Transcriptome meta-analysis-based identification of hub transcription factors and RNA-binding proteins potentially orchestrating gene regulatory cascades and crosstalk in response to abiotic stresses in Arabidopsis thaliana.

Deteriorating climatic conditions and increasing human population necessitate the development of robust plant varieties resistant to harsh environments. Manipulation of regulatory proteins such as transcription factors (TFs) and RNA-binding proteins (RBPs) would be a beneficial strategy in this regard. Further, understanding the complex interconnections between different classes of regulatory molecules would be essential for the identification of candidate genes/proteins for trait improvement. Most studies to date have analysed the roles of TFs or RBPs individually, in conferring stress resilience. However, it would be important to identify dominant/upstream TFs and RBPs inducing widespread transcriptomic alterations through other regulators (i.e., other TFs/RBPs targeted by the upstream regulators). To this end, the present study employed a transcriptome meta-analysis and computational approaches to obtain a comprehensive overview of regulatory interactions. This work identified dominant TFs and RBPs potentially influencing stress-mediated differential expression of other regulators, which could in turn influence gene expression, and consequently, physiological responses. Twenty transcriptomic studies [related to (i) UV radiation, (ii) wounding, (iii) salinity, (iv) cold, and (v) drought stresses in Arabidopsis thaliana] were analysed for differential gene expression, followed by the identification of differentially expressed TFs and RBPs. Subsequently, other TFs and RBPs which could be influencing these regulators were identified, and their interaction networks and hub nodes were analysed. As a result, an interacting module of Basic Leucine Zipper (bZIP) family TFs as well as Heterogeneous nuclear ribonucleoproteins (hnRNP) and Glycine-rich protein (GRP) family RBPs (among other TFs and RBPs) were shown to potentially influence the stress-induced differential expression of other TFs and RBPs under all the considered stress conditions. Some of the identified hub TFs and RBPs are known to be of major importance in orchestrating stress-induced transcriptomic changes influencing a variety of physiological processes from seed germination to senescence. This study highlighted the gene/protein candidates that could be considered for multiplexed genetic manipulation - a promising approach to develop robust, multi-stress-resilient plant varieties.

Computational analysis of crosstalk between transcriptional regulators and RNA-binding proteins suggests mutual regulation of polycomb proteins and SRSF1 influencing adult hippocampal neurogenesis.

BACKGROUND: Adult hippocampal neurogenesis (AHN) is a clinically significant neural phenomenon. Understanding its molecular regulation would be important. In this regard, most studies have focused on transcriptional regulators (TRs), epigenetic modifiers, or non-coding RNAs. RNA-binding proteins (RBPs) have emerged as dominant molecular regulators. It would be significant to understand the potential cross-talk between RBPs and TRs, which could influence AHN. METHODS: The present study employed computational analyses to identify RBPs and TRs regulating AHN, followed by the analysis of their interaction networks and detection of hub proteins. Next, the potential mutual regulation of hub TRs and RBPs was analyzed. Additionally, hippocampal genes differentially expressed upon exercise were analyzed for potential regulation by the identified TRs and RBPs. RESULTS: 105 TRs and 26 RBPs were found to influence AHN, which could also form interactive networks. Polycomb complex proteins were among the TR network hubs, while HNRNP and SRSF family members were among the hub RBPs. Further, the polycomb complex proteins and SRSF1 could have a mutual regulatory relationship, suggesting a cross-talk between epigenetic/transcriptional and post-transcriptional regulatory pathways. A number of exercise-induced hippocampal genes were also found to be potential targets of the identified TRs and RBPs. CONCLUSION: SRSF1 may influence post-transcriptional stability, localization, and alternative splicing patterns of polycomb complex transcripts, and the polycomb proteins may in turn epigenetically influence the SRSF1. Further experimental validation of these regulatory loops/networks could provide novel insights into the molecular regulation of AHN, and unravel new targets for disease-treatment.

Understanding the possible influence of Pumilio RNA binding proteins on terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Catharanthus roseus is a clinically significant medicinal plant; the sole source of chemotherapy agents, vincristine and vinblastine (specialized metabolites, terpenoid indole alkaloids/TIAs). Owing to large clinical demand and low bioavailability, several studies have focused on biosynthesis and regulation of TIA biosynthesis in C. roseus. However, transcription factor mediated regulation has been a major research focus, and the impact of post-transcriptional regulation remains under-explored. RNA binding proteins (RBPs) are an emerging class of post-transcriptional regulators having a profound influence on transcript stability. Pumilio (Pum) RBPs are evolutionarily conserved post-transcriptional regulators, involved in RNA degradation across eukaryotes. However, their potential influence on TIA biosynthesis has not been studied till date in any medicinal plants including C. roseus. Thus, the present study aimed at identification and computational characterization of Pum in C. roseus, followed by expression and functional analyses. The genome-wide identification and characterization revealed twelve CrPum isoforms. The effect of CrPum2, 3, and 5 knockdown on TIA biosynthesis (specifically vindoline and catharanthine) was analyzed via high performance liquid chromatography. CrPum5 knockdown was associated with increased TIA levels and upregulation of key TIA pathway genes. Thus, the present study is the first to report the potential influence of Pum on TIA biosynthesis in C. roseus. Further studies to elucidate the mechanism of Pum activity could provide new insights into the molecular regulation of TIA biosynthesis. A holistic understanding of regulatory mechanisms could benefit the metabolic engineering programs aimed at higher productivity of plant specialized metabolites. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01193-5.

Global Exploration of RNA-Binding Proteins in Exercise-Induced Adult Hippocampal Neurogenesis: A Transcriptome Meta-analysis and Computational Study.

Voluntary physical exercise is a robust enhancer of adult hippocampal neurogenesis (AHN). A complete understanding of the molecular regulation of AHN is important in order to exploit the benefits of the process toward therapeutic approaches. Several factors such as epigenetic modifiers, non-coding RNAs, and transcription factors have been reported to regulate AHN. However, there is a limited understanding of the impact of RNA-binding proteins (RBPs) on exercise-mediated AHN, in spite of their well-documented significance in embryonic neurogenesis. The present study is the first global analysis to catalog the potential RBPs influencing exercise-mediated AHN. Here, a transcriptome meta-analysis was conducted to study exercise-mediated gene expression modulation in hippocampi of adult mice. Next, potential RBPs influencing transcriptome-wide expression changes via untranslated regions (UTRs) were identified. Among other RBPs, MATR3, Musashi, TIA1, and FXR2 (known critical modulators of neurogenesis) were found to potentially regulate gene expression patterns. Subsequently, binding sites of known neurogenesis-regulating RBPs were identified in the UTRs of AHN-associated genes modulated by exercise. Finally, a number of RBPs including RBFOX1, RBFOX3, and QKI (known regulators of neurogenesis) were found to be highly expressed in mouse hippocampal formation and also potentially interact with other RBPs, suggesting their combinatorial functioning in exercise-induced AHN. Thus, the present meta-analysis-based computational study identified several RBPs potentially important in exercise-induced AHN, which could form a foundation for further experiments to unravel RBP-mediated regulation of AHN.

Understanding the neural basis of survival instinct vs. suicidal behavior: a key to decode the biological enigma of human suicidal behavior.

Suicidal behavior is a globally widespread psychiatric disorder with a high rate of mortality. Suicide causes psychological and economic hardships for the families and societies, necessitating the development of effective prevention and treatment programs. However, a clear understanding of the neural basis of suicidal behavior would be essential to develop clinically effective therapies. To date, several neurobiological studies have reported the genetic and epigenetic factors, brain regions, and neurotransmitters involved in suicidal behavior; but, a clear understanding of the origins of self-destructive tendencies is lacking. The high prevalence of self-destructive tendency, a potential hallmark of suicidal behavior presents a biological enigma in light of the evolutionarily pervasive struggle for existence and survival (self-preservation instinct). The potential neural correlates of suicidality and survival behavior have been separately investigated. Several regions of prefrontal cortex were implicated in suicide, while the survival circuits regulating the life-processes (defense, thermoregulation, energy and nutrition, fluid balance, and reproduction) include hypothalamus, amygdala, and parabrachial nucleus, among other structures. Future research to understand the possible influence of malfunctioning survival circuits in suicide could provide valuable insights into suicidal behavior. In addition, understanding the possible evolutionary significance of suicidal traits can help us understand the mechanisms of evolution, and also serve towards alleviation of social stigma around suicide. Thus, future research to unravel the biological correlates of survival vs. suicidal instincts, equipped with high-resolution neuroimaging techniques, would be clinically and socially advantageous towards suicide prevention and treatment.

Functions, mechanisms and regulation of Pumilio/Puf family RNA binding proteins: a comprehensive review.

The Pumilio (Pum)/Puf family proteins are ubiquitously present across eukaryotes, including yeast, plants and humans. They generally bind to the 3' untranslated regions of single stranded RNA targets in a sequence specific manner and destabilize them, although a few reports suggest their role in stabilizing the target transcripts. The Pum isoforms are implicated in a wide array of biological processes including stem cell maintenance, development, ribosome biogenesis as well as human diseases. Further studies on Pum would be interesting and important to understand their evolutionarily conserved and divergent features across species, which can have potential implications in medicine, plant sciences as well as basic molecular and cell biological studies. A large number of research reports exists, pertaining to various aspects of Pum, in individual experimental systems. This review is a comprehensive summary of the functions, types, mechanism of action as well as the regulation of Pum in various species. Also, the research questions to be addressed in future are discussed.

Expression analysis of Cell wall invertase under abiotic stress conditions influencing specialized metabolism in Catharanthus roseus.

Catharanthus roseus is a commercial source for anti-cancer terpenoid indole alkaloids (TIAs: vincristine and vinblastine). Inherent levels of these TIAs are very low, hence research studies need to focus on enhancing their levels in planta. Since primary metabolism provides precursors for specialized-metabolism, elevating the former can achieve higher amounts of the latter. Cell Wall Invertase (CWIN), a key enzyme in sucrose-metabolism catalyses the breakdown of sucrose into glucose and fructose, which serve as carbon-skeleton for specialized-metabolites. Understanding CWIN regulation could unravel metabolic-engineering approaches towards enhancing the levels of TIAs in planta. Our study is the first to characterize CWIN at gene-expression level in the medicinal plant, C. roseus. The CWINs and their inter-relationship with sucrose and TIA metabolism was studied at gene and metabolite levels. It was found that sucrose-supplementation to C. roseus leaves significantly elevated the monomeric TIAs (vindoline, catharanthine) and their corresponding genes. This was further confirmed in cross-species, wherein Nicotiana benthamiana leaves transiently-overexpressing CrCWIN2 showed significant upregulation of specialized-metabolism genes: NbPAL2, Nb4CL, NbCHS, NbF3H, NbANS, NbHCT and NbG10H. The specialized metabolites- cinnamic acid, coumarin, and fisetin were significantly upregulated. Thus, the present study provides a valuable insight into metabolic-engineering approaches towards augmenting the levels of therapeutic TIAs.

Effect of Melatonin on the stability and expression of reference genes in Catharanthus roseus.

The role of Melatonin in influencing diverse genes in plants has gained momentum in recent years and many reports have employed qRT-PCR for their quantification. Relative quantification of gene expression relies on accurate normalization of qRT-PCR data against a stably-expressing internal reference-gene. Although researchers have been using commonly available reference-genes to assess Melatonin-induced gene expression, but to-date, there have been no attempts to validate the reference-gene stability under Melatonin-supplementation in planta. In this study, we performed stability assessment of common reference-genes under Melatonin-supplementation and abiotic stress in leaves and seedlings of Catharanthus roseus using geNorm, NormFinder, BestKeeper, ΔCt and RefFinder algorithms. Nine candidate reference-genes were tested for stability in C. roseus (FBOX, CACS, TIP, RSP9, EXP, EXPR, SAND, F17M5, ACT) and our study inferred that while EXP and EXPR were the most-stable, F17M5 was the lowest-stable gene in the leaf-fed samples. Among seedlings of C. roseus, F17M5 and TIP were the most, while ACT was the least-stable gene. The suitability of selected stable reference-gene pairs was demonstrated by assessing the transcript levels of the Melatonin-biosynthesis gene SNAT under same conditions. Our study is the first to comprehensively analyze the stability of commonly-used reference-genes under Melatonin-induced conditions in C. roseus.

Comparative genome based cis-elements analysis in the 5' upstream and 3' downstream region of cell wall invertase and Phenylalanine ammonia lyase in Nicotiana benthamiana.

Plant secondary metabolites are widely used in human disease treatment; though primary metabolism provides precursors for secondary metabolism, not much has been studied to unravel the link connecting both the processes. Most common form of gene regulation interconnecting diverse metabolism occurs at the transcriptional and/or posttranscriptional level mediated by regulatory cis-elements. The present study aims at understanding the common cis-elements network connecting the major primary metabolic enzyme, cell wall invertase (CWIN) and secondary metabolism genes in Nicotiana benthamiana (N. benthamiana). The CWIN and thirty one other gene sequences were extracted from N. benthamiana genome, followed by cis-element analysis of their 5' upstream and 3' downstream region using different programs (Genomatix software suite; PLACE and PlantCARe). Comparative cis-element analysis of CWIN (N. benthamiana and other plant species) and other primary, secondary metabolism and transcription factor genes (N. benthamiana) revealed the occurrence of common stress associated cis-elements. Predominantly, AHBP, L1BX, MYBL, MADS, MYBS, GTBX, DOFF and CCAF were found in the 5' upstream region of all genes, whereas AHBP, MYBL, L1BX, HEAT, CCAF and KAN1 were largely occurring in the 3' downstream region of all genes; indicating common function of these elements in transcriptional and posttranscriptional gene regulation. Further, genomic analysis using FGENESH, GenScan and homology based methods (BlastX and BlastN) was performed on the N. benthamiana contigs harboring CWIN and PAL, in an attempt to identify genomic neighborhood genes. The 5' upstream and 3' downstream region of genes in the genomic neighborhood of CWIN and PAL were also subjected to similar cis-element analysis, and the results indicated cis-elements profile similar to CWIN, PAL and other primary, secondary metabolism and transcription factor genes. The results of evolutionary studies confirmed that the 5' upstream region of NbCWINs significantly showed more proximity to secondary metabolism genes 4CL and the redox gene SOD, followed by the phenylpropanoid pathway gene CHI. The 3' downstream regions of NbCWINs were more closely related to other plant CWINs, followed by the redox gene, SOD and primary metabolism gene FBA. Thus, the commonly found stress responsive cis-elements in our study can play a vital role in modulating key pathways of both primary and secondary metabolism; thereby postulating their role in regulating plant growth and metabolisms under unfavourable growth conditions.

Stress-Mediated cis-Element Transcription Factor Interactions Interconnecting Primary and Specialized Metabolism in planta.

Plant specialized metabolites are being used worldwide as therapeutic agents against several diseases. Since the precursors for specialized metabolites come through primary metabolism, extensive investigations have been carried out to understand the detailed connection between primary and specialized metabolism at various levels. Stress regulates the expression of primary and specialized metabolism genes at the transcriptional level via transcription factors binding to specific cis-elements. The presence of varied cis-element signatures upstream to different stress-responsive genes and their transcription factor binding patterns provide a prospective molecular link among diverse metabolic pathways. The pattern of occurrence of these cis-elements (overrepresentation/common) decipher the mechanism of stress-responsive upregulation of downstream genes, simultaneously forming a molecular bridge between primary and specialized metabolisms. Though many studies have been conducted on the transcriptional regulation of stress-mediated primary or specialized metabolism genes, but not much data is available with regard to cis-element signatures and transcription factors that simultaneously modulate both pathway genes. Hence, our major focus would be to present a comprehensive analysis of the stress-mediated interconnection between primary and specialized metabolism genes via the interaction between different transcription factors and their corresponding cis-elements. In future, this study could be further utilized for the overexpression of the specific transcription factors that upregulate both primary and specialized metabolism, thereby simultaneously improving the yield and therapeutic content of plants.