Publications of NIPGR Scientists
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Item MEDIATOR SUBUNIT17 is required for transcriptional optimization of root system architecture in Arabidopsis(Oxford University Press, 2023) Agrawal, Rekha; Singh, Amrita; Giri, Jitender; Magyar, Zoltan; Thakur, Jitendra K.Sucrose and auxin are well-known determinants of root system architecture (RSA). However, the factors that connect the signaling pathways evoked by these two critical factors during root development are poorly understood. In this study, we report the role of MEDIATOR SUBUNIT17 (MED17) in RSA and its involvement in the transcriptional integration of sugar and auxin signaling pathways in Arabidopsis (Arabidopsis thaliana). Sucrose regulates root meristem activation through the TARGET OF RAPAMYCIN-E2 PROMOTER BINDING FACTOR A TOR-E2FA pathway, and auxin regulates lateral root (LR) development through AUXIN RESPONSE FACTOR-LATERAL ORGAN BOUNDARIES DOMAIN ARF-LBDs. Both sucrose and auxin play a vital role during primary and LR development. However, there is no clarity on how sucrose is involved in the ARF-dependent regulation of auxin-responsive genes. This study establishes MED17 as a nodal point to connect sucrose and auxin signaling. Transcription of MED17 was induced by sucrose in an E2FA/B-dependent manner. Moreover, E2FA/B interacted with MED17, which can aid in the recruitment of the Mediator complex on the target promoters. Interestingly, E2FA/B and MED17 also occupied the promoter of ARF7, but not ARF19, leading to ARF7 expression, which then activates auxin signaling and thus initiates LR development. MED17 also activated cell division in the root meristem by occupying the promoters of cell-cycle genes, thus regulating their transcription. Thus, MED17 plays an important role in relaying the transcriptional signal from sucrose to auxin-responsive and cell-cycle genes to regulate primary and lateral root development, highlighting the role of the Mediator as the transcriptional processor for optimal root system architecture in Arabidopsis.Item MedProDB: A database of mediator proteins(Elsevier B.V., 2021) Bhardwaj, Rohan; Thakur, Jitendra K.; Kumar, ShaileshIn the last three decades, the multi-subunit Mediator complex has emerged as the key component of transcriptional regulation of eukaryotic gene expression. Although there were initial hiccups, recent advancements in bioinformatics tools contributed significantly to in-silico prediction and characterization of Mediator subunits from several organisms belonging to different eukaryotic kingdoms. In this study, we have developed the first database of Mediator proteins named MedProDB with 33,971 Mediator protein entries. Out of those, 12531, 11545, and 9895 sequences belong to metazoans, plants, and fungi, respectively. Apart from the core information consisting of sequence, length, position, organism, molecular weight, and taxonomic lineage, additional information of each Mediator sequence like aromaticity, hydropathy, instability index, isoelectric point, functions, interactions, repeat regions, diseases, sequence alignment to Mediator subunit family, Intrinsically Disordered Regions (IDRs), Post-translation modifications (PTMs), and Molecular Recognition Features (MoRFs) may be of high utility to the users. Furthermore, different types of search and browse options with four different tools namely BLAST, Smith-Waterman Align, IUPred, and MoRF-Chibi_Light are provided at MedProDB to perform different types of analysis. Being a critical component of the transcriptional machinery and regulating almost all the aspects of transcription, it generated lots of interest in structural and functional studies of Mediator functioning. So, we think that the MedProDB database will be very useful for researchers studying the process of transcription. This database is freely available at http://14.139.61.8/MedProDB/index.htmlItem Kinase module of mediator complex: important signalling processor for development and survival of plants(Oxford University Press, 2021) Agrawal, Rekha; Jiří, Fajkus; Thakur, Jitendra K.Mediator, a multisubunit protein complex is a signal processor that conveys regulatory information from transcription factors (TFs) to RNA Polymerase II (RNA Pol II) and therefore, plays an important role in the regulation of gene expression. This mega Dalton complex comprises four modules namely Head, Middle, Tail, and Kinase. The first three modules form the core part of the complex whereas association of the Kinase module is facultative. Being able to tweak the function of Mediator, the Kinase module has established itself as a major transcriptional regulator of numerous developmental and biochemical processes. The Kinase module consists of MED12, MED13, CycC, and kinase CDK8. Upon association with Mediator, the Kinase module can alter its structure and function dramatically. In the last decade, it has been established that the Kinase module is very important for plant growth and development and the fight against biotic and abiotic challenges. However, there is no comprehensive review discussing these findings in detail and depth. In this review, we have surveyed the regulation of Kinase module subunits and highlighted the multitude of their functions in plants. Coordination between the subunits to process different signals for optimum plant growth and development is also discussed.Item The Mediator subunit OsMED15a is a transcriptional co-regulator of seed size/weight-modulating genes in rice(Elsevier B.V., 2019) Dwivedi, Nidhi; Maji, Sourobh; Waseem, Mohd; Thakur, Pallabi; Kumar, Vinay; Parida, Swarup K.; Thakur, Jitendra K.Although several transcription factors (TFs) that regulate seed size/weight in plants are known, the molecular landscape regulating this important trait is unclear. Here, we report that a Mediator subunit, OsMED15a, links rice grain size/weight-regulating TFs to their target genes. Expression analysis and high-resolution quantitative trait loci (QTL) mapping suggested that OsMED15a is involved in rice seed development. OsMED15a has an N-terminal, three-helical KIX domain. Two of these helices, α1 and α3, and three amino acids, 76LRC78, within OsMED15a helix α3 were important for its interaction with several proteins, including interactions with the transactivation domains of two NAC-type TFs, OsNAC024 and OsNAC025. Moreover, OsMED15a, OsNAC024, and OsNAC025 all exhibited increased expression during seed development, and we identified several grain size/weight-associated SNPs in these genes in 509 low- and high-grain-weight rice genotypes. RNAi-mediated repression of OsMED15a expression down-regulated the expression of the grain size/weight regulating genes GW2, GW5 and DR11 and reduced grain length, weight, and yield. Of note, both OsNAC024 and OsNAC025 bound to the promoters of these three genes. We conclude that the transactivation domains of OsNAC024 and OsNAC025 target the KIX domain of OsMED15a in the regulation of grain size/weight-associated genes such as GW2, GW5, and D11. We propose that the integrated molecular-genetics approach used here could help identify networks of functional alleles of other regulator and co-regulator genes and thereby inform efforts for marker-assisted introgression of useful alleles in rice crop improvement.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 Shared functions of plant and mammalian StAR-related lipid transfer (START) domains in modulating transcription factor activity(BioMed Central Ltd, 2014) Schrick, Kathrin; Bruno, Michael; Khosla, Aashima; Cox, Paige N.; Marlatt, Sara A.; Roque, Remigio A.; Nguyen, Henry C.; He, Cuiwen; Snyder, Michael P.; Singh, Daljit; Yadav, GitanjaliBackground Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity. Results Our results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein–metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts. Conclusions The START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein’s nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity.Item MYC2, a bHLH transcription factor, modulates the adult phenotype of SPA1(Landes Bioscience, 2010) Gangappa, Sreeramaiah N.; Chattopadhyay, SudipMYC2 and SPA1 are two key regulatory proteins that negatively regulate light-controlled Arabidopsis seedling development. We have recently demonstrated the genetic and molecular relationships of MYC2 and SPA1 in light and JA (jasmonic acid) signaling pathways. Here, we have further shown the genetic interactions between these two proteins in flowering time and lateral root development.Item Leader RNA binding ability of chandipura virus P protein is regulated by its phosphorylation status: A possible role in genome transcription-replication switch(Elsevier B.V., 2003) Basak, Soumen; Raha, Tamal; Chattopadhyay, Debasis; Majumder, Amitabha; Shaila, M.S.; Chattopadhyay, D.J.The molecular events associated with the transcriptive and replicative cycle of negative-stranded RNA viruses are still an enigma. We took Chandipura virus, a member of the Rhabdoviridae family, as our model system to demonstrate that Phosphoprotein P, besides Nucleocapsid protein N, also acts as a leader RNA-binding protein in its unphosphorylated form, whereas CKII-mediated phosphorylation totally abrogates its RNA-binding ability. However, interaction between P protein and leader RNA can be distinguished from N-mediated encapsidation of viral sequences. Furthermore, P protein bound to leader chain can successively recruit N protein on RNA while itself being replaced. We also observed that the accumulation of phosphorylation null mutant of P protein in cells results in enhanced genome RNA replication with concurrent increase in the viral yield. All these results led us to propose a model explaining viral transcription-replication switch where Phosphoprotein P acts as a modulator of genome transcription and replication by its ability to bind to the nascent leader RNA in its unphosphorylated form, promoting read-through of the transcription termination signals and initiating nucleocapsid assembly on the nascent RNA chain.