Improvement of C-to-U RNA editing using an artificial MS2-APOBEC system.

RNA cytidine deamination (C-to-U editing) has been achieved using the MS2-apolipoprotein B-editing catalytic polypeptide-like (APOBEC)1 editing system. Here, we fused the cytidine deaminase (CDA) enzymes APOBEC3A and APOBEC3G with the MS2 system and examined their RNA editing efficiencies in transfected HEK 293T cells. Given the single-stranded RNA preferences of APOBEC3A and APOBEC3G, we designed unconventional guide RNAs that induced a loop at the target sequence, allowing the target to form a single-stranded structure. Because APOBEC3A and APOBEC3G have different base preferences (5'-TC and 5'-CC, respectively), we introduced the D317W mutation into APOBEC3G to convert its base preference to that of APOBEC3A. Upon co-transfection with a guide RNA that induced the formation of a 14 nt loop on the target sequence, MS2-fused APOBEC3A and APOBEC3G showed high editing efficiency. While the D317W mutation of APOBEC3G led to a slight improvement in editing efficiency, the difference was not statistically significant. These findings indicate that APOBEC3A and APOBEC3G can induce C-to-U RNA editing when transfected with a loop guide RNA. Moreover, the editing efficiency of APOBEC3G can be enhanced by site-specific mutation to alter the base preference. Overall, our results demonstrate that the MS2 system can fuse and catalyze reactions with different enzymes, suggesting that it holds an even greater potential for RNA editing than is utilized currently.

The Role of the Exonic lncRNA PRKDC-210 in Transcription Regulation.

In recent years, long noncoding RNAs (lncRNAs) have received increasing attention and have been reported to be associated with various genetic abnormalities. However, the functions of many lncRNAs, including those of long exonic noncoding RNAs (lencRNAs), have not yet been elucidated. Here, we used a novel tethering luciferase assay to analyze the transcriptional regulatory functions of five lencRNAs that are upregulated in cancer. We found that the lencRNA PRKDC-210 interacts with MED12, a component of the CDK8 complex, to regulate the transcription of several genes. The transcriptional activation ability of PRKDC-210 was abolished in siRNA-treated CDK8-depleted cells. We also confirmed the enrichment of PRKDC-210 on RNA polymerase II. RNA-seq analysis of cells in which PRKDC-210 or PRKDC mRNA was knocked down using antisense oligonucleotides revealed that PRKDC-210 can affect the expression levels of genes related to fatty acid metabolism. Finally, we used a ChIRP assay to examine PRKDC-210-enriched sites in the genome. Overall, our findings demonstrate that the lencRNA PRKDC-210 promotes transcription through the CDK8 complex pathway at the transcription initiation site. We propose that PRKDC-210 can affect the transcription of adjacent genes after its transcription and splicing.

Genome-Wide Identification of U-To-C RNA Editing Events for Nuclear Genes in Arabidopsis thaliana.

Cytosine-to-Uridine (C-to-U) RNA editing involves the deamination phenomenon, which is observed in animal nucleus and plant organelles; however, it has been considered the U-to-C is confined to the organelles of limited non-angiosperm plant species. Although previous RNA-seq-based analysis implied U-to-C RNA editing events in plant nuclear genes, it has not been broadly accepted due to inadequate confirmatory analyses. Here we examined the U-to-C RNA editing in Arabidopsis tissues at different developmental stages of growth. In this study, the high-throughput RNA sequencing (RNA-seq) of 12-day-old and 20-day-old Arabidopsis seedlings was performed, which enabled transcriptome-wide identification of RNA editing sites to analyze differentially expressed genes (DEGs) and nucleotide base conversions. The results showed that DEGs were expressed to higher levels in 12-day-old seedlings than in 20-day-old seedlings. Additionally, pentatricopeptide repeat (PPR) genes were also expressed at higher levels, as indicated by the log2FC values. RNA-seq analysis of 12-day- and 20-day-old Arabidopsis seedlings revealed candidates of U-to-C RNA editing events. Sanger sequencing of both DNA and cDNA for all candidate nucleotide conversions confirmed the seven U-to-C RNA editing sites. This work clearly demonstrated presence of U-to-C RNA editing for nuclear genes in Arabidopsis, which provides the basis to study the mechanism as well as the functions of the unique post-transcriptional modification.

Effective RNA Knockdown Using CRISPR-Cas13a and Molecular Targeting of the EML4-ALK Transcript in H3122 Lung Cancer Cells.

RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA-guide RNA complex residing at the 5' of the crRNA. The activity of Cas13a was maximal for guide RNAs 24-30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70-76%), mCherry fluorescence (72%), and EML4-ALK at the protein (>80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.

RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code.

Many genetic diseases are caused by T-to-C point mutations. Hence, editing of mutated genes represents a promising strategy for treating these disorders. We engineered an artificial RNA editase by combining the deaminase domain of APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1) with a guideRNA (gRNA) which is complementary to target mRNA. In this artificial enzyme system, gRNA is bound to MS2 stem-loop, and deaminase domain, which has the ability to convert mutated target nucleotide C-to-U, is fused to MS2 coat protein. As a target RNA, we used RNA encoding blue fluorescent protein (BFP) which was derived from the gene encoding GFP by 199 T > C mutation. Upon transient expression of both components (deaminase and gRNA), we observed GFP by confocal microscopy, indicating that mutated 199C in BFP had been converted to U, restoring original sequence of GFP. This result was confirmed by PCR-RFLP and Sanger's sequencing using cDNA from transfected cells, revealing an editing efficiency of approximately 21%. Although deep RNA sequencing result showed some off-target editing events in this system, we successfully developed an artificial RNA editing system using artificial deaminase (APOBEC1) in combination with MS2 system could lead to therapies that treat genetic disease by restoring wild-type sequence at the mRNA level.

Development of a Single Construct System for Site-Directed RNA Editing Using MS2-ADAR.

Site-directed RNA editing (SDRE) technologies have great potential for treating genetic diseases caused by point mutations. Our group and other researchers have developed SDRE methods utilizing adenosine deaminases acting on RNA (ADARs) and guide RNAs recruiting ADARs to target RNAs bearing point mutations. In general, efficient SDRE relies on introducing numerous guide RNAs relative to target genes. However, achieving a large ratio is not possible for gene therapy applications. In order to achieve a realistic ratio, we herein developed a system that can introduce an equal number of genes and guide RNAs into cultured cells using a fusion protein comprising an ADAR fragment and a plasmid vector containing one copy of each gene on a single construct. We transfected the single construct into HEK293T cells and achieved relatively high efficiency (up to 42%). The results demonstrate that efficient SDRE is possible when the copy number is similar for all three factors (target gene, guide RNA, and ADAR enzyme). This method is expected to be capable of highly efficient gene repair in vivo, making it applicable for gene therapy.

The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB.

N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-ß-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-ß-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-ß-induced EMT of lung cancer cells through the regulation of JUNB.

Androgen receptor promotes sex-independent angiogenesis in response to ischemia and is required for activation of vascular endothelial growth factor receptor signaling.

BACKGROUND: Hypoandrogenemia is associated with an increased risk of ischemic diseases. Because actions of androgens are exerted through androgen receptor (AR) activation, we studied hind-limb ischemia in AR knockout mice to elucidate the role of AR in response to ischemia. METHODS AND RESULTS: Both male and female AR knockout mice exhibited impaired blood flow recovery, more cellular apoptosis, and a higher incidence of autoamputation after ischemia. In ex vivo and in vivo angiogenesis studies, AR-deficient vascular endothelial cells showed reduced angiogenic capability. In ischemic limbs of AR knockout mice, reductions in the phosphorylation of the Akt protein kinase and endothelial nitric oxide synthase were observed despite a robust increase in hypoxia-inducible factor 1α and vascular endothelial cell growth factor (VEGF) gene expression. In in vitro studies, siRNA-mediated ablation of AR in vascular endothelial cells blunted VEGF-stimulated phosphorylation of Akt and endothelial nitric oxide synthase. Immunoprecipitation experiments documented an association between AR and kinase insert domain protein receptor that promoted the recruitment of downstream signaling components. CONCLUSIONS: These results document a physiological role of AR in sex-independent angiogenic potency and provide evidence of novel cross-talk between the androgen/AR signaling and VEGF/kinase insert domain protein receptor signaling pathways.

The androgen receptor in health and disease.

Androgens play pivotal roles in the regulation of male development and physiological processes, particularly in the male reproductive system. Most biological effects of androgens are mediated by the action of nuclear androgen receptor (AR). AR acts as a master regulator of downstream androgen-dependent signaling pathway networks. This ligand-dependent transcriptional factor modulates gene expression through the recruitment of various coregulator complexes, the induction of chromatin reorganization, and epigenetic histone modifications at target genomic loci. Dysregulation of androgen/AR signaling perturbs normal reproductive development and accounts for a wide range of pathological conditions such as androgen-insensitive syndrome, prostate cancer, and spinal bulbar muscular atrophy. In this review we summarize recent advances in understanding of the epigenetic mechanisms of AR action as well as newly recognized aspects of AR-mediated androgen signaling in both men and women. In addition, we offer a perspective on the use of animal genetic model systems aimed at eventually developing novel therapeutic AR ligands.

Testis-specific protein on Y chromosome (TSPY) represses the activity of the androgen receptor in androgen-dependent testicular germ-cell tumors.

Testis-specific protein on Y chromosome (TSPY) is an ampliconic gene on the Y chromosome, and genetic interaction with gonadoblastoma has been clinically established. However, the function of the TSPY protein remains to be characterized in physiological and pathological settings. In the present study, we observed coexpression of TSPY and the androgen receptor (AR) in testicular germ-cell tumors (TGCTs) in patients as well as in model cell lines, but such coexpression was not seen in normal testis of humans or mice. TSPY was a repressor for androgen signaling because of its trapping of cytosolic AR even in the presence of androgen. Androgen treatment stimulated cell proliferation of a TGCT model cell line, and TSPY potently attenuated androgen-dependent cell growth. Together with the finding that TSPY expression is reduced in more malignant TGCTs in vivo, the present study suggests that TSPY serves as a repressor in androgen-induced tumor development in TGCTs and raises the possibility that TSPY could be used as a clinical marker to assess the malignancy of TGCTs.

The growth of a vascular network inside a collagen-citric acid derivative hydrogel in rats.

Three-dimensional regenerative tissue with a certain bulk cannot survive without sufficient blood perfusion in vivo, so construction of a vascular system in regenerative tissue is a key technology in tissue engineering. In order to construct such a vascular system, we attempted to create a scaffold material that induces neovascular growth from the recipient bed into the material. This material, an ion complex gel matrix (IC gel) consisting of collagen and a citric acid derivative, enabled it to associate with basic fibroblast growth factor (bFGF). The IC gel was implanted in the subfascial space of the rat rectus muscle and excised 5 days later. Cross-sections of the excised samples were stained for von Willebrand factor, and then neovascular development into the gel was observed and also quantified by image analysis. These data showed that the IC gel markedly induced growth of vascular-rich tissue into the inside of the gel by day 5, which surpassed that after implantation of Matrigel or gelated collagen. Further, combination with bFGF significantly enhanced the vascularization ability of IC gel. These findings suggest that IC gel functioned as a scaffold material for neovascular ingrowth and a reservoir of bFGF.

Non-steroidal antiandrogens act as AF-1 agonists under conditions of high androgen-receptor expression.

BACKGROUND: The mechanism of resistance acquisition to antiandrogens in prostate cancer is not fully understood. Numerous clinical and basic research studies have shown expression of androgen receptors (ARs) increases in hormone-refractory prostate cancer and therefore we explored possible molecular mechanisms by which prostate cancer acquires resistance to antiandrogens under conditions of increased AR expression. METHODS: In order to study resistance to antiandrogens at the AR transactivation level we used a human AR (hAR) reporter assay system. In addition, we utilized an hAR deletion mutant to determine the functional domain responsible for the acquisition of resistance. RESULTS: Increased hAR protein expression enhanced the sensitivity of AR transactivation to low concentrations of DHT, and also reduced the inhibitory activity of the non-steroidal antiandrogens, hydroxyflutamide, and bicalutamide on DHT-induced AR transactivation. Moreover, these antiandrogens acquired agonistic activity under conditions of high hAR protein expression. Such agonistic activity of antiandrogens was not detected in an hAR deletion mutant (hAR-DeltaA/B) that lacked an A/B domain with AF-1 activity. CONCLUSIONS: We found that non-steroidal antiandrogens act as AF-1 agonists under conditions of high AR protein expression. This partial antagonistic property of antiandrogens may be a molecular mechanism by which prostate cancer develops resistance to these drugs.

Premature ovarian failure in androgen receptor-deficient mice.

Premature ovarian failure (POF) syndrome, an early decline of ovarian function in women, is frequently associated with X chromosome abnormalities ranging from various Xq deletions to complete loss of one of the X chromosomes. However, the genetic locus responsible for the POF remains unknown, and no candidate gene has been identified. Using the Cre/LoxP system, we have disrupted the mouse X chromosome androgen receptor (Ar) gene. Female AR(-/-) mice appeared normal but developed the POF phenotype with aberrant ovarian gene expression. Eight-week-old female AR(-/-) mice are fertile, but they have lower follicle numbers and impaired mammary development, and they produce only half of the normal number of pups per litter. Forty-week-old AR(-/-) mice are infertile because of complete loss of follicles. Genome-wide microarray analysis of mRNA from AR(-/-) ovaries revealed that a number of major regulators of folliculogenesis were under transcriptional control by AR. Our findings suggest that AR function is required for normal female reproduction, particularly folliculogenesis, and that AR is a potential therapeutic target in POF syndrome.

Splicing potentiation by growth factor signals via estrogen receptor phosphorylation.

Mitogen-activated protein kinase-mediated growth factor signals are known to augment the ligand-induced transactivation function of nuclear estrogen receptor alpha (ERalpha) through phosphorylation of Ser-118 within the ERalpha N-terminal transactivation (activation function-1) domain. We identified the spliceosome component splicing factor (SF)3a p120 as a coactivator specific for human ERalpha (hERalpha) activation function-1 that physically associated with ERalpha dependent on the phosphorylation state of Ser-118. SF3a p120 potentiated hERalpha-mediated RNA splicing, and notably, the potentiation of RNA splicing by SF3a p120 depended on hER Ser-118 phosphorylation. Thus, our findings suggest a mechanism by which growth factor signaling can regulate gene expression through the modulation of RNA splicing efficiency via phosphorylation of sequence-specific activators, after association between such activators and the spliceosome.

Transcriptional regulation of the mouse steroid 5alpha-reductase type II gene by progesterone in brain.

The steroid 5alpha-reductase (5alpha-R) plays an important physiological role in the conversion of steroid hormones such as androgen and progesterone to their 5alpha-reduced derivatives. 5alpha-R type II (5alpha-R2), one of two 5alpha-R isoforms, is thought to be a key enzyme in the generation of neuroactive steroids in the brain, particularly allopregnanolone (AP), via the production of its precursor dihydroprogesterone from progesterone. In the present study, we investigated possible regulatory mechanisms of 5alpha-R2 gene expression by steroid hormones in the female mouse brain. We first cloned mouse 5alpha-R2 (m5alpha-R2) cDNA by degenerate PCR, and found that progesterone induced 5alpha-R2 gene expression to levels detectable by in situ hybridization in female mouse brains. Functional analysis of the m5alpha-R2 gene promoter by a transient expression assay with human progesterone receptor (PR) and androgen receptor (AR) expression vectors identified a progesterone and androgen regulatory element (m5alpha-R2 PRE/ARE). Results of an electrophoretic mobility shift assay revealed that both PR and AR homodimers bound directly to m5alpha-R2 PRE/ARE sequence. These findings suggest that the gene expression of m5alpha-R2 is transcriptionally regulated by progesterone in female brains.