Between the vast lands of DNA and proteins lies the ephemeral and dynamic world of chromatin-associated RNAs, a less-explored but utterly critical domain of the eukaryotic nucleus. Thankfully, RNA-focused researchers can now turn to a tagmentation-based profiling technique (“RT&Tag”) to map the landscape of regulatory chromatin-associated RNAs to aid this quest.
In their fascinating study, Nadiya Khyzha, Steven Henikoff, and Kami Ahmad (Fred Hutchinson Cancer Center) describe Reverse Transcribe and Tagment (RT&Tag) – an adaption of an enzyme-tethering strategy to profile protein binding sites in chromatin (CUT&Tag) – as a rapid, low-cost, proximity labeling tool that maps chromatin-associated RNAs. In this novel approach, RNAs associated with a chromatin epitope are targeted by an antibody, which is recognized by the protein A-Tn5 transposase; next, localized reverse transcription generates RNA/cDNA hybrids that are then tagmented by Tn5 transposases for downstream next-generation sequencing.
Let’s hear from Khyzha, Henikoff, and Ahmad about their RT&Tag approach to chromatin-associated RNA mapping:
- The high efficiency of in-situ antibody tethering and tagmentation by RT&Tag means fewer input cells (~100,000) and a smaller number of sequencing reads (4–8 million per sample) when compared to conventional RNA immunoprecipitation-based methodologies
- RT&Tag forgoes crosslinking and RNA fragmentation and captures interactions within intact nuclei
- RT&Tag in Drosophila S2 nuclei using an antibody against the MSL complex subunit 2 (MSL2) captures the known interaction of the roX2 non-coding RNA with the dosage compensation complex of proteins and detects nascent mRNA transcripts across the X chromosome
- These proof-of-concept assays provide evidence that RT&Tag detects trans-acting RNAs in chromatin regulatory complexes and cis-acting RNAs arising from stimulated genes
- RT&Tag using an antibody against H3K27me3 (to target Polycomb domains) detects candidate silencing RNAs and rare unprocessed transcripts from silenced target genes
- RT&Tag can also detect post-transcriptionally N6-methyladenosine-modified (m6A) mRNAs using a specific antibody
- METTL3 binding alone does not support mRNA methylation, suggesting a need for additional helping factors; instead, promoter pausing of RNA polymerase II characterizes genes producing methylated transcripts
Overall, these pioneering experiments highlight the efficacy of RT&Tag when mapping RNA-protein interactions, RNA-chromatin interactions, and RNA modifications within eukaryotic cells. Future developments in RT&Tag hope to enable the capture of additional functional RNA families (siRNAs, piRNAs, miRNAs, and snoRNAs) while future studies may focus on situations in which low amounts of biological material have classically limited most investigations, such as developmental processes. Take a glimpse of the ephemeral world of regulatory chromatin-associated RNAs over at Nature Methods, October 2022.