Figure 1.
Structure model of RNA editing PPR proteins and their alignment to the RNA editing target sequence.
The RNA editing PPR proteins are extended at their C-termini by E and often also by DYW domains. Different from P-type PPR proteins, the RNA editing PPR proteins contain alternating P-L-S type elements. The positions of the amino acid identities at positions 6 and 1′ are not given in the structurally correct position. These two amino acid positions have here been correlated to nucleotide identities (Figure S1). Dashed lines indicate their presumed connection to target nucleotide identities. Position 1′ is the first amino acid of the respective C-terminally adjacent repeat. For element S2 this position corresponds to amino acid 33 of this repeat while the E domain begins by convention only after amino acid 36. To illustrate this unclear assignment we placed position 1′ for the S2 element between the S2 and E domains. Question marks indicate the connections to the L, S2 and L2 domains investigated here for correlations with the opposite nucleotides. The nucleotide sequence is arbitrary and is spelled out solely to indicate the specific order of nucleotides here.
Figure 2.
Amino acids in RNA editing PPR protein S2 motifs correlate with target nucleotides.
(A) Sequence logos were constructed for each of the four nucleotides facing the respective S2 domains in the predicted PPR-RNA interaction at position –4 relative to the edited C (Figure 1). Coincidences between nucleotide and amino acid identities are seen for position 1′ (also labelled as amino acid 33). No coinciding amino acid preference is seen with the C nucleotide. (B) The amino acid identity at position 1′ shows the most prominent correlation between D (aspartic acid) and nucleotide G, N (asparagine) and A, T (threonine) and U. In the bar diagram, percentages of nucleotide identities coinciding with the respective amino acid are indicated. Nucleotide percentages are normalized by calculations with taking the A, C, G and U percentage into account as detailed in the methods section. Sequence logos were derived with the web-based software at weblogo.berkeley.edu [25].
Figure 3.
Amino acids at position 6 in RNA editing PPR protein L motifs correlate with nucleotide identities.
(A) Sequence logos opposite each of the four nucleotides show the amino acid identities in L domains of predicted PPR-RNA interactions at position 6. Amino acid V (valine) is prominent at all nucleotide identities and thus possibly represents non-discriminatory spacer elements. (B) Correlations between amino acid identities at position 6 are most prominent for amino acid P and to a lower extent also for L, I, T and M with nucleotide U and amino acid T (threonine) with A or G. Position 1′ shows no discernible correlation when amino acids I, L, P, T or M are present at position 6. When these amino acid identities are excluded (ex.), a weak correlation can be seen with amino acid N to nucleotide identity A or U.
Figure 4.
The L2 motifs in RNA editing PPR proteins correlate with nucleotide identities.
(A) Correlations between amino acid and respective nucleotide identities in the target RNAs reveal preferential combinations with amino acid position 6 in the sequence logos. (B) Amino acid identities leucine and isoleucine at position 6 correlate with C, U or A (respectively G) in descending frequency, whereas threonine at this position is prevalent opposite nucleotide A. As in the L repeats, amino acid V occurs with any nucleotide. Preferences at position 1′ are not apparent in the sample size available.
Figure 5.
Positions 6 or 1′ in P and S motifs in RNA editing PPRs correlate with specific nucleotides.
Depicted are individual connections of positions 6 or 1′ in those instances, where the most prominent combinatory amino acid identity correlations between positions 6 and 1′ are excluded as indicated (ex.). In these instances single amino acid positions correlate with distinct nucleotide preferences in S and P elements, respectively. For the S elements non-random distributions are found at positions 6 and 1′, for the P elements only at position 1′. The most prominent combinatory amino acid – nucleotide identity correlations which are excluded here have been identified previously (8–10).
Figure 6.
Prediction of nucleotide target sequences for MEF11 and the novel RNA editing PPR protein MEF32.
(A) For MEF11 (At4g14850), the RNA editing sites ranked at positions 1, 2 and 3 out of 430 sites have been previously identified as target sites (shaded light blue) [17]. When we analysed all 20 top ranked editing sites in a MEF11 mutant, sites ranked 10, 14 and 17 turned out to be also targets of MEF11 (shaded yellow). (B) Target sequence predictions for the previously unassigned mitochondrial RNA editing factor encoded by At4g14170 are shown for the top ranked twenty sites. When we investigated these in a T-DNA mutant of the gene At4g14170, the three top ranked sites were identified as bona fide targets, at these nucleotides editing is absent in the mutant. This locus has been accordingly renamed to indicate that it codes for the novel RNA editing protein MEF32. The respective top parts in panels A and B show the PPR motifs considered (shaded light green; including the L2 and S2 elements) and their alignment to nucleotide positions which are counted 3′ to 5′ from the edited C (from right to left, −4 to −17 and −4 to −13, respectively). Amino acid identities at positions 6 and 1′ are given (shaded blue) and the respective scores are shown. In the box below, the locations of the top twenty ranked sites are indicated and the assigned specificity factor is given for experimentally confirmed targets, here MEF11 or MEF32. For each repeat the score at each site is given (shaded ocre with the color intensity reflecting the score) and the p-value of FIMO progaram as shown in the far right column is used for the ranking.
Figure 7.
Inclusion of L, L2 and S2 repeats generally improves the prediction accuracy of RNA editing targets.
Although the bona fide target sites are listed in the top ranks even without including the L, L2 and S2 repeats, their consideration mostly improves the prediction accuracy if only slightly. This suggests that these repeats also connect to target RNA sequences. Shown here are only the data for Arabidopsis. For Physcomitrella mitochondria, prediction ranks target sites always at the top, but then there are only very few editing sites in this moss (Figure S3). (A) Prediction of the target sites for the known chloroplast editing factors finds the identified targets within the top ranks out of the 34 RNA editing sites in chloroplasts of Arabidopsis. Prediction from only the P and S repeats (□) is usually sufficient, but inclusion of the L, L2 and S2 elements (•) often improves the ranking. (B) Analogous improvements of the predictions are seen within the 430 editing sites considered for mitochondrial PPR proteins. In a few instances the predicted PPR-RNA interaction drops in rank when the L, L2 and S2 elements are included (e.g. the targets of MEF18 and MEF19; further details are given in Figure S3). The nad6-95 target site of MEF8 (asterisk) cannot be ranked since the p-value is >1 in the FIMO program evaluation.