Rosenkranz, I.; Mermet, S.; Zacharaki, V.; Kindgren, P.

Long non-coding RNAs (lncRNAs) are emerging as key regulatory players of coding gene expression in eukaryotes. Here, we investigate the roles of the lncRNAs SVALKA (SVK) and SVALNA (SVN) in regulating CBF1 and CBF3 gene expression in Arabidopsis under cold stress conditions. We used Native Elongation Transcript Sequencing, CRISPR-Cas9 deletion strategies, and RT-qPCR to analyze the transcriptional dynamics and regulatory mechanisms of SVK and SVN. Our results demonstrate that SVK functions as a cis- and trans-acting lncRNA, regulating both CBF1 and CBF3 through RNAPII collision and chromatin remodeling, while SVN serves a cis role by negatively regulating CBF3 via a RNAPII collision mechanism. We identified isoforms of SVK, originating from distinct transcription start sites and undergo alternative splicing to adapt structural stability, crucial for their regulatory functions. This study elucidates the complex interplay of lncRNAs in gene regulation, highlighting their essential roles in modulating responses to environmental stresses. Our findings contribute to a deeper understanding of the mechanisms underlying lncRNA functionality and their significance in gene regulatory networks in eukaryotes.