Long non-coding RNAs (lncRNAs) are key regulators of gene expression, yet their functional roles and mechanistic diversity remain poorly understood. We developed an integrated platform combining genome-wide CRISPR interference (CRISPRi), CasRx-mediated knockdown, and nanopore direct RNA sequencing (DRS) to dissect lncRNA function at multiple regulatory layers.
A CRISPRi screen targeting 744 annotated lncRNAs dysregulated in pediatric leukemias identified five candidates essential for proliferation. CasRx knockdowns validated these hits and revealed widespread transcriptional and isoform-level changes. Using nanopore DRS, we captured full-length isoforms, alternative splicing events, and m⁶A methylation dynamics, uncovering distinct regulatory signatures for each lncRNA. These findings demonstrate how lncRNAs influence gene expression through transcriptional, post-transcriptional, and epitranscriptomic mechanisms.
Two candidates, DHRS4-AS1 and PCBP1-AS1, localize to p-bodies as shown by smFISH, implicating them in cytoplasmic condensate organization. These structures act as hubs for RNA decay and m⁶A reader activity, positioning these lncRNAs as scaffolds that seed post-transcriptional complexes. Knockdown of PCBP1-AS1 triggered isoform switching and hypermethylation of transcripts linked to translation and RNA stability, while DHRS4-AS1 influenced isoform usage in proliferation-related genes such as CIT and SUN2. These insights reveal spatially organized lncRNA functions coupling condensate biology with epitranscriptomic regulation.
To extend this approach, we applied de novo assembly of bulk and long-read single-cell transcriptomes from patient samples, revealing disease subtype and tumour-specific lncRNA isoforms absent in normal hematopoietic cells. This forward-looking analysis highlights opportunities for precision RNA therapeutics targeting lncRNAs.
Our study showcases how direct RNA sequencing enables multi-modal functional transcriptomics, bridging experimental and computational RNA biology. By resolving isoform diversity and RNA modifications in a disease-relevant context, this work advances understanding of lncRNA biology and establishes a scalable framework for future therapeutic exploration.