Biological replicates exhibit a high correlation of editing extent.

<p>Each graph represents a pairwise comparison of editing extent that was measured on two libraries obtained from cDNAs of two plants belonging to the same genotype grown in the same conditions and harvested at the same time.</p

Relative importance of RIPs on plastid editing.

<p>(A) Number of plastid sites under the control of RIPs (ΔEE≥0.1, P 2.7<10⁻⁵). <i>RIP2</i> and <i>RIP9</i> results were obtained from VIGS. (B) Examples of plastid sites falling into one of the eight categories described in the Venn diagram shown in (A). The background color reflects the range of editing extent from red (low: 0–0.2) to dark green (high: 0.8–1).</p

Comprehensive High-Resolution Analysis of the Role of an Arabidopsis Gene Family in RNA Editing

<div><p>In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP) family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq) to explore the effect of mutation or silencing of every <i>RIP</i> gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.</p></div

The <i>rip1</i> mutant is the only mutant in our study with a severe defective phenotype.

<p><i>rip1</i>, <i>rip1</i>-wt, <i>rip3-2</i> and <i>rip3-2</i>-wt were grown under long-day growth room conditions (14 h light/10 h dark) while the other plants were grown in short day conditions (10 k light/14 h dark). <i>rip3-2</i> mutant plants show a slight delay in development compared to their wild-type siblings. At the time of photography, 2 out of 8 (1/4) <i>rip3-2</i> seedlings (30 days post-sowing) have flowered while all the wild-type seedlings have flowered (white spots in the middle of each plant are the flowers). <i>rip4-1</i>, <i>rip4-2</i>, and <i>rip5-1</i> do not show any phenotypic difference with their wild-type (a <i>rip5-1</i> wild-type was not available for the picture, but its genetic background is Columbia like <i>rip4-2</i>).</p

Examples of newly identified editing sites having sequences in their putative <i>cis</i> elements similar to known editing sites.

<p>The sequences shown are 20 nt upstream and 5 nt downstream around the target C for editing (underlined and capitalized). Other Cs that are edited are underlined. The upper (lower) sequence belongs to a new (known) editing site. The names of the sites are given on the right of each sequence with the average editing extent found in the wild-type in between parentheses. Identical sequences are highlighted by red squares. Upon visual inspection, gaps were introduced in order to increase the similarity between <i>cis</i> elements.</p

Statistics of number of reads/editing site for the 30 Illumina libraries analyzed in this study.

<p>s18 and a13 were obtained with the same cDNA.</p

Number (percentage) of <i>RIP</i>-dependent (ΔEE≥0.1) and <i>RIP</i>-independent (ΔEE<0.1) mitochondrial sites in the <i>RIP</i> mutant and <i>RIP</i>-silenced plants.

<p>619 (586) sites were analyzed in the mutant (silenced) plants.</p

Number (percentage) of <i>RIP</i>-dependent (ΔEE≥0.1) and <i>RIP</i>-independent (ΔEE<0.1) plastid sites in the <i>RIP</i> mutant and <i>RIP</i>-silenced plants.

<p>37 (36) sites were analyzed in the mutant (silenced) plants.</p

Overlap of genes assessed in the three tissues overall and in the CCT-AB gene list.

<p>Each compartment of the Venn diagram contains the tissue combination on top, number of genes overall in the middle, and number of genes from the CCT-AB gene list on bottom. CCT-AB overlap numbers marked by an “*” indicate significantly more overlap than expected by chance (permutation tests, p<1e-5). In the overall analysis the vast majority of genes (82%) were assayed in all three tissues. While this percent is much smaller for the CCT-AB candidate gene list (∼7%), this is still more of an overlap than expected by chance. The much higher degree of overlap of CCT-AB genes than expected suggests some CREs act in multiple tissues. Additionally, there are also many single tissue CCT-AB genes, which points towards the many <i>cis</i> elements that appear to function in tissue specific patterns.</p

SupplementalDataset2

Supplemental Dataset 2: Gene level counts for the three experimental tissues for each individual maize x teosinte comparison. Data includes depth at segregating sites, number of SNPs, log2 maize:teosinte ratios, binomial test p-values, and amount of sequence obtained