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Nair, R. R.

Publications and source records attributed to Nair, R. R..

27 records · Page 2Linked to original sources
bioRxiv2020

RNA-binding protein network alteration causes aberrant axon branching and growth phenotypes in FUS ALS mutant motoneurons

Mutations in the RNA-binding protein (RBPs) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS.

neuroscience↗
bioRxiv2020

Keratin 17 regulates nuclear morphology and chromatin organization

Keratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wildtype K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17 null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization, and RNA processing. Key histone modifications and LAP2{beta} localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells. SummaryKeratin 17 (K17) is one of two non-lamin intermediate filament proteins recently identified to localize to and function in the cell nucleus. K17 is here shown to regulate nuclear morphology, chromatin organization, LAP2 localization, and cell proliferation.

cell biology↗
bioRxiv2020

Multiplexed mRNA assembly into ribonucleoprotein particles plays an operon-like role in the control of yeast cell physiology

Prokaryotes utilize polycistronic messages (operons) to co-translate proteins involved in the same biological process. Whether eukaryotes achieve similar regulation by selectively assembling monocistronic messages derived from different chromosomes is unclear. We employed transcript-specific RNA pulldowns and RNA-seq/RT-PCR to identify mRNAs that co-precipitate into ribonucleoprotein (RNP) particles in yeast. Consistent with the hypothesis of eukaryotic RNA operons, mRNAs encoding components of the mating pathway, heat shock proteins, and mitochondrial outer membrane proteins multiplex in trans to form discrete mRNP particles, termed transperons. Chromatin-capture experiments reveal that genes encoding multiplexed mRNAs physically interact, thus, RNA assembly may result from co-regulated gene expression. Transperon assembly and function depends upon H4 histones and their depletion leads to defects in RNA multiplexing, resulting in decreased pheromone responsiveness and mating, and increased heat shock sensitivity. We propose that intergenic associations and non-canonical H4 histone functions contribute to transperon formation in eukaryotic cells to regulate cell physiology. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗
bioRxiv2020

Structural and Functional Implications of Non-synonymous Mutations in the Spike protein of 2,954 SARS-CoV-2 Genomes

Protein-protein interactions between virus and host are crucial for infection. SARS-CoV-2, the causative agent of COVID-19 pandemic is an RNA virus prone to mutations. Formation of a stable binding interface between the Spike (S) protein Receptor Binding Domain (RBD) of SARS-CoV-2 and Angiotensin-Converting Enzyme 2 (ACE2) of host actuates viral entry. Yet, how this binding interface evolves as virus acquires mutations during pandemic remains elusive. Here, using a high fidelity bioinformatics pipeline, we analysed 31,403 SARS-CoV-2 genomes across the globe, and identified 444 non-synonymous mutations that cause 49 distinct amino acid substitutions in the RBD. Molecular phylogenetic analysis suggested independent emergence of these RBD mutants during pandemic. In silico structure modelling of interfaces induced by mutations on residues which directly engage ACE2 or lie in the near vicinity revealed molecular rearrangements and binding energies unique to each RBD mutant. Comparative structure analysis using binding interface from mouse that prevents SARS-CoV-2 entry uncovered minimal molecular determinants in RBD necessary for the formation of stable interface. We identified that interfacial interaction involving amino acid residues N487 and G496 on either ends of the binding scaffold are indispensable to anchor RBD and are well conserved in all SARS-like corona viruses. All other interactions appear to be required to locally remodel binding interface with varying affinities and thus may decide extent of viral transmission and disease outcome. Together, our findings propose the modalities and variations in RBD-ACE2 interface formation exploited by SARS-CoV-2 for endurance. ImportanceCOVID-19, so far the worst hit pandemic to mankind, started in January 2020 and is still prevailing globally. Our study identified key molecular arrangements in RBD-ACE2 interface that help virus to tolerate mutations and prevail. In addition, RBD mutations identified in this study can serve as a molecular directory for experimental biologists to perform functional validation experiments. The minimal molecular requirements for the formation of RBD-ACE2 interface predicted using in silico structure models may help precisely design neutralizing antibodies, vaccines and therapeutics. Our study also proposes the significance of understanding evolution of protein interfaces during pandemic.

evolutionary biology↗
bioRxiv2020

The rifampicin-inactivating mono-ADP-ribosyl transferase of Mycobacterium smegmatis significantly influences reactive oxygen species levels in the actively growing cells

A classic example of antibiotic inactivating function in bacteria is the Mycobacterium smegmatis (Msm) encoded rifampicin-inactivating mono-ADP-ribosyl transferase (arr). Since its probable biological role has been proposed to be in DNA damage response, which is inflicted by reactive oxygen species (ROS), in the present study, we examined whether Msm Arr influences ROS levels. For this purpose, the levels of the ROS, hydroxyl radical and superoxide, were determined in the mid-log phase (MLP) cells of Msm arr knockout (arr-KO) strain, in comparison to those in the equivalently grown Msm arr+ wild-type (WT) strain. The MLP arr-KO cells generated significantly elevated levels of superoxide and hydroxyl radical, unlike the equivalently grown WT MLP cells. Complementation of arr-KO with arr, but not with empty vector, restored the ROS levels comparable to those in the WT strain. Elevated ROS levels in the arr-KO strain enabled selection of rifampicin-resistant mutants at 10-7 cfu/ml from the rifampicin-unexposed MLP cells of arr-KO, which is one-log10 higher than that for WT cells (10-8). Upon prolonged exposure to rifampicin, the susceptibility, persister formation, generation of elevated levels of hydroxyl radical by the persisters, rifampicin-resister generation frequency of the persisters and regrowth of the rifampicin-resistant mutants from the respective persisters were all comparable between the arr-KO and WT strains. These observations revealed that Arr influences ROS levels in the actively growing M. smegmatis cells but not in the rifampicin-exposed cells. We proposed the probable pathway through which Arr might be influencing ROS levels in the actively growing M. smegmatis cells. IMPORTANCEDiverse genera of bacteria consisting of pathogens, opportunistic pathogens and non-pathogens, possess Arr-type activities that confer equally efficient rifampicin resistance, thereby posing serious health hazard. Acquisition of this function by other bacteria through horizontal gene transfer enhances the hazard posed by the bacteria possessing it. M. smegmatis is an opportunistic human pathogen that causes infections of skin and soft tissues. Moreover, M. smegmatis is a genetically tractable model organism for M. tuberculosis with the potential to function even as tuberculosis vaccine. In view of these significant aspects of Arr and M. smegmatis, the study to find out the natural physiological role of Arr in M. smegmatis, gains importance for designing strategies to prevent antibiotic inactivation and to target the cellular function to contain the bacterium. Above all, the three-dimensional structure of M. smegmatis Arr reveals significant structural homology with eukaryotic ADP-ribosyltransferases and bacterial toxins, thereby giving the study broad significance.

microbiology↗
bioRxiv2019

Unveiling the physiological mechanisms that drive the emergence of antibiotic resisters from antibiotic persister population of mycobacteria

The physiological mechanisms behind the emergence of antibiotic-resistant bacteria from their antibiotic-persister population are beginning to be explored. Here we delineate the sequential physiological events that drive the emergence of rifampin-resistant rpoB mutants from rifampin-persister population of mycobacteria during prolonged exposure. The rifampin-persister population generated elevated levels of hydroxyl radical, which inflicted mutations, enabling regrowth of the persister cells to form multi-septated, multi-nucleated elongated cells. These cells, through multiple divisions, produced multiple sister-daughter cells, causing an abrupt, unexpectedly high increase of rifampin-resistant colonies. Similar response was observed against moxifloxacin also. Earlier studies on the rifampin/moxifloxacin-exposed laboratory/clinical M. tuberculosis strains from in-vitro cultures and infected mouse-lung also showed cfu spurt, but went unnoticed/unreported. It is likely that these sequential physiological events may be driving the emergence of antibiotic-resistant tubercle bacilli in TB patients also. Escherichia coli also has been found to respond similarly against subminimal inhibitory concentrations of ciprofloxacin. Thus, the present findings attain broad significance as a general physiological mechanism used by diverse bacterial genera to emerge as drug-resistant strains against antibiotics.

microbiology↗
bioRxiv2019

Generation of viral vectors specific to neuronal subtypes of targeted brain regions by Enhancer-Driven Gene Expression (EDGE)

Understanding brain function requires understanding neural circuits at the level of specificity at which they operate. While recent years have seen the development of a variety of remarkable molecular tools for the study of neural circuits, their utility is currently limited by the inability to deploy them in specific elements of native neural circuits, i.e. particular neuronal subtypes. One can obtain a degree of specificity with neuron-specific promoters, but native promoters are almost never sufficiently specific restricting this approach to transgenic animals. We recently showed that one can obtain transgenic mice with augmented anatomical specificity in targeted brain regions by identifying cis-regulatory elements (i.e. enhancers) uniquely active in those brain regions and combining them with a heterologous promoter, an approach we call EDGE (Enhancer-Driven Gene Expression). Here we extend this strategy to the generation of viral (rAAV) vectors, showing that when combined with the right minimal promoter they largely recapitulate the specificity seen in the corresponding transgenic lines in wildtype animals, even of another species. Because active enhancers can be identified in any tissue sample, this approach promises to enable the kind of circuit-specific manipulations in any species. This should not only greatly enhance our understanding of brain function, but may one day even provide novel therapeutic avenues to correct the imbalances in neural circuits underlying many disorders of the brain.

neuroscience↗
bioRxiv2019

Targeting the dysfunctional mitochondrial respiration in drug resistant B-cell acute lymphoblastic leukemia

Reprogramming of cellular pathways is a crucial mechanism of drug resistance and survival in refractory acute lymphoblastic leukemia (ALL) cells. In the present study, we performed an unbiased gene expression analysis and identified a dysfunctional mitochondrial respiration program in drug-resistant ALL cells grown in a co-culture system with bone marrow stromal cells (BMSC). Specifically, the activity of the complexes within the electron transport chain was significantly downregulated, correlated with decreased mitochondrial mass and ATP production in drug-resistant ALL cells. To validate mitochondrial respiration as a druggable target, we utilized pyrvinium pamoate (PP), a known inhibitor of mitochondrial respiration and documented its anti-leukemic activity in several ALL cell lines grown alone or in co-culture with BMSC. To increase the bioavailability profile of PP, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. PP anti-leukemic activity was decreased by the addition of sodium pyruvate, and furthermore, PP was found to have an additive anti-leukemic effect when used in combination with rotenone, a mitochondrial complex I inhibitor with activity similar to PP on the mitochondrial respiration. Importantly, PPs cell death activity was found to be specific for leukemic cells as primary normal immune cells were resistant to PP-mediated cell death. In conclusion, we have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells.

cancer biology↗