DOI: 10.5061/dryad.mcvdnck4z
title: Massive RNA editing in ascetosporean mitochondria

description: Adenosine-to-inosine(A-to-I) substitution type of RNA editing is mediated
 by adenosine deaminase acting on RNA (ADAR) and is involved in various
 essential cellular functions. As ADAR functions in the metazoan nucleus,
 it may have evolved from adenosine deaminase acting on tRNA in the
 metazoan ancestor. Although ADAR has not been detected in fungi and
 early-branching opisthokonts, it has not been explored in other eukaryotic
 lineages. Here, we detected and analyzed ADAR from two novel rhizarian
 amoebae and determined that other protists also possess ADAR. This finding
 indicates that ADAR may have originated from the last common ancestor of
 eukaryotes (LECA). Furthermore, the ADAR of the rhizarian amoeba is
 localized in its mitochondria and may be involved in the massive RNA
 editing in the mitochondria. Such complex RNA editing in the rhizarian
 amoeba may have a function to mask some lethal mutations in the
 mitochondrial genomes, which possibly contributes to the acceleration of
 diversification instead of the increase of future extinction risk.
description: <strong>Sequencing
 analyses</strong> Approximately 200 ml of mid-exponential phase
 cell cultures of Paradinida spp. FC901 and SRM-001 were centrifuged at
 2,400 × g for 5 min. The cell pellets were frozen and
 sent to the sequencing company (Azenta, Tokyo, Japan), and the library
 reconstruction and sequencing analyses were conducted using the default
 setting. The details of the analyses and the sequence outputs are
 summarized in Table S4. The raw fastq data of DNA-seq was divided into
 100 subsets using SeqKit,
 and three subsets of each species were subjected to the contig assembly
 using SPAdes 3.13 with
 default settings. From each assembly data, a single possible mitochondrial
 genomic fragment was detected by BLASTN using the mitochondrial genome
 sequence of Ophirina
 amphinema (GenBank
 accession number: LC369600.1) as the query sequence. The detected
 sequences were identical among the three subset analyses of each species,
 while the starting position of each sequence differed. By comparing these
 sequences, a circular mitochondrial genome of FC901 and SRM-001 was
 reconstructed. The same assembly analyses were also conducted using the
 RNA-seq data. The obtained mitochondrial sequences, which were
 reconstructed from RNA-seq data, were subjected to annotation using
 MFannot (https://megasun.bch.umontreal.ca/apps/mfannot/) and compared with
 those assembled from DNA-seq data using Mesquite 3.10. For analysing the transcriptome data, three
 RNA-seq datasets of Paradinida sp. FC901 was combined into a single dataset.
 The fastq data of each species was subjected to contig assembly using
 SPAdes 3.13 with the
 ‘--rna’ option. From the reconstructed contigs, their ADAR and ADAT
 sequences were searched by TBLASTN using the ADAR sequence of
 Symbiodinium
 microadriaticum (OLQ07757;
 E-value cut-off was set to 10−10). We also searched publicly available
 sequencing data (Table S3) for ADAR and ADAT sequences of other protists
 using the same approach. The detected sequences (e.g., ADAR and ADAT
 of Phaeodactylum
 tricornutum) were also
 used as the query in further searches for identifying more ADARs and
 ADATs. The obtained sequences were aligned with the metazoan ADARs and
 ADATs and then subjected to automated alignment using MAFFT v7.471 with
 the ‘L-INS-’ option. The
 aligned sequences were masked for the phylogenetic analysis using trimAl
 v1.4 with the ‘strict’ option. This initial dataset contained all the
 detected sequences, including the partial short and highly divergent
 sequences, and only 94 positions were included in the phylogenetic
 analysis. The tree topology and branch lengths were inferred using the
 maximum-likelihood (ML) methods using IQ-TREE 2.2.0 with the LG+F+I+G4 model. The robustness of the
 ML phylogenetic tree was evaluated using a non-parametric ML bootstrap
 analysis with the LG+F+I+G4 model (100 replicates). We also conducted
 Bayesian phylogenetic analysis with the CAT + GTR model using PhyloBayes
 MPI v. 1.8a. The analysis
 included two Markov chain Monte Carlo runs of 100,000 cycles with a
 ‘burn-in’ of 25,000 cycles. The consensus tree with branch lengths and
 Bayesian posterior probabilities was calculated from the remaining trees.
 Based on these findings, we revised the main dataset, excluding 14 partial
 and divergent ADAR sequences from an initial alignment. The main dataset
 was prepared using the same method used for the initial dataset and
 comprised 209 positions. The same methods were used to infer the
 phylogenetic tree and statistical support. Of the newly detected ADAR
 sequences, 12 sequences were retained in the main dataset, as well as the
 ADAR of Paradinida sp. FC901 were subjected to motif identification by
 HMMER 3.3 (http://hmmer.org) against the Pfam
 database. The 18S rRNA gene sequences of Paradinida sp.
 FC901 and SRM-001 were determined using the DNA that was extracted with
 Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from 20 ml of
 culture. The primers used were Euk1A and EukB. The sequences were added to the alignment that
 was created based on the method proposed by Ward et al. and aligned using MAFFT v7.471 with the default
 settings. The ML tree with the non-parametric bootstrap analyses of 1,000
 replicates and the Bayesian tree were reconstructed using the same methods
 described earlier.
description: # Massive RNA editing in ascetosporean mitochondria Dataset DOI:
 [https://doi.org/10.5061/dryad.mcvdnck4z](https://doi.org/10.5061/dryad.mcvdnck4z) ## Description of the data and file structure The dataset for molecular phylogenetic analysis used in the paper "*Massive RNA editing in ascetosporean mitochondria*." ### Files and variables #### File: dtst1\_for\_18S **Description:** The 18S rRNA gene sequences of Paradinida sp. FC901 and SRM-001 #### File: ADAR-ADAT\_allEuk\_4\_FC901-SRM\_strict.trimal.fst **Description:**  Main dataset for molecular phylogenetic analysis of ADAR-ADAT ## Code/software It can be viewed with a text editor. ## Access information Other publicly accessible locations of the data: * NCBI