Researchers from the University of Cambridge introduce RNA G-quadruplex sequencing (rG4-seq), a transcriptome-wide RNA G-quadruplex (rG4) profiling method that couples rG4-mediated reverse transcriptase stalling with next-generation sequencing. Using rG4-seq on polyadenylated-enriched HeLa RNA, the researchers generated a global in vitro map of thousands of canonical and noncanonical rG4 structures. They characterize rG4 formation relative to cytosine content and alternative RNA structure stability, uncover rG4-dependent differences in RNA folding and show evolutionarily conserved enrichment in transcripts mediating RNA processing and stability.
Overview of rG4-seq and chemical structures of rG4 and PDS
(a) Chemical structure of G-quartet and schematic of an intramolecular RNA G-quadruplex (rG4). The presence of K+ stabilises this RNA structural motif. (b) Working flowchart of rG4-seq. RNA is ligated to a 3’ adapter, followed by RNA folding under Li+ (rG4 non-stabilizing), K+ (physiological) or K++PDS (rG4-stabilizing) conditions. rG4 induces reverse transcriptase (RTase) stalling, leading to cDNA fragments of different lengths. cDNAs are ligated to a 5’ adapter, followed by PCR and next generation sequencing (NGS). The BASP1 (chr5:17,276,185-17,276,254) example here shows a drop in coverage (from 3’ to 5’ direction) in K+ and K++PDS conditions due to rG4 formation, whereas coverage is generally uniform in Li+. (c) Chemical structure of pyridostatin (PDS), an rG4 stabilising ligand.
Availability – The computer code and scripts to analyze the data are available in Supplementary Software.