The Polycistronic Transcription Landscape of the Populus Genome

Polycistronic transcripts, which span multiple gene loci, are common in prokaryotes but have rarely been observed in eukaryotic genes. This long-standing view, however, has been challenged by recent advances in long-read transcriptome sequencing technologies. In this study, we employed nanopore direct RNA sequencing (DRS) to investigate polycistronic transcription in the nuclear genome of Populus under drought. We detected widespread polycistronic-like RNAs associated with approximately 60% of transcribed genes in two Populus species: black cottonwood and the hybrid 717. Thousands of incomplete dicistronic gene loci were identified, many of which encode modified or fusion open reading frames (ORFs), and are drought-responsive and functionally linked to organelles and cellular membrane systems. These polycistronic RNAs are frequently alternatively spliced and exhibit longer poly(A) tails and distinct m ⁶ A base modifications compared to their monocistronic counterparts. Additionally, we observed a positive expression correlation between monocistronic gene pairs within dicistronic loci, with a particularly strong correlation between polycistronic RNAs and the monocistronic gene at the 5′ end of the polycistronic locus. This suggests a complex regulatory mechanism governing transcription from incomplete polycistronic gene loci in Populus . Together, these findings highlight the unique structural and regulatory features of polycistronic RNAs and their potential roles in plant adaptation to environmental stress.