Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs

Abstract

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.

Exonuclease-enhanced prime editors

“…At 48 h after transfection, cells were selected for 2 weeks with 0.5 μg ml −1 puromycin dihydrochloride (Gibco, Thermo Fisher Scientific) in the presence of 0.5 μM AZD7648 (MedChemExpress, catalog no. HY-111783) (a DNA-PKcs inhibitor 32 ) and a CAG-promoter-driven i53 (a 53BP1 inhibitor) to inhibit NHEJ and shift the DNA repair towards HDR 33,34 . The surviving polyclonal population carrying the eTLR system stably in the AAVS1 locus was monoclonalized using limiting dilution in 96-well plates.…”

A Peptide Encoded by Long Non-coding RNA NEAT1 Suppresses Cancer Growth through Interfering RAF-HSP90β Complex Stability

“…Paraspeckles cannot be formed or maintained upon loss of NEAT1 [7][8][9] . Although NEAT1 is primarily located in the nucleus and plays important regulatory roles, studies have shown that NEAT1 is also localized in the cytoplasm 10 . As a long non-coding RNA (lncRNA), NEAT1 acts as a competitive endogenous RNA (ceRNA) in the cytoplasm, exerting sponge-like effects by interacting with microRNA molecules and regulating their activity and availability [11][12][13][14] .…”