Biology subjects

Das, U.

Publications and source records attributed to Das, U..

25 records · Page 2Linked to original sources

bioRxiv2024

5-Aza-Cytidine Enhances Terminal Polyadenylation Site Usage for Full-Length Transcripts in Cells

As an inhibitor of DNA methyltransferases (DNMTs) and an anti-cancer drug, 5-aza-cytidine (5-azaC)s many effects on gene expression remains unclear. Here, we show that 5-azaC treatment of cultured GH3 pituitary tumour cells increases relative usage of genomic terminal exons (GTEs) across the transcriptome. This effect is largely achieved by shifting mRNA polyadenylation from proximal poly(A) sites to GTEs, which harbour a more optimal consensus motif of poly(A) signals. Consistent with this shift, 5-azaC upregulates the mRNA anti-termination factors Scaf4 and Scaf8 while downregulating the early termination enhancer E2f2. In MOLM-13 leukaemia cells, 5-azaC similarly promotes the production of full-length transcripts and regulates alternative polyadenylation factors, some of which in the same direction as observed in GH3 cells. Moreover, PCF11, a factor known to promote proximal poly(A) site usage, is upregulated in both cell lines, suggesting a homeostatic response by these cells to counteract transcript lengthening during 5-azaC treatment. Together, these findings uncover a previously unrecognized effect of 5-azaC on gene expression: directional promotion of terminal polyadenylation site usage, driving a transcriptome-wide switch from shortened to full-length mRNAs in tumour or cancer cells and consequently altering the alternative usage of multiple 3' exons.

cell biology↗

bioRxiv2024

Epigenetic Control of Adaptive or Homeostatic Splicing of Alternative Exons and Prolactin Gene Expression During Interval-Training Activities of Pituitary Cells

Interval-training activities induce adaptive cellular changes without altering their fundamental identity, but the precise underlying molecular mechanisms are not fully understood. In this study, we demonstrate that interval-training depolarization (ITD) of pituitary cells triggers distinct adaptive or homeostatic splicing responses of alternative exons. This occurs while preserving the steady-state expression of the Prolactin and other hormone genes. The nature of these splicing responses depends on the exons DNA methylation status, the methyl-C-binding protein MeCP2 and its associated CA-rich motif-binding hnRNP L. Interestingly, the steady expression of the Prolactin gene is also reliant on MeCP2, whose disruption during ITD leads to exacerbated overexpression and multi-exon aberrant splicing of the hormone gene transcripts, similar to the observed hyperprolactinemia or activity-dependent aberrant splicing in Rett Syndrome. Therefore, depending on how many times cells are stimulated, exons may exhibit different splicing responses to cell activities. During the ITD, epigenetic control is crucial for both adaptive or homeostatic splicing and the steady expression of the Prolactin hormone gene. Disruption in this regulation may have significant implications for the development of progressive diseases.

cell biology↗

bioRxiv2022

Serine-129 phosphorylation of α-synuclein is a trigger for physiologic protein-protein interactions and synaptic function

Phosphorylation of -synuclein at the Serine-129 site (-syn Ser129P) is an established pathologic hallmark of synucleinopathies, and also a therapeutic target. In physiologic states, only a small fraction of total -syn is phosphorylated at this site, and consequently, almost all studies to date have focused on putative pathologic roles of this post-translational modification. We noticed that unlike native (total) -syn that is widely expressed throughout the brain, the overall pattern of -syn Ser129P is restricted, suggesting intrinsic regulation and putative physiologic roles. Surprisingly, preventing phosphorylation at the Ser-129 site blocked the ability of -syn to attenuate activity-dependent synaptic vesicle (SV) recycling - widely thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments -syn Ser-129P, and this phosphorylation is required for -syn binding to VAMP2 and synapsin - two functional binding-partners that are necessary for -syn function. AlphaFold2-driven modeling suggests a scenario where Ser129P induces conformational changes in the C-terminus that stabilizes this region and facilitates protein-protein interactions. Our experiments indicate that the pathology-associated Ser129P is an unexpected physiologic trigger of -syn function, which has broad implications for pathophysiology and drug-development.

neuroscience↗

bioRxiv2021

Self-transfecting GMO-PMO and PMO-GMO chimeras enable gene silencing in vitro and in vivo zebrafish model and NANOG Inhibition Induce the Apoptosis in Breast and Prostate Cancer Cells

Phosphorodiamidate Morpholino Oligonucleotides (PMOs)-based antisense reagents cannot enter inside cells by itself without the help of any delivery technique which is the last hurdle for their clinical applications. To overcome this limitation, a self-transfecting GMO-PMO or PMO-GMO chimeras has been explored as a gene silencing reagent where GMO stands for guanidinium morpholino oligonucleotides which linked either at the OH- or NH-end of PMOs. GMO not only facilitates cellular internalization of such chimeras but also participates in Watson-Crick base pairing during gene silencing in ShhL2 cells when designed against mGli1 and compared with scrambled GMO-PMO where mutations were made only to the GMO part. GMO-PMO-mediated knockdown of no tail gene resulted no tail-dependent phenotypes in zebrafish and worked even after the delivery at 16-, 32- and 64-cell stages which were previously unachievable by regular PMO. Furthermore, GMO-PMO chimeras has shown the inhibition of NANOG, a key regulator of self-renewal and pluripotency of both embryonic and cancer stem cells. Its inhibition influences on the expression of other cancer related proteins and the respective phenotypes in breast cancer cells and increases the therapeutic potential of taxol. To the best of our knowledge, this is the first report on the self-transfecting antisense reagents since the discovery of guanidinium linked DNA (DNG) and most effective among the all cell-penetrating PMOs reported till date expected to solve the longstanding problem of PMO delivery. In principle, this technology could be useful for the inhibition of any target gene without using any delivery vehicle and should have applications in the fields of antisense therapy, diagnostic and nanotechnology area. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/447039v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1374f3forg.highwire.dtl.DTLVardef@33ca57org.highwire.dtl.DTLVardef@292959org.highwire.dtl.DTLVardef@599b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets

Clathrin has established roles in endocytosis, with clathrin-cages enclosing membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin synthesized in cell-bodies is conveyed into axons and synapses via slow axonal transport; as shown by classic pulse-chase radiolabeling. What is the cargo-structure, and mechanisms underlying transport and presynaptic-targeting of clathrin? What is the precise organization at synapses? Combining live-imaging, mass-spectrometry (MS), Apex-labeled EM-tomography and super-resolution, we found that unlike dendrites where clathrin transiently assembles/disassembles as expected, axons contain stable transport-packets that move intermittently with an anterograde bias; with actin/myosin-VI as putative tethers. Transport-packets are unrelated to endocytosis, and the overall kinetics generate a slow biased flow of axonal clathrin. Synapses have integer-numbers of clathrin-packets circumferentially abutting the synaptic-vesicle cluster, advocating a model where delivery of clathrin-packets by slow axonal transport generates a radial organization of clathrin at synapses. Our experiments reveal novel trafficking mechanisms, and an unexpected nanoscale organization of synaptic clathrin.

neuroscience↗

bioRxiv2019

Demonstration of altruistic behaviour in rats

A pilot study demonstrating complex cognitive abilities of altruism in rats by observing if rats would voluntarily put themselves in distress solely in order to help another conspecific irrespective of kinship and without any expectation of reward in terms of food or mate in return.

animal behavior and cognition↗

bioRxiv2018

A CRISPR/Cas9 based strategy to manipulate the Alzheimer’s amyloid pathway

The gradual accumulation of amyloid-{beta} (A{beta}) is a neuropathologic hallmark of Alzheimers disease (AD); playing a key role in disease progression. A{beta} is generated by the sequential cleavage of amyloid precursor protein (APP) by {beta}- and {gamma}-secretases, with BACE-1 ({beta}-site APP cleaving enzyme-1) cleavage as the rate limiting step 1-3. CRISPR/Cas9 guided gene-editing is emerging as a promising tool to edit pathogenic mutations and hinder disease progression 4,5,6 However, few studies have applied this technology to neurologic diseases 7-9. Besides technical caveats such as low editing efficiency in brains and limited in vivo validation 7, the canonical approach of mutation-correction would only be applicable to the small fraction of neurodegenerative cases that are inherited (i.e. < 10% of AD, Parkinsons, ALS); with a new strategy needed for every gene. Moreover, feasibility of CRISPR/Cas9 as a therapeutic possibility in sporadic AD has not been explored. Here we introduce a strategy to edit endogenous APP at the extreme C-terminus and reciprocally manipulate the amyloid pathway - attenuating {beta}-cleavage and A{beta}, while up-regulating neuroprotective a-cleavage. APP N-terminus, as well as compensatory APP homologues remain intact, and key physiologic parameters remain unaffected. Robust APP-editing is seen in cell lines, cultured neurons, human embryonic stem cells/iPSC-neurons, and mouse brains. Our strategy works by limiting the physical association of APP and BACE-1, and we also delineate the mechanism that abrogates APP/BACE-1 interaction in this setting. Our work offers an innovative cut and silence gene-editing strategy that could be a new therapeutic paradigm for AD.

neuroscience↗