Characteristics and prediction of RNA editing sites in transcripts of the Moss Takakia lepidozioides chloroplast

Abstract

RNA editing in land plant organelles is a process primarily involving the conversion of cytidine to uridine in pre-mRNAs. The process is required for gene expression in plant organelles, because this conversion alters the encoded amino acid residues and improves the sequence identity to homologous proteins. A recent study uncovered that proteins encoded in the nuclear genome are essential for editing site recognition in chloroplasts; the mechanisms by which this recognition occurs remain unclear. To understand these mechanisms, we determined the genomic and cDNA sequences of moss Takakia lepidozioides chloroplast genes, then computationally analyzed the sequences within -30 to +10 nucleotides of RNA editing sites (neighbor sequences) likely to be recognized by trans-factors. As the T. lepidozioides chloroplast has many RNA editing sites, the analysis of these sequences provides a unique opportunity to perform statistical analyses of chloroplast RNA editing sites. We divided the 302 obtained neighbor sequences into eight groups based on sequence similarity to identify group-specific patterns. The patterns were then applied to predict novel RNA editing sites in T. lepidozioides transcripts; approximately 60% of these predicted sites are true editing sites. The success of this prediction algorithm suggests that the obtained patterns are indicative of key sites recognized by trans-factors around editing sites of T. lepidozioides chloroplast genes.

Figure 1

Gene arrangement of the region spanning rps4 to rps11 in the T. lepidozioides plastid genome. The filled boxes indicate the translated regions for each gene, and the open boxes represent introns. The genes over the thick horizontal bar were transcribed from left to right, whereas those below the bar were transcribed in the opposite direction. Fragments 1–19, shown under the bar, were amplified by PCR. Sequences for the dotted fragments under the bar were taken from Genbank/EMBL/DDBJ.

Figure 2

Classification of neighbor sequences for RNA editing sites. We drew the classification dendrogram for 41-nt sequences based on sequence identity. The sequences were grouped at the second or third branching points. Each group, named from G-1 to G-8, was depicted by a background color. Each sequence was named based on the sequenced region and number of nucleotides. Note that the figure does not express the phylogenetic relation of the sequences.

Figure 3

(A) Singlet propensities of eight groups at each position for every nucleotide. The preference was color-coded from red to blue on a log₂ scale, with the exception of the RNA editing site (position 0). When the value was not statistically significant, the singlet value was set to 0 and colored as white. The number of sequences in each group is noted at the bottom of each diagram. (B) All 53 neighbor sequences in group G-2. On the basis of this alignment, we calculated the singlet propensities for G-2 in A. Strong tendencies for specific nucleotides, found in A, at positions −10, −8, −5, −2 and +2 as well as the RNA editing site are boxed. At positions −10 and +2, G is favored, whereas T is favored at −2. At −8, G is avoided, and at −5, C is avoided. C at 0 is edited to U.

Figure 4

The magnitude of doublet propensities for the eight groups in a log₂ scale at each pair of positions. At each position pair, there are 16 possible combinations of nucleotides. Each combination has a value for doublet propensity. When the value was not statistically significant, it was set to 0. A square sum of positive doublet propensity at each position was color-coded from white to red in the upper triangular portion of the matrix. A square sum of negative doublet propensity at each position was color-coded from white to blue in the lower triangular portion of the matrix. The number of statistically significant values for doublet propensity is noted at the top of each matrix. The maximum number of doublet propensities should be 12 480 (=40 nt × 39 nt/2 × 16 pairs).

Figure 5

Prediction score histogram for group G-2. The horizontal axis is the score in a bin of scale 10, and the vertical axis is the count of the prediction score in the bin. A red bar indicates the score for the edited C, and a bar in cyan indicates the score for a non-edited C. The prediction was performed for all transcripts (coding regions) between clpP and rpoA and between rps4 and psbE in the T. lepidozioides chloroplast genome. A bar for scores >70 has two numbers separated by a slash on the top. The number on the left is a count of the edited C, and the number on the right is the whole count in the bar.