A single mutation in the ACTR8 gene associated with lineage-specific expression in primates.

BACKGROUND: Alternative splicing (AS) generates various transcripts from a single gene and thus plays a significant role in transcriptomic diversity and proteomic complexity. Alu elements are primate-specific transposable elements (TEs) and can provide a donor or acceptor site for AS. In a study on TE-mediated AS, we recently identified a novel AluSz6-exonized ACTR8 transcript of the crab-eating monkey (Macaca fascicularis). In the present study, we sought to determine the molecular mechanism of AluSz6 exonization of the ACTR8 gene and investigate its evolutionary and functional consequences in the crab-eating monkey. RESULTS: We performed RT-PCR and genomic PCR to analyze AluSz6 exonization in the ACTR8 gene and the expression of the AluSz6-exonized transcript in nine primate samples, including prosimians, New world monkeys, Old world monkeys, and hominoids. AluSz6 integration was estimated to have occurred before the divergence of simians and prosimians. The Alu-exonized transcript obtained by AS was lineage-specific and expressed only in Old world monkeys and apes, and humans. This lineage-specific expression was caused by a single G duplication in AluSz6, which provides a new canonical 5' splicing site. We further identified other alternative transcripts that were unaffected by the AluSz6 insertion. Finally, we observed that the alternative transcripts were transcribed into new isoforms with C-terminus deletion, and in silico analysis showed that these isoforms do not have a destructive function. CONCLUSIONS: The single G duplication in the TE sequence is the source of TE exonization and AS, and this mutation may suffer a different fate of ACTR8 gene expression during primate evolution.

Cooperative evolution of two different TEs results in lineage-specific novel transcripts in the BLOC1S2 gene.

BACKGROUND: The BLOC1S2 gene encodes the multifunctional protein BLOS2, a shared subunit of two lysosomal trafficking complexes: i) biogenesis of lysosome-related organelles complex-1 and i) BLOC-1-related complex. In our previous study, we identified an intriguing unreported transcript of the BLOC1S2 gene that has a novel exon derived from two transposable elements (TEs), MIR and AluSp. To investigate the evolutionary footprint and molecular mechanism of action of this transcript, we performed PCR and RT-PCR experiments and sequencing analyses using genomic DNA and RNA samples from humans and various non-human primates. RESULTS: The results showed that the MIR element had integrated into the genome of our common ancestor, specifically in the BLOC1S2 gene region, before the radiation of all primate lineages and that the AluSp element had integrated into the genome of our common ancestor, fortunately in the middle of the MIR sequences, after the divergence of Old World monkeys and New World monkeys. The combined MIR and AluSp sequences provide a 3' splice site (AG) and 5' splice site (GT), respectively, and generate the Old World monkey-specific transcripts. Moreover, branch point sequences for the intron removal process are provided by the MIR and AluSp combination. CONCLUSIONS: We show for the first time that sequential integration into the same location and sequence divergence events of two different TEs generated lineage-specific transcripts through sequence collaboration during primate evolution.

Successful application of human-based methyl capture sequencing for methylome analysis in non-human primate models.

BACKGROUND: The characterization of genomic or epigenomic variation in human and animal models could provide important insight into pathophysiological mechanisms of various diseases, and lead to new developments in disease diagnosis and clinical intervention. The African green monkey (AGM; Chlorocebus aethiops) and cynomolgus monkey (CM; Macaca fascicularis) have long been considered important animal models in biomedical research. However, non-human primate-specific methods applicable to epigenomic analyses in AGM and CM are lacking. The recent development of methyl-capture sequencing (MC-seq) has an unprecedented advantage of cost-effectiveness, and further allows for extending the methylome coverage compared to conventional sequencing approaches. RESULTS: Here, we used a human probe-designed MC-seq method to assay DNA methylation in DNA obtained from 13 CM and three AGM blood samples. To effectively adapt the human probe-designed target region for methylome analysis in non-human primates, we redefined the target regions, focusing on regulatory regions and intragenic regions with consideration of interspecific sequence homology and promoter region variation. Methyl-capture efficiency was controlled by the sequence identity between the captured probes based on the human reference genome and the AGM and CM genome sequences, respectively. Using reasonable guidelines, 56 and 62% of the human-based capture probes could be effectively mapped for DNA methylome profiling in the AGM and CM genome, respectively, according to numeric global statistics. In particular, our method could cover up to 89 and 87% of the regulatory regions of the AGM and CM genome, respectively. CONCLUSIONS: Use of human-based MC-seq methods provides an attractive, cost-effective approach for the methylome profiling of non-human primates at the single-base resolution level.

Quantitative expression analysis of APP pathway and tau phosphorylation-related genes in the ICV STZ-induced non-human primate model of sporadic Alzheimer's disease.

The accumulation and aggregation of misfolded proteins in the brain, such as amyloid-ß (Aß) and hyperphosphorylated tau, is a neuropathological hallmark of Alzheimer's disease (AD). Previously, we developed and validated a novel non-human primate model for sporadic AD (sAD) research using intracerebroventricular administration of streptozotocin (icv STZ). To date, no characterization of AD-related genes in different brain regions has been performed. Therefore, in the current study, the expression of seven amyloid precursor protein (APP) pathway-related and five tau phosphorylation-related genes was investigated by quantitative real-time PCR experiments, using two matched-pair brain samples from control and icv STZ-treated cynomolgus monkeys. The genes showed similar expression patterns within the control and icv STZ-treated groups; however, marked differences in gene expression patterns were observed between the control and icv STZ-treated groups. Remarkably, other than ß-secretase (BACE1) and cyclin-dependent kinase 5 (CDK5), all the genes tested showed similar expression patterns in AD models compared to controls, with increased levels in the precuneus and occipital cortex. However, significant changes in gene expression patterns were not detected in the frontal cortex, hippocampus, or posterior cingulate. Based on these results, we conclude that APP may be cleaved via the general metabolic mechanisms of increased α- and γ-secretase levels, and that hyperphosphorylation of tau could be mediated by elevated levels of tau protein kinase, specifically in the precuneus and occipital cortex.

Transcriptome analysis of cynomolgus macaques throughout their lifespan reveals age-related immune patterns.

Despite the different perspectives by diverse research sectors spanning several decades, aging research remains uncharted territory for human beings. Therefore, we investigated the transcriptomic characteristics of eight male healthy cynomolgus macaques, and the annual sampling was designed with two individuals in four age groups. As a laboratory animal, the macaques were meticulously shielded from all environmental factors except aging. The results showed recent findings of certain immune response and the age-associated network of primate immunity. Three important aging patterns were identified and each gene clusters represented a different immune response. The increased expression pattern was predominantly associated with innate immune cells, such as Neutrophils and NK cells, causing chronic inflammation with aging whereas the other two decreased patterns were associated with adaptive immunity, especially "B cell activation" affecting antibody diversity of aging. Furthermore, the hub gene network of the patterns reflected transcriptomic age and correlated with human illness status, aiding in future human disease prediction. Our macaque transcriptome profiling results offer systematic insights into the age-related immunological features of primates.

Utilization of nicking properties of CRISPR-Cas12a effector for genome editing.

The CRISPR-Cas nickase system for genome editing has attracted considerable attention owing to its safety, efficiency, and versatility. Although alternative effectors to Cas9 have the potential to expand the scope of genome editing, their application has not been optimized. Herein, we used an enhanced CRISPR-Cas12a nickase system to induce mutations by targeting genes in a human-derived cell line. The optimized CRISPR-Cas12a nickase system effectively introduced mutations into target genes under a specific directionality and distance between nickases. In particular, the single-mode Cas12a nickase system can induce the target-specific mutations with less DNA double-strand breaks. By inducing mutations in the Thymine-rich target genes in single- or dual-mode, Cas12a nickase compensates the limitations of Cas9 nickase and is expected to contribute to the development of future genome editing technologies.

Identification of microRNAs Derived from Transposable Elements in the Macaca mulatta (Rhesus Monkey) Genome.

Transposable elements (TEs) are mobile DNA entities that can move within the host genome. Over long periods of evolutionary time, TEs are typically silenced via the accumulation of mutations in the genome, ultimately resulting in their immobilization. However, they still play an important role in the host genome by acting as regulatory elements. They influence host transcription in various ways, one of which as the origin of the generation of microRNAs (miRNAs), which are so-called miRNAs derived from TEs (MDTEs). miRNAs are small non-coding RNAs that are involved in many biological processes by regulating gene expression at the post-transcriptional level. Here, we identified MDTEs in the Macaca mulatta (rhesus monkey) genome, which is phylogenetically close species to humans, based on the genome coordinates of miRNAs and TEs. The expression of 5 out of 17 MDTEs that were exclusively registered in M. mulatta from the miRBase database (v22) was examined via quantitative polymerase chain reaction (qPCR). Moreover, Gene Ontology analysis was performed to examine the functional implications of the putative target genes of the five MDTEs.

Large-scale transcriptome sequencing and gene analyses in the crab-eating macaque (Macaca fascicularis) for biomedical research.

BACKGROUND: As a human replacement, the crab-eating macaque (Macaca fascicularis) is an invaluable non-human primate model for biomedical research, but the lack of genetic information on this primate has represented a significant obstacle for its broader use. RESULTS: Here, we sequenced the transcriptome of 16 tissues originated from two individuals of crab-eating macaque (male and female), and identified genes to resolve the main obstacles for understanding the biological response of the crab-eating macaque. From 4 million reads with 1.4 billion base sequences, 31,786 isotigs containing genes similar to those of humans, 12,672 novel isotigs, and 348,160 singletons were identified using the GS FLX sequencing method. Approximately 86% of human genes were represented among the genes sequenced in this study. Additionally, 175 tissue-specific transcripts were identified, 81 of which were experimentally validated. In total, 4,314 alternative splicing (AS) events were identified and analyzed. Intriguingly, 10.4% of AS events were associated with transposable element (TE) insertions. Finally, investigation of TE exonization events and evolutionary analysis were conducted, revealing interesting phenomena of human-specific amplified trends in TE exonization events. CONCLUSIONS: This report represents the first large-scale transcriptome sequencing and genetic analyses of M. fascicularis and could contribute to its utility for biomedical research and basic biology.

Intron Retention and TE Exonization Events in ZRANB2.

The Zinc finger, RAN-binding domain-containing protein 2 (ZRANB2), contains arginine/serine-rich (RS) domains that mediate its function in the regulation of alternative splicing. The ZRANB2 gene contains 2 LINE elements (L3b, Plat_L3) between the 9th and 10th exons. We identified the exonization event of a LINE element (Plat_L3). Using genomic PCR, RT-PCR amplification, and sequencing of primate DNA and RNA samples, we analyzed the evolutionary features of ZRANB2 transcripts. The results indicated that 2 of the LINE elements were integrated in human and all of the tested primate samples (hominoids: 3 species; Old World monkey: 8 species; New World monkey: 6 species; prosimian: 1 species). Human, rhesus monkey, crab-eating monkey, African-green monkey, and marmoset harbor the exon derived from LINE element (Plat_L3). RT-PCR amplification revealed the long transcripts and their differential expression patterns. Intriguingly, these long transcripts were abundantly expressed in Old World monkey lineages (rhesus, crab-eating, and African-green monkeys) and were expressed via intron retention (IR). Thus, the ZRANB2 gene produces 3 transcript variants in which the Cterminus varies by transposable elements (TEs) exonization and IR mechanisms. Therefore, ZRANB2 is valuable for investigating the evolutionary mechanisms of TE exonization and IR during primate evolution.

Generation of induced pluripotent stem cells (cmESF-iPS-C5) derived from cynomolgus monkey ear skin fibroblasts (cmESF).

Cynomolgus monkeys, due to their close anatomical, genetic and physiological similarity to humans, have been employed as a popular laboratory non-human primate model over rodents. Primate animal induced pluripotent stem cell (iPSC) have been used to aid on the investigation of autologous regenerative therapies. Here, we reprogrammed cynomolgus monkey ear skin fibroblasts (cmESFs) into iPSCs as a starting material for autologous based study. The resulting cmESF-iPSCs with canonical features of PSCs will advance the development of autologous transplantation.

Hsa-miR-422a Originated from Short Interspersed Nuclear Element Increases ARID5B Expression by Collaborating with NF-E2.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of target messenger RNA (mRNA) complementary to the 3' untranslated region (UTR) at the post-transcriptional level. Hsa-miR-422a, which is commonly known as miRNA derived from transposable element (MDTE), was derived from short interspersed nuclear element (SINE). Through expression analysis, hsa-miR-422a was found to be highly expressed in both the small intestine and liver of crab-eating monkey. AT-Rich Interaction Domain 5 B (ARID5B) was selected as the target gene of hsa-miR-422a, which has two binding sites in both the exon and 3'UTR of ARID5B. To identify the interaction between hsa-miR-422a and ARID5B, a dual luciferase assay was conducted in HepG2 cell line. The luciferase activity of cells treated with the hsa-miR-422a mimic was upregulated and inversely downregulated when both the hsa-miR-422a mimic and inhibitor were administered. Nuclear factor erythroid-2 (NF-E2) was selected as the core transcription factor (TF) via feed forward loop analysis. The luciferase expression was downregulated when both the hsa-miR-422a mimic and siRNA of NF-E2 were treated, compared to the treatment of the hsa-miR-422a mimic alone. The present study suggests that hsa-miR-422a derived from SINE could bind to the exon region as well as the 3'UTR of ARID5B. Additionally, hsa-miR-422a was found to share binding sites in ARID5Bwith several TFs, including NF-E2. The hsa-miR-422a might thus interact with TF to regulate the expression of ARID5B, as demonstrated experimentally. Altogether, hsa-miR-422a acts as a super enhancer miRNA of ARID5Bby collaborating with TF and NF-E2.

Generation and characterization of cynomolgus monkey kidney fibroblasts (cmKF)-derived induced pluripotent stem cells (cmKF-iPS-C5).

Cynomolgus monkeys, a non-human primate species, are genetically and physiologically similar to humans; hence, they have been employed as an ideal developmental and biomedical model. Non-human primate animals and their induced pluripotent stem cell (iPSC) derivatives have been used as a research tool to investigate autologous regenerative medicine. Here, we reprogrammed cynomolgus monkey kidney fibroblasts (cmKFs) as a control for animal iPSCs and to study autologous transplant. The resulting cmKF-iPSCs, which displayed canonical characteristics of PSCs, could be used as a unique model for autologous cell-based therapy.

Bioinformatic analysis of TE-spliced new exons within human, mouse and zebrafish genomes.

Recent studies indicate major roles for transposable elements (TEs) in alternative splicing. In this study, we conducted genome-wide alternative splicing analyses focusing on new internal exon birth derived from TEs in human, mouse, and zebrafish genomes. We identified two different exon sets, TE-spliced exons and non-TE-spliced exons. The proportion of TE-spliced exons was nearly twice as high as the proportion of non-TE-spliced exons in the coding sequence (CDS) region. Detailed analysis of various families of TEs in three different species of TE-spliced exons revealed a different pattern in zebrafish. In our analysis, we could identify the functional role of TE insertions in the vertebrate genome affecting mRNA splicing machinery. Their effects can be directly linked to the shift from constitutive to alternative splicing during primate evolution. Our results indicate that TEs have a significant effect on shaping new internal exons in human, mouse, and zebrafish transcriptomes.

The enhancer activity of long interspersed nuclear element derived microRNA 625 induced by NF-κB.

Transposable elements (TEs) are DNA sequences that cut or introduced into the genome, and they represent a massive portion of the human genome. TEs generate a considerable number of microRNAs (miRNAs) are derived from TEs (MDTEs). Numerous miRNAs are related to cancer, and hsa-miRNA-625 is a well-known oncomiR derived from long interspersed nuclear elements (LINEs). The relative expression of hsa-miRNA-625-5p differs in humans, chimpanzees, crab-eating monkeys, and mice, and four primers were designed against the 3'UTR of GATAD2B to analyze the different quantities of canonical binding sites and the location of miRNA binding sites. Luciferase assay was performed to score for the interaction between hsa-miRNA-625 and the 3'UTR of GATAD2B, while blocking NF-κB. In summary, the different numbers of canonical binding sites and the locations of miRNA binding sites affect gene expression, and NF-κB induces the enhancer activity of hsa-miRNA-625-5p by sharing the binding sites.

Sense-oriented AluYRa1 elements provide a lineage-specific transcription environment for polyadenylation.

Transposable elements cause alternative splicing (AS) in different ways, contributing to transcript diversification. Alternative polyadenylation (APA), one of the AS events, is related to the generation of mRNA isoforms in 70% of human genes. In this study, we tried to investigate AluYRa1s located at the terminal region of cynomolgus monkey genes, utilizing both computational analysis and molecular experimentation. We found that ten genes had AluYRa1 at their 3' end, and nine of these AluYRa1s were sense-oriented. Furthermore, in seven genes, AluYRa1s were expected to have a similar consensus sequence for polyadenylation cleavage. Additional computational analysis using the annotation files from the UCSC database showed that AluYRa1 was more involved in polyadenylation than in open reading frame exon splicing. To examine the extent of AluYRa1 involvement in polyadenylation, RNA-seq data from 30 normal cynomolgus monkeys were analyzed using TAPAS, a recently devised software that detects all the promising polyadenylation sites including APA sites. We observed that approximately 74% of possible polyadenylation sites in the analyzed genes were provided by sense-oriented AluYRa1. In conclusion, AluYRa1 is an Old-World monkey-specific TE, and its sense-oriented insertion at the 3'UTR region tends to provide a favorable environment for polyadenylation, diversifying gene transcripts.

Anti-Obesity Effect of Pine Needle Extract on High-Fat Diet-Induced Obese Mice.

BACKGROUND: Obesity due to an excessive intake of nutrient disturbs the hypothalamus-mediated energy metabolism subsequently develops metabolic disorders. In this study, we investigated the effect of pine needle extract (PNE) on the hypothalamic proopiomelanocortin (POMC) neurons involved in the regulation of energy balance via melanocortin system and fat tissue metabolism. METHODS: We performed electrophysiological and immunohistochemical analyses to determine the effect of PNE on POMC neurons. Mice were fed a normal or high-fat diet for 12 weeks, then received PNE for the last 2 weeks to measure the following physiological indices: Body weight, food intake, fat/lean mass, glucose metabolism, and plasma leptin levels. In addition, changes of thermogenic, lipolytic, and lipogenetic markers were evaluated in brown adipose tissue (BAT) and white adipose tissue (WAT) by western blotting, respectively. RESULTS: PNE increased hypothalamic POMC neuronal activity, and the effect was abolished by blockade of melanocortin 3/4 receptors (MC3/4Rs). PNE decreased body weight, fat mass, plasma leptin levels, and improved glucose metabolism after high-fat-induced obesity. However, PNE did not change the expression of thermogenic markers of the BAT in HFD fed groups, but decreased only the lipogenetic markers of WAT. This study suggests that PNE has a potent anti-obesity effect, inhibiting lipogenesis in WAT, even though HFD-induced leptin resistance-mediated disruption of POMC neuronal activity.

Full-length cDNA sequences from Rhesus monkey placenta tissue: analysis and utility for comparative mapping.

BACKGROUND: Rhesus monkeys (Macaca mulatta) are widely-used as experimental animals in biomedical research and are closely related to other laboratory macaques, such as cynomolgus monkeys (Macaca fascicularis), and to humans, sharing a last common ancestor from about 25 million years ago. Although rhesus monkeys have been studied extensively under field and laboratory conditions, research has been limited by the lack of genetic resources. The present study generated placenta full-length cDNA libraries, characterized the resulting expressed sequence tags, and described their utility for comparative mapping with human RefSeq mRNA transcripts. RESULTS: From rhesus monkey placenta full-length cDNA libraries, 2000 full-length cDNA sequences were determined and 1835 rhesus placenta cDNA sequences longer than 100 bp were collected. These sequences were annotated based on homology to human genes. Homology search against human RefSeq mRNAs revealed that our collection included the sequences of 1462 putative rhesus monkey genes. Moreover, we identified 207 genes containing exon alterations in the coding region and the untranslated region of rhesus monkey transcripts, despite the highly conserved structure of the coding regions. Approximately 10% (187) of all full-length cDNA sequences did not represent any public human RefSeq mRNAs. Intriguingly, two rhesus monkey specific exons derived from the transposable elements of AluYRa2 (SINE family) and MER11B (LTR family) were also identified. CONCLUSION: The 1835 rhesus monkey placenta full-length cDNA sequences described here could expand genomic resources and information of rhesus monkeys. This increased genomic information will greatly contribute to the development of evolutionary biology and biomedical research.

Four different ways of alternative transcripts generation mechanism in ADRA1A gene.

The ADRA1A (Alpha-1-adrenergic receptor) gene is one of the members of G protein-coupled receptor superfamily. Alternative splicing of this gene was known to generate four transcript variants which code four isoforms with various C-terminal regions. In this study, we conducted expression analysis and evolutionary characterization of alternative transcripts of the ADRA1A gene. In total, 10 alternative transcripts were identified using experimental approaches and in silico analysis. Among them, 6 alternative transcripts (T1, T2, T3, T4, T4-1, and T8) were validated by RT-PCR approaches and sequencing procedures. From the alternative splicing analysis, it could be assumed that there were three different alternative transcripts generation mechanisms and unknown mechanism. First one is the integration event of three different TEs (AluSc, L1MC5, and MIR3) as seen on the last exons of T3, T4, T4-1, T5, T6, and T7 transcripts. The second mechanism is a differential promoter usage on T8. The third one is a substitution event of the 3' splicing site during the primate evolution on T3. The last one is an unknown mechanism which was identified in the T4-1 transcript. Therefore, alternative transcripts of human ADRA1A gene occurred by four different ways, such as integration of TEs, differential promoter usage, substitution of splicing sites, and unknown mechanism.

Enhancer Function of MicroRNA-3681 Derived from Long Terminal Repeats Represses the Activity of Variable Number Tandem Repeats in the 3' UTR of SHISA7.

microRNAs (miRNAs) are non-coding RNA molecules involved in the regulation of gene expression. miRNAs inhibit gene expression by binding to the 3' untranslated region (UTR) of their target gene. miRNAs can originate from transposable elements (TEs), which comprise approximately half of the eukaryotic genome and one type of TE, called the long terminal repeat (LTR) is found in class of retrotransposons. Amongst the miRNAs derived from LTR, hsa-miR-3681 was chosen and analyzed using bioinformatics tools and experimental analysis. Studies on hsa-miR-3681 have been scarce and this study provides the relative expression analysis of hsa-miR-3681-5p from humans, chimpanzees, crab-eating monkeys, and mice. Luciferase assay for hsa-miR-3681-5p and its target gene SHISA7 supports our hypothesis that the number of miRNA binding sites affects target gene expression. Especially, the variable number tandem repeat (VNTR) and hsa-miR-3681-5p share the binding sites in the 3' UTR of SHISA7, which leads the enhancer function of hsa-miR-3681-5p to inhibit the activity of VNTR. In conclusion, hsa-miR-3681-5p acts as a super-enhancer and the enhancer function of hsa-miR-3681-5p acts as a repressor of VNTR activity in the 3' UTR of SHISA7.

Longitudinal profiling of the blood transcriptome in an African green monkey aging model.

African green monkeys (AGMs, Chlorocebus aethiops) are Old World monkeys which are used as experimental models in biomedical research. Recent technological advances in next generation sequencing are useful for unraveling the genetic mechanisms underlying senescence, aging, and age-related disease. To elucidate the normal aging mechanisms in older age, the blood transcriptomes of nine healthy, aged AGMs (15‒23 years old), were analyzed over two years. We identified 910‒1399 accumulated differentially expressed genes (DEGs) in each individual, which increased with age. Aging-related DEGs were sorted across the three time points. A major proportion of the aging-related DEGs belonged to gene ontology (GO) categories involved in translation and rRNA metabolic processes. Next, we sorted common aging-related DEGs across three time points over two years. Common aging-related DEGs belonged to GO categories involved in translation, cellular component biogenesis, rRNA metabolic processes, cellular component organization, biogenesis, and RNA metabolic processes. Furthermore, we identified 29 candidate aging genes that were upregulated across the time series analysis. These candidate aging genes were linked to protein synthesis. This study describes a changing gene expression pattern in AGMs during aging using longitudinal transcriptome sequencing. The candidate aging genes identified here may be potential targets for the treatment of aging.