Browsing by Author "Thakur, Jitendra K."

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Analysis of differential expression of mediator subunit genes in Arabidopsis
(Landes Bioscience, 2012) Pasrija, Richa; Thakur, Jitendra K.
Mediator is a conserved eukaryotic multiprotein complex required by RNA polymerase II for transcription of its target genes. Till date, there is no report explaining the signals that affect the overall concentration of individual Med subunits. In this report, we have analyzed the effect of different phytohormones and stresses on the transcript level of Med genes in Arabidopsis. Hormones like auxin and JA, and cold stress did not show significant effect. ABA moderately increased the transcript abundance of more than 70% of AtMed genes analyzed in this study. However, there was noticeable change in the transcript level of several AtMed genes in response to BR. Stresses like high light, dark and salt also caused significant change in the transcript abundance of many AtMed genes. These data reveal that different environmental cues can affect stoichiometric concentration of Med subunits by affecting the transcription of their respective genes. This may, in turn, affect the overall arrangement of functional Mediator complex. This also suggests that some subunits may have some specific functions to play in response different signals.
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.
Co-overexpression of SWEET sucrose transporters modulates sucrose synthesis and defence responses to enhance immunity against bacterial blight in rice
(John Wiley & Sons, 2024) Singh, Jitender; James, Donald; Das, Shubhashis; Patel, Manish Kumar; Sutar, Rashmi Ranjan; Achary, V. Mohan Murali; Goel, Naveen; Gupta, Kapuganti Jagadis; Reddy, Malireddy K.; Jha, Gopaljee; Sonti, Ramesh V.; Foyer, Christine H.; Thakur, Jitendra K.; Tripathy, Baishnab C.
Enhancing carbohydrate export from source to sink tissues is considered to be a realistic approach for improving photosynthetic efficiency and crop yield. The rice sucrose transporters OsSUT1, OsSWEET11a and OsSWEET14 contribute to sucrose phloem loading and seed filling. Crucially, Xanthomonas oryzae pv. oryzae (Xoo) infection in rice enhances the expression of OsSWEET11a and OsSWEET14 genes, and causes leaf blight. Here we show that co‐overexpression of OsSUT1, OsSWEET11a and OsSWEET14 in rice reduced sucrose synthesis and transport leading to lower growth and yield but reduced susceptibility to Xoo relative to controls. The immunity‐related hypersensitive response (HR) was enhanced in the transformed lines as indicated by the increased expression of defence genes, higher salicylic acid content and presence of HR lesions on the leaves. The results suggest that the increased expression of OsSWEET11a and OsSWEET14 in rice is perceived as a pathogen (Xoo) attack that triggers HR and results in constitutive activation of plant defences that are related to the signalling pathways of pathogen starvation. These findings provide a mechanistic basis for the trade‐off between plant growth and immunity because decreased susceptibility against Xoo compromised plant growth and yield.
Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP
(Cold Spring Harbor Laboratory Press, 2010) Walker, Amy K.; Yang, Fajun; Jiang, Karen; Ji, Jun-Yuan; Watts, Jennifer L.; Purushotham, Aparna; Boss, Olivier; Hirsch, Michael L.; Ribich, Scott; Smith, Jesse J.; Israelian, Kristine; Westphal, Christoph H.; Rodgers, Joseph T.; Shioda, Toshi; Elson, Sarah L.; Mulligan, Peter; Najafi-Shoushtari, Hani; Black, Josh C.; Thakur, Jitendra K.; Kadyk, Lisa C.; Whetstine, Johnathan R.; Mostoslavsky, Raul; Puigserver, Pere; Li, Xiaoling; Dyson, Nicholas J.; Hart, Anne C.; Naar, Anders M.
The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD(+)-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.
Designing a mini-core collection effectively representing 3004 diverse rice accessions
(Elsevier B.V., 2020) Kumar, Angad; Kumar, Shivendra; Singh, Kajol B.M.; Prasad, Manoj; Thakur, Jitendra K.
Genetic diversity provides the foundation for plant breeding and genetic research. Over 3000 rice genomes were recently sequenced as part of the 3K Rice Genome (3KRG) Project. We added four additional Indian rice accessions to create a panel of 3004 accessions. However, such a large collection of germplasm is difficult to preserve and evaluate. The construction of core and mini-core collections is an efficient method for the management of genetic resources. In this study, we developed a mini-core comprising 520 accessions that captured most of the SNPs and represented all of the phenotypes and geographic regions from the original panel. The mini-core was validated using different statistical analyses and contained representatives from all major rice groups, including japonica, indica, aus/boro, and aromatic/basmati. Genomewide association analyses of the mini-core panel efficiently reproduced the marker–trait associations identified in the original panel. Haplotype analysis validated the utility of the mini-core panel. In the current era with many ongoing large-scale sequencing projects, such a strategy for mini-core design should be useful in many crops. The rice mini-core collection developed in this study would be valuable for agronomic trait evaluation and useful for rice improvement via marker-assisted molecular breeding.
Endosperm ontogeny through the lens of epigenetics
(Elsevier B.V., 2023) Singh, Kajol B.M.; Thakur, Jitendra K.
Endosperm tissue is crucial to seed development. Monocots and dicots have evolved different fates for their endosperm: it is usually consumed in dicots during seed development, while monocots seeds retain the endosperm till maturity. Recently, Wu et al. addressed the role of divergent epigenetic regulation over functionally conserved IKU2 gene contributing to silenced or persistent endosperm proliferation and has provided insights into diverged seed ontogeny pattern in plants. Here, we highlight the novel findings of this study and their potential significance. Seed production represents a remarkable life-history adaptation that has been established during plant evolution. In flowering plants, a double fertilization event initiates seed development, producing embryo and the endosperm. Endosperm (3n) is a triploid outcome of second fertilization, which occurs when a female central cell (2n) fuses with one of the two male gametes (n) carried by the pollen tube (Ingram, 2020). It develops precociously and is primed to perform nutritional and developmental functions for embryo growth (Povilus & Gehring, 2022). Different taxa have evolved specific seed formation patterns that elicit different levels of endosperm perseverance during development (Baroux et al., 2002). As a result of this, the endosperm-to-embryo ratio in mature seeds has varied gradually among different plants. As an example, endosperm in cereals is formed and retained as a nutritive reserve till seed maturity. In several eudicots such as Arabidopsis, early formed endosperm is substantially devoured to serve during embryo maturation. In peas with non-persistent endosperm, the tissue appears to be absorbed in the early free nuclear division phase before cell wall formation. The Podostomenaceae and Orchidaceae are the two endosperm-free lineages because their seeds lack or terminate endosperm nuclear divisions during early stages. Endosperm develops through two major phases: first syncytial involving continuous nuclear divisions, and the subsequent cellularization in which the free nuclear state develops cell wall (Baroux et al., 2002). Despite that the embryo produces the next generation progeny, endosperm proliferation and cellularization timing is crucial to embryo viability.
Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer
(Springer Nature, 2018) Nagpal, Neha; Sharma, Shivani; Maji, Sourobh; Durante, Giorgio; Ferracin, Manuela; Thakur, Jitendra K.; Kulshreshtha, Ritu
Mediator complex has been extensively shown to regulate the levels of several protein-coding genes; however, its role in the regulation of miRNAs in humans remains unstudied so far. Here we show that MED1, a Mediator subunit in the Middle module of Mediator complex, is overexpressed in breast cancer and is a negative prognostic factor. The levels of several miRNAs (miR-100-5p, -191-5p, -193b-3p, -205-5p, -326, -422a and -425-5p) were found to be regulated by MED1. MED1 induces miR-191/425 cluster in an estrogen receptor-alpha (ER-α) dependent manner. Occupancy of MED1 on estrogen response elements (EREs) upstream of miR-191/425 cluster is estrogen and ER-α-dependent and ER-α-induced expression of these miRNAs is MED1-dependent. MED1 mediates induction of cell proliferation and migration and the genes associated with it (JUN, FOS, EGFR, VEGF, MMP1, and ERBB4) in breast cancer, which is abrogated when used together with miR-191-inhibition. Additionally, we show that MED1 also regulates the levels of direct miR-191 target genes such as SATB1, CDK6 and BDNF. Overall, the results show that MED1/ER-α/miR-191 axis promotes breast cancer cell proliferation and migration and may serve as a novel target for therapy.
Evolution of disorder in Mediator complex and its functional relevance
(Oxford University Press, 2016) Nagulapalli, Malini; Maji, Sourobh; Dwivedi, Nidhi; Dahiya, Pradeep; Thakur, Jitendra K.
Mediator, an important component of eukaryotic transcriptional machinery, is a huge multisubunit complex. Though the complex is known to be conserved across all the eukaryotic kingdoms, the evolutionary topology of its subunits has never been studied. In this study, we profiled disorder in the Mediator subunits of 146 eukaryotes belonging to three kingdoms viz., metazoans, plants and fungi, and attempted to find correlation between the evolution of Mediator complex and its disorder. Our analysis suggests that disorder in Mediator complex have played a crucial role in the evolutionary diversification of complexity of eukaryotic organisms. Conserved intrinsic disordered regions (IDRs) were identified in only six subunits in the three kingdoms whereas unique patterns of IDRs were identified in other Mediator subunits. Acquisition of novel molecular recognition features (MoRFs) through evolution of new subunits or through elongation of the existing subunits was evident in metazoans and plants. A new concept of ‘junction-MoRF’ has been introduced. Evolutionary link between CBP and Med15 has been provided which explain the evolution of extended-IDR in CBP from Med15 KIX-IDR junction-MoRF suggesting role of junction-MoRF in evolution and modulation of protein–protein interaction repertoire. This study can be informative and helpful in understanding the conserved and flexible nature of Mediator complex across eukaryotic kingdoms.
Expression of AtMed15 of Arabidopsis in yeast causes flocculation and increases ethanol production in yeast culture
(Nature Publishing Group, 2016) Dahiya, Pradeep; Bhat, Divya S.; Thakur, Jitendra K.
Mediator, a multiprotein complex involved in transcription of class II genes, was first discovered in yeast and then characterized in many metazoans revealing a striking structural conservation of the complex. However, sequences of Mediator subunits are not well conserved raising a question on the functional conservation of these individual subunits. In this study, expression of Med15 of Arabidopsis (AtMed15) in gal11∆ yeast could not complement the function of ScGal11 in galactose metabolism and resistance against cycloheximide. Surprisingly, AtMed15 changed the morphology of the yeast cells. The cells adhered strongly on the surface of the agar media, and showed robust flocculation in the liquid media without affecting the growth. The AtMed15-induced adhesion and flocculation were observed in different carbon sources. Calcium-assisted cell wall-bound mannan-binding proteins were found to be involved in this flocculation, which was unaffected by wide fluctuation of pH or temperatures revealing its constitutive robust nature. Expression of few flocculation related Flo genes was up-regulated in these cells. Interestingly, there was significant increase in ethanol production by the yeast expressing AtMed15. Robust and constitutive flocculation and increased ethanol production by yeast cells harbouring AtMed15 indicate an opportunity of its important usage in biotechnology industries.
GC-MS-based analysis of methanol: chloroform-extracted fatty acids from plant tissues
(Bio-protocol LLC., 2018) Patel, Manish Kumar; Das, Shubhashis; Thakur, Jitendra K.
Fatty acids (FAs) are carboxylic acids with long aliphatic chains that may be straight, branched and saturated or unsaturated. Most of the naturally occurring plant FAs contains an even number of carbon (C4-C24). FAs are used in food and pharmacological industries due to their nutritional importance. In addition, FAs are considered as a promising alternative for the production of biodiesel from terrestrial plant biomass. To establish commercial applications, more reliable analytical methods are needed for the identification, quantification, and composition determination of FAs. Here, we describe a relatively rapid and sensitive method for the extraction, identification, and quantification of FAs from a small quantity of plant tissue. The method includes steps of lipid extraction, conversion of lipid to fatty acid methyl esters (FAMEs) by transmethylation, identification and quantification of FAMEs using gas chromatography-mass spectrometry (GC-MS). In this protocol, an internal standard is added prior to GC-MS analysis. The amount of each FA is calculated from its peak area relative to the peak area of the internal standard.
Genome wide investigation and transcriptional profiling of SWEET genes in two contrasting cultivars of foxtail millet under abiotic stresses
(Elsevier B.V., 2025) Singh, Jitender; Singh, Kajol BM.; Sutar, Rashmi Ranjan; Kumar, Angad; Prasad, Manoj; Thakur, Jitendra K.
The SWEET (Sugars will eventually be exported transporter) gene family is an important class of sugar transporters that regulates diverse aspects of plant physiology such as apoplastic phloem loading, plant-pathogen interactions and plant responses to abiotic stresses. While majority of the studies on SWEET family in plants have been performed in C3 species, there are limited reports on C4 plants. In this study we conducted genome wide investigation of the SWEET gene family in foxtail millet, a naturally stress tolerant C4 crop. In-silico analysis identified 24 SWEET genes in foxtail millet genome that were classified into 4 distinct clades. Domain analysis revealed the presence of conserved MtN3_slv/PQ-loop domains in all identified SWEET proteins. Interestingly, many SWEET proteins also harboured the prokaryotic SemiSWEET/PQ-loop domain suggesting an evolutionary link to their prokaryotic Semi-SWEET ancestors. In-silico analysis predicted the presence of abscisic acid and drought responsive cis-elements in the promoter region of SWEET genes. Transcriptional analysis under control, drought, and salinity stress revealed differential expression patterns of SWEET genes in stress resistant and stress susceptible foxtail millet cultivars. Moreover, the differential expression of SWEET genes altered the soluble sugar content in leaves and roots under stress conditions suggesting altered carbon re-allocation between source and sink tissues. This study significantly advances our understanding of the SWEET gene family in C4 plants, particularly in foxtail millet, and provides insights into its role in stress tolerance mechanisms and carbohydrate re-allocation under stress conditions.
Genome-wide analysis of polymorphisms identified domestication-associated long low diversity region carrying important rice grain size/weight QTL
(John Wiley & Sons, 2020) Kumar, Angad; Daware, Anurag; Kumar, Arvind; Kumar, Vinay; Krishnan S, Gopala; Mondal, Subhasish; Patra, Bhaskar Chandra; Singh, Ashok. K.; Tyagi, Akhilesh K.; Parida, Swarup K.; Thakur, Jitendra K.
Rice grain size and weight are major determinants of grain quality and yield and so have been under rigorous selection since domestication. However, genetic basis for contrasting grain size/weight trait among Indian germplasms and their association with domestication‐driven evolution is not well understood. In this study, two long (LGG) and two short grain (SGG) genotypes were resequenced. LGG (LGR and PB 1121) differentiated from SGG (Sonasal and Bindli) by 504,439 SNPs and 78,166 InDels. The LRK gene cluster was different and a truncation mutation in the LRK8 kinase domain was associated with LGG. Phylogeny with 3000 diverse rice accessions revealed that the four sequenced genotypes belonged to japonica group and were at the edge of the clades indicating them to be the potential source of genetic diversity available in Indian rice germplasm. Six SNPs were significantly associated with grain size/weight and top four of them could be validated in mapping population, suggesting this study as a valuable resource for high‐throughput genotyping. A contiguous ~ 6 Mb long low diversity region (LDR) carrying a major grain weight QTL (harbouring OsTOR gene) was identified on chromosome 5. This LDR was identified as an evolutionary important site with significant positive selection and multiple selection sweeps, and showed association with many domestication‐related traits including grain size/weight. The aus population retained more allelic variations in the LDR than japonica and indica populations, suggesting it to be one of the divergence loci. All the data and analyses can be accessed from RiceSzWtBase database.
Genome-wide association study for phosphate deficiency responsive root hair elongation in chickpea
(Springer Nature Publishing AG, 2020) Kohli, Pawandeep Singh; Verma, Pankaj Kumar; Verma, Rita; Parida, Swarup K.; Thakur, Jitendra K.; Giri, Jitender
Root hairs (RHs) are single-celled elongated epidermal cells and play a vital role in nutrient absorption, particularly for immobile minerals like phosphorus (P). As an adaptive response to P deficiency, an increase in RH length enhances root-soil contact and absorptive area for P absorption. Genetic variations have been reported for RH length and its response to P deficiency in plants. However, only a few association studies have been conducted to identify genes and genetic loci associated with RH length. Here, we screened desi chickpea accessions for RH length and its plasticity under P deficiency. Further, the genome-wide association study (GWAS) was conducted to identify the genetic loci associated with RH length in P deficient and sufficient conditions. Although high variability was observed in terms of RH length in diverse genotypes, majority of the accessions showed typical response of increase in RH length in low P. Genome-wide association mapping identified many SNPs with significant associations with RH length in P-sufficient and P-deficient conditions. A few candidate genes for RH length in P deficient (SIZ1-like and HAD superfamily protein) and sufficient (RSL2-like and SMAP1-like) conditions were identified which have known roles in RH development and P deficiency response or both. Highly associated loci and candidate genes identified in this study would be useful for genomic-assisted breeding to develop P-efficient chickpea.
Histone acetylation dynamics regulating plant development and stress responses
(Springer Nature Publishing AG, 2021) Kumar, Verandra; Thakur, Jitendra K.; Prasad, Manoj
Crop productivity is directly dependent on the growth and development of plants and their adaptation during different environmental stresses. Histone acetylation is an epigenetic modification that regulates numerous genes essential for various biological processes, including development and stress responses. Here, we have mainly discussed the impact of histone acetylation dynamics on vegetative growth, flower development, fruit ripening, biotic and abiotic stress responses. Besides, we have also emphasized the information gaps which are obligatory to be examined for understanding the complete role of histone acetylation dynamics in plants. A comprehensive knowledge about the histone acetylation dynamics will ultimately help to improve stress resistance and reduce yield losses in different crops due to climate changes.
Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum
(Nature Publishing Group, 2017) Prakash, Prem; Zeeshan, Mohammad; Saini, Ekta; Muneer, Azhar; Khurana, Sachin; Chourasia, Bishwanath Kumar; Deshmukh, Arunaditya; Kaur, Inderjeet; Dabral, Surabhi; Singh, Niharika; Anam, Zille; Chaurasiya, Ayushi; Kaushik, Shikha; Dahiya, Pradeep; Kalamuddin, Md.; Thakur, Jitendra K.; Mohmmed, Asif; Ranganathan, Anand; Malhotra, Pawan
Invasion of human erythrocytes by Plasmodium falciparum merozoites involves multiple interactions between host receptors and their merozoite ligands. Here we report human Cyclophilin B as a receptor for PfRhopH3 during merozoite invasion. Localization and binding studies show that Cyclophilin B is present on the erythrocytes and binds strongly to merozoites. We demonstrate that PfRhopH3 binds to the RBCs and their treatment with Cyclosporin A prevents merozoite invasion. We also show a multi-protein complex involving Cyclophilin B and Basigin, as well as PfRhopH3 and PfRh5 that aids the invasion. Furthermore, we report identification of a de novo peptide CDP3 that binds Cyclophilin B and blocks invasion by up to 80%. Collectively, our data provide evidence of compounded interactions between host receptors and merozoite surface proteins and paves the way for developing peptide and small-molecules that inhibit the protein-protein interactions, individually or in toto, leading to abrogation of the invasion process.
Identification of novel inhibitors against Med15a KIX domain of Candida glabrata
(Elsevier B.V., 2023) Waseem, Mohd; Das, Shubhashis; Mondal, Debarati; Jain, Monika; Thakur, Jitendra K.; Subbarao, Naidu
Candida glabrata, the second most common cause of invasive fungal infections, exhibits multi-drug resistance to commonly used antifungal drugs. To counter this resistance, there is a critical need for novel antifungals. This study identifies small molecule inhibitors that target a three-helix bundle KIX domain in the Med15a Mediator subunit of Candida glabrata (CgMed15a KIX). This domain plays a crucial role by interacting with the Pleiotropic Drug Resistance transcription factor Pdr1, a key regulator of the multidrug resistance pathway in Candida glabrata. We performed high throughput computational screening of large chemical datasets against the binding sites of the CgMed15a KIX domain to identify novel inhibitors. We selected six potential candidates with high affinity and confirmed their binding with the CgMed15a KIX domain. A phytochemical compound, Chebulinic acid binds to the CgMed15a KIX domain with a KD value of 0.339 μM and shows significant inhibitory effects on the growth of Candida glabrata. Molecular dynamics simulation studies further revealed the structural stability of the CgMed15a KIX-Chebulinic acid complex. Thus, in conclusion, this study highlights Chebulinic acid as a novel potential antifungal compound against Candida glabrata.
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.
An integrated genomic strategy delineates candidate mediator genes regulating grain size and weight in rice
(Nature Publishing Group, 2016) Malik, Naveen; Dwivedi, Nidhi; Singh, Ashok K.; Parida, Swarup K.; Agarwal, Pinky; Thakur, Jitendra K.; Tyagi, Akhilesh K.
The present study deployed a Mediator (MED) genes-mediated integrated genomic strategy for understanding the complex genetic architecture of grain size/weight quantitative trait in rice. The targeted multiplex amplicon resequencing of 55 MED genes annotated from whole rice genome in 384 accessions discovered 3971 SNPs, which were structurally and functionally annotated in diverse coding and non-coding sequence-components of genes. Association analysis, using the genotyping information of 3971 SNPs in a structured population of 384 accessions (with 50–100 kb linkage disequilibrium decay), detected 10 MED gene-derived SNPs significantly associated (46% combined phenotypic variation explained) with grain length, width and weight in rice. Of these, one strong grain weight-associated non-synonymous SNP (G/A)-carrying OsMED4_2 gene was validated successfully in low- and high-grain weight parental accessions and homozygous individuals of a rice mapping population. The seed-specific expression, including differential up/down-regulation of three grain size/weight-associated MED genes (including OsMED4_2) in six low and high-grain weight rice accessions was evident. Altogether, combinatorial genomic approach involving haplotype-based association analysis delineated diverse functionally relevant natural SNP-allelic variants in 10 MED genes, including three potential novel SNP haplotypes in an OsMED4_2 gene governing grain size/weight differentiation in rice. These molecular tags have potential to accelerate genomics-assisted crop improvement in rice.
Interaction map of Arabidopsis mediator complex expounding its topology
(Oxford University Press, 2019) Maji, Sourobh; Dahiya, Pradeep; Waseem, Mohd; Dwivedi, Nidhi; Bhat, Divya S.; Dar, Tanvir H.; Thakur, Jitendra K.
Understanding of mechanistic details of Mediator functioning in plants is impeded as the knowledge of subunit organization and structure is lacking. In this study, an interaction map of Arabidopsis Mediator complex was analyzed to understand the arrangement of the subunits in the core part of the complex. Combining this interaction map with homologybased modeling, probable structural topology of core part of the Arabidopsis Mediator complex was deduced. Though the overall topology of the complex was similar to that of yeast, several differences were observed. Many interactions discovered in this study are not yet reported in other systems. AtMed14 and AtMed17 emerged as the key component providing important scaffold for the whole complex. AtMed6 and AtMed10 were found to be important for linking head with middle and middle with tail, respectively. Some Mediator subunits were found to form homodimers and some were found to possess transactivation property. Subcellular localization suggested that many of the Mediator subunits might have functions beyond the process of transcription. Overall, this study reveals role of individual subunits in the organization of the core complex, which can be an important resource for understanding the molecular mechanism of functioning of Mediator complex and its subunits in plants.
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, Ritu
MicroRNAs 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.