Browsing by Author "Chitkara, Pragya"

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BURP domain-containing genes in legumes: genome-wide identification, structure, and expression analysis under stresses and development
(Springer Nature Publishing AG, 2022) Chitkara, Pragya; Poddar, Nikita; Singh, Amarjeet; Kumar, Shailesh
BURP domain-containing proteins are a plant-specific protein family which play an important role in plant metabolism and development. These proteins have also been involved in various abiotic and biotic stress responses. In this study, genome-wide identification and characterization of BURP domain protein encoding gene family is performed in four important legumes, Phaseolus vulgaris, Cicer arietinum, Cajanus cajan, and Vigna radiata. BURP genes were distributed randomly across chromosomes in all four legume plants. The phylogenetic analysis classified all BURP proteins into five major subfamilies, namely, USP-like, RD22-like, BNM2-like, PG1β-like, and BURPV. Our findings revealed that BURP gene family descended from common ancestors with segmental gene duplication events playing a critical role in their evolution and expansion in legumes. The intron–exon and conserved protein motifs analysis revealed that BURP genes are structurally conserved in legumes. The promoter analysis revealed the presence of hormone, and stress-responsive cis-regulatory elements in BURP promoters, implying that BURP functions in both hormone and abiotic stress signaling. Global expression analysis revealed that several BURP genes in all four legumes express differentially during plant development, and under biotic and abiotic stresses. This indicates crucial role of BURP proteins in regulating the development of legumes and adaptation to different abiotic/biotic stresses. This study will provide the starter for cloning and detail functional investigation of BURP proteins in legume crops.
A comprehensive investigation of lipid-transfer proteins from Cicer arietinum disentangles their role in plant defense against Helicoverpa armigera-infestation
(Frontiers Media S.A., 2023) Saxena, Harshita; Negi, Harshita; Keshan, Radhika; Chitkara, Pragya; Kumar, Shailesh; Chakraborty, Amrita; Roy, Amit; Singh, Indrakant K.; Singh, Archana
Lipid Transfer Proteins (LTPs) play a crucial role in synthesizing lipid barrier polymers and are involved in defense signaling during pest and pathogen attacks. Although LTPs are conserved with multifaceted roles in plants, these are not yet identified and characterized in Cicer arietinum. In this study, a genome-wide analysis of LTPs was executed and their physiochemical properties, biochemical function, gene structure analysis, chromosomal localization, promoter analysis, gene duplication, and evolutionary analysis were performed using in silico tools. Furthermore, tissue-specific expression analysis and gene expression analysis during pest attack was also conducted for the LTPs. A total of 48 LTPs were identified and named as CaLTPs. They were predicted to be small unstable proteins with "Glycolipid transfer protein" and "Alpha-Amylase Inhibitors, Lipid Transfer and Seed Storage" domains, that are translocated to the extracellular region. CaLTPs were predicted to possess 3-4 introns and were located on all the eight chromosomes of chickpea with half of the CaLTPs being localized on chromosomes 4, 5, and 6, and found to be closely related to LTPs of Arabidopsis thaliana and Medicago trancatula. Gene duplication and synteny analysis revealed that most of the CaLTPs have evolved due to tandem or segmental gene duplication and were subjected to purifying selection during evolution. The promoters of CaLTPs had development-related, phytohormone-responsive, and abiotic and biotic stress-related cis-acting elements. A few CaLTP transcripts exhibited differential expression in diverse tissue types, while others showed no/very low expression. Out of 20 jasmonate-regulated CaLTPs, 14 exhibited differential expression patterns during Helicoverpa armigera-infestation, indicating their role in plant defense response. This study identified and characterized CaLTPs from an important legume, C. arietinum, and indicated their involvement in plant defense against H. armigera-infestation, which can be further utilized to explore lipid signaling during plant-pest interaction and pest management.
The landscape of fusion transcripts in plants: a new insight into genome complexity
(BioMed Central Ltd, 2024) Chitkara, Pragya; Singh, Ajeet; Gangwar, Rashmi; Bhardwaj, Rohan; Zahra, Shafaque; Arora, Simran; Hamid, Fiza; Arya, Ajay; Sahu, Namrata; Chakraborty, Srija; Ramesh, Madhulika; Kumar, Shailesh
Background Fusion transcripts (FTs), generated by the fusion of genes at the DNA level or RNA-level splicing events significantly contribute to transcriptome diversity. FTs are usually considered unique features of neoplasia and serve as biomarkers and therapeutic targets for multiple cancers. The latest findings show the presence of FTs in normal human physiology. Several discrete reports mentioned the presence of fusion transcripts in planta, has important roles in stress responses, morphological alterations, or traits (e.g. seed size, etc.). Results In this study, we identified 169,197 fusion transcripts in 2795 transcriptome datasets of Arabidopsis thaliana, Cicer arietinum, and Oryza sativa by using a combination of tools, and confirmed the translational activity of 150 fusion transcripts through proteomic datasets. Analysis of the FT junction sequences and their association with epigenetic factors, as revealed by ChIP-Seq datasets, demonstrated an organised process of fusion formation at the DNA level. We investigated the possible impact of three-dimensional chromatin conformation on intra-chromosomal fusion events by leveraging the Hi-C datasets with the incidence of fusion transcripts. We further utilised the longread RNA-Seq datasets to validate the most reoccurring fusion transcripts in each plant species followed by further authentication through RT-PCR and Sanger sequencing. Conclusions Our findings suggest that a significant portion of fusion events may be attributed to alternative splicing during transcription, accounting for numerous fusion events without a proportional increase in the number of RNA pairs. Even non-nuclear DNA transcripts from mitochondria and chloroplasts can participate in intra- and inter-chromosomal fusion formation. Genes in close spatial proximity are more prone to undergoing fusion formation, especially in intra-chromosomal FTs. Most of the fusion transcripts may not undergo translation and serve as long non-coding RNAs. The low validation rate of FTs in plants indicated that the fusion transcripts are expressed at very low levels, like in the case of humans. FTs often originate from parental genes involved in essential biological processes, suggesting their relevance across diverse tissues and stress conditions. This study presents a comprehensive repository of fusion transcripts, offering valuable insights into their roles in vital physiological processes and stress responses.
Molecular and expression analysis indicate the role of CBL interacting protein kinases (CIPKs) in abiotic stress signaling and development in chickpea
(Springer Nature Publishing AG, 2022) Poddar, Nikita; Deepika, Deepika; Chitkara, Pragya; Singh, Amarjeet; Kumar, Shailesh
Calcineurin B-like proteins (CBL)-interacting protein kinases (CIPKs) regulate the developmental processes, hormone signal transduction and stress responses in plants. Although the genome sequence of chickpea is available, information related to the CIPK gene family is missing in this important crop plant. Here, a total of 22 CIPK genes were identified and characterized in chickpea. We found a high degree of structural and evolutionary conservation in the chickpea CIPK family. Our analysis showed that chickpea CIPKs have evolved with dicots such as Arabidopsis and soybean, and extensive gene duplication events have played an important role in the evolution and expansion of the CIPK gene family in chickpea. The three-dimensional structure of chickpea CIPKs was described by protein homology modelling. Most CIPK proteins are localized in the cytoplasm and nucleus, as predicted by subcellular localization analysis. Promoter analysis revealed various cis-regulatory elements related to plant development, hormone signaling, and abiotic stresses. RNA-seq expression analysis indicated that CIPKs are significantly expressed through a spectrum of developmental stages, tissue/organs that hinted at their important role in plant development. The qRT-PCR analysis revealed that several CaCIPK genes had specific and overlapping expressions in different abiotic stresses like drought, salt, and ABA, suggesting the important role of this gene family in abiotic stress signaling in chickpea. Thus, this study provides an avenue for detailed functional characterization of the CIPK gene family in chickpea and other legume crops.
Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA
(Springer Nature Publishing AG, 2023) Singh, Sandhya; Gaur, Aakash; Sharma, Rakesh Kumar; Kumari, Renu; Prakash, Shakti; Kumari, Sunaina; Chaudhary, Ayushi Devendrasingh; Prasun, Pankaj; Pant, Priyanka; Hunkler, Hannah; Thum, Thomas; Jagavelu, Kumaravelu; Bharati, Pragya; Hanif, Kashif; Chitkara, Pragya; Kumar, Shailesh; Mitra, Kalyan; Gupta, Shashi Kumar
Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 is known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains largely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the heart cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified Msi2 isoforms 1, 4, and 5, and two novel putative isoforms labeled as Msi2 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 exhibited a significant increase in a pressure-overload model of cardiac hypertrophy. We selected isoforms 4 and 7 to validate the hypertrophic effects due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, seahorse, and transmembrane potential measurement assays, increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh and Smyd1 inhibited Msi2-induced cardiac malfunction and mitochondrial dysfunction. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.
A protocol for the detection of fusion transcripts using RNA-sequencing data
(Springer Nature Publishing AG, 2024) Hamid, Fiza; Arora, Simran; Chitkara, Pragya; Kumar, Shailesh
Fusion transcripts are formed when two genes or their mRNAs fuse to produce a novel gene or chimeric transcript. Fusion genes are well-known cancer biomarkers used for cancer diagnosis and as therapeutic targets. Gene fusions are also found in normal physiology and lead to the evolution of novel genes that contribute to better survival and adaptation for an organism. Various in vitro approaches, such as FISH, PCR, RT-PCR, and chromosome banding techniques, have been used to detect gene fusion. However, all these approaches have low resolution and throughput. Due to the development of high-throughput next-generation sequencing technologies, the detection of fusion transcript becomes feasible using whole genome sequencing, RNA-Seq data, and bioinformatics tools. This chapter will overview the general computational protocol for fusion transcript detection from RNA-sequencing datasets.
The RNA-binding protein Quaking is essential for cardiac homeostasis and function by regulating Morf4l2 splicing
(Elsevier B.V., 2026) Kumari, Sunaina; Shashi; Singh, Sandhya; Swain, Abinash; Prakash, Shakti; Chitkara, Pragya; Sharma, Rakesh Kumar; Agarwal, Pratyush; Kundu, Samprikta; Gaur, Aakash; Kumari, Renu; Sinha, Abhipsa; Chatterjee, Shambhabi; Prasun, Pankaj; Hummel, Oliver; Pant, Bhaskar; Srivastava, Kinshuk Raj; Hübner, Norbert; Datta, Dipak; Mitra, Kalyan; Mishra, Durga Prasad; Guha, Rajdeep; Thum, Thomas; Kumar, Shailesh; Gupta, Shashi Kumar
Background: Lower levels of Qki were reported in human and mouse-failing hearts, implicating its involvement in cardiac diseases. However, the molecular and functional effects of its downregulation in adult myocardium remain largely unknown. Objective: We aim to uncover the effects of Qki knockdown in adult hearts. Methods & results: Here we show that AAV9-mediated knockdown of Qki by shRNAs in the hearts of adult BALB/c mice led to cardiac malfunction, atrophy, apoptosis, heart failure, and death within two weeks. Global transcriptomic analysis of Qki knockdown hearts revealed significant dysregulation of 996 alternative splicing events upon Qki knockdown. Mechanistically, we discovered that loss of Qki promotes the exclusion of the third exon of Morf4l2, leading to higher expression of exon three excluded variant (Morf4l2Δex3). Like rodents, the RNA-seq dataset from 108 human hearts revealed a lower splice junction count of MORF4L2 exon three in hearts with low levels of QKI compared to subjects with higher QKI levels. Specific knockdown of Morf4l2Δex3 rescues Qki knockdown-induced cardiac cachexia and improves cardiac function. Moreover, Morf4l2Δex3 was increased in the colon cancer-induced cardiac cachexia mouse model, and its inhibition prevented cardiac cachexia and improved cardiac function. Mechanistically, exon three of Morf4l2 lies in the 5'UTR, and its exclusion leads to higher expression of MORF4L2 upon Qki knockdown due to the lack of a G2-quadruplex. Importantly, MORF4L2 protein sequence and localization were not affected by alternative splicing as exon three lies in the 5'UTR. We found that MORF4L2 is a chromatin-bound protein and regulates H3K27ac. Conclusion: Qki knockdown in the adult heart leads to cardiac cachexia due to the alteration of Morf4l2 splicing. Inhibition of Morf4l2Δex3 inhibits cancer-induced cardiac cachexia, demonstrating it as a potential therapeutic target.