Suman Ghosal

Competing Endogenous RNA: The Key to Posttranscriptional Regulation

Competing endogenous RNA, ceRNA, vie with messenger RNAs (mRNAs) for microRNAs (miRNAs) with shared miRNAs responses elements (MREs) and act as modulator of miRNA by influencing the available level of miRNA. It has recently been discovered that, apart from protein-coding ceRNAs, pseudogenes, long noncoding RNAs (lncRNAs), and circular RNAs act as miRNA “sponges” by sharing common MRE, inhibiting normal miRNA targeting activity on mRNA. These MRE sharing elements form the posttranscriptional ceRNA network to regulate mRNA expression. ceRNAs are widely implicated in many biological processes. Recent studies have identified ceRNAs associated with a number of diseases including cancer. This brief review focuses on the molecular mechanism of ceRNA as part of the complex post-transcriptional regulatory circuit in cell and the impact of ceRNAs in development and disease.

lnCeDB: database of human long noncoding RNA acting as competing endogenous RNA.

Long noncoding RNA (lncRNA) influences post-transcriptional regulation by interfering with the microRNA (miRNA) pathways, acting as competing endogenous RNA (ceRNA). These lncRNAs have miRNA responsive elements (MRE) in them, and control endogenous miRNAs available for binding with their target mRNAs, thus reducing the repression of these mRNAs. ln Ce DB provides a database of human lncRNAs (from GENCODE 19 version) that can potentially act as ceRNAs. The putative mRNA targets of human miRNAs and the targets mapped to AGO clipped regions are collected from TargetScan and StarBase respectively. The lncRNA targets of human miRNAs (up to GENCODE 11) are downloaded from miRCode database. miRNA targets on the rest of the GENCODE 19 lncRNAs are predicted by our algorithm for finding seed-matched target sites. These putative miRNA-lncRNA interactions are mapped to the Ago interacting regions within lncRNAs. To find out the likelihood of an lncRNA-mRNA pair for actually being ceRNA we take recourse to two methods. First, a ceRNA score is calculated from the ratio of the number of shared MREs between the pair with the total number of MREs of the individual candidate gene. Second, the P-value for each ceRNA pair is determined by hypergeometric test using the number of shared miRNAs between the ceRNA pair against the number of miRNAs interacting with the individual RNAs. Typically, in a pair of RNAs being targeted by common miRNA(s), there should be a correlation of expression so that the increase in level of one ceRNA results in the increased level of the other ceRNA. Near-equimolar concentration of the competing RNAs is associated with more profound ceRNA effect. In lnCeDB one can not only browse for lncRNA-mRNA pairs having common targeting miRNAs, but also compare the expression of the pair in 22 human tissues to estimate the chances of the pair for actually being ceRNAs. Availability: Downloadable freely from http://gyanxet-beta.com/lncedb/.

Long Noncoding RNAs: New Players in the Molecular Mechanism for Maintenance and Differentiation of Pluripotent Stem Cells

Maintenance of the pluripotent state or differentiation of the pluripotent state into any germ layer depends on the factors that orchestrate expression of thousands of genes through epigenetic, transcriptional, and post-transcriptional regulation. Long noncoding RNAs (lncRNAs) are implicated in the complex molecular circuitry in the developmental processes. The ENCODE project has opened up new avenues for studying these lncRNA transcripts with the availability of new datasets for lncRNA annotation and regulation. Expression studies identified hundreds of long noncoding RNAs differentially expressed in the pluripotent state, and many of these lncRNAs are found to control the pluripotency and stemness in embryonic and induced pluripotent stem cells or, in the reverse way, promote differentiation of pluripotent cells. They are generally transcriptionally activated or repressed by pluripotency-associated transcription factors and function as molecular mediators of gene expression that determine the pluripotent state of the cell. They can act as molecular scaffolds or guides for the chromatin-modifying complexes to direct them to bind into specific genomic loci to impart a repressive or activating effect on gene expression, or they can transcriptionally or post-transcriptionally regulate gene expression by diverse molecular mechanisms. This review focuses on recent findings on the regulatory role of lncRNAs in two main aspects of pluripotency, namely, self renewal and differentiation into any lineage, and elucidates the underlying molecular mechanisms that are being uncovered lately.

Dynamic changes in global microRNAome and transcriptome reveal complex miRNA-mRNA regulated host response to Japanese Encephalitis Virus in microglial cells.

Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation.

HNOCDB: A comprehensive database of genes and miRNAs relevant to head and neck and oral cancer

In spite of the wide prevalence of head, neck and oral cancer, HNOC, there is no integrated database on genes and miRNAs associated with all the carcinoma subtypes of HNOC. The objective is to compile a multilayered and comprehensive database of HNOC as a user-friendly resource for researchers devising novel therapeutic strategies. We present HNOCDB, the head, neck and oral cancer database, with the following key features: (i) it tabulates all the different categories of HNOC separately under appropriate subtype-names, and then puts them together in a table headlined All; (ii) the oncogenes/oncomiRs that cause HNOC are listed; their mutations, methylations and polymorphisms loci are marked, and the variations in their expression profiles relative to the normal are recorded; (iii) HNOCDB contains a chromosomal map of HNOC genes and miRNA; (iv) contains references that experimentally validate the reason for the inclusion of the genes and the miRNAs in HNOCDB. HNOCDB is freely accessible for academic and non-profit users via http://gyanxet.com/hno.html.

HumanViCe: host ceRNA network in virus infected cells in human.

Host-virus interaction via host cellular components has been an important field of research in recent times. RNA interference mediated by short interfering RNAs and microRNAs (miRNA), is a widespread anti-viral defense strategy. Importantly, viruses also encode their own miRNAs. In recent times miRNAs were identified as key players in host-virus interaction. Furthermore, viruses were shown to exploit the host miRNA networks to suite their own need. The complex cross-talk between host and viral miRNAs and their cellular and viral targets forms the environment for viral pathogenesis. Apart from protein-coding mRNAs, non-coding RNAs may also be targeted by host or viral miRNAs in virus infected cells, and viruses can exploit the host miRNA mediated gene regulatory network via the competing endogenous RNA effect. A recent report showed that viral U-rich non-coding RNAs called HSUR, expressed in primate virus herpesvirus saimiri (HVS) infected T cells, were able to bind to three host miRNAs, causing significant alteration in cellular level for one of the miRNAs. We have predicted protein coding and non protein-coding targets for viral and human miRNAs in virus infected cells. We identified viral miRNA targets within host non-coding RNA loci from AGO interacting regions in three different virus infected cells. Gene ontology (GO) and pathway enrichment analysis of the genes comprising the ceRNA networks in the virus infected cells revealed enrichment of key cellular signaling pathways related to cell fate decisions and gene transcription, like Notch and Wnt signaling pathways, as well as pathways related to viral entry, replication and virulence. We identified a vast number of non-coding transcripts playing as potential ceRNAs to the immune response associated genes; e.g., APOBEC family genes, in some virus infected cells. All these information are compiled in HumanViCe (http://gyanxet-beta.com/humanvice), a comprehensive database that provides the potential ceRNA networks in virus infected human cells.

An siRNA designing tool with a unique functional off-target filtering approach.

Investigations have revealed that silencing unwanted transcripts or off-targeting can induce false positive phenotype during RNA interference (RNAi)-based gene function study. But still the standard computational approaches towards small interfering RNA (siRNA) off-target minimization fall short in terms of addressing this false positive phenotype issue. Some of these off-targets may interfere with the biochemical pathway being investigated. It may also inadvertently target cell’s metabolic pathways with unquantifiable consequences on the processes of user’s interest. Here, we report the development of a siRNA selection tool that, for the first time, implements a functional off-target filtering that aims to minimize false positive phenotypes arising from inadvertent targets that are functionally similar or related to the direct target gene, along with a multi-parametric classifier (support vector machine) for optimized selection of potent siRNAs. The functional off-target filtering minimizes the number of off-target genes which are functionally related to the direct target gene, i.e. involved in a common biological process and may have similar phenotype. A text-mining algorithm is used to find related biological processes associated with the direct target and each off-target transcript by comparison of the biological processes associated with these genes. It also gives the user a choice to select one or more off-targets that may be potentially more harmful, from a predicted off-target gene list to be filtered out. Testing with huge set of biologically validated siRNAs from three different sources showed consistent good performance of our tool in terms of effective siRNA selection. It outperformed four potent siRNA selection algorithms of present day in terms of specificity in the selection of highly efficient siRNAs when compared on a common test set. A genome wide testing with potent siRNAs used in high-content screening confirmed validation of 2767 designed siRNAs in terms of phenotypic output. This tool presently supports siRNA designs for human genes and is freely available at http://gyanxet-beta.com.

SeedSeq: Off-Target Transcriptome Database

Detection of potential cross-reaction between a short oligonucleotide sequence and a longer (unintended) sequence is crucial for many biological applications, such as high content screening (HCS), microarray nucleotide probes, or short interfering RNAs (siRNAs). However, owing to a tolerance for mismatches and gaps in base-pairing with target transcripts, siRNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs (off-targets) besides their intended target transcripts (on-targets). The reference sequence (RefSeq) collection aims to provide a comprehensive, integrated, nonredundant, well-annotated set of sequences, including mRNA transcripts. We performed a detailed off-target analysis of three most commonly used kinome siRNA libraries based on the latest RefSeq version. To simplify the access to off-target transcripts, we created a SeedSeq database, a new unique format to store off-target information.

miRepress: modelling gene expression regulation by microRNA with non-conventional binding sites

Some earlier studies have reported an alternative mode of microRNA-target interaction. We detected target regions within mRNA transcripts from AGO PAR-CLIP that did not contain any conventional microRNA seed pairing but only had non-conventional binding sites with microRNA 3′ end. Our study from 7 set of data that measured global protein fold change after microRNA transfection pointed towards the association of target protein fold change with 6-mer and 7-mer target sites involving microRNA 3′ end. We developed a model to predict the degree of microRNA target regulation in terms of protein fold changes from the number of different conventional and non-conventional target sites present in the target, and found significant correlation of its output with protein expression changes. We validated the effect of non-conventional interactions with target by modulating the abundance of microRNA in a human breast cancer cell line MCF-7. The validation was done using luciferase assay and immunoblot analysis for our predicted non-conventional microRNA-target pair WNT1 (3′ UTR) and miR-367-5p and immunoblot analysis for another predicted non-conventional microRNA-target pair MYH10 (coding region) and miR-181a-5p. Both experiments showed inhibition of targets by transfection of microRNA mimics that were predicted to have only non-conventional sites.

Alteration of MicroRNA Biogenesis Pathways in Cancers

MicroRNAs (miRNAs) are tiny noncoding RNAs known for their critical role in posttranscriptional gene regulation. They play a major role in cell growth, proliferation, differentiation, immune response, and apoptosis. Aberration in the expressions of key oncogenic or tumor suppressor miRNAs is related to tumorigenesis. Global change in miRNA expression due to alterations in the miRNA processing machinery is also not uncommon in cancer. In this chapter, we will discuss the importance of miRNA biogenesis pathways in context of cancer.