LncRTPred: Predicting RNA-RNA mode of interaction mediated by lncRNA.

Long non-coding RNAs (lncRNAs) play a significant role in various biological processes. Hence, it is utmost important to elucidate their functions in order to understand the molecular mechanism of a complex biological system. This versatile RNA molecule has diverse modes of interaction, one of which constitutes lncRNA-mRNA interaction. Hence, identifying its target mRNA is essential to understand the function of an lncRNA explicitly. Existing lncRNA target prediction tools mainly adopt thermodynamics approach. Large execution time and inability to perform real-time prediction limit their usage. Further, lack of negative training dataset has been a hindrance in the path of developing machine learning (ML) based lncRNA target prediction tools. In this work, we have developed a ML-based lncRNA-mRNA target prediction model- 'LncRTPred'. Here we have addressed the existing problems by generating reliable negative dataset and creating robust ML models. We have identified the non-interacting lncRNA and mRNAs from the unlabelled dataset using BLAT. It is further filtered to get a reliable set of outliers. LncRTPred provides a cumulative_model_score as the final output against each query. In terms of prediction accuracy, LncRTPred outperforms other popular target prediction protocols like LncTar. Further, we have tested its performance against experimentally validated disease-specific lncRNA-mRNA interactions. Overall, performance of LncRTPred is heavily dependent on the size of the training dataset, which is highly reflected by the difference in its performance for human and mouse species. Its performance for human species shows better as compared to that for mouse when applied on an unknown data due to smaller size of the training dataset in case of mouse compared to that of human. Availability of increased number of lncRNA-mRNA interaction data for mouse will improve the performance of LncRTPred in future. Both webserver and standalone versions of LncRTPred are available. Web server link: http://bicresources.jcbose.ac.in/zhumur/lncrtpred/index.html. Github Link: https://github.com/zglabDIB/LncRTPred.

Defense Surveillance System at the Interface: Response of Rice Towards Rhizoctonia solani During Sheath Blight Infection.

Sheath blight of rice caused by necrotrophic plant pathogen Rhizoctonia solani is one of the most common fungal diseases of rice leading to significant yield loss. Among the defense responses exhibited by the host plants towards fungal infections, those functional within the apoplast contribute significantly. Here, we have studied apoplastic defense response of rice towards R. solani during sheath blight infection. The transcriptome of R. solani-infected rice plants was compared with that of uninfected rice, to identify the set of defense genes that undergo differential expression and code for proteins with a predicted N-terminal signal peptide. Significant changes in the stress-responsive, molecular signal perception, protein modification, and metabolic process pathways represented by a group of differentially expressed genes were observed. Our data also revealed two secreted protease inhibitors from rice that exhibit increased expression during R. solani infection and induce disease resistance when expressed in Nicotiana benthamiana. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Heightened miR6024-NLR interactions facilitate necrotrophic pathogenesis in tomato.

KEY MESSAGE: miR6024 acts as a negative regulator of R genes, hence of Tomato plant immunity, and facilitates disease by the necrotrophic pathogen A. solani. Plant resistance genes or Nucleotide-binding leucine-rich repeat (NLR) genes, integral components of plant disease stress-signaling are targeted by variable groups of miRNAs. However, the significance of miRNA-mediated regulation of NLRs during a pathogen stress response, specifically for necrotrophic fungus, is poorly understood. A thorough examination of Tomato NLRs and miRNAs could map substantial interactions of which half the annotated NLRs were targets of Solanaceae-specific and conserved miRNAs, at the NB subdomain. The Solanaceae-specific miR6024 and its NLR targets analysed in different phytopathogenic stresses revealed differential and mutually antagonistic regulation. Interestingly, miR6024-targeted cleavage of a target NLR also triggered the generation of secondary phased siRNAs which could potentially amplify the defense signal. RNA-seq analysis of leaf tissues from miR6024 overexpressing Tomato plants evidenced a perturbation in the defense transcriptome with the transgenics showing unwarranted immune response-related genes' expression with or without infection with necrotrophic Alternaria solani, though no adverse effect could be observed in the growth and development of the transgenic plants. Transgenic plants exhibited constitutive downregulation of the target NLRs, aggravated disease phenotype with an enhanced lesion, greater ROS generation and hypersusceptibility to A. solani infection, thus establishing that miR6024 negatively impacts plant immune response during necrotrophic pathogenesis. Limited knowledge about the outcome of NLR-miRNA interaction during necrotrophic pathogenesis is a hindrance to the deployment of miRNAs in crop improvement programs. With the elucidation of the necrotrophic disease-synergistic role played by miR6024, it becomes a potent candidate for biotechnological manipulation for the rapid development of pathogen-tolerant solanaceous plants.

piRNAQuest V.2: an updated resource for searching through the piRNAome of multiple species.

PIWI interacting RNAs (piRNAs) have emerged as important gene regulators in recent times. Since the release of our first version of piRNAQuest in 2014, lots of novel piRNAs have been annotated in different species other than human, mouse and rat. Such new developments in piRNA research have led us to develop an updated database piRNAQuest V.2. It consists of 92,77,689 piRNA entries for 25 new species of different phylum along with human, mouse and rat. Besides providing primary piRNA features which include their genomic location, with further information on piRNAs overlapping with repeat elements, pseudogenes and syntenic regions, etc., the novel features of this version includes (i) density based cluster prediction, (ii) piRNA expression profile across various healthy and disease systems and (iii) piRNA target prediction. The concept of density-based piRNA cluster identification is robust as it does not consider parametric distribution in its model. The piRNA expression profile for 21 disease systems including cancer have been hosted in addition to 32 tissue specific piRNA expression profile for various species. Further, the piRNA target prediction section includes both predicted and curated piRNA targets within eight disease systems and developmental stages of mouse testis. Further, users can visualize the piRNA-target duplex structure and the ping-pong signature pattern for all the ping-pong piRNA partners in different species. Overall, piRNAQuest V.2 is an updated user-friendly database which will serve as a useful resource to survey, search and retrieve information on piRNAs for multiple species. This freely accessible database is available at http://dibresources.jcbose.ac.in/zhumur/pirnaquest2.

Repurposed drugs and nutraceuticals targeting envelope protein: A possible therapeutic strategy against COVID-19.

COVID-19 pandemic caused by SARS-CoV-2 has already claimed millions of lives worldwide due to the absence of a suitable anti-viral therapy. The CoV envelope (E) protein, which has not received much attention so far, is a 75 amino acid long integral membrane protein involved in assembly and release of the virus inside the host. Here we have used artificial intelligence (AI) and pattern recognition techniques for initial screening of FDA approved pharmaceuticals and nutraceuticals to target this E protein. Subsequently, molecular docking simulations have been performed between the ligands and target protein to screen a set of 9 ligand molecules. Finally, we have provided detailed insight into their mechanisms of action related to the varied symptoms of infected patients.

The Natural Antisense Transcript DONE40 Derived from the lncRNA ENOD40 Locus Interacts with SET Domain Protein ASHR3 During Inception of Symbiosis in Arachis hypogaea.

The long noncoding RNA ENOD40 is required for cortical cell division during root nodule symbiosis (RNS) of legumes, though it is not essential for actinorhizal RNS. Our objective was to understand whether ENOD40 was required for aeschynomenoid nodule formation in Arachis hypogaea. AhENOD40 express from chromosome 5 (chr5) (AhENOD40-1) and chr15 (AhENOD40-2) during symbiosis, and RNA interference of these transcripts drastically affected nodulation, indicating the importance of ENOD40 in A. hypogaea. Furthermore, we demonstrated several distinct characteristics of ENOD40. (i) Natural antisense transcript (NAT) of ENOD40 was detected from the AhENOD40-1 locus (designated as NAT-AhDONE40). (ii) Both AhENOD40-1 and AhENOD40-2 had two exons, whereas NAT-AhDONE40 was monoexonic. Reverse-transcription quantitative PCR analysis indicated both sense and antisense transcripts to be present in both cytoplasm and nucleus, and their expression increased with the progress of symbiosis. (iii) RNA pull-down from whole cell extracts of infected roots at 4 days postinfection indicated NAT-AhDONE40 to interact with the SET (Su(var)3-9, enhancer of Zeste and Trithorax) domain containing absent small homeotic disc (ASH) family protein AhASHR3 and this interaction was further validated using RNA immunoprecipitation and electrophoretic mobility shift assay. (iv) Chromatin immunoprecipitation assays indicate deposition of ASHR3-specific histone marks H3K36me3 and H3K4me3 in both of the ENOD40 loci during the progress of symbiosis. ASHR3 is known for its role in optimizing cell proliferation and reprogramming. Because both ASHR3 and ENOD40 from legumes cluster away from those in actinorhizal plants and other nonlegumes in phylogenetic distance trees, we hypothesize that the interaction of DONE40 with ASHR3 could have evolved for adapting the nodule organogenesis program for legumes.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Activating transcription factor 3 modulates the macrophage immune response to Mycobacterium tuberculosis infection via reciprocal regulation of inflammatory genes and lipid body formation.

Infection of macrophages by Mycobacterium tuberculosis elicits an immune response that clears the bacterium. However, the bacterium is able to subvert the innate immune response. Differential expression of transcription factors (TFs) is central to the dynamic balance of this interaction. Among other functions, TFs regulate the production of antibacterial agents such as nitric oxide, pro-inflammatory cytokines and neutral lipids which are stored in lipid bodies (LBs) and favour bacterial survival. Here, we demonstrate that the TF activating transcription factor 3 (ATF3) is upregulated early during infection of macrophages or mice. Depletion of ATF3 enhances mycobacterial survival in macrophages suggesting its host-protective role. ATF3 interacts with chromatin remodelling protein brahma-related gene 1 and both associate with the promoters of interleukin-12p40, interleukin-6 and nitric oxide synthase 2, to activate expression of these genes. Strikingly, ATF3 downregulates LB formation by associating at the promoters of positive regulators of LB formation such as cholesterol 25 hydroxylase and the microRNA-33 locus. ATF3 represses the association of the activating mark, acetyl histone H4 lysine 8 at the promoter of cholesterol 25 hydroxylase. Our study suggests opposing roles of ATF3 in regulation of distinct sets of macrophage genes during infection, converging on a host-protective immune response.

LncRBase V.2: an updated resource for multispecies lncRNAs and ClinicLSNP hosting genetic variants in lncRNAs for cancer patients.

The recent discovery of long non-coding RNA as a regulatory molecule in the cellular system has altered the concept of the functional aptitude of the genome. Since our publication of the first version of LncRBase in 2014, there has been an enormous increase in the number of annotated lncRNAs of multiple species other than Human and Mouse. LncRBase V.2 hosts information of 549,648 lncRNAs corresponding to six additional species besides Human and Mouse, viz. Rat, Fruitfly, Zebrafish, Chicken, Cow and C.elegans. It provides additional distinct features such as (i) Transcription Factor Binding Site (TFBS) in the lncRNA promoter region, (ii) sub-cellular localization pattern of lncRNAs (iii) lnc-pri-miRNAs (iv) Possible small open reading frames (sORFs) within lncRNA. (v) Manually curated information of interacting target molecules and disease association of lncRNA genes (vi) Distribution of lncRNAs across multiple tissues of all species. Moreover, we have hosted ClinicLSNP within LncRBase V.2. ClinicLSNP has a comprehensive catalogue of lncRNA variants present within breast, ovarian, and cervical cancer inferred from 561 RNA-Seq data corresponding to these cancers. Further, we have checked whether these lncRNA variants overlap with (i)Repeat elements,(ii)CGI, (iii)TFBS within lncRNA loci (iv)SNP localization in trait-associated Linkage Disequilibrium(LD) region, (v)predicted the potentially pathogenic variants and (vi)effect of SNP on lncRNA secondary structure. Overall, LncRBaseV.2 is a user-friendly database to survey, search and retrieve information about multi-species lncRNAs. Further, ClinicLSNP will serve as a useful resource for cancer specific lncRNA variants and their related information. The database is freely accessible and available at http://dibresources.jcbose.ac.in/zhumur/lncrbase2/.

Elucidating the gene regulatory networks modulating cancer stem cells and non-stem cancer cells in high grade serous ovarian cancer.

The origin and pathogenesis of epithelial ovarian cancer have perplexed investigators for decades. The most prevalent type of it is the high-grade serous ovarian carcinoma (HGSOv) which is a highly aggressive disease with high relapse rates and insurgence of chemo-resistance at later stages of treatment. These are driven by a rare population of stem cell like cancer cells called cancer stem cells (CSCs). We have taken up a systems approach to find out the common gene interaction paths between non-CSC tumor cells (CCs) and CSCs in HGSOv. Detailed investigation reveals a set of 17 Transcription Factors (named as pivot-TFs) which can govern changes in the mode of gene regulation along these paths. Overall, this work highlights a divergent road map of functional information relayed by these common key players in the two cell states, which might aid towards designing novel therapeutic measures to target the CSCs for ovarian cancer therapy.

ParStream-seq: An improved method of handling next generation sequence data.

The exponential growth of next generation sequencing (NGS) data has put forward the challenge for its storage as well as its efficient and faster analysis. Storing the entire amount of data for a particular experiment and its alignment to the reference genome is an essential step for any quantitative analysis of NGS data. Here, we introduce streaming access technique 'ParStream-seq' that splits the bulk sequence data, accessed from a remote repository into short manageable packets followed by executing their alignment process in parallel in each of the compute core. The optimal packet size with fixed number of reads is determined in the stream that maximizes system utilization. Result shows a reduction in the execution time and improvement in the memory footprint. Overall, this streaming access technique provides means to overcome the hurdle of storing the entire volume of sequence data corresponding to a particular experiment, prior to its analysis.

A systemic insight into astrocytoma biology across different grades.

Astrocytomas are the most common type of brain tumors, which originate from glial cells, and are classified into specific grades based on their histopathological behavior. To develop precise therapeutic strategies for the disease, it is important to identify the molecular signatures specific to each grade as well as the key factors responsible for the transition from one grade to the next. In this study, we have taken a systems approach to investigate the gene expression profiles of each grades of the disease by mapping shortest paths of gene interaction in each grade and also between one grade and the next. Module core genes govern the topology of these networks and serve as important functional players in each grade as well as help in grade transition events. Shortlisted among these module core genes are well-characterized players of glioma as well as novel molecules (32 grade-specific genes and 15 grade transition-specific genes), which influence important prooncogenic functions but have not been linked to glioma biology yet. These module core genes provide interesting insight into the biology of astrocytic tumors and are potential therapeutic targets for astrocytoma.

MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPß regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection.

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 ß (IL-1ß) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPß signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPßsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

Genome-wide mRNA-miRNA profiling uncovers a role of the microRNA miR-29b-1-5p/PHLPP1 signalling pathway in Helicobacter pylori-driven matrix metalloproteinase production in gastric epithelial cells.

Aberrant expression of microRNAs (miRNAs) is associated with tumour progression, extracellular matrix remodelling, and cell proliferation. miRNAs modulate host gene expression during infection by pathogens such as Helicobacter pylori, which is associated with varying degrees of gastric pathology. In order to gain insight into the regulation of gene expression by miRNAs during H. pylori infection of gastric epithelial cells and its likely downstream consequences, we analysed the transcriptomes and miRnomes of AGS cells infected with H. pylori. In silico analysis of miRNA-mRNA interactions suggested that miR-29b-1-5p was a likely regulator of pathways associated with gastric epithelial cell pathology. We validated PH domain leucine rich phosphatase 1 (PHLPP1), a negative regulator of the Akt signalling pathway, as a target of miR-29b-1-5p. In an in vivo mouse model, we observed that infection with H. pylori was associated with upregulation of miR-29b-1-5p and downregulation of PHLPP1. Transfection with either a mimic or an inhibitor of miR-29b-1-5p confirmed that downregulation of PHLPP1 upregulates Akt-dependent NF-κB signalling leading to activation of matrix metalloproteinases 2 and 9, players in the degradation of extracellular matrix during H. pylori infection. The secreted antigen HP0175 was associated with upregulation of miR-29b-1-5p, regulation of metalloproteinase activity, and migration of AGS cells. Our study suggests that targeting the miR-29b-1-5p/PHLPP1 signalling axis could be a potential host-directed approach for regulating the outcome of H. pylori infection.

Exploring the major cross-talking edges of competitive endogenous RNA networks in human Chronic and Acute Myeloid Leukemia.

BACKGROUND: Human Chronic and Acute Myeloid Leukemia are myeloproliferative disorders in myeloid lineage of blood cells characterized by accumulation of aberrant white blood cells. In cancer, the anomalous transcriptome includes deregulated expression of non-coding RNAs in conjunction with protein-coding mRNAs in human genome. The coding or non-coding RNA transcripts harboring miRNA-binding sites can converse with and regulate each other by explicitly contending for a limited pool of shared miRNAs and act as competitive endogenous RNAs (ceRNAs). An unifying hypothesis attributing 'modulation of expression of transcripts' in this fashion had been defined as 'competitive endogenous RNA hypothesis'. Network built with ceRNAs evidently offers a platform to elucidate complex regulatory interactions at post-transcriptional level in human cancers. METHODS: Contemplating cancers of human myeloid lineage we constructed ceRNA networks for CML and AML coding and non-coding repertoire utilizing patient sample data. Through functional enrichment analysis we selected the significant functional modules for transcripts being differentially expressed in Blastic phases of each cancer types with respect to Normal. After retrieving free energy of binding and duplex formation of shared miRNAs on ceRNAs, we performed statistical averaging of energy values over the ensemble of populations considering cellular system as in canonical (Iso-thermal) situation. RESULTS AND CONCLUSIONS: We aimed to shed light on 'Sibling Rivalry' in ceRNA partners from the perspective of statistical thermodynamics, identified major cross-talking tracks and ceRNAs influencing transcripts concerned in myeloid cancer systems. GENERAL SIGNIFICANCE: Insights into ceRNA-regulation will shed light on progression and prognosis of human Chronic and Acute Myeloid Leukemia.

Ganglioside GM2 mediates migration of tumor cells by interacting with integrin and modulating the downstream signaling pathway.

The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor.

Gene expression profiling of Mycobacterium tuberculosis Lipoarabinomannan-treated macrophages: A role of the Bcl-2 family member A1 in inhibition of apoptosis in mycobacteria-infected macrophages.

Macrophages play an important role in the establishment of infection by intracellular pathogens. Mycobacterium tuberculosis is known to inhibit apoptosis and to downregulate immune responses of host cells using various strategies, including activation of peroxisome proliferator-activated receptor (PPAR)γ. Mannose-capped lipoarabinomannan (ManLAM) is one of the known bacterial effectors that plays a role in subversion of host immunity and activation of PPARγ. Here, we have used an unbiased global gene expression profiling approach to understand (a) how ManLAM regulates host cell immune responses and (b) the role of PPARγ in modulating ManLAM-induced host cell signaling. We have demonstrated that ManLAM-dependent inhibition of macrophage apoptosis is mediated by the upregulation of the antiapoptotic B-cell CLL/lymphoma 2 (Bcl2) family member A1. Our in silico analyses suggested that ManLAM-mediated PPARγ signaling is linked to important functions such as phagocytosis, cytoskeleton remodeling, cell survival, and autophagy. We have validated that ManLAM upregulates signal transducer and activator of transcription (STAT5)α, an important transcriptional regulator of cell survival in a PPARγ-dependent manner.

PVT: an efficient computational procedure to speed up next-generation sequence analysis.

BACKGROUND: High-throughput Next-Generation Sequencing (NGS) techniques are advancing genomics and molecular biology research. This technology generates substantially large data which puts up a major challenge to the scientists for an efficient, cost and time effective solution to analyse such data. Further, for the different types of NGS data, there are certain common challenging steps involved in analysing those data. Spliced alignment is one such fundamental step in NGS data analysis which is extremely computational intensive as well as time consuming. There exists serious problem even with the most widely used spliced alignment tools. TopHat is one such widely used spliced alignment tools which although supports multithreading, does not efficiently utilize computational resources in terms of CPU utilization and memory. Here we have introduced PVT (Pipelined Version of TopHat) where we take up a modular approach by breaking TopHat's serial execution into a pipeline of multiple stages, thereby increasing the degree of parallelization and computational resource utilization. Thus we address the discrepancies in TopHat so as to analyze large NGS data efficiently. RESULTS: We analysed the SRA dataset (SRX026839 and SRX026838) consisting of single end reads and SRA data SRR1027730 consisting of paired-end reads. We used TopHat v2.0.8 to analyse these datasets and noted the CPU usage, memory footprint and execution time during spliced alignment. With this basic information, we designed PVT, a pipelined version of TopHat that removes the redundant computational steps during 'spliced alignment' and breaks the job into a pipeline of multiple stages (each comprising of different step(s)) to improve its resource utilization, thus reducing the execution time. CONCLUSIONS: PVT provides an improvement over TopHat for spliced alignment of NGS data analysis. PVT thus resulted in the reduction of the execution time to ~23% for the single end read dataset. Further, PVT designed for paired end reads showed an improved performance of ~41% over TopHat (for the chosen data) with respect to execution time. Moreover we propose PVT-Cloud which implements PVT pipeline in cloud computing system.

piRNAQuest: searching the piRNAome for silencers.

BACKGROUND: PIWI-interacting RNA (piRNA) is a novel and emerging class of small non-coding RNA (sncRNA). Ranging in length from 26-32 nucleotides, this sncRNA is a potent player in guiding the vital regulatory processes within a cellular system. Inspite of having such a wide role within cellular systems, piRNAs are not well organized and classified, so that a researcher can pool out the biologically relevant information concerning this class. DESCRIPTION: Here we present piRNAQuest- a unified and comprehensive database of 41749 human, 890078 mouse and 66758 rat piRNAs obtained from NCBI and different small RNA sequence experiments. This database provides piRNA annotation based on their localization in gene, intron, intergenic, CDS, 5/UTR, 3/UTR and repetitive regions which has not been done so far. We have also annotated piRNA clusters and have elucidated characteristic motifs within them. We have looked for the presence of piRNAs and piRNA clusters in pseudogenes, which are known to regulate the expression of protein coding transcripts by generating small RNAs. All these will help researchers progress towards solving the unanswered queries on piRNA biogenesis and their mode of action. Further, expression profile for piRNA in different tissues and from different developmental stages has been provided. In addition, we have provided several tools like 'homology search', 'dynamic cluster search' and 'pattern search'. Overall, piRNAQuest will serve as a useful resource for exploring human, mouse and rat piRNAome. The database is freely accessible and available at http://bicresources.jcbose.ac.in/zhumur/pirnaquest/. CONCLUSION: piRNAs play a remarkable role in stem cell self-renewal and various vital processes of developmental biology. Although researchers are mining different features on piRNAs, the exact regulatory mechanism is still fuzzy. Thus, understanding the true potential of these small regulatory molecules with respect to their origin, localization and mode of biogenesis is crucial. piRNAQuest will provide us with a better insight on piRNA origin and function which will help to explore the true potential of these sncRNAs.

MicroRNA-302 increases reprogramming efficiency via repression of NR2F2.

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.

NSAID targets SIRT3 to trigger mitochondrial dysfunction and gastric cancer cell death.

Gastric cancer (GC) is a deadly malignancy that demands effective therapeutic intervention capitalizing unique drug target/s. Here, we report that indomethacin, a cyclooxygenase non-selective non-steroidal anti-inflammatory drug, arrests GC cell growth by targeting mitochondrial deacetylase Sirtuin 3 (SIRT3). Interaction study revealed that indomethacin competitively inhibited SIRT3 by binding to nicotinamide adenine dinucleotide (NAD)-binding site. The Cancer Genome Atlas data meta-analysis indicated poor prognosis associated with high SIRT3 expression in GC. Further, transcriptome sequencing data of human gastric adenocarcinoma cells revealed that indomethacin treatment severely downregulated SIRT3. Indomethacin-induced SIRT3 downregulation augmented SOD2 and OGG1 acetylation, leading to mitochondrial redox dyshomeostasis, mtDNA damage, respiratory chain failure, bioenergetic crisis, mitochondrial fragmentation, and apoptosis via blocking the AMPK/PGC1α/SIRT3 axis. Indomethacin also downregulated SIRT3 regulators ERRα and PGC1α. Further, SIRT3 knockdown aggravated indomethacin-induced mitochondrial dysfunction as well as blocked cell-cycle progression to increase cell death. Thus, we reveal how indomethacin induces GC cell death by disrupting SIRT3 signaling.