Mukesh Kumar Lalwani

Human cellular microRNA hsa-miR-29a interferes with viral nef protein expression and HIV-1 replication

BackgroundCellular miRNAs play an important role in the regulation of gene expression in eukaryotes. Recently, miRNAs have also been shown to be able to target and inhibit viral gene expression. Computational predictions revealed earlier that the HIV-1 genome includes regions that may be potentially targeted by human miRNAs. Here we report the functionality of predicted miR-29a target site in the HIV-1 nef gene.ResultsWe find that the human miRNAs hsa-miR-29a and 29b are expressed in human peripheral blood mononuclear cells. Expression of a luciferase reporter bearing the nef miR-29a target site was decreased compared to the luciferase construct without the target site. Locked nucleic acid modified anti-miRNAs targeted against hsa-miR-29a and 29b specifically reversed the inhibitory effect mediated by cellular miRNAs on the target site. Ectopic expression of the miRNA results in repression of the target Nef protein and reduction of virus levels.ConclusionOur results show that the cellular miRNA hsa-miR29a downregulates the expression of Nef protein and interferes with HIV-1 replication.

Systematic Transcriptome Wide Analysis of lncRNA-miRNA Interactions

Background Long noncoding RNAs (lncRNAs) are a recently discovered class of non-protein coding RNAs, which have now increasingly been shown to be involved in a wide variety of biological processes as regulatory molecules. The functional role of many of the members of this class has been an enigma, except a few of them like Malat and HOTAIR. Little is known regarding the regulatory interactions between noncoding RNA classes. Recent reports have suggested that lncRNAs could potentially interact with other classes of non-coding RNAs including microRNAs (miRNAs) and modulate their regulatory role through interactions. We hypothesized that lncRNAs could participate as a layer of regulatory interactions with miRNAs. The availability of genome-scale datasets for Argonaute targets across human transcriptome has prompted us to reconstruct a genome-scale network of interactions between miRNAs and lncRNAs. Results We used well characterized experimental Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) datasets and the recent genome-wide annotations for lncRNAs in public domain to construct a comprehensive transcriptome-wide map of miRNA regulatory elements. Comparative analysis revealed that in addition to targeting protein-coding transcripts, miRNAs could also potentially target lncRNAs, thus participating in a novel layer of regulatory interactions between noncoding RNA classes. Furthermore, we have modeled one example of miRNA-lncRNA interaction using a zebrafish model. We have also found that the miRNA regulatory elements have a positional preference, clustering towards the mid regions and 3′ ends of the long noncoding transcripts. We also further reconstruct a genome-wide map of miRNA interactions with lncRNAs as well as messenger RNAs. Conclusions This analysis suggests widespread regulatory interactions between noncoding RNAs classes and suggests a novel functional role for lncRNAs. We also present the first transcriptome scale study on miRNA-lncRNA interactions and the first report of a genome-scale reconstruction of a noncoding RNA regulatory interactome involving lncRNAs.

Dynamic expression of long non-coding RNAs (lncRNAs) in adult zebrafish.

Long non-coding RNAs (lncRNA) represent an assorted class of transcripts having little or no protein coding capacity and have recently gained importance for their function as regulators of gene expression. Molecular studies on lncRNA have uncovered multifaceted interactions with protein coding genes. It has been suggested that lncRNAs are an additional layer of regulatory switches involved in gene regulation during development and disease. LncRNAs expressing in specific tissues or cell types during adult stages can have potential roles in form, function, maintenance and repair of tissues and organs. We used RNA sequencing followed by computational analysis to identify tissue restricted lncRNA transcript signatures from five different tissues of adult zebrafish. The present study reports 442 predicted lncRNA transcripts from adult zebrafish tissues out of which 419 were novel lncRNA transcripts. Of these, 77 lncRNAs show predominant tissue restricted expression across the five major tissues investigated. Adult zebrafish brain expressed the largest number of tissue restricted lncRNA transcripts followed by cardiovascular tissue. We also validated the tissue restricted expression of a subset of lncRNAs using independent methods. Our data constitute a useful genomic resource towards understanding the expression of lncRNAs in various tissues in adult zebrafish. Our study is thus a starting point and opens a way towards discovering new molecular interactions of gene expression within the specific adult tissues in the context of maintenance of organ form and function.

Reverse genetics screen in zebrafish identifies a role of miR-142a-3p in vascular development and integrity.

MicroRNAs are a well-studied class of non-coding RNA and are known to regulate developmental processes in eukaryotes. Their role in key biological processes such as vasculature development has attracted interest. However, a comprehensive understanding of molecular regulation of angiogenesis and vascular integrity during development remains less explored. Here we identified miRNAs involved in the development and maintenance of vasculature in zebrafish embryos using a reverse genetics approach. Using a combination of bioinformatics predictions and literature based evidences we mined over 701 Human and 329 Zebrafish miRNAs to derive a list of 29 miRNAs targeting vascular specific genes in zebrafish. We shortlisted eight miRNAs and investigated their potential role in regulating vascular development in zebrafish transgenic model. In this screen we identified three miRNAs, namely miR-1, miR-144 and miR-142a-3p that have the potential to influence vascular development in zebrafish. We show that miR-142a-3p mediates vascular integrity and developmental angiogenesis in vivo. Overexpression of miR-142a-3p results in loss of vascular integrity, hemorrhage and vascular remodeling during zebrafish embryonic development, while loss of function of miR-142a-3p causes abnormal vascular remodeling. MiR-142a-3p functions in part by directly repressing cdh5 (VE-cadherin). The vascular abnormalities that results from modulation of miR-142a-3p are reminiscent of cdh5 perturbation in zebrafish embryos. We also demonstrate that the action of miR-142a on cdh5 is potentially regulated by Lmo2, an important transcription factor, known for its role in vasculature development. The miR142a-3p mediated control of cdh5 constitutes an additional layer of regulation for maintaining vascular integrity and developmental angiogenesis. These findings have implications in development, wound repair and tumor growth.

FishMap: a community resource for zebrafish genomics.

An enormous amount of information on a genomics scale is available for zebrafish (Danio rerio), which is a well-studied model organism for human diseases. However, a majority of this annotation is scattered in obscure data sources. There have been limited efforts to present it on a unified and integrated platform, which would help to understand the biological processes in this organism better. FishMap is a unified and centralized resource for storage, retrieval, and display of genomic information of zebrafish. The datasets have been methodically collected from various resources and supplementary information of publications and mapped to the zebrafish genome. The data are organized into nine major sections, which include comparative genomics, mapping and sequencing, gene and gene predictions, expression and regulation, and variation and repeats. A number of unique sections have been incorporated, which include tracks on noncoding gene annotation, location of retrovirus/transposon integrations in the genome, and their flanking genomic sequences and novel transcripts. The datasets are linked to related data sources. FishMap is built on the Gbrowse, which is a part of the Generic Model Organism Database Consortium Project. The resource also features a Web-based BLAST server for sequence homology search and a gene ID converter that would enable users to sift through different interchangeable gene annotation identifier systems. The database is amenable to programmatic access through the Distributed Annotation System as well as BioMoby protocols, thus making it a central community resource that can be integrated with existing data mining and analysis workflows. We hope that FishMap would be an integral resource for community participation in zebrafish genomics. The resource is freely available at http://miracle.igib.res.in/fishmap, or at http://fishmap.igib.res.in.

Exome sequencing of patients with histiocytoid cardiomyopathy reveals a de novo NDUFB11 mutation that plays a role in the pathogenesis of histiocytoid cardiomyopathy

Histiocytoid cardiomyopathy (Histiocytoid CM) is a rare form of cardiomyopathy observed predominantly in newborn females that is fatal unless treated early in life. We have performed whole exome sequencing on five parent‐proband trios and identified nuclear‐encoded mitochondrial protein mutations in three cases. The molecular genetic basis of Histiocytoid CM remains unknown despite several hypotheses in medical literature. The findings presented in this manuscript may represent components of genetic etiologies for this heterogeneous disease. Two probands had de novo non‐sense mutations in the second exon of the X‐linked nuclear gene NDUFB11. A third proband was doubly heterozygous for inherited rare variants in additional components of complex I, NDUFAF2 and NDUFB9, confirming that Histiocytoid CM is genetically heterogeneous. In a fourth case, the proband with Histiocytoid CM inherited a mitochondrial mutation from her heteroplasmic mother, as did her brother who presented with cardiac arrhythmia. Strong candidate recessive or compound heterozygous variants were not found for this individual or for the fifth case. Although NDUFB11 has not been implicated before in cardiac pathology, morpholino‐mediated knockdown of ndufb11 in zebrafish embryos generated defective cardiac tissue with cardiomegaly, looping defects, and arrhythmia which suggests the role of NDUFB11 in the pathogenesis of this abnormal cardiac pathology. Taken together, the unbiased whole exome sequencing approach confirms the suspected genetic heterogeneity of Histiocytoid CM. Therefore, the novel NDUFB11 mutation may cause a complex 1 deficiency in synergy with additional unknown mtDNA variants.

Proteomic analysis of zebrafish (Danio rerio) embryos exposed to cyclosporine A

Cyclosporine A, a potent immunosuppressive agent extensively used to prevent allograft rejections, is under scrutiny due to severe toxic effects. CsA therapy is often continued during pregnancy in conditions such as organ transplantations and autoimmune diseases. Herein, we investigated the effects of CsA on early morphogenesis of zebrafish and identified a spectrum of proteins whose expression was altered in the drug treated embryos. Time-lapse fluorescence imaging of germ-line double transgenic zebrafish embryos treated with CsA revealed severe blood regurgitation in heart chambers, absence of blood circulation in vessels, pericardial and yolk sac edema. We also observed lack of mature blood vessels and down-regulation of endothelial markers in CsA treated embryos. Proteomic analysis using 2D-DIGE followed by mass-spectrometry led to the identification of 37 proteins whose expression was significantly modulated in presence of the drug. These proteins were mostly associated with cytoskeletal/structural assembly, lipid-binding, stress response and metabolism. Furthermore, mRNA expression analysis of eight proteins and Western blotting of actin revealed consistency between the changes observed in protein expression and its corresponding mRNA levels. Our findings demonstrate that CsA administration during early morphogenesis in zebrafish modulates the expression of some proteins which are known to be involved in important physiological processes.

Morphological effects of G-quadruplex stabilization using a small molecule in zebrafish.

Zebrafish (Danio rerio) embryos are transparent and advantageous for studying early developmental changes due to ex utero development, making them an appropriate model for studying gene expression changes as a result of molecular targeting. Zebrafish embryos were injected with a previously reported G-quadruplex selective ligand, and the phenotypic changes were recorded. We report marked discrepancies in the development of intersegmental vessels. In silico analysis determined that the putative G-quadruplex motif occur in the upstream promoter region of the Cdh5 (N-cadherin) gene. A real-time polymerase chain reaction-based investigation indicated that in zebrafish, CDH-2 (ZN-cad) was significantly downregulated in the ligand-treated embryos. Biophysical characterization of the interaction of the ligand with the G-quadruplex motif found in this promoter yielded strong binding and stabilization of the G-quadruplex with this ligand. Hence, we report for the first time the phenotypic impact of G-quadruplex targeting with a ligand in a vertebrate organism. This study has unveiled not only G-quadruplex targeting in non-human animal species but also the potential that G-quadruplexes can provide a ready tool for understanding the phenotypic effects of targeting certain important genes involved in differentiation and developmental processes in a living eukaryotic organism.

Antagonism of microRNA function in zebrafish embryos by using locked nucleic acid enzymes (LNAzymes).

MicroRNAs (miRNAs) have crucial functions in many cellular processes, such as differentiation, proliferation and apoptosis; aberrant expression of miRNAs has been linked to human diseases, including cancer. Tools that allow specific and efficient knockdown of miRNAs would be of immense importance for exploring miRNA function. Zebrafish serves as an excellent vertebrate model system to understand the functions of miRNAs involved in a variety of biological processes. We designed and employed a strategy based on locked nucleic acid enzymes (LNAzymes) for in vivo knockdown of miRNA in zebrafish embryos. We demonstrate that LNAzyme can efficiently knockdown miRNAs with minimal toxicity to the zebrafish embryos.

The Zebrafish GenomeWiki: a crowdsourcing approach to connect the long tail for zebrafish gene annotation.

A large repertoire of gene-centric data has been generated in the field of zebrafish biology. Although the bulk of these data are available in the public domain, most of them are not readily accessible or available in nonstandard formats. One major challenge is to unify and integrate these widely scattered data sources. We tested the hypothesis that active community participation could be a viable option to address this challenge. We present here our approach to create standards for assimilation and sharing of information and a system of open standards for database intercommunication. We have attempted to address this challenge by creating a community-centric solution for zebrafish gene annotation. The Zebrafish GenomeWiki is a ‘wiki’-based resource, which aims to provide an altruistic shared environment for collective annotation of the zebrafish genes. The Zebrafish GenomeWiki has features that enable users to comment, annotate, edit and rate this gene-centric information. The credits for contributions can be tracked through a transparent microattribution system. In contrast to other wikis, the Zebrafish GenomeWiki is a ‘structured wiki’ or rather a ‘semantic wiki’. The Zebrafish GenomeWiki implements a semantically linked data structure, which in the future would be amenable to semantic search. Database URL: http://genome.igib.res.in/twiki