Biology subjects

Vasu, K.

Publications and source records attributed to Vasu, K..

3 recordsLinked to original sources

bioRxiv2026

Leaky scanning translates a conserved ORF in the 3'UTR of RPL36A and regulates the expression of ribosomal protein L36a.

The 3'UTRs of eukaryotic mRNAs are major hubs of post-transcriptional regulation that govern the spatial and temporal regulation of gene expression. Although 3'UTRs were long considered non-coding, recent studies have revealed translation within downstream open reading frames (dORFs) located in 3'UTRs. In contrast to upstream ORFs (uORFs) in 5'UTRs, the mechanisms governing dORF translation and their functional significance remain poorly understood. Here, we identify and characterize a dORF within the 3'UTR of RPL36A, which encodes ribosomal protein L36a. Analysis of ribosome profiling datasets revealed ribosome footprints aligned with the dORF reading frame across multiple human tissues and cell lines. Conservation analysis together with luciferase- and GFP-based reporter assays further supported active translation of the dORF. Using strategically placed RNA hairpin structures and mutational analyses, we show that dORF translation is consistent with ribosomal leaky scanning. Disruption of dORF translation, either by mutating its start codon in exogenous constructs or by CRISPR-mediated deletion of the endogenous dORF-containing region, reduced RPL36A mRNA and protein levels and resulted in impaired global translation. We further identified a miR-5701-binding site within the dORF region and provide evidence that dORF translation antagonizes miR-5701-mediated repression of RPL36A. Finally, we identify a similar dORF-associated regulatory mechanism in RPS3, which encodes ribosomal protein S3. Together, our findings uncover dORF translation as a regulatory mechanism controlling ribosomal protein expression.

molecular biology↗

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↗