Detection and Analysis of C-to-U RNA Editing in Rice Mitochondria-Encoded ORFs

Abstract

Cytidine to uridine (C-to-U) RNA editing is an important type of substitutional RNA modification and is almost omnipresent in plant chloroplasts and mitochondria. In rice mitochondria, 491 C-to-U editing sites have been identified previously, and case studies have elucidated the function of several C-to-U editing sites in rice, but the functional consequence of most C-to-U alterations needs to be investigated further. Here, by means of Sanger sequencing and publicly available RNA-seq data, we identified a total of 569 C-to-U editing sites in rice mitochondria-encoded open reading frames (ORFs), 85.41% of these editing sites were observed on the first or the second base of a codon, resulting in the alteration of encoded amino acid. Moreover, we found some novel editing sites and several inaccurately annotated sites which may be functionally important, based on the highly conserved amino acids encoded by these edited codons. Finally, we annotated all 569 C-to-U RNA editing sites in their biological context. More precise information about C-to-U editing sites in rice mitochondria-encoded ORFs will facilitate our investigation on the function of C-to-U editing events in rice and also provide a valid benchmark from rice for the analysis of mitochondria C-to-U editing in other plant species.

Keywords: C-to-U RNA editing; edited codon; mitochondria-encoded ORFs; rice.

Figure 1

Confirming and correcting the annotations in REDIdb. (A) Annotation of RNA editing sites in atp1 transcript in REDIdb. Red box indicates the incorrectly annotated RNA editing site at atp1-1291. (B) Sequence chromatogram of PCR-amplified atp1 cDNA. Arrow indicates the editing site atp1-1292. Amino acids before and after editing are shown below the codons indicated by black lines. (C) Alignment of amino acid sequences of rice Atp1 and its orthologs from other plants species around the amino acid Atp1-431. The sequences were retrieved from REDIdb and aligned with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) [34]. Arrow indicates the amino acid for rice Atp1-431 encoded by the edited codon. Note that amino acid sequences from REDIdb are translated from edited transcripts. (D) Annotations retrieved from REDIdb on the amino acid alteration at Nad7-149 in rice and its counterpart in other species. Red box indicates the alteration of amino acid 149 resulting from incorrectly annotated RNA editing site of rice nad7-446. (E) Sequence chromatogram of PCR-amplified nad7 cDNA. Arrow indicates the editing site nad7-445. Amino acids before and after editing are shown below the codons indicated by black line.

Figure 2

Principal statistics of C-to-U RNA editing sites in ORFs of rice mitochondria. (A) Distribution of editing sites at each codon position. (B) Number of nonsynonymous (nsy) and synonymous (syn) amino acid substitutions by RNA editing. (C) Number of codons with different number of editing sites. (D) Number of amino acid changes induced by C-to-U RNA editing. (E) Distribution of editing events in each codon.

Figure 3

Identification of novel RNA editing sites in rice mitochondria-encoded ORFs. (A) Sequence chromatogram of PCR-amplified ccmFN cDNA. Arrow indicates the novel editing sites ccmFN-151, ccmFN-263, ccmFN-356, and ccmFN-365. Amino acids before and after editing are shown below the codons indicated by black lines. (B) Alignment of amino acid sequences from rice CcmFN and its orthologs from other plant species. The sequences of Oryza sativa, Citrullus lanatus, Lotus japonicus, Cucurbita pepo, Oenothera berteroana, Arabidopsis thaliana, Silene latifolia, Silene latifolia, Nelumbo nucifera and Millettia pinnata were retrieved from REDIdb and sequences of Nicotiana tabacum (BAD83458.2), Beta vulgaris (NP_063987.2) and Solanum tuberosum (QEQ76363.1) were downloaded from NCBI. All the sequences were aligned with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) [34]. Arrows indicate the amino acids for rice CcmFN-51, CcmFN-88, CcmFN-119, and CcmFN-122 encoded by edited codons. Note that amino acid sequences from NCBI are translated from unedited transcripts.