PDIVAS: Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing.

BACKGROUND: Deep-intronic variants that alter RNA splicing were ineffectively evaluated in the search for the cause of genetic diseases. Determination of such pathogenic variants from a vast number of deep-intronic variants (approximately 1,500,000 variants per individual) represents a technical challenge to researchers. Thus, we developed a Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing (PDIVAS) to easily detect pathogenic deep-intronic variants. RESULTS: PDIVAS was trained on an ensemble machine-learning algorithm to classify pathogenic and benign variants in a curated dataset. The dataset consists of manually curated pathogenic splice-altering variants (SAVs) and commonly observed benign variants within deep introns. Splicing features and a splicing constraint metric were used to maximize the predictive sensitivity and specificity, respectively. PDIVAS showed an average precision of 0.92 and a maximum MCC of 0.88 in classifying these variants, which were the best of the previous predictors. When PDIVAS was applied to genome sequencing analysis on a threshold with 95% sensitivity for reported pathogenic SAVs, an average of 27 pathogenic candidates were extracted per individual. Furthermore, the causative variants in simulated patient genomes were more efficiently prioritized than the previous predictors. CONCLUSION: Incorporating PDIVAS into variant interpretation pipelines will enable efficient detection of disease-causing deep-intronic SAVs and contribute to improving the diagnostic yield. PDIVAS is publicly available at https://github.com/shiro-kur/PDIVAS .

CCL21-Ser expression in melanoma cells recruits CCR7+ naïve T cells to tumor tissues and promotes tumor growth.

CCL21-Ser, a chemokine encoded by the Ccl21a gene, is constitutively expressed in the thymic epithelial cells and stromal cells of secondary lymphoid organs. It regulates immune cell migration and survival through its receptor CCR7. Herein, using CCL21-Ser-expressing melanoma cells and the Ccl21a-deficient mice, we demonstrated the functional role of cancer cell-derived CCL21-Ser in melanoma growth in vivo. The B16-F10 tumor growth was significantly decreased in Ccl21a-deficient mice compared with that in wild-type mice, indicating that host-derived CCL21-Ser contributes to melanoma proliferation in vivo. In Ccl21a-deficient mice, tumor growth of melanoma cells expressing CCL21-Ser was significantly enhanced, suggesting that CCL21-Ser from melanoma cells promotes tumor growth in the absence of host-derived CCL21-Ser. The increase in tumor growth was associated with an increase in the CCR7+ CD62L+ T cell frequency in the tumor tissue but was inversely correlated with Treg frequency, suggesting that naïve T cells primarily promote tumor growth. Adoptive transfer experiments demonstrated that naïve T cells are preferentially recruited from the blood into tumors with melanoma cell-derived CCL21-Ser expression. These results suggest that CCL21-Ser from melanoma cells promotes the infiltration of CCR7+ naïve T cells into the tumor tissues and creates a tumor microenvironment favorable for melanoma growth.

Comparison of Measured Data between Pre- and Post-Radiotherapy in a Patient with Cardiac Resynchronization Therapy Defibrillator.

Although some researches proved the influence of radiation therapy (RT) on pacemakers and implantable cardioverter defibrillators, little has been reported on cardiac resynchronization therapy defibrillators (CRTDs). We experienced a case of RT on CRTD and had a new finding.A patient with CRTD implanted for dilated cardiomyopathy was diagnosed with lung squamous cell carcinoma and started receiving RT. All the implanted devices, including the main body of CRTD, left ventricular lead (LV), right ventricular lead with high-voltage conductor, and right atrial lead, were from the same manufacturer. The radiation targeted the tumor of 67 mm in diameter in the right superior lobe for 5 min per session. The CRTD was outside the radiation field, which is 65 mm, but the leads were inside. Plan 1 used 2 Gy/fr with 8 megavolt photons, and Plan 1 was irradiated at 0° and 180° for 16 RT sessions. The dosage was increased to 3 Gy for Plan 2 for 4 sessions. Plan 3 used 2 Gy with 6 and 8 megavolt photons, and Plan 3 was irradiated at 27.7° and 200.7° for 11 RT sessions. Changes in measured parameters were assessed before and after RT.Changes in impedance of LV and high-voltage lead exceeded prespecified threshold. However, no significant errors were detected in the CRTD on the dosages and energy we used.We hypothesize that the lead insulator could have been affected by radiation.

Integrative network analysis reveals biological pathways associated with Williams syndrome.

BACKGROUND: Williams syndrome (WS) is a neurodevelopmental disorder that has been attributed to heterozygous deletions in chromosome 7q11.23 and exhibits a variety of physical, cognitive, and behavioral features. However, the genetic basis of this phenotypic variability is unclear. In this study, we identified genetic clues underlying these complex phenotypes. METHODS: Neurobehavioral function was assessed in WS patients and healthy controls. Total RNA was extracted from peripheral blood and subjected to microarray analysis, RNA-sequencing, and qRT-PCR. Weighted gene co-expression network analysis was performed to identify specific alterations related to intermediate disease phenotypes. To functionally interpret each WS-related module, gene ontology and disease-related gene enrichment were examined. We also investigated the micro (mi)RNA expression profiles and miRNA co-expression networks to better explain the regulation of the transcriptome in WS. RESULTS: Our analysis identified four significant co-expression modules related to intermediate WS phenotypes. Notably, the three upregulated WS-related modules were composed exclusively of genes located outside the 7q11.23 region. They were significantly enriched in genes related to B-cell activation, RNA processing, and RNA transport. BCL11A, which is known for its association with speech disorders and intellectual disabilities, was identified as one of the hub genes in the top WS-related module. Finally, these key upregulated mRNA co-expression modules appear to be inversely correlated with a specific downregulated WS-related miRNA co-expression module. CONCLUSIONS: Dysregulation of the mRNA/miRNA network involving genes outside of the 7q11.23 region is likely related to the complex phenotypes observed in WS patients.

Use of direct oral anti-coagulants for the treatment of venous thromboembolism in patients with advanced cancer: a prospective observational study.

BACKGROUND: The efficacy of direct oral anti-coagulants (DOACs) for the treatment of venous thromboembolism (VTE) in Japanese patients with advanced cancer is largely unknown. METHODS: This prospective single-center observational study enrolled Japanese patients with unresectable advanced cancer who started DOAC treatment for new-onset VTE between December 2015 and May 2018. Patients were followed for 3 months to evaluate bleeding and VTE recurrences. The primary study endpoint was major and non-major bleeding. RESULTS: One hundred and forty-five of 147 enrolled patients were analyzed. Of these, 8 [5.5%, 95% confidence interval (CI) 2.8-10.5] and 29 patients (20%, 95% CI 14.3-27.2) experienced major and non-major bleeding, respectively. Patients with bleeding were more likely to have a poor performance status (PS) [hazard rate (HR) 2.04, 95% CI 1.15-3.63] and more frequent use of non-steroidal anti-inflammatory drugs (NSAIDs) (HR 2.75, 95% CI 1.62-4.67) relative to those without bleeding. In a multivariate analysis, combined DOAC and NSAID use correlated significantly with bleeding (odds ratio 4.63, 95% CI 1.70-12.9, p = 0.003). Among 105 of 145 patients included in the VTE recurrence assessment, 9 experienced a VTE recurrence (8.6%, 95% CI 4.6-15.5). CONCLUSIONS: Our findings confirm the risk of bleeding during DOAC treatment for VTE in Japanese patients with advanced cancer, particularly those with poor PS and those using NSAIDs. The risk of bleeding in these patients may be reduced by avoiding the combined use of DOACs and NSAIDs.

Novel Stress-Inducible Antisense RNAs of Protein-Coding Loci Are Synthesized by RNA-Dependent RNA Polymerase.

Our previous study identified approximately 6,000 abiotic stress-responsive noncoding transcripts existing on the antisense strand of protein-coding genes and implied that a type of antisense RNA was synthesized from a sense RNA template by RNA-dependent RNA polymerase (RDR). Expression analyses revealed that the expression of novel abiotic stress-induced antisense RNA on 1,136 gene loci was reduced in the rdr1/2/6 mutants. RNase protection indicated that the RD29A antisense RNA and other RDR1/2/6-dependent antisense RNAs are involved in the formation of dsRNA. The accumulation of stress-inducible antisense RNA was decreased and increased in dcp5 and xrn4, respectively, but not changed in dcl2/3/4, nrpd1a and nrpd1b RNA-seq analyses revealed that the majority of the RDR1/2/6-dependent antisense RNA loci did not overlap with RDR1/2/6-dependent 20-30 nt RNA loci. Additionally, rdr1/2/6 mutants decreased the degradation rate of the sense RNA and exhibited arrested root growth during the recovery stage following a drought stress, whereas dcl2/3/4 mutants did not. Collectively, these results indicate that RDRs have stress-inducible antisense RNA synthesis activity and a novel biological function that is different from the known endogenous small RNA pathways from protein-coding genes. These data reveal a novel mechanism of RNA regulation during abiotic stress response that involves complex RNA degradation pathways.

Chemotherapy-induced cardiomyopathy caused by Pemetrexed.

Chemotherapy-related cardiac toxicity is a rare but serious adverse event in patients with cancer. Thus far, no case of serious cardiac toxicity of pemetrexed has been reported. We describe the case of a patient with advanced lung cancer and cardiomyopathy due to pemetrexed. A 59-year-old woman visited our hospital, and we found abnormal findings on a chest radiograph. She was diagnosed as having stage IV lung adenocarcinoma. Chemotherapy with cisplatin and pemetrexed every 3 weeks was initiated. After four cycles of chemotherapy, maintenance chemotherapy with pemetrexed was administered every 3 weeks. During the seventeenth cycle of pemetrexed, she had shortness of breath in her daily life. A chest radiograph showed an enlarged cardiothoracic ratio (66%), and the transthoracic echocardiogram demonstrated expansion of the left ventricle (diastolic diameter, 67 mm), severe global hypokinesis, and reduced left ventricular ejection fraction (28%). The coronary angiogram showed no coronary constriction. There was no delayed accumulation on the contrast-enhanced cardiac magnetic resonance imaging scan. After right heart catheterization, pathological results of a myocardial biopsy from the ventricular septum indicated no cardiac muscle hypertrophy, cardiac fibrosis, inflammatory cell infiltration, or myocyte disarray. Eventually, she was diagnosed as having pemetrexed-induced cardiomyopathy. Pemetrexed was discontinued, and furosemide, enalapril, and carvedilol were started. Then her symptoms and cardiac function improved. Early detection and discontinuation of causative agents are the most important treatment strategies in similar patients. Diuretics, angiotensin-conversion enzyme inhibitors, and beta-blockers may be effective for treating heart failure.

Crizotinib-induced simultaneous multiple cardiac toxicities.

Crizotinib is a receptor tyrosine kinase inhibitor that has several targets, including c-ros oncogene 1 and the MET proto-oncogene. Considering its known cardiac toxicity, bradycardia is often investigated following treatment with crizotinib. Our patients had bradycardia, QT prolongation, ventricular rhythm, ventricular fibrillation, and pericarditis simultaneously. The cardiotoxicity of crizotinib can sometimes be simultaneous; thus, intensive observation is needed.

Characteristics of cellular composition in malignant pericardial effusion and its association with the clinical course of carcinomatous pericarditis.

To date, the cellular composition of malignant pericardial effusion (MPE) and its association with the clinical course of carcinomatous pericarditis remain unclear. We aimed to determine the MPE cellular composition and its association with carcinomatous pericarditis. Forty-four cases indicated for pericardial drainage due to symptomatic carcinomatous pericarditis were retrospectively reviewed; the blood cell count and composition of MPE were examined. The most dominant cells in MPE were neutrophils. The appearance ratio of an atypical cell in cytologically positive MPE was 95.5%. Low neutrophil and high lymphocyte counts were significantly associated with good effusion failure-free survival at 1 month. The survival after pericardial drainage was significantly shorter when the neutrophil/lymphocyte ratio was 3.5 or more (P = 0.041). Patients whose performance status improved due to drainage had significantly high leukocyte counts in MPE (P = 0.02). Prediction of the course of drainage through basic examination of MPE cellular composition might be beneficial in clinical practice.

Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia.

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD.

Next-generation sequencing-based analysis of reverse transcriptase fidelity.

In this study, we devised a simple and rapid method to analyze fidelity of reverse transcriptase (RT) using next-generation sequencing (NGS). The method comprises a cDNA synthesis reaction from standard RNA with a primer containing a tag of 14 randomized bases and the RT to be tested, PCR using high-fidelity DNA polymerase, and NGS. By comparing the sequence of each read with the reference sequence, mutations were identified. The mutation can be identified to be due to an error introduced by either cDNA synthesis, PCR, or NGS based on whether the sequence reads with the same tag contain the same mutation or not. The error rates in cDNA synthesis with Moloney murine leukemia virus (MMLV) RT thermostable variant MM4 or the recently developed 16-tuple variant of family B DNA polymerase with RT activity, RTX, from Thermococcus kodakarensis, were 0.75-1.0 × 10-4 errors/base, while that in the reaction with the wild-type human immunodeficiency virus type 1 (HIV-1) RT was 2.6 × 10-4 errors/base. Overall, our method could precisely evaluate the fidelity of various RTs with different reaction conditions in a high-throughput manner without the use of expensive optics and troublesome adaptor ligation.

Retinoid derivative Tp80 exhibits anti-hepatitis C virus activity through restoration of GI-GPx expression.

Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus with an estimated infection in ∼180 million people worldwide, and its chronic infection leads to development of cirrhosis and hepatocellular carcinoma. Although recent development of direct acting antiviral (DAA) compounds improved anti-HCV regimens, alternative therapeutic compounds are still demanded due to an expected emergence of escape mutants for those DAAs. In order to identify novel anti-HCV agents, we conducted chemical library screening for 2086 compounds using HCV Rep-Feo reporter replicon in Huh7 hepatoma cells. Our screening identified retinoid derivative Tp80, which inhibits replication of HCV Rep-Feo (genotype 1b) and JFH1 HCV (genotype 2a) with 0.62 µM and 1.0 µM, respectively, of 50% effective concentration (EC50 ), at which cytotoxicity is not evident for host hepatocytes. Subsequent transcriptome profiling revealed Tp80 exhibits anti-HCV activity through restoration of gastrointestinal glutathione peroxidase (GI-GPx), suppression of which is responsible for HCV-induced oxidative stress to facilitate HCV replication. Furthermore, comparison of Tp80 with other retinoid derivatives revealed Tp80 shows best potency in both GI-GPx restoration and anti-HCV activity among compounds we examined. In conclusion, our current study provides Tp80 as a promising candidate of anti-HCV compound, suppressing host cellular oxidative stress through a restoration of GI-GPx.

Synthetic ligand promotes gene expression by affecting GC sequence in promoter.

A naphthyridine carbamate tetramer (NCT8) is a synthetic compound, which selectively binds to nucleic acids containing CGG/CGG sequence. Although NCT8 is a promising compound for a wide range of DNA and RNA based biotechnology such as modulation of specific gene expression, little is known about its behavior in human cells. In the present study, we investigated the changes induced in gene expression by NCT8. Genes differentially expressed in the presence of NCT8 in HeLa cells were identified by whole-transcriptome analysis. The whole-transcriptome analysis showed that NCT8 significantly induced up-regulation of specific genes, whose promoter region has GC-rich sequence.

Prevalence of deep venous thrombosis detected by ultrasonography before surgery in patients with gastric cancer: a retrospective study of 1140 consecutive patients.

BACKGROUND: The prevalence of deep venous thrombosis (DVT) in patients with gastric cancer before surgery is unknown. This study aimed to clarify the risk factors for DVT of the lower extremities in patients with gastric cancer before surgery and to evaluate the usefulness of ultrasonographic screening for prevention of postoperative pulmonary thromboembolism (PTE). METHODS: Patients who had undergone lower-extremity venous ultrasonography before surgery for gastric cancer were retrospectively analyzed. Univariate and multivariate logistic regression analyses were performed to identify the predictors of DVT before surgery. Perioperative management of patients with DVTs and the incidence of postoperative PTE were investigated. RESULTS: Of the total 1140 patients, 86 had DVT preoperatively. On univariate analysis, the incidence of DVT was significantly higher with: female sex; age ≥80 years; PS ≥ 1 (vs. PS = 0); stage IV (vs. stages I-III); history of preoperative chemotherapy; and the presence of a central venous catheter (CVC). Multivariate logistic regression analysis demonstrated that sex, age ≥80 years, PS ≥ 1, history of preoperative chemotherapy, and the presence of CVC were significantly correlated with DVT before surgery. Postoperative PTE occurred in 2 patients with proximal DVT. No patients in whom DVT was not detected developed PTE. CONCLUSIONS: Female sex, older age, worse PS, the presence of CVC, and a history of preoperative chemotherapy were the independent risk factors for DVT. Routine lower-extremity venous ultrasonographic screening is useful for prevention of PTE because it can identify patients at high or low risk for PTE.

Next-generation sequencing-based comprehensive molecular analysis of 43 Japanese patients with cone and cone-rod dystrophies.

PURPOSE: To investigate the efficacy of targeted exome sequencing for mutational screening of Japanese patients with cone dystrophy (CD) or cone-rod dystrophy (CRD). METHODS: DNA samples from 43 Japanese patients with CD or CRD were sequenced using an exome-sequencing panel targeting all 193 known inherited eye disease genes and next-generation sequencing methodologies. Subsequently, candidate variants were screened using systematic data analyses, and their potential pathogenicity was assessed using distinct filtering approaches, which included the frequency of the variants in normal populations, in silico prediction tools, and cosegregation. RESULTS: Causative mutations were detected in 12 patients with CD or CRD (27.9%). In total, 14 distinct mutations were identified in the genes ABCA4, CDHR1, CRB1, CRX, GUCY2D, KCNV2, PROM1, PRPH2, and RDH5, including four novel mutations, c.3050+1G>A in ABCA4, c.386A>G in CDHR1, c.652+1_652+4del in CRB1, and c.454G>A in KCNV2. Moreover, a putative pathogenic mutation was identified in RGS9BP, a gene recognized as the source of bradyopsia. CONCLUSIONS: Targeted exome sequencing effectively identified causative mutations in Japanese patients with CD or CRD. The results confirmed the heterogeneity of the genes responsible for CD and CRD in Japanese populations, as well as the efficacy of targeted exome sequencing-based screening of patients with inherited retinal degeneration.

Small open reading frames associated with morphogenesis are hidden in plant genomes.

It is likely that many small ORFs (sORFs; 30-100 amino acids) are missed when genomes are annotated. To overcome this limitation, we identified ∼8,000 sORFs with high coding potential in intergenic regions of the Arabidopsis thaliana genome. However, the question remains as to whether these coding sORFs play functional roles. Using a designed array, we generated an expression atlas for 16 organs and 17 environmental conditions among 7,901 identified coding sORFs. A total of 2,099 coding sORFs were highly expressed under at least one experimental condition, and 571 were significantly conserved in other land plants. A total of 473 coding sORFs were overexpressed; ∼10% (49/473) induced visible phenotypic effects, a proportion that is approximately seven times higher than that of randomly chosen known genes. These results indicate that many coding sORFs hidden in plant genomes are associated with morphogenesis. We believe that the expression atlas will contribute to further study of the roles of sORFs in plants.

Deciphering targeting rules of splicing modulator compounds: case of TG003.

BACKGROUND: Recent advances in the development of small chemical compounds that can modulate RNA splicing brought excitement to the field of splicing-targeting therapy. Splicing-targeting therapy tries to ameliorate the disease by altering the exon combination of transcripts to reduce the undesired effect of genetic mutations. However, the knowledge and tools to understand factors contributing to splicing modulator compound sensitivity have been lacking. Our goal was to establish a method to characterize sequence features found in compound sensitive exons. RESULTS: Here we developed a comparative transcriptomic approach to explore features that make an exon sensitive to a chemical compound. In this study, we chose TG003, a potential drug for Duchenne muscular dystrophy, and performed RNA-sequencing on samples from human and mouse skeletal muscle cells, with and without TG003 treatments. We compared TG003 responsiveness between homologous exon pairs and identified 21 pairs in which human exons were skip-enhanced but not mouse exons. We compared the sequence features; splice site scores, number of splicing factor binding sites, and properties of branch sequence and polypyrimidine tracts, and found that polypyrimidine tracts were stronger (longer stretches and richer content of consecutive polypyrimidine) in the mouse TG003 insensitive exons. We also compared the features between TG003 skip-enhanced and insensitive exons within the species, and discovered that human TG003 skip-enhanced exons were shorter and had less splicing factor binding sites than the group of human TG003 insensitive exons. Mouse insensitive exons homologous to human TG003 skip-enhanced exons shared these properties. Our results suggested that these features are prerequisites for TG003 skip-enhanced exons and weak polypyrimidine tracts are defining features, which were supported by a decision tree analysis on all cassette exons in human. CONCLUSIONS: In this study we established a comparative transcriptomic approach, which shed lights on how small chemical compounds modulate RNA splicing. The results described here was the first attempt to decipher the targeting rules of a splicing modulator compound. We expect that this approach would contribute to the precise understanding of the mechanism of TG003-induced splicing modulation, expand target diseases of splicing modulators in general, as well as the development of new splicing modulators.

Loss of Arabidopsis 5'-3' Exoribonuclease AtXRN4 Function Enhances Heat Stress Tolerance of Plants Subjected to Severe Heat Stress.

mRNA degradation plays an important role in the rapid and dynamic alteration of gene expression in response to environmental stimuli. Arabidopsis 5'-3' exoribonuclease (AtXRN4), a homolog of yeast Xrn1p, functions after a de-capping step in the degradation of uncapped RNAs. While Xrn1p-dependent degradation of mRNA is the main process of mRNA decay in yeast, information pertaining to the targets of XRN4-based degradation in plants is limited. In order to better understand the biological function of AtXRN4, the current study examined the survivability of atxrn4 mutants subjected to heat stress. The results indicated that atxrn4 mutants, compared with wild-type plants, exhibited an increased survival rate when subjected to a short-term severe heat stress. A microarray and mRNA decay assay showed that loss of AtXRN4 function caused a reduction in the degradation of heat shock factor A2 (HSFA2) and ethylene response factor 1 (ERF1) mRNA. The heat stress tolerance phenotype of atxrn4 mutants was significantly reduced or lost by mutation of HSFA2, a known key regulator of heat acclimation, thus indicating that HSFA2 is a target gene of AtXRN4-mediated mRNA degradation both under non-stress conditions and during heat acclimation. These results demonstrate that AtXRN4-mediated mRNA degradation is linked to the suppression of heat acclimation.

RARGE II: an integrated phenotype database of Arabidopsis mutant traits using a controlled vocabulary.

Arabidopsis thaliana is one of the most popular experimental plants. However, only 40% of its genes have at least one experimental Gene Ontology (GO) annotation assigned. Systematic observation of mutant phenotypes is an important technique for elucidating gene functions. Indeed, several large-scale phenotypic analyses have been performed and have generated phenotypic data sets from many Arabidopsis mutant lines and overexpressing lines, which are freely available online. Since each Arabidopsis mutant line database uses individual phenotype expression, the differences in the structured term sets used by each database make it difficult to compare data sets and make it impossible to search across databases. Therefore, we obtained publicly available information for a total of 66,209 Arabidopsis mutant lines, including loss-of-function (RATM and TARAPPER) and gain-of-function (AtFOX and OsFOX) lines, and integrated the phenotype data by mapping the descriptions onto Plant Ontology (PO) and Phenotypic Quality Ontology (PATO) terms. This approach made it possible to manage the four different phenotype databases as one large data set. Here, we report a publicly accessible web-based database, the RIKEN Arabidopsis Genome Encyclopedia II (RARGE II; http://rarge-v2.psc.riken.jp/), in which all of the data described in this study are included. Using the database, we demonstrated consistency (in terms of protein function) with a previous study and identified the presumed function of an unknown gene. We provide examples of AT1G21600, which is a subunit in the plastid-encoded RNA polymerase complex, and AT5G56980, which is related to the jasmonic acid signaling pathway.

ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis.

DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms. Recent studies suggest that the methylation status of a number of genes is dynamically regulated by methylation and demethylation. In Arabidopsis, active DNA demethylation is mediated by the ROS1 (repressor of silencing 1) subfamily of 5-methylcytosine DNA glycosylases through a base excision repair pathway. These demethylases have critical roles in erasing DNA methylation and preventing TGS of target genes. However, it is not known how the demethylases are targeted to specific sequences. Here we report the identification of ROS3, an essential regulator of DNA demethylation that contains an RNA recognition motif. Analysis of ros3 mutants and ros1 ros3 double mutants suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS. Gel mobility shift assays and analysis of ROS3 immunoprecipitate from plant extracts shows that ROS3 binds to small RNAs in vitro and in vivo. Immunostaining shows that ROS3 and ROS1 proteins co-localize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.