Publications of NIPGR Scientists
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Item Saccharomyces cerevisiae polo-like kinase, Cdc5 exhibits ATP-dependent Mg2+ -enhanced kinase activity in vitro(Elsevier B.V., 2019) Chauhan, Sujata; Samanta, Subhasis; Sharma, Nitin; Thakur, Jitendra K.; Dev, Kamal; Sourirajan, AnuradhaPhosphorylation of proteins on serine/threonine residues represents an important biochemical mechanism to regulate several cellular processes. Polo-like kinases (PLKs) are a family of serine-threonine kinases that play an imminent role in cell cycle regulation in yeast to humans, and thus an important therapeutic target for cancers. The present study provides insights into the enzymatic features of Saccharomyces cerevisiae PLK, Cdc5 using in vitro casein phosphorylation assays. The recombinant yeast PLK, GST-Cdc5 showed maximum casein phosphorylation activity at 30 °C, pH 9 and 45 min of incubation period. GST-Cdc5 exhibited a KM of 1.35 μM for casein, and high affinity for ATP, since addition of non-radioactive ATP chased out casein phosphorylation by radiolabeled ATP. The recombinant enzyme showed maximum kinase activity at 2.7 μM of GST-Cdc5. Casein was found to be the best in vitro substrate of GST-Cdc5 followed by BSA (Bovine Serum Albumin) and MBP (Myelin Basic Protein). Of the metal ions tested, Mg2+ (at 20 mM) was found to enhance GST-Cdc5 kinase activity, while Ca2+ (at 5 mM) and Mn2+ (at 10 mM) inhibited the same. The presence of EDTA, SDS and PMSF inhibited phosphorylation by GST-Cdc5, while DTT had no effect. The recombinant GST-Cdc5 can be used as a tool for deciphering PLKs’ structure and functions, which are still at infancy.Item Characterization of mediator complex and its associated proteins from rice(Springer, 2017) Samanta, Subhasis; Thakur, Jitendra K.The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.Item Role of plant mediator complex in stress response(Springer, 2015) Samanta, Subhasis; Thakur, Jitendra K.Class II gene loci of eukaryotes are transcribed by RNA Polymerase II, which functions in coordination with several other proteins like transcription factors, general transcription factors, and cofactors. Recently, Mediator complex, a multi-subunit, megadalton size protein complex has gained lots of attention as an important component of RNA pol II transcriptional machinery because of its essentiality in the regulation of most of the class II genes. Like yeast and other metazoans, plants also possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Recent times have experienced a flurry of scientific papers containing the functional information of individual Mediator subunits in plants, although many were reported earlier without consideration of their association with the Mediator complex. Among its diverse functional aspects, several reports have established the Mediator complex as an important integrative hub of different biotic and abiotic stress signaling pathways, which have been discussed in this chapter from the functional genomics perspectives. Although reports are emerging in support of its inclusion as a component of the basic transcriptional machinery, the gene selective roles of the individual Mediator subunits are proven and indisputably accepted.Item Importance of mediator complex in the regulation and integration of diverse signaling pathways in plants(Frontiers Media S.A., 2015) Samanta, Subhasis; Thakur, Jitendra K.Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes.Item The interplay of HuR and miR-3134 in regulation of AU rich transcriptome(Taylor & Francis Group, 2013) Sharma, Shivani; Verma, Suneer; Vasudevan, Madavan; Samanta, Subhasis; Thakur, Jitendra K.; Kulshreshtha, RituMicroRNAs and AU Rich element (ARE)-mediated degradation of transcripts are thought to be two independent means of gene regulation at the post-transcriptional level. However, since their site of action is the same (3'UTR of mRNA), there exists a high probability that specific miRNAs may bind to AREs and, thus, interact with ARE-binding proteins (ARE-BPs) to regulate transcript levels. In this study, we have characterized AREs as potential targets of hsa-miR-3134. An analysis of the global gene expression profile of breast cancer cell line MCF7 overexpressing miR-3134 revealed the presence of at least one AUUUA element in the 3'-UTRs of 63% of miR-3134 regulated protein coding genes. Quantitative RT-PCR or 3'UTR luciferase assays show that miR-3134 mediates an up to 4-8-fold increase in the levels of ARE bearing transcripts-SOX9, VEGFA, and EGFR, while mutated miR-3134 shows a decreased effect. The miR-3134-mediated increase in transcript levels was unaffected by treatment with transcription inhibitor (actinomycin D), indicating that miR-3134 enhances transcript stability. To investigate a possible interplay between miR-3134 and a prototype ARE-BP, HuR, we compared their overexpression transcriptome profiles. Interestingly, up to 80% of miR-3134-regulated genes were also regulated by HuR. Overexpression studies of HuR alone or in combination with miR-3134 shows that wt miR-3134 but not a mutated miR-3134 promotes stabilization of HuR-regulated transcripts SOX9, VEGFA, and EGFR as confirmed by qRT-PCR or RNA-immunoprecipitation experiments. Overall, this report suggests that collaboration between ARE-binding microRNAs and ARE-binding proteins could be a general mechanism of 3'-UTR mediated regulation of gene expression in human cells.