RanGTPase links nucleo-cytoplasmic transport to the recruitment of cargoes into small extracellular vesicles.

Small extracellular vesicle (sEV)-mediated intercellular communication regulates multiple aspects of growth and development in multicellular organisms. However, the mechanism underlying cargo recruitment into sEVs is currently unclear. We show that the key nucleo-cytoplasmic transport (NCT) protein-RanGTPase, in its GTP-bound form (RanGTP), is enriched in sEVs secreted by mammalian cells. This recruitment of RanGTP into sEVs depends on the export receptor CRM1 (also called XPO1). The recruitment of GAPDH, a candidate cargo protein, into sEVs is regulated by the RanGTP-CRM1axis in a nuclear export signal (NES)-dependent manner. Perturbation of NCT through overexpression or depletion of nuclear transport components affected the recruitment of Ran, CRM1 and GAPDH into sEVs. Our studies, thus, suggest a link between NCT, particularly the Ran-CRM1 axis, and recruitment of NES-containing cargoes into the sEVs. Collectively, these findings implicate RanGTPase as a link between NCT and sEV mediated intercellular communication.

Correction to: A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR.

In Volume 46 of the Journal of Biosciences, in the article titled 'A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR' by Ridim D Mote, V Shinde Laxmikant, Surya Bansi Singh, Mahak Tiwari, Hemant Singh, Juhi Srivastava, Vidisha Tripathi,Vasudevan Seshadri, Amitabha Majumdar and Deepa Subramanyam, published on 27 November 2021 (https://doi.org/10.1007/s12038-021- 00231-w), the second author's name was incorrectly set as V Shinde Laxmikant. The correct name should read as Shinde Laxmikant V.

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR.

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

Conserved RNA Binding Activity of Phosphatidyl Inositol 5-Phosphate 4-Kinase (PIP4K2A).

Plasmodium falciparum is a causative agent for malaria and has a complex life cycle in human and mosquito hosts. During its life cycle, the malarial parasite Plasmodium goes through different asexual and sexual stages, in humans and mosquitoes. Expression of stage-specific proteins is important for successful completion of its life cycle and requires tight gene regulation. In the case of Plasmodium, due to relative paucity of the transcription factors, it is postulated that posttranscriptional regulation plays an important role in stage-specific gene expression. Translation repression of specific set of mRNA has been reported in gametocyte stages of the parasite. A conserved element present in the 3'UTR of some of these transcripts was identified. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K2A) was identified as the protein that associates with these RNA. We now show that the RNA binding activity of PIP4K2A is independent of its kinase activity. We also observe that PIP4K2A is imported into the parasite from the host on Plasmodium berghei and Toxoplasma gondii. The RNA binding activity of PIP4K2A seems to be conserved across species from Drosophila and C. elegans to humans, suggesting that the RNA binding activity of PIP4K may be important, and there may be host transcripts that may be regulated by PIP4K2A. These results identify a novel RNA binding role for PIP4K2A that may not only play a role in Plasmodium propagation but may also function in regulating gene expression in multicellular organisms.

Structural insights into histone chaperone Asf1 and its characterization from Plasmodium falciparum.

Asf1 is a highly conserved histone chaperone that regulates tightly coupled nucleosome assembly/disassembly process. We observed that Plasmodium falciparum Asf1 (PfAsf1) is ubiquitously expressed in different stages of the life cycle of the parasite. To gain further insight into its biological activity, we solved the structure of N-terminal histone chaperone domain of PfAsf1 (1-159 amino acids) by X-ray crystallography to a resolution of 2.4 Å. The structure is composed of two beta-sheet to form a beta-sandwich, which resembles an immunoglobulin-like fold. The surface-charge distribution of PfAsf1 is distinct from yAsf1 and hAsf1 although the core-structure shows significant similarity. The crystal-structure indicated that PfAsf1 may exist in a dimeric-state which was further confirmed by solution cross-linking experiment. PfAsf1 was found to specifically interact with Plasmodium histone H3 and H4 and was able to deposit H3/H4 dimer onto DNA-template to form disomes, showing its characteristic histone chaperone activity. We mapped the critical residues of PfAsf1 involved in histone H3/H4 interaction and confirmed by site-directed mutagenesis. Further analysis indicates that histone interacting surface of Asf1 is highly conserved while the dimerization interface is variable. Our results identify the role of PfAsf1 as a mediator of chromatin assembly in Plasmodium falciparum, which is the causative agent of malignant malaria in humans.

Application of mass spectrometry based proteomics to understand diabetes: A special focus on interactomics.

Diabetes, a multifactorial disorder is characterized by elevated blood glucose levels resulting from changes in lifestyle, genetic and epigenetic changes or aberrations in proteome. In addition, alterations in post-translational modifications (PTMs) and protein-protein interactions (PPIs) also contribute to the development of diabetes pathogenesis. Recent advances in omics technologies have broadened the perspective for systematic investigation of proteome alterations in understanding the pathogenesis of diabetes. Further, PPIs are central to cellular signaling in all living organisms and deranged PPIs lead to diabetic complications. In this context, affinity purification mass spectrometry (AP-MS) along with diverse bioinformatic approaches has proven to be competent in mapping large-scale PPI networks around the critical players in the glucose homeostasis. In this review, we revisit the application of proteomic approaches in investigating proteome alterations and probing PPI networks for a better understanding of the underlying intricacies of the major signaling pathways in altered glucose homeostasis.

Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development.

The chromatin organizer SATB1 is highly enriched in thymocytes and is essential for T-cell development. Although SATB1 regulates a large number of genes important for T-cell development, the mechanism(s) regulating expression of SATB1 during this process remain elusive. Using chromatin immune precipitation-seq-based occupancy profiles of H3K4me3 and H3Kme1 at Satb1 gene locus, we predicted four different alternative promoters of Satb1 in mouse thymocytes and characterized them. The expression of Satb1 transcript variants with distinct 5' UTRs occurs in a stage-specific manner during T-cell development and is dependent on TCR signaling. The observed discrepancy between the expression levels of SATB1 mRNA and protein in developing thymocytes can be explained by the differential translatability of Satb1 transcript variants as confirmed by polysome profiling and in vitro translation assay. We show that Satb1 alternative promoters exhibit lineage-specific chromatin accessibility during T-cell development from progenitors. Furthermore, TCF1 regulates the Satb1 P2 promoter switch during CD4SP development, via direct binding to the Satb1 P2 promoter. CD4SP T cells from TCF1 KO mice exhibit downregulation of P2 transcript variant expression as well as low levels of SATB1 protein. Collectively, these results provide unequivocal evidence toward alternative promoter switch-mediated developmental stage-specific regulation of SATB1 in thymocytes.

Translation of insulin granule proteins are regulated by PDI and PABP.

Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain elusive. The trans-acting factors like PABP (Poly A Binding Protein) and PDI (Protein Disulphide Isomerize) binds to a conserved sequence in the 5'UTR of insulin mRNA and regulates its translation. Here, we demonstrate that 5'UTR of PC1/3 and PC2 also associate with PDI and PABP. We show that a' and RRM 3-4 domains of PDI and PABP respectively, are necessary for RNA binding activity to the 5'UTRs of insulin and its processing enzymes.

MicroRNA, Proteins, and Metabolites as Novel Biomarkers for Prediabetes, Diabetes, and Related Complications.

Type 2 diabetes mellitus (T2DM) is no more a lifestyle disease of developed countries. It has emerged as a major health problem worldwide including developing countries. However, how diabetes could be detected at an early stage (prediabetes) to prevent the progression of disease is still unclear. Currently used biomarkers like glycated hemoglobin and assessment of blood glucose level have their own limitations. These classical markers can be detected when the disease is already established. Prognosis of disease at early stages and prediction of population at a higher risk require identification of specific markers that are sensitive enough to be detected at early stages of disease. Biomarkers which could predict the risk of disease in people will be useful for developing preventive/proactive therapies to those individuals who are at a higher risk of developing the disease. Recent studies suggested that the expression of biomolecules including microRNAs, proteins, and metabolites specifically change during the progression of T2DM and related complications, suggestive of disease pathology. Owing to their omnipresence in body fluids and their association with onset, progression, and pathogenesis of T2DM, these biomolecules can be potential biomarker for prognosis, diagnosis, and management of disease. In this article, we summarize biomolecules that could be potential biomarkers and their signature changes associated with T2DM and related complications during disease pathogenesis.

Curcumin reverses diabetes-induced endothelial progenitor cell dysfunction by enhancing MnSOD expression and activity in vitro and in vivo.

Diabetes mellitus (DM) causes dysfunction of endothelial progenitor cells (EPCs), resulting in impaired wound healing. EPC therapy is a potential substitute to the current treatments of chronic wounds. Because EPCs isolated from diabetic patients are dysfunctional and therefore pose an obstacle in their efficacious employment in autologous cell therapy, a strategy to rescue them prior to transplantation would be expected to improve the efficacy of autologous cell therapy multifold. Compromised reactive oxygen species scavenging ability being the main cause of EPC dysfunction (EPCD), reactive oxygen species scavengers are likely to reverse or rescue EPCD. Therefore, in this study, we evaluated the potential of curcumin in reversing DM-induced EPCD. We found that in vitro treatment of bone marrow EPCs from diabetic mice (D-EPC) with curcumin restored their functionality, as judged by colony formation, tubule formation, and migration assays. Most importantly, autologous transplantation of curcumin-treated D-EPCs onto diabetic wounds also resulted in accelerated wound healing. Furthermore, curcumin-treated diabetic mice exhibited improved wound healing, as compared with their vehicle-treated diabetic counterparts, underscoring the efficacy of curcumin in vivo as well. The levels and activity of manganese superoxide dismutase (MnSOD) in D-EPCs treated in vitro with curcumin or those isolated from curcumin-treated diabetic mice were comparable with those in non-diabetic EPCs. Addition of methyl mercury chloride to inhibit MnSOD activity during curcumin treatment abolished the salutary effects of curcumin. Our data demonstrate that curcumin reverses DM-induced EPCD by boosting MnSOD expression and activity and emphasizes its potential for use in autologous cell therapy for diabetic wound management.

Identification of human Phosphatidyl Inositol 5-Phosphate 4-Kinase as an RNA binding protein that is imported into Plasmodium falciparum.

Plasmodium falciparum is a causative agent for malaria and has a complex life cycle in human and mosquito hosts. Translation repression of specific set of mRNA has been reported in gametocyte stages of this parasite. A conserved element present in the 3'UTR of some of these transcripts was identified. Biochemical studies have identified components of the RNA storage and/or translation inhibitor complex but it is not yet clear how the complex is specifically recruited on the RNA targeted for translation regulation. We used the 3'UTR region of translationally regulated transcripts to identify Phosphatidyl-inositol 5-phosphate 4-kinase (PIP4K2A) as the protein that associates with these RNAs. We further show that recombinant PIP4K2A has the RNA binding activity and can associate specifically with Plasmodium 3'UTR RNAs. Immunostainings show that hPIP4K2A is imported into the Plasmodium parasite from RBC. These results identify a novel RNA binding role for PIP4K2A that may play a role in Plasmodium propagation.

Interaction of HuDA and PABP at 5'UTR of mouse insulin2 regulates insulin biosynthesis.

Understanding the regulation of insulin biosynthesis is important as it plays a central role in glucose metabolism. The mouse insulin gene2 (Ins2) has two splice variants; long (Ins2L) and short (Ins2S), that differ only in their 5'UTR sequence and Ins2S is the major transcript which translate more efficiently as compared to Ins2L. Here, we show that cellular factors bind preferentially to the Ins2L 5'UTR, and that PABP and HuD can bind to Ins2 splice variants and regulate its translation. In vitro binding assay with insulin 5'UTR and different HuD isoforms indicate that the 'N' terminal region of HuD is important for RNA binding and insulin translation repression. Using reporter assay we showed that specifically full-length HuD A isoform represses translation of reporter containing insulin 5'UTR. We further show that PABP and HuD interact with each other in RNA-dependent manner and this interaction is affected by glucose and PDI (5'UTR associated translation activator). These results suggest that PABP interacts with HuD in basal glucose conditions making translation inhibitory complex, however upon glucose stimulation this association is affected and PABP is acted upon by PDI resulting in stimulation of insulin translation. Together, our findings snapshot the mechanism of post-transcriptional regulation of insulin biosynthesis.

A MicroRNA/Ubiquitin Ligase Feedback Loop Regulates Slug-Mediated Invasion in Breast Cancer.

The transformation of a normal cell to cancer requires the derail of multiple pathways. Normal signaling in a cell is regulated at multiple stages by the presence of feedback loops, calibration of levels of proteins by their regulated turnover, and posttranscriptional regulation, to name a few. The tumor suppressor protein FBXO31 is a component of the SCF E3 ubiquitin ligase and is required to arrest cells at G1 following genotoxic stresses. Due to its growth-suppression activity, it is underexpressed in many cancers. However, the molecular mechanism underlying the translational regulation of FBXO31 remains unclear. Here we show that the oncogenic microRNAs miR-93 and miR-106a repress FBXO31, resulting in the upregulation of Slug, which is involved in epithelial-mesenchymal transition and cell invasion. FBXO31 targets and ubiquitylates Slug for proteasomal degradation. However, this mechanism is repressed in breast tumors where miR-93 and miR-106a are overexpressed. Our study further unravels an interesting mechanism whereby Slug drives the expression of miR-93 and miR-106a, thus establishing a positive feedback loop to maintain an invasive phenotype. Together, these results establish the presence of interplay between microRNAs and the ubiquitination machinery, which together regulate cancer cell invasion.

Nup358 binds to AGO proteins through its SUMO-interacting motifs and promotes the association of target mRNA with miRISC.

MicroRNA (miRNA)-guided mRNA repression, mediated by the miRNA-induced silencing complex (miRISC), is an important component of post-transcriptional gene silencing. However, how miRISC identifies the target mRNA in vivo is not well understood. Here, we show that the nucleoporin Nup358 plays an important role in this process. Nup358 localizes to the nuclear pore complex and to the cytoplasmic annulate lamellae (AL), and these structures dynamically associate with two mRNP granules: processing bodies (P bodies) and stress granules (SGs). Nup358 depletion disrupts P bodies and concomitantly impairs the miRNA pathway. Furthermore, Nup358 interacts with AGO and GW182 proteins and promotes the association of target mRNA with miRISC A well-characterized SUMO-interacting motif (SIM) in Nup358 is sufficient for Nup358 to directly bind to AGO proteins. Moreover, AGO and PIWI proteins interact with SIMs derived from other SUMO-binding proteins. Our study indicates that Nup358-AGO interaction is important for miRNA-mediated gene silencing and identifies SIM as a new interacting motif for the AGO family of proteins. The findings also support a model wherein the coupling of miRISC with the target mRNA could occur at AL, specialized domains within the ER, and at the nuclear envelope.

Prolonged exposure to insulin with insufficient glucose leads to impaired Glut4 translocation.

Insulin maintains glucose homeostasis by stimulating glucose uptake from extracellular environment to adipose and muscle tissue through glucose transporter (GLUT4). Insulin resistance plays a significant role in pathologies associated with type2 diabetes. It has been previously shown that hyperinsulinemia can lead to insulin resistance. In these studies very high levels of insulin was used to achieve insulin resistance. We hypothesized that one of the causes of type 2 diabetes could be insulin synthesis in the absence of glucose stimulation. We used CHO cell line, stably expressing Myc-GLUT4-GFP along with human insulin receptor to study the effect of hyperinsulinemia in the presence of low glucose (6.5 mM) or high glucose (20 mM). The insulin responsiveness of these cells was assessed by FRAP, FACS and subcellular fractionation. The results suggest that exposure of cells to insulin in low glucose conditions made these cells insulin resistant within 10 passages, while the same level of insulin in the presence of high glucose did not result in insulin resistance. These results clearly suggest that hyperinsulinemia combined with hypoglycaemia may lead to insulin resistance and may be one of the causes for the typ2 diabetes.

miR-196b-mediated translation regulation of mouse insulin2 via the 5'UTR.

The 5' and the 3' untranslated regions (UTR) of the insulin genes are very well conserved across species. Although microRNAs (miRNAs) are known to regulate insulin secretion process, direct regulation of insulin biosynthesis by miRNA has not been reported. Here, we show that mouse microRNA miR-196b can specifically target the 5'UTR of the long insulin2 splice isoform. Using reporter assays we show that miR-196b specifically increases the translation of the reporter protein luciferase. We further show that this translation activation is abolished when Argonaute 2 levels are knocked down after transfection with an Argonaute 2-directed siRNA. Binding of miR-196b to the target sequence in insulin 5'UTR causes the removal of HuD (a 5'UTR-associated translation inhibitor), suggesting that both miR-196b and HuD bind to the same RNA element. We present data suggesting that the RNA-binding protein HuD, which represses insulin translation, is displaced by miR-196b. Together, our findings identify a mechanism of post-transcriptional regulation of insulin biosynthesis.

Vimentin is a component of a complex that binds to the 5'-UTR of human heme-regulated eIF2α kinase mRNA and regulates its translation.

The human heme-regulated eIF2α kinase, also called the human heme-regulated inhibitor (hHRI) is significantly up-regulated particularly at the level of translation during stress. In this report we show that during lead-stress, the regulation of hHRI mRNA translation is mediated through its 5'-untranslated region (UTR) that interacts with specific trans-acting factors. Further, vimentin has been identified as one of the trans-acting factors that contribute to this regulation.

Obesity, insulin resistance, and metabolic syndrome: a study in WNIN/Ob rats from a pancreatic perspective.

Alterations in pancreatic milieu to adapt to physiological shifts occurring in conditions of obesity and metabolic syndrome (MS) have been documented, though mechanisms leading to such a state have remained elusive so far. The data presented here tries to look at the gravity of metabolic insult during the early and prolonged phases of obesity/insulin resistance (IR) depicted in WNIN/Ob strain of rats-an obese euglycemic mutant rat model developed indigenously at our institute which is highly vulnerable for a variety of degenerative diseases. The present results in situ show the participation of several confounding factors in the pancreatic milieu that collectively coprecipitates for a state of profound inflammation in the pancreas (among Mutant compared to Lean/Control) which gets worsened with age. These include hypertrophy, macrophage infiltration (CD11b/TNFα/IL6), apoptosis, ß-cell vacuolation, hyperinsulinemia (HI), and stress markers (RL-77/HSP104/TBARS) all of which correlated well with indices for obesity (2-3 fold), IR (1.5-3 fold), and HI (2-3 fold). Further, supportive data was also obtained from in vitro studies using islet cell cultures amongst phenotypes. Taken together, these results advocate that inflammation was the major precipitating factor to cause islet cell dysfunctions (in situ and in vitro) in these Mutant rats compared to their Lean littermates and parental Control.

Wnt signalling antagonizes stress granule assembly through a Dishevelled-dependent mechanism.

Cells often respond to diverse environmental stresses by inducing stress granules (SGs) as an adaptive mechanism. SGs are generally assembled as a result of aggregation of mRNAs stalled in a translational pre-initiation complex, mediated by a set of RNA-binding proteins such as G3BP and TIA-1. SGs may serve as triage centres for storage, translation re-initiation or degradation of specific mRNAs. However, the mechanism involved in the modulation of their assembly/disassembly is unclear. Here we report that Wnt signalling negatively regulates SG assembly through Dishevelled (Dvl), a cytoplasmic Wnt effector. Overexpression of Dvl2, an isoform of Dvl, leads to impairment of SG assembly through a DEP domain dependent mechanism. Intriguingly, the Dvl2 mutant K446M, which corresponds to an analogous mutation in Drosophila Dishevelled DEP domain (dsh(1)) that results in defective PCP pathway, fails to antagonize SG assembly. Furthermore, we show that Dvl2 exerts the antagonistic effect on SG assembly through a mechanism involving Rac1-mediated inhibition of RhoA. Dvl2 interacts with G3BP, a downstream component of Ras signalling involved in SG assembly, and functional analysis suggests a model wherein the Dvl-Rac1-RhoA axis regulates G3BP's SG-nucleating activity. Collectively, these results define an antagonistic effect of Wnt signalling on SG assembly, and reveal a novel role for Wnt/Dvl pathway in the modulation of mRNA functions.