Identification of Prognostic and Tumor Microenvironment by Shelterin Complex-Related Signatures in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the oral cavity. Shelterin complex gene (SG) has an important role in regulating telomere structure and length. SG is considered promising as a novel prognostic marker for cancer and a potential target for tumor therapy. However, SGs have not been systematically studied in OSCC. We analyzed SGs based on public data from OSCC patients and showed that SGs are closely associated with the prognosis of OSCC patients. Two different subtypes of SGs were identified in the TCGA and GEO cohorts, and LASSO regression analysis was used to further construct an SGs-related prognostic model. Randomized cohorts and different clinical subgroups validated the model's accuracy. The assessment of clinical characteristics, tumor mutational burden (TMB), and tumor microenvironment (TME) between high- and low-risk scores groups showed lower TMB, more abundant immune cell infiltration, and better prognosis in the low-risk group. According to the IPS analysis, patients in the low-risk group were more responsive to immunotherapy. This study establishes a foundation for research on SG and confirms that risk scores can predict prognosis and guide clinical treatment in OSCC patients.

2-Methoxyestradiol combined with ascorbic acid facilitates the apoptosis of chronic myeloid leukemia cells via the microRNA-223/Fms-like tyrosine kinase 3/phosphatidylinositol-3 kinase/protein kinase B axis.

Chronic myeloid leukemia (CML) is a malignant myeloproliferative tumor. 2-Methoxyestradiol (2-ME) is an endogenous estrogen metabolite that shows efficacy in human malignancies. Ascorbic acid (AA) possesses antioxidant activity. This study explored the mechanism of 2-ME combined with AA in the apoptosis of CML cells. Firstly, human CML cell lines were treated with 2-ME and AA. The cell viability, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were detected. miR-223 expression in CML cells was detected. In addition, CML cells were transfected with miR-223 inhibitor. The binding relationship between miR-223 and FLT3 was verified. Subsequently, the FLT3 was overexpressed or silenced for the function rescue experiment to confirm the role of FLT3 in CML cell apoptosis. The expression levels of key factors of the PI3K/AKT pathway were detected. Finally, xenograft nude mouse models were established for in vivo verification. 2-ME + AA treatment inhibited CML cell viability and promoted apoptosis, elevated ROS content, and reduced MMP. 2-ME + AA treatment promoted miR-223 expression in CML cells. miR-223 targeted FLT3. Moreover, miR-223 inhibitor or FLT3 overexpression partially annulled the effect of 2-ME + AA on CML cells. 2-ME + AA inhibited the PI3K/AKT pathway via the miR-223/FLT3 axis. Furthermore, 2-ME + AA suppressed CML xenograft growth in mice. Collectively, 2-ME + AA promoted miR-223 expression and suppressed FLT3 and the PI3K/AKT pathway, thereby facilitating the apoptosis of CML cells and inhibiting CML xenograft growth in mice.

The miR169b/NFYA1 module from the halophyte Halostachys caspica endows salt and drought tolerance in Arabidopsis through multi-pathways.

Salt and drought are the major abiotic stress factors plaguing plant growth, development and crop yields. Certain abiotic-stress tolerant plants have developed special mechanisms for adapting to adverse environments in the long process of evolution. Elucidating the molecular mechanisms by which they can exert resistance to abiotic stresses is beneficial for breeding new cultivars to guide agricultural production. Halostachys caspica, a perennial halophyte belonging to Halostachys in Amaranthaceae, is extremely tolerant to harsh environments, which is commonly grown in the saline-alkali arid desert area of Northwest, China. However, the molecular mechanism of stress tolerance is unclear. Nuclear Factor Y-A (NFYA) is a transcription factor that regulates the expression of downstream genes in plant response to adverse environments. It has also been reported that some members of the NFYA family are the main targets of miR169 in plants. In this study, we mainly focused on exploring the functions and preliminary mechanism of the miR169b/NFYA1 module from H. caspica to abiotic stress. The main results showed that RLM-RACE technology validated that HcNFYA1 was targeted by HcmiR169b, qRT-PCR revealed that HcmiR169b was repressed and HcNFYA1 was induced in the H. caspica branches under various abiotic stress as well ABA treatment and Arabidopsis stable transformation platform with molecular methods was applied to elucidate that the HcmiR169b/HcNFYA1 module conferred the salt and drought tolerance to plants by enhancing ABA synthesis and ABA signal transduction pathways, maintaining ROS homeostasis and the stability of cell membrane. HcNFYA1 is expected to be a candidate gene to improve plant resistance to salt and drought stresses.

The Role of Fecal Fusobacterium nucleatum and pks+ Escherichia coli as Early Diagnostic Markers of Colorectal Cancer.

BACKGROUND: Accurate analysis of intestinal microbiota will facilitate establishment of an evaluating system for assessing colorectal cancer (CRC) risk and prognosis. This study evaluates the potential role of Fusobacterium nucleatum (F. nucleatum) and Escherichia coli with a pks gene (pks+ E. coli) in early CRC diagnosis. METHODS: We recruited 139 patients, including CRC (n = 60), colorectal adenomatous polyposis (CAP) (n = 37), and healthy individuals (n = 42) based on their colonoscopy examinations. We collected stool and serum samples from the participants and measured the relative abundance of F. nucleatum and pks+ E. coli in fecal samples by quantitative PCR. Receiver operating characteristic curve (ROC) analyses were used to analyze the diagnostic value of single or combined biomarkers. RESULTS: Fecal F. nucleatum and pks+ E. coli levels were higher in the CRC group in either the CAP group or healthy controls (P = 0.02; 0.01). There was no statistical difference in the distribution of F. nucleatum and pks+ E. coli in patients with different tumor sites (P > 0.05). The combination of F. nucleatum+pks+ E. coli+CEA+CA19-9+FOBT was chosen as the optimal panel in differentiating both CRC and CAP from the controls. The combination of F. nucleatum, pks+ E. coli, and FOBT improved diagnostic efficiency. However, there was difficulty in differentiating CRC from CAP. CONCLUSION: Our results suggested that combining bacterial markers with conventional tumor markers improves the diagnostic efficiency for noninvasive diagnosis of CRC.

Enhanced Piezocatalytic Activity of Sr0.5Ba0.5Nb2O6 Nanostructures by Engineering Surface Oxygen Vacancies and Self-Generated Heterojunctions.

Piezocatalysis provides a promising strategy for directly converting weak mechanical energy into chemical energy. In this work, we report a simple one-step hydrogen reduction route for the simultaneous generation of surface defects and heterojunctions in Sr0.5Ba0.5Nb2O6 nanorods fabricated by a molten salt synthesis method. The as-fabricated Sr0.5Ba0.5Nb2O6/Sr2Nb2O7 nanocomposites with controllable oxygen vacancies exhibited excellent piezocatalytic activity under ultrasonic vibration, with an about 7 times enhancement of the rate constant (k = 0.0395 min-1) for rhodamine B degradation and an about 10 times enhancement of the water-splitting efficiency for hydrogen generation (109.4 µmol g-1 h-1) for the optimized sample (H2 annealed at 500 °C) compared to pristine Sr0.5Ba0.5Nb2O6 nanorods. This work demonstrates the essential role of a well-modulated oxygen vacancy concentration in the piezocatalytic activity and provides an inspiring guide for designing self-generated heterojunction piezocatalysts.

Bisphenol A triggers the malignancy of acute myeloid leukemia cells via regulation of IL-4 and IL-6.

Acute myeloid leukemia (AML) is a cancer of hematopoietic stem cells with a rapid progression. The progression of AML can be regulated by estrogenic signals. Our present data showed that an industrial endocrine-disrupting chemical, bisphenol A (BPA), can promote the proliferation of AML cells and decrease their sensitivity to daunorubicin and cytarabine treatment. Among the tested cytokines, BPA treatment can decrease the expression of interleukin-4 (IL-4) while increasing the expression of IL-6. Overexpression of IL-4 or neutralization antibody of IL-6 (anti-IL-6) can attenuate BPA-induced proliferation of AML cells and reverse BPA-suppressed chemosensitivity. Furthermore, activation of nuclear factor kappa B is essential for BPA-induced upregulation of IL-6 in AML cells. As to IL-4, BPA can increase the expression of NFAT1 to inhibit its transcription. Collectively, our data showed that BPA can trigger the malignancy of AML cells via regulation of IL-4 and IL-6.

Ginsenoside Rg3 protects mouse leydig cells against triptolide by downregulation of miR-26a.

BACKGROUND: Ginsenoside Rg3 has been reported to exert protection function on germ cells. However, the mechanisms by which Rg3 regulates apoptosis in mouse Leydig cells remain unclear. In addition, triptolide (TP) has been reported to induce infertility in male rats. Thus, this study aimed to investigate the protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells. METHODS: CCK-8, immunofluorescence assay, Western blotting and flow cytometry were used to detect cell proliferation and cell apoptosis, respectively. In addition, the dual luciferase reporter system assay was used to detect the interaction between miR-26a and GSK3ß in MLTC-1 cells. RESULTS: TP significantly inhibited the proliferation of MLTC-1 cells, while the inhibitory effect of TP was reversed by Rg3. In addition, TP markedly induced apoptosis in MLTC-1 cells via increasing the expressions of Bax, active caspase 3, Cyto c and active caspase 9, and decreasing the level of Bcl-2. However, Rg3 alleviated TP-induced apoptosis of MLTC-1 cells. Moreover, the level of miR-26a was obviously downregulated by Rg3 treatment. The protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells was abolished by miR-26a upregulation. Meanwhile, dual-luciferase assay showed GSK3ß was the direct target of miR-26a in MLTC-1 cells. Overexpression of miR-26a markedly decreased the level of GSK3ß. As expected, upregulation of miR-26a could abrogate the protective effects of Rg3 against TP-induced cytotoxicity via inhibiting the expression of GSK3ß. CONCLUSION: These results indicated that Rg3 could protect MLTC-1 against TP by downregulation of miR-26a. Therefore, Rg3 might serve as a potential agent for the treatment of male hypogonadism.

Overlapped differentially expressed genes between acute lymphoblastic leukemia and chronic lymphocytic leukemia revealed potential key genes and pathways involved in leukemia.

Common differentially expressed genes (DEGs) in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) might play critical roles in the pathogenesis and process of leukemia. We collected RNA sequencing (RNA-seq) data of human CLL, ALL samples, and normal peripheral blood CD19+ B cells as well as thymus samples, and analyzed similarities and differences between their transcriptomes using Cuffdiff2, DESeq, and edgeR. Compared with the RNA-seq data of normal peripheral blood CD19+ B cells and thymus samples, there were a large number of DEGs in ALL and CLL. DEGs in ALL and CLL not only have their distinguished features but also have a similar pattern. To figure out the common DEGs between CLL and ALL, we further identified 26 overlapped genes between CLL and ALL, among which 10 genes showed similar expression variation profiles whereas 16 genes showed opposite variation. The expression levels of 10 genes (SCML4, TNF-α, CD1C, FGFR1, MYO7B, DUSP1, PAP1GAP, MAN1C1, SLFN5, and CD8A) among the 26 genes were further confirmed by experiments, which was consistent with the results obtained by analyzing the RNA-seq data. The current study contributes to better understanding the pathophysiology of leukemia and unearthing novel potential prognostic markers and therapeutic targets of leukemia.

Copper-nanoparticle-dispersed amorphous BaTiO3 thin films as hole-trapping centers: enhanced photocatalytic activity and stability.

Nobel metal (Au and Ag) nanoparticles are often used in semiconductor photocatalysis to enhance the photocatalytic activity, while inexpensive Cu attracts less attention due to its easy oxidization. Herein, an elaborate study was conducted using Cu-nanoparticle-dispersed amorphous BaTiO3 films as photocatalysts. Photocatalytic and photoelectrochemical measurements demonstrated that the degradation efficiency and photocurrent density of the nanocomposite films are approximately 3.5 and 10 times as high as the pristine BaTiO3 film, respectively, which can be ascribed to a synergetic effect of the surface plasmon resonance and interband excitation. In addition, a good stability was also demonstrated by cyclic tests for the degradation of rhodamine B, which may be due to the amorphous nature of the BaTiO3 matrix providing hole-trapping centers. The high photocatalytic stability suggests that Cu is a promising alternative metal to replace Au and Ag for the development of cost-effective photocatalysts. Our work demonstrates a simple and promising strategy for improving the photostability of Cu nanomaterials and may provide a useful guideline for designing Cu-based composite materials toward various photocatalytic applications such as water pollution treatment.

Microarray is an efficient tool for circRNA profiling.

Circular RNAs (circRNAs) are emerging as a new class of endogenous and regulatory noncoding RNAs in latest years. With the widespread application of RNA sequencing (RNA-seq) technology and bioinformatics prediction, large numbers of circRNAs have been identified. However, at present, we lack a comprehensive characterization of all these circRNAs in interested samples. In this study, we integrated 87 935 circRNAs sequences that cover most of circRNAs identified till now represented in circBase to design microarray probes targeting back-splice site of each circRNA to profile expression of those circRNAs. By comparing the circRNA detection efficiency of RNA-seq with this circRNA microarray, we revealed that microarray is more efficient than RNA-seq for circRNA profiling. Then, we found ∼80 000 circRNAs were expressed in cervical tumors and matched normal tissues, and ∼25 000 of them were differently expressed. Notably, many of these circRNAs detected by this microarray can be validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) or RNA-seq. Strikingly, as many as ∼18 000 circRNAs could be robustly detected in cell-free plasma samples, and the expression of ∼2700 of them differed after surgery for tumor removal. Our findings provided a comprehensive and genome-wide characterization of circRNAs in paired normal tissues and tumors and plasma samples from multiple individuals. In addition, we also provide a rich resource with 41 microarray data sets and 10 RNA-seq data sets and strong evidences for circRNA expression in cervical cancer. In conclusion, circRNAs could be efficiently profiled by circRNA microarray to target their reported back-splice sites in interested samples.

Particulate matter pollution in Kunshan High-Tech zone: Source apportionment with trace elements, plume evolution and its monitoring.

Particulate matter (PM) in the Kunshan High-Tech zone is studied during a three-month campaign. PM and trace elements are measured by the online pollution monitoring, forecast-warning and source term retrieval system AS3. Hourly measured concentrations of PM10, PM2.5 and 16 trace elements in the PM2.5 section (Ca, Pb, Cu, Cl, V, Cr, Fe, Ti, Mn, Ni, Zn, Ga, As, Se, Sr, Ba) are focused. Source apportionment of trace elements by Positive Matrix Factorization modeling indicates that there are five major sources, including dust, industrial processing, traffic, combustion, and sea salt with contribution rate of 23.68%, 21.66%, 14.30%, 22.03%, and 6.89%, respectively. Prediction of plume dispersion from concrete plant and traffic emissions shows that PM10 pollution of concrete plant is three orders of magnitude more than that of the traffic. The influence range can extend to more than 3km in 1hr. Because the footprint of the industrial plumes is constantly moving according to the local meteorological conditions, the fixed monitoring sites scattered in a few hundred meters haven't captured the heaviest pollution plume at the local scale of a few km2. As a more intensive monitoring network is not operationally possible, the use of online modeling gives accurate and quantitative information of plume location, which increases the spatial pollution monitoring capacity and improves the understanding of measurement data. These results indicate that the development of the AS3 system, which combines monitoring equipment and air pollution modeling systems, is beneficial to the real-time pollution monitoring in the industrial zone.

MiR-182-5p regulates BCL2L12 and BCL2 expression in acute myeloid leukemia as a potential therapeutic target.

The importance of microRNAs (miRNAs) are shown during various cancers including acute myeloid leukemia (AML). MiR-182-5p functions as an oncogene or a potential suppressive miRNA in cancers, but its expression and function in AML is unknown. The purpose is to investigate the roles of miR-182-5p in AML in this study. MiR-182-5p was examined in the blood samples of AML and it was found that miR-182-5p expression levels were higher in AML tissues than it in their normal controls, so did in the AML cells. BCL2L12 and BCL2 were predicted as target genes of miR-182-5p and verified using luciferase reporter assay. BCL2L12 and BCL2 mRNA and protein levels were up-regulated in the AML cells with miR-182-5p inhibition. Cellular function of miR-182-5p indicated that miR-182-5p suppression in AML cells could decrease cell proliferation and reverse cisplatin (DDP) resistance via targeting BCL2L12 and BCL2 expression. Inhibition of miR-182-5p promoted AML cell apoptosis by targeting BCL2 or BCL2L12. The study demonstrates that high levels of miR-182-5p in AML promotes cell proliferation and suppresses cell apoptosis by targeting BCL2L12 and BCL2.

Selection of suitable reference genes for quantitative RT-PCR normalization in the halophyte Halostachys caspica under salt and drought stress.

The plants are always subjected to various environmental stress, because of plant sessile growth. qRT-PCR is a sensitive and reliable technology, and the normalization of target gene expression with suitable reference genes is very important for obtaining accurate data. Halostachys caspica is an extremely salt-tolerant halophyte belonging to Chenopodiaceae and a good candidate to explore the stress-physiological and molecular mechanism. To get truly the expression profiles of coding genes and miRNAs in H. caspica in response to salt and drought stress using qRT-PCR, suitable reference genes need to be confirmed. In this study, 10 candidate genes including ACT, UBC10, UBC13, TUB2, TUB3, EF1α, 5S rRNA, tRNA, U6 and miR1436 from H. caspica are chosen, and among them, the former nine are commonly used as internal control genes, and miR1436 with high sequence copies is no significant difference expression in high salinity-treated and untreated small RNA libraries of this species. The three softwares are used to analyze expression stability. The results showed that EF1α and TUB3 were the most stable under salt and drought stress, respectively, and UBC10 was the most constant aross all the samples with the both stressed combination. This work will benefit deep studies on abiotic tolerance in H. caspica.

The Splicing Factor PRP31 Is Involved in Transcriptional Gene Silencing and Stress Response in Arabidopsis.

Although DNA methylation is known to play an important role in the silencing of transposable elements (TEs) and introduced transgenes, the mechanisms that generate DNA methylation-independent transcriptional silencing are poorly understood. Previous studies suggest that RNA-directed DNA methylation (RdDM) is required for the silencing of the RD29A-LUC transgene in the Arabidopsis ros1 mutant background with defective DNA demethylase. Loss of function of ARGONAUTE 4 (AGO4) gene, which encodes a core RdDM component, partially released the silencing of RD29A-LUC in the ros1/ago4 double mutant plants. A forward genetic screen was performed to identify the mutants with elevated RD29A-LUC transgene expression in the ros1/ago4 mutant background. We identified a mutation in the homologous gene of PRP31, which encodes a conserved pre-mRNA splicing factor that regulates the formation of the U4/U6.U5 snRNP complex in fungi and animals. We previously demonstrated that the splicing factors ZOP1 and STA1 contribute to transcriptional gene silencing. Here, we reveal that Arabidopsis PRP31 associates with ZOP1, STA1, and several other splicing-related proteins, suggesting that these splicing factors are both physically and functionally connected. We show that Arabidopsis PRP31 participates in transcriptional gene silencing. Moreover, we report that PRP31, STA1, and ZOP1 are required for development and stress response. Under cold stress, PRP31 is not only necessary for pre-mRNA splicing but also for regulation of cold-responsive gene expression. Our results suggest that the splicing machinery has multiple functions including pre-mRNA splicing, gene regulation, transcriptional gene silencing, and stress response.

SUVR2 is involved in transcriptional gene silencing by associating with SNF2-related chromatin-remodeling proteins in Arabidopsis.

The SU(VAR)3-9-like histone methyltransferases usually catalyze repressive histone H3K9 methylation and are involved in transcriptional gene silencing in eukaryotic organisms. We identified a putative SU(VAR)3-9-like histone methyltransferase SUVR2 by a forward genetic screen and demonstrated that it is involved in transcriptional gene silencing at genomic loci targeted by RNA-directed DNA methylation (RdDM). We found that SUVR2 has no histone methyltransferase activity and the conserved catalytic sites of SUVR2 are dispensable for the function of SUVR2 in transcriptional silencing. SUVR2 forms a complex with its close homolog SUVR1 and associate with three previously uncharacterized SNF2-related chromatin-remodeling proteins CHR19, CHR27, and CHR28. SUVR2 was previously thought to be a component in the RdDM pathway. We demonstrated that SUVR2 contributes to transcriptional gene silencing not only at a subset of RdDM target loci but also at many RdDM-independent target loci. Our study suggests that the involvement of SUVR2 in transcriptional gene silencing is related to nucleosome positioning mediated by its associated chromatin-remodeling proteins.

The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci.

RNA-directed DNA methylation (RdDM) is required for transcriptional silencing of transposons and other DNA repeats in Arabidopsis thaliana. Although previous research has demonstrated that the SET domain-containing SU(VAR)3-9 homologs SUVH2 and SUVH9 are involved in the RdDM pathway, the underlying mechanism remains unknown. Our results indicated that SUVH2 and/or SUVH9 not only interact with the chromatin-remodeling complex termed DDR (DMS3, DRD1, and RDM1) but also with the newly characterized complex composed of two conserved Microrchidia (MORC) family proteins, MORC1 and MORC6. The effect of suvh2suvh9 on Pol IV-dependent siRNA accumulation and DNA methylation is comparable to that of the Pol V mutant nrpe1 and the DDR complex mutant dms3, suggesting that SUVH2 and SUVH9 are functionally associated with RdDM. Our CHIP assay demonstrated that SUVH2 and SUVH9 are required for the occupancy of Pol V at RdDM loci and facilitate the production of Pol V-dependent noncoding RNAs. Moreover, SUVH2 and SUVH9 are also involved in the occupancy of DMS3 at RdDM loci. The putative catalytic active site in the SET domain of SUVH2 is dispensable for the function of SUVH2 in RdDM and H3K9 dimethylation. We propose that SUVH2 and SUVH9 bind to methylated DNA and facilitate the recruitment of Pol V to RdDM loci by associating with the DDR complex and the MORC complex.

DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV.

DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase IV (Pol IV) transcribes target sequences to initiate 24-nt siRNA production and action. The putative DNA binding protein DTF1/SHH1 of Arabidopsis has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. DTF1 interacts directly with the chromatin remodeling protein CLASSY 1 (CLSY1), and both DTF1 and CLSY1 are associated in vivo with Pol IV but not Pol V, which functions downstream in the RdDM effector complex. DTF1 and DTF2 (a DTF1-like protein) contain a SAWADEE domain, which was found to bind specifically to histone H3 containing H3K9 methylation. Taken together, our results show that DTF1 is a core component of the RdDM pathway, and suggest that DTF1 interacts with CLSY1 to assist in the recruitment of Pol IV to RdDM target loci where H3K9 methylation may be an important feature. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci.

The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis.

DNA methylation in transposons and other DNA repeats is conserved in plants as well as in animals. In Arabidopsis thaliana, an RNA-directed DNA methylation (RdDM) pathway directs de novo DNA methylation. We performed a forward genetic screen for suppressors of the DNA demethylase mutant ros1 and identified a novel Zinc-finger and OCRE domain-containing Protein 1 (ZOP1) that promotes Pol IV-dependent siRNA accumulation, DNA methylation, and transcriptional silencing. Whole-genome methods disclosed the genome-wide effects of zop1 on Pol IV-dependent siRNA accumulation and DNA methylation, suggesting that ZOP1 has both RdDM-dependent and -independent roles in transcriptional silencing. We demonstrated that ZOP1 is a pre-mRNA splicing factor that associates with several typical components of the splicing machinery as well as with Pol II. Immunofluorescence assay revealed that ZOP1 overlaps with Cajal body and is partially colocalized with NRPE1 and DRM2. Moreover, we found that the other development-defective splicing mutants tested including mac3a3b, mos4, mos12 and mos14 show defects in RdDM and transcriptional silencing. We propose that the splicing machinery rather than specific splicing factors is involved in promoting RdDM and transcriptional silencing.

IDN2 and its paralogs form a complex required for RNA-directed DNA methylation.

IDN2/RDM12 has been previously identified as a component of the RNA-directed DNA methylation (RdDM) machinery in Arabidopsis thaliana, but how it functions in RdDM remains unknown. By affinity purification of IDN2, we co-purified two IDN2 paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2). The coiled-coil domain between the XS and XH domains of IDN2 is essential for IDN2 homodimerization, whereas the IDN2 C-terminal XH domain but not the coiled-coil domain is required for IDN2 interaction with IDP1 and IDP2. By introducing the wild-type IDN2 sequence and its mutated derivatives into the idn2 mutant for complementation testing, we demonstrated that the previously uncharacterized IDN2 XH domain is required for the IDN2-IDP1/IDP2 complex formation as well as for IDN2 function. IDP1 is required for de novo DNA methylation, siRNA accumulation, and transcriptional gene silencing, whereas IDP2 has partially overlapping roles with IDP1. Unlike IDN2, IDP1 and IDP2 are incapable of binding double-stranded RNA, suggesting that the roles of IDP1 and IDP2 are different from those of IDN2 in the IDN2-IDP1/IDP2 complex and that IDP1 and IDP2 are essential for the functioning of the complex in RdDM.

An atypical component of RNA-directed DNA methylation machinery has both DNA methylation-dependent and -independent roles in locus-specific transcriptional gene silencing.

RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. RdDM mediates the transcriptional silencing of many endogenous genomic loci, most of which are transposon related. A forward genetics screen identified DTF1 (DNA-binding transcription factor 1) as a new component for RdDM in Arabidopsis. Loss-of-function mutations in DTF1 release the transcriptional silencing of RdDM target loci and reduce the accumulation of 24-nt small interfering RNAs (siRNAs) from some of the targets. Interestingly, in the dtf1 mutant plants, the release of transcriptional gene silencing at solo-LTR is accompanied by decreased siRNA accumulation but not by reduced DNA methylation. These results suggest that DTF1 is an atypical component of RdDM and has both DNA methylation-dependent and -independent roles in transcriptional gene silencing. We suggest that besides DNA methylation, siRNAs may cause some other uncharacterized epigenetic modifications that lead to transcriptional gene silencing.