Defining expansions and perturbations to the RNA polymerase III transcriptome and epitranscriptome by modified direct RNA nanopore sequencing

RNA polymerase III (Pol III) transcribes cytosolic transfer RNAs (tRNAs) and other non-coding RNAs (ncRNAs) essential to cellular function. However, many aspects of Pol III transcription and processing, including RNA modifications, remain poorly understood, mainly due to a lack of available sensitive and systematic methods for their analysis. Here, we present DRAP3R (Direct Read and Analysis of Polymerase III transcribed RNAs), a modified nanopore direct RNA sequencing approach and analysis framework that enables the specific and sensitive capture of pre-mature Pol III transcribed RNAs. Applying DRAP3R to distinct cell types, we identify previously unconfirmed tRNA genes and other novel Pol III transcribed RNAs, thus expanding the known Pol III transcriptome. Critically, DRAP3R also enables discrimination between co- and post-transcriptional RNA modifications such as pseudouridine (Ψ) and N⁶-methyladenosine (m⁶A) at single-nucleotide resolution across all examined transcript types and reveals differential Ψ installation patterns across tRNA isodecoders and other ncRNAs. Finally, applying DRAP3R to epithelial cells infected with Herpes Simplex Virus Type 1 reveals an extensive remodelling of both the Pol III transcriptome and epitranscriptome. Our findings thus establish DRAP3R as a powerful tool for systematically studying Pol III transcribed RNAs and their modifications in diverse cellular contexts.