The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Arms race between anti-silencing and RdDM in noncoding regions of transposable elements.

Transposable elements (TEs) are among the most dynamic parts of genomes. Since TEs are potentially deleterious, eukaryotes silence them through epigenetic mechanisms such as repressive histone modifications and DNA methylation. We previously reported that Arabidopsis TEs, called VANDALs, counteract epigenetic silencing through a group of sequence-specific anti-silencing proteins, VANCs. VANC proteins bind to noncoding regions of specific VANDAL copies and induce loss of silent chromatin marks. The VANC-target regions form tandem repeats, which diverge rapidly. Sequence-specific anti-silencing allows these TEs to proliferate with minimum host damage. Here, we show that RNA-directed DNA methylation (RdDM) efficiently targets noncoding regions of VANDAL TEs to silence them de novo. Thus, escape from RdDM could be a primary event leading to the rapid evolution and diversification of sequence-specific anti-silencing systems. We propose that this selfish behavior of TEs paradoxically could make them diverse and less harmful to the host.

A divergent RWP-RK transcription factor determines mating type in heterothallic Closterium.

The Closterium peracerosum-strigosum-littorale complex (Closterium, Zygnematophyceae) has an isogamous mating system. Members of the Zygnematophyceae are the closest relatives to extant land plants and are distantly related to chlorophytic models, for which a genetic basis of mating type (MT) determination has been reported. We thus investigated MT determination in Closterium. We sequenced genomes representing the two MTs, mt+ and mt-, in Closterium and identified CpMinus1, a gene linked to the mt- phenotype. We analyzed its function using reverse genetics methods. CpMinus1 encodes a divergent RWP-RK domain-containing-like transcription factor and is specifically expressed during gamete differentiation. Introduction of CpMinus1 into an mt+ strain was sufficient to convert it to a phenotypically mt- strain, while CpMinus1-knockout mt- strains were phenotypically mt+. We propose that CpMinus1 is the major MT determinant that acts by evoking the mt- phenotype and suppressing the mt+ phenotype in heterothallic Closterium. CpMinus1 likely evolved independently in the Zygnematophyceae lineage, which lost an egg-sperm anisogamous mating system. mt- specific regions possibly constitute an MT locus flanked by common sequences that undergo some recombination.

MoG+: a database of genomic variations across three mouse subspecies for biomedical research.

Laboratory mouse strains have mosaic genomes derived from at least three major subspecies that are distributed in Eurasia. Here, we describe genomic variations in ten inbred strains: Mus musculus musculus-derived BLG2/Ms, NJL/Ms, CHD/Ms, SWN/Ms, and KJR/Ms; M. m. domesticus-derived PGN2/Ms and BFM/Ms; M. m. castaneus-derived HMI/Ms; and JF1/Ms and MSM/Ms, which were derived from a hybrid between M. m. musculus and M. m. castaneus. These strains were established by Prof. Moriwaki in the 1980s and are collectively named the "Mishima Battery". These strains show large phenotypic variations in body size and in many physiological traits. We resequenced the genomes of the Mishima Battery strains and performed a comparative genomic analysis with dbSNP data. More than 81 million nucleotide coordinates were identified as variant sites due to the large genetic distances among the mouse subspecies; 8,062,070 new SNP sites were detected in this study, and these may underlie the large phenotypic diversity observed in the Mishima Battery. The new information was collected in a reconstructed genome database, termed MoG+ that includes new application software and viewers. MoG+ intuitively visualizes nucleotide variants in genes and intergenic regions, and amino acid substitutions across the three mouse subspecies. We report statistical data from the resequencing and comparative genomic analyses and newly collected phenotype data of the Mishima Battery, and provide a brief description of the functions of MoG+, which provides a searchable and unique data resource of the numerous genomic variations across the three mouse subspecies. The data in MoG+ will be invaluable for research into phenotype-genotype links in diverse mouse strains.

Gene-body chromatin modification dynamics mediate epigenome differentiation in Arabidopsis.

Heterochromatin is marked by methylation of lysine 9 on histone H3 (H3K9me). A puzzling feature of H3K9me is that this modification localizes not only in promoters but also in internal regions (bodies) of silent transcription units. Despite its prevalence, the biological significance of gene-body H3K9me remains enigmatic. Here we show that H3K9me-associated removal of H3K4 monomethylation (H3K4me1) in gene bodies mediates transcriptional silencing. Mutations in an Arabidopsis H3K9 demethylase gene IBM1 induce ectopic H3K9me2 accumulation in gene bodies, with accompanying severe developmental defects. Through suppressor screening of the ibm1-induced developmental defects, we identified the LDL2 gene, which encodes a homolog of conserved H3K4 demethylases. The ldl2 mutation suppressed the developmental defects, without suppressing the ibm1-induced ectopic H3K9me2. The ectopic H3K9me2 mark directed removal of gene-body H3K4me1 and caused transcriptional repression in an LDL2-dependent manner. Furthermore, mutations of H3K9 methylases increased the level of H3K4me1 in the gene bodies of various transposable elements, and this H3K4me1 increase is a prerequisite for their transcriptional derepression. Our results uncover an unexpected role of gene-body H3K9me2/H3K4me1 dynamics as a mediator of heterochromatin silencing and epigenome differentiation.

The genomic landscape at a late stage of stickleback speciation: High genomic divergence interspersed by small localized regions of introgression.

Speciation is a continuous process and analysis of species pairs at different stages of divergence provides insight into how it unfolds. Previous genomic studies on young species pairs have revealed peaks of divergence and heterogeneous genomic differentiation. Yet less known is how localised peaks of differentiation progress to genome-wide divergence during the later stages of speciation in the presence of persistent gene flow. Spanning the speciation continuum, stickleback species pairs are ideal for investigating how genomic divergence builds up during speciation. However, attention has largely focused on young postglacial species pairs, with little knowledge of the genomic signatures of divergence and introgression in older stickleback systems. The Japanese stickleback species pair, composed of the Pacific Ocean three-spined stickleback (Gasterosteus aculeatus) and the Japan Sea stickleback (G. nipponicus), which co-occur in the Japanese islands, is at a late stage of speciation. Divergence likely started well before the end of the last glacial period and crosses between Japan Sea females and Pacific Ocean males result in hybrid male sterility. Here we use coalescent analyses and Approximate Bayesian Computation to show that the two species split approximately 0.68-1 million years ago but that they have continued to exchange genes at a low rate throughout divergence. Population genomic data revealed that, despite gene flow, a high level of genomic differentiation is maintained across the majority of the genome. However, we identified multiple, small regions of introgression, occurring mainly in areas of low recombination rate. Our results demonstrate that a high level of genome-wide divergence can establish in the face of persistent introgression and that gene flow can be localized to small genomic regions at the later stages of speciation with gene flow.

Centromere-targeted de novo integrations of an LTR retrotransposon of Arabidopsis lyrata.

The plant genome evolves with rapid proliferation of LTR-type retrotransposons, which is associated with their clustered accumulation in gene-poor regions, such as centromeres. Despite their major role for plant genome evolution, no mobile LTR element with targeted integration into gene-poor regions has been identified in plants. Here, we report such targeted integrations de novo. We and others have previously shown that an ATCOPIA93 family retrotransposon in Arabidopsis thaliana is mobilized when the DNA methylation machinery is compromised. Although ATCOPIA93 family elements are low copy number in the wild-type A. thaliana genome, high-copy-number related elements are found in the wild-type Arabidopsis lyrata genome, and they show centromere-specific localization. To understand the mechanisms for the clustered accumulation of the A. lyrata elements directly, we introduced one of them, named Tal1 (Transposon of Arabidopsis lyrata 1), into A. thaliana by transformation. The introduced Tal1 was retrotransposed in A. thaliana, and most of the retrotransposed copies were found in centromeric repeats of A. thaliana, suggesting targeted integration. The targeted integration is especially surprising because the centromeric repeat sequences differ considerably between A. lyrata and A. thaliana. Our results revealed unexpectedly dynamic controls for evolution of the transposon-rich heterochromatic regions.

Functional divergence of a heterochromatin-binding protein during stickleback speciation.

Intragenomic conflict, the conflict of interest between different genomic regions within an individual, is proposed as a mechanism driving both the rapid evolution of heterochromatin-related proteins and the establishment of intrinsic genomic incompatibility between species. Although molecular studies of laboratory model organisms have demonstrated the link between heterochromatin evolution and hybrid abnormalities, we know little about their link in natural systems. Previously, we showed that F1 hybrids between the Japan Sea stickleback and the Pacific Ocean stickleback show hybrid male sterility and found a region responsible for hybrid male sterility on the X chromosome, but did not identify any candidate genes. In this study, we first screened for genes rapidly evolving under positive selection during the speciation of Japanese sticklebacks to find genes possibly involved in intragenomic conflict. We found that the region responsible for hybrid male sterility contains a rapidly evolving gene encoding a heterochromatin-binding protein TRIM24B. We conducted biochemical experiments and showed that the binding affinity of TRIM24B to a heterochromatin mark found at centromeres and transposons, histone H4 lysine 20 trimethylation (H4K20me3), is reduced in the Japan Sea stickleback. In addition, mRNA expression levels of Trim24b were different between the Japan Sea and the Pacific Ocean testes. Further expression analysis of genes possibly in the TRIM24B-regulated pathway showed that some gypsy retrotransposons are overexpressed in the F1 hybrid testes. We, therefore, demonstrate that a heterochromatin-binding protein can evolve rapidly under positive selection and functionally diverge during stickleback speciation.

Time-Course Transcriptomics Analysis Reveals Key Responses of Submerged Deepwater Rice to Flooding.

Water submergence is an environmental factor that limits plant growth and survival. Deepwater rice (Oryza sativa) adapts to submergence by rapidly elongating its internodes and thereby maintaining its leaves above the water surface. We performed a comparative RNA sequencing transcriptome analysis of the shoot base region, including basal nodes, internodes, and shoot apices of seedlings at two developmental stages from two varieties with contrasting deepwater growth responses. A transcriptomic comparison between deepwater rice cv C9285 and nondeepwater rice cv Taichung 65 revealed both similar and differential expression patterns between the two genotypes during submergence. The expression of genes related to gibberellin biosynthesis, trehalose biosynthesis, anaerobic fermentation, cell wall modification, and transcription factors that include ethylene-responsive factors was significantly different between the varieties. Interestingly, in both varieties, the jasmonic acid content at the shoot base decreased during submergence, while exogenous jasmonic acid inhibited submergence-induced internode elongation in cv C9285, suggesting that jasmonic acid plays a role in the submergence response of rice. Furthermore, a targeted de novo transcript assembly revealed transcripts that were specific to cv C9285, including submergence-induced biotic stress-related genes. Our multifaceted transcriptome approach using the rice shoot base region illustrates a differential response to submergence between deepwater and nondeepwater rice. Jasmonic acid metabolism appears to participate in the submergence-mediated internode elongation response of deepwater rice.

Effects of androgen and oestrogen on IGF pathways controlling phallus growth.

The development of the mammalian phallus involves hormone-dependent mesenchymal-epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

Germline mutation rates and the long-term phenotypic effects of mutation accumulation in wild-type laboratory mice and mutator mice.

The germline mutation rate is an important parameter that affects the amount of genetic variation and the rate of evolution. However, neither the rate of germline mutations in laboratory mice nor the biological significance of the mutation rate in mammalian populations is clear. Here we studied genome-wide mutation rates and the long-term effects of mutation accumulation on phenotype in more than 20 generations of wild-type C57BL/6 mice and mutator mice, which have high DNA replication error rates. We estimated the base-substitution mutation rate to be 5.4 × 10(-9) (95% confidence interval = 4.6 × 10(-9)-6.5 × 10(-9)) per nucleotide per generation in C57BL/6 laboratory mice, about half the rate reported in humans. The mutation rate in mutator mice was 17 times that in wild-type mice. Abnormal phenotypes were 4.1-fold more frequent in the mutator lines than in the wild-type lines. After several generations, the mutator mice reproduced at substantially lower rates than the controls, exhibiting low pregnancy rates, lower survival rates, and smaller litter sizes, and many of the breeding lines died out. These results provide fundamental information about mouse genetics and reveal the impact of germline mutation rates on phenotypes in a mammalian population.

Hormone-responsive genes in the SHH and WNT/ß-catenin signaling pathways influence urethral closure and phallus growth.

Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during phallus development to cause hypospadias or a masculinized clitoris. We treated developing male tammar wallabies with estrogen and females with androgen from day 20-40 postpartum (pp) during the androgen imprinting window of sensitivity. Estrogen inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment in females did not promote phallus elongation but initiated urethral closure. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two signaling pathways, sonic hedgehog (SHH) and wingless-type MMTV integration site family (WNT)/ß-catenin. SHH mRNA and ß-catenin were predominantly expressed in the urethral epithelium in the tammar phallus, as in eutherian mammals. Estrogen treatment and castration of males induced an upregulation of SHH, while androgen treatment downregulated SHH. These effects appear to be direct since we detected putative estrogen receptor α (ERα) and androgen receptor (AR) binding sites near SHH. WNT5A, like SHH, was downregulated by androgen, while WNT4 was upregulated in female phalluses after androgen treatment. After estrogen treatment, WIF1 and WNT7A were both downregulated in male phalluses. After castration, WNT9A was upregulated. These results suggest that SHH and WNT pathways are regulated by both estrogen and androgen to direct the proliferation and elongation of the phallus during differentiation. Their response to exogenous hormones makes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias.

A map of rice genome variation reveals the origin of cultivated rice.

Crop domestications are long-term selection experiments that have greatly advanced human civilization. The domestication of cultivated rice (Oryza sativa L.) ranks as one of the most important developments in history. However, its origins and domestication processes are controversial and have long been debated. Here we generate genome sequences from 446 geographically diverse accessions of the wild rice species Oryza rufipogon, the immediate ancestral progenitor of cultivated rice, and from 1,083 cultivated indica and japonica varieties to construct a comprehensive map of rice genome variation. In the search for signatures of selection, we identify 55 selective sweeps that have occurred during domestication. In-depth analyses of the domestication sweeps and genome-wide patterns reveal that Oryza sativa japonica rice was first domesticated from a specific population of O. rufipogon around the middle area of the Pearl River in southern China, and that Oryza sativa indica rice was subsequently developed from crosses between japonica rice and local wild rice as the initial cultivars spread into South East and South Asia. The domestication-associated traits are analysed through high-resolution genetic mapping. This study provides an important resource for rice breeding and an effective genomics approach for crop domestication research.

Genome-wide negative feedback drives transgenerational DNA methylation dynamics in Arabidopsis.

Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3' regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome.

A Genome Scan for Genes Underlying Microgeographic-Scale Local Adaptation in a Wild Arabidopsis Species.

Adaptive divergence at the microgeographic scale has been generally disregarded because high gene flow is expected to disrupt local adaptation. Yet, growing number of studies reporting adaptive divergence at a small spatial scale highlight the importance of this process in evolutionary biology. To investigate the genetic basis of microgeographic local adaptation, we conducted a genome-wide scan among sets of continuously distributed populations of Arabidopsis halleri subsp. gemmifera that show altitudinal phenotypic divergence despite gene flow. Genomic comparisons were independently conducted in two distinct mountains where similar highland ecotypes are observed, presumably as a result of convergent evolution. Here, we established a de novo reference genome and employed an individual-based resequencing for a total of 56 individuals. Among 527,225 reliable SNP loci, we focused on those showing a unidirectional allele frequency shift across altitudes. Statistical tests on the screened genes showed that our microgeographic population genomic approach successfully retrieve genes with functional annotations that are in line with the known phenotypic and environmental differences between altitudes. Furthermore, comparison between the two distinct mountains enabled us to screen out those genes that are neutral or adaptive only in either mountain, and identify the genes involved in the convergent evolution. Our study demonstrates that the genomic comparison among a set of genetically connected populations, instead of the commonly-performed comparison between two isolated populations, can also offer an effective screening for the genetic basis of local adaptation.

MVH in piRNA processing and gene silencing of retrotransposons.

VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.

Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes.

BACKGROUND: Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. RESULTS: Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. CONCLUSIONS: We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.

Mobilization of a plant transposon by expression of the transposon-encoded anti-silencing factor.

Transposable elements (TEs) have a major impact on genome evolution, but they are potentially deleterious, and most of them are silenced by epigenetic mechanisms, such as DNA methylation. Here, we report the characterization of a TE encoding an activity to counteract epigenetic silencing by the host. In Arabidopsis thaliana, we identified a mobile copy of the Mutator-like element (MULE) with degenerated terminal inverted repeats (TIRs). This TE, named Hiun (Hi), is silent in wild-type plants, but it transposes when DNA methylation is abolished. When a Hi transgene was introduced into the wild-type background, it induced excision of the endogenous Hi copy, suggesting that Hi is the autonomously mobile copy. In addition, the transgene induced loss of DNA methylation and transcriptional activation of the endogenous Hi. Most importantly, the trans-activation of Hi depends on a Hi-encoded protein different from the conserved transposase. Proteins related to this anti-silencing factor, which we named VANC, are widespread in the non-TIR MULEs and may have contributed to the recent success of these TEs in natural Arabidopsis populations.

A biochemical landscape of A-to-I RNA editing in the human brain transcriptome.

Inosine is an abundant RNA modification in the human transcriptome and is essential for many biological processes in modulating gene expression at the post-transcriptional level. Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosines to inosines (A-to-I editing) in double-stranded regions. We previously established a biochemical method called "inosine chemical erasing" (ICE) to directly identify inosines on RNA strands with high reliability. Here, we have applied the ICE method combined with deep sequencing (ICE-seq) to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome of human adult brain. Taken together with the sites identified by the conventional ICE method, we mapped 19,791 novel sites and newly found 1258 edited mRNAs, including 66 novel sites in coding regions, 41 of which cause altered amino acid assignment. ICE-seq detected novel editing sites in various repeat elements as well as in short hairpins. Gene ontology analysis revealed that these edited mRNAs are associated with transcription, energy metabolism, and neurological disorders, providing new insights into various aspects of human brain functions.