Kharel Lab

Research

RNA in the cellular stress response pathways

Cells constantly encounter environmental and physiological stressors that threaten their survival, and RNA plays a central role in orchestrating the cellular stress response. Beyond its traditional role as a messenger, RNA dynamically participates in sensing stress, regulating gene expression, and forming membraneless organelles such as stress granules. These granules sequester specific RNAs and RNA-binding proteins (RBPs), helping the cell prioritize survival pathways while pausing non-essential translation. Our lab investigates how RNA sequence and structural features, and their associated binding partners contribute to stress adaptation mechanisms. By dissecting these RNA-centered regulatory networks, we aim to uncover novel therapeutic targets for diseases where stress response pathways are dysregulated, such as neurodegeneration and cancers.

Stress granules (SGs) are dynamic, membraneless cytoplasmic organelles composed of nonpolysomal mRNAs, specific RBPs, 40S ribosomal subunits, and a subset of translation initiation factors. Our research aims to elucidate the molecular determinants that govern the selective enrichment of specific mRNAs within SGs and to understand how this selective partitioning contributes to cellular adaptation under stress versus the development of disease.

G-quadruplexes (G4s) in RNA biology and G4 interacting proteins

G-quadruplexes are unique four stranded secondary structures formed by guanine-rich nucleic acid sequences. There are thousands of putative G4-forming motifs in the human genome and transcriptome, only a fraction of such motifs fold into G4 structures under a given cellular environment. We use biochemical, biophysical, molecular, cellular, and next-generation sequencing tools to identify folded G4s in normal vs pathological cellular environment with the aim to understand the mechanism and function of such folding. We are also interested in studying different G4 binding proteins that regulate G-rich motif ↔ G4 folding-unfolding dyanmics.

Figure 1. G-quadruplexes (G4s) play critical role in RNA biology, that ranges from transcription, splicing, transport, function, and decay. We use state up the art techniques to study biologically relevant RNA G4s and their interaction with RNA binding proteins, such as DHX36 and G3BP1. — G-quadruplexes (G4s) play critical role in RNA biology, that ranges from transcription, splicing, transport, function, and decay. We use state up the art techniques to study biologically relevant RNA G4s and their interaction with RNA binding proteins, such as DHX36 and G3BP1.

DUX4 RNA biology in FSHD

Facioscapulohumeral Muscular Dystrophy (FSHD) is one of the most common genetic muscle disorder primarily driven by the abnormal expression of the DUX4 gene, which encodes a transcription factor toxic to skeletal muscle cells. Normally, DUX4 is active only during early embryonic development and mostly remains silenced in adult tissues except in testis and thymus. In FSHD, this silencing mechanism fails due to genetic and epigenetic alterations, leading to inappropriate DUX4 activation in skeletal muscle cells. Our lab investigates how G4s and other nucleic acid secondary structures contribute to DUX4 transcription and translation regulation, with the goal of developing targeted therapeutics that disrupt DUX4 mRNA synthesis and stabilization.

Facioscapulo humeral muscular dystrophy (FSHD) is caused by the derepression of DUX4 gene in human skeletal muscles. We use biochemical and cellular approaches to identify molecular processes contributing to the disease and identify novel therapeutic avenues. — Facioscapulo humeral muscular dystrophy (FSHD) is caused by the derepression of DUX4 gene in human skeletal muscles. We use biochemical and cellular approach to identify molecular processes contributing to the disease and identify novel therapeutic avenues.

Mechanism of non-canonical mRNA translation

Nucleotide-repeat expansions are a major cause of several neurological disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These expansions can disrupt cellular function in multiple ways, for example, by producing toxic proteins, altering gene expression, or forming abnormal RNA-protein assemblies. GGGGCC (G4C2) hexanucleotide repeat expansion mutation in the open reading frame 72 of chromosome 9 (C9ORF72) is the most common genetic cause of ALS and FTD, accounting for ~25%-40% of familial ALS cases, ~6% of sporadic ALS cases, and ~25% of FTD. Normal function of C9ORF72 and exact mechanism of pathogenecity because of the repeat expansion mutation has not been clearly understood yet. However, it is evident that G4C2 repeats can form complex G4s and undergo repeat-associated non-AUG (RAN) translation, generating toxic dipeptide repeat proteins. Our lab uses molecular, cellular, and synthetic biology approaches to dissect how C9ORF72 G4C2 repeat expansions drive ALS/FTD pathogenesis, with a focus on the mechanisms of RAN translation, its associated toxicity, and the cellular factors that enable this process.

G4C2 repeat mutation within the introns of C9ORF72 gene is one of the most common genetic cause of ALS/FTD. We use biochemical, biophysical, molecular and cellular tools to decipher the molecular mechanism of repeat associated non-canonical (RAN) translation that drives G4C2 repeat mediated pathogenesis. — G4C2 repeat mutation within the introns of C9ORF72 gene is the most common genetic cause of ALS/FTD. We use biochemical, biophysical, molecular and cellular tools to decipher the molecular mechanism of repeat associated non-canonical (RAN) translation that drives G4C2 repeat mediated pathogenesis.