Biology subjects

Kleiner, R. E.

Publications and source records attributed to Kleiner, R. E..

3 recordsLinked to original sources

bioRxiv2022

Profiling dynamic RNA-protein interactions using small molecule-induced RNA editing

RNA binding proteins (RBPs) play an important role in biology and characterizing dynamic RNA-protein interactions in their native context is essential for understanding RBP function. Here, we develop targets of RNA-binding proteins identified by editing induced through dimerization (TRIBE-ID), a facile strategy for identifying and quantifying state-specific RNA-protein interactions based upon rapamycin-mediated chemically induced dimerization and RNA editing. We perform TRIBE-ID with G3BP1, an abundant RBP and core component of stress granules, to study transcriptome-wide G3BP1-RNA interactions during normal conditions and upon oxidative stress-induced liquid-liquid phase separation (LLPS). We quantify editing kinetics in order to infer interaction persistence and show that stress granule formation strengthens preexisting G3BP1-RNA interactions and induces new RNA-protein binding events. Further, we demonstrate that G3BP1 stabilizes its RNA clients in a dose-dependent manner, suggesting that stress granules function as RNA storage depots. Finally, we apply our method to characterize small molecule modulators of G3BP1-RNA binding. Taken together, our work provides a general approach to profile RNA-protein binding events with temporal control and illuminates the role of LLPS in organizing G3BP1-RNA interactions in the cell.

biochemistry↗

bioRxiv2022

Live-cell RNA imaging with metabolically incorporated fluorescent nucleosides

Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems, however approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2 (UCK2). In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with quantitative confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO2 treatment. Further, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.

biochemistry↗

bioRxiv2021

A chemical method to sequence 5-formylcytosine on RNA

Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method to sequence 5-formylcytosine (f5C) modifications on RNA based upon selective and efficient malononitrile-mediated labeling of f5C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and PCR amplification. We apply Mal-Seq to characterize the prevalence of f5C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f5C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f5C epitranscriptome.

biochemistry↗