Poster abstracts
Poster number 112 submitted by Alexa Schnell
Non-invasive cellular extraction of RNA (NICER-seq) for continuous monitoring of live cell states
Alexa L. Schnell (Department of Pharmacology and Department of Biochemistry, Center for RNA Science and Therapeutics, Case Western Reserve University), Joseph M. Luna (Department of Biochemistry, Center for RNA Science and Therapeutics, Case Western Reserve University)
Abstract:
RNA-seq has revolutionized biomedical research by enabling efficient analysis of the transcriptome, but most RNA extraction methods are inherently destructive, requiring cell lysis or fixation, thereby eliminating the possibility for dynamic studies of cell populations over time. Yet, temporal resolution of transcriptomic changes arising from disease progression, drug resistance, and immune activation requires a scalable, non-invasive technology. Inspired by the packaging of viral RNA genomes, we demonstrate that self-assembling viral capsid proteins can be repurposed to package RNA-RNA-binding protein (RBP) complexes from cells. Here, we fuse HIV Gag with poly(A)-binding protein (PABPC1) to facilitate mRNA packaging inside virus-like particles (VLPs), serving as a continuous reporter of the coding transcriptome. We demonstrate that Gag-PABP fusion proteins are expressed and released into the supernatant of HEK293 cells. Further, RT-qPCR shows that mRNA is enriched in the supernatant of cells transfected with Gag-PABP constructs. Encouraged by these results, we performed RNA-seq which revealed that mRNAs packaged in supernatant reflect the mRNA transcriptome found in the cell. Termed NICER-seq (non-invasive cellular extraction of RNA), our preliminary results suggest that the technology is functional, yet we still need to validate the temporal resolution of NICER-seq against published results from static timepoints. Beyond the coding transcriptome, we plan to concretely define the pools of RNA molecules accessible for interrogation via NICER-seq. We anticipate NICER-seq’s utility in contexts such as drug treatment, disease, and other environmental stressors that link the dynamically regulated RBPome to changing cell states.
Keywords: Transcriptomics, RNA-binding protein