Biology subjects

Eswarappa, S.

Publications and source records attributed to Eswarappa, S..

5 recordsLinked to original sources

bioRxiv2026

Translation of a four-codon ORF in the 3'UTR of MAP3K10 regulates its expression

Several eukaryotic mRNAs are polycistronic because of translatable open reading frames (ORFs) embedded in their untranslated regions (UTRs), in addition to the primary ORF (i.e., the coding sequence). While 5'UTR upstream ORFs (uORFs) are mechanistically and functionally well studied, 3'UTR downstream ORFs (dORFs) remain poorly understood. Here, we identify and characterize a short, translatable dORF in the 3'UTR of MAP3K10, which encodes a serine/threonine kinase involved in JNK signalling. A stringent computational screen predicted a conserved RNA G-quadruplex (rG4) within the 3'UTR of MAP3K10. Biophysical assays provided more evidence for rG4 formation, which drives translation of a conserved four-codon dORF. Disruption of the rG4 by point mutations or by an rG4-binding ligand reduced dORF translation. Reporter assays using constructs with strategically placed hairpin structures show that translation of the dORF is independent of both the 5 cap and the translation of the canonical ORF. Notably, deletion of the rG4-dORF module, either in exogenous constructs or in CRISPR-edited cells, led to reduced MAP3K10 expression. Together, these results provide evidence for the regulation of MAP3K10 expression by rG4-driven translation of a dORF. Thus, our study contributes to the growing body of evidence suggesting that dORF translation can regulate gene expression.

molecular biology↗

bioRxiv2024

IVISc-L: A quick and simple in vivo assay to study the regulation of gene expression

Several methods are available to study the regulation of gene expression at cellular and molecular levels. Adaptation of these methods in vivo is cumbersome and often requires animal sacrifice. Here, we report an assay (IVISc-L, In Vivo Imaging of Subcutaneous Luminescence) to study gene regulation in vivo. This assay involves subcutaneous injection of a plasmid DNA encoding firefly luciferase, whose expression is under the regulatory mechanism to be investigated. We could infer its regulated expression by detecting the subcutaneous luminescence using an in vivo imaging system. Using this assay, we have demonstrated the regulation of gene expression mediated by a promoter, micro-RNAs, stop codon readthrough, and rare codons. This minimally invasive assay does not require animal sacrifice or any tissue extraction. The entire assay can be completed within 24 hours. Therefore, this assay will be useful in investigating the mechanisms of gene expression regulation, and screening molecules that can alter gene expression in vivo.

molecular biology↗

bioRxiv2023

Transcript-specific induction of stop codon readthrough using CRISPR-dCas13 system

Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using CRISPR-dCas13 system. Using specific guide RNAs, we targeted dCas13 to the downstream region of the canonical stop codons of mammalian AGO1 and VEGFA, which are known to exhibit natural SCR. Results of readthrough assays revealed the enhancement of SCR of these mRNAs (both exogenous and endogenous) caused by dCas13. This effect was associated with ribosomal pausing, which has been reported in several SCR events. Furthermore, our results show that CRISPR-dCas13 can induce SCR across premature termination codons (PTC) in the mRNAs of green fluorescent protein and TP53. Finally, we demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.

molecular biology↗

bioRxiv2021

Drug targeting Nsp1-ribosomal complex shows antiviral activity against SARS-CoV-2

The SARS-Cov-2 non-structural protein 1 (Nsp1) contains an N-terminal domain and C-terminal helices connected by a short linker region. The C-terminal helices of Nsp1 (Nsp1-C-ter) from SARS-Cov-2 bind in the mRNA entry channel of the 40S ribosomal subunit and block the entry of mRNAs thereby shutting down host protein synthesis. Nsp1 suppresses the host immune function and is vital for viral replication. Hence, Nsp1 appears to be an attractive target for therapeutics. In this study, we have in silico screened Food and Drug Administration (FDA)-approved drugs against Nsp1-C-ter and find that montelukast sodium hydrate binds to Nsp1-C-ter with a binding affinity (KD) of 10.8{+/-}0.2 M in vitro and forms a stable complex with it in simulation runs with a binding energy of -76.71{+/-}8.95 kJ/mol. The drug also rescues the inhibitory effect of Nsp1 in host protein synthesis as demonstrated by the expression of firefly luciferase reporter gene in cells. Importantly, montelukast sodium hydrate demonstrates antiviral activity against SARS-CoV-2 with reduced viral replication in HEK cells expressing ACE2 and Vero-E6 cells. We therefore propose montelukast sodium hydrate may help in combatting SARS-CoV-2 infection.

biophysics↗

bioRxiv2019

Naturally occurring fluorescence protects the eutardigrade Paramacrobiotus sp. from ultraviolet radiation

Naturally occurring fluorescence has been observed in multiple species ranging from bacteria to birds. In macroscopic animals such as birds and fishes, fluorescence provides a visual communication signal. However, the functional significance of this phenomenon is not known in most cases. Though photoprotection is attributed to fluorescence under ultraviolet (UV) light in some organisms, it lacks direct experimental evidence. Here, we have identified a new species of eutardigrade belonging to the genus Paramacrobiotus, which exhibits fluorescence under UV light. Using a natural variant of the same species that lacks fluorescence, we show that the fluorescence confers tolerance to lethal UV radiation. Remarkably, we could transfer this property to UV-sensitive Hypsibius exemplaris, another eutardigrade, and also to C. elegans, a nematode. Using high performance liquid chromatography (HPLC) we isolated the fluorescent compound from Paramacrobiotus sp. This compound has excitation maxima ({lambda}ex) at 370 nm and emission maxima ({lambda}em) at 420-430 nm. We propose that Paramacrobiotus sp. uses a fluorescent shield that absorbs harmful UV radiation, and emits harmless blue light, thereby protecting itself from the lethal effects of UV radiation. Summary statementTardigrades are well known for their tolerance to extreme environmental conditions. In this study, we have identified a new tardigrade species that employs a fluorescent shield to protect itself from the germicidal ultra violet radiation.

ecology↗