graphs show ribo-seq reads in the ISR. Red arrows indicate the position of the two stop codons. Adjacent parts of the coding sequence and the 3′UTR are also shown for comparison. The data shown are from the dataset SRP074840. Inset show zoomed-in graphs. CDS, coding sequence; ISR, interstop codon region; SCR, stop codon readthrough; UTR, untranslated region.

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  Figure 3. Three-nucleotide periodicity at the ISR of five SCR-positive mRNAs—RPS15AD, CURT1B, CAM1, MUB6, and DXR. Graphs show fraction of ribo-seq reads in three translation frames. The three-nucleotide periodicity profile of coding sequences of all protein-coding genes is shown for comparison (All CDS; see Fig. S3). Graphs are derived from the dataset SRP160376 (for RPS15AD) and SRP074840 (for others). CDS, coding sequence; ISR, interstop codon region; SCR, stop codon readthrough; UTR, untranslated region.

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  Figure 4. Ribosomal density in the ISR of SCR-positive mRNAs.A, the graph shows the increase in ribosome density in the ISRs after each round of screening. ∗p < 0.001 Mann–Whitney rank sum test (compared to ‘All mRNAs’). B, graph shows the comparison of ribosomal density in the ISR versus that in the 3′UTR. This comparison is shown for SCR-positive mRNAs and for all mRNAs. p values were calculated using Mann–Whitney rank sum test. Numbers at the bottom of the graph indicate the mean value. The box represents 25% and 75% values, and the horizontal line within the box shows the median value. The analysis shown is for the dataset SRP074840. ISR, interstop codon region; SCR, stop codon readthrough; UTR, untranslated region.

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  Figure 5. The canonical stop codon and its context in SCR-positive mRNAs.A, distribution of the three stop codons in SCR-positive mRNAs. Expected values were obtained based on their occurrence in all mRNAs of A. thaliana. B, sequence logo of the stop codon context of SCR-positive mRNAs. The analysis was performed using WebLogo. SCR, stop codon readthrough.

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  Figure 6. Evolutionary conservation of the ISRs of SCR-positive genes. Amino acid sequences encoded by the CDS, ISR, and 3′UTR (excluding the ISR) of SCR-positive genes belonging to the plant species of Brassicacae family were aligned, and the alignment score was calculated using T-Coffee multiple sequence alignment program. Student’s t test with Welch’s correction was used to calculate the p value. Horizontal lines represent mean with 95% confidence interval. CDS, coding sequence; ISR, interstop codon region; SCR, stop codon readthrough; UTR, untranslated region.

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  Figure 7. Gene ontology analysis of SCR-positive genes of A. thaliana. Results of gene ontology (GO) functional enrichment analysis on SCR-positive genes using PANTHER web server. The X-axis shows false discovery rate, and the Y-axis shows multiple functional classes enriched in SCR-positive genes. Color of the circle indicates fold enrichment. The number of SCR-positive genes showing enrichment in a functional group is shown next to the circle. Size of the circle is proportional to this number. SCR-positive genes with more than 4-fold enrichment are shown here. SCR, stop codon readthrough.

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  Figure 8. Experimental validation of SCR in five A. thaliana mRNAs—DXR, RPS15AD, CURT1B, CAM1, and MUB6. Luminescence-based SCR assay. cDNA of a test gene along with the ISR was cloned upstream of and in-frame with the cDNA of firefly luciferase (FLuc) such that FLuc is expressed only if there is SCR across the stop codon of the test cDNA (see the schematic). ADF1 and UPL7 were used as negative controls. Constructs were subjected to in vitro transcription followed by in vitro translation using wheat germ extract with equal amount of RNA. Expression of Fluc was measured by its luminescence activity, which is shown in the graphs. Constructs without ISR were used to measure background signal (first bar), and constructs without any stop codon between the test cDNA and the FLuc were used to measure the maximum luminescence activity (last bar). Second bar in all graphs represents SCR activity across the canonical stop codon. Activity across mutated stop codons are also shown. Statistical significance was calculated using Student’s t test. p-values (two-sided) shown are in comparison with the constructs without the ISR. Welch’s correction was applied wherever required. Bars show mean ± SD (n =…

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  Figure 9. Nuclear localization of the SCR product of CURT1B (CURT1Bx).A, confocal microscopy images of N. benthamiana leaves expressing G3GFP-CURT1B or G3GFP-CURT1Bx showing their localization with respect to the nucleus (DAPI). Leaves were infiltrated with Agrobacterium harboring G3GFP-CURT1B or G3GFP-CURT1Bx constructs. Brightness and contrast were increased equally across the two channels. B, HeLa cells expressing GFP-CURT1B or GFP-CURT1Bx showing their localization with respect to the nucleus (DAPI). Cells were transfected with plasmids expressing GFP-CURT1B or GFP-CURT1Bx. Images are representatives from two independent experiments. SCR, stop codon readthrough.