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 m6A 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.