The diversity of long log-noncoding RNAs is paralleled by their abundant molecular functions, which hinders their systematic functional characterization. Numerous known and novel lncRNAs have been identified in pediatric leukemias, which serve as potent diagnostic and prognostic biomarkers. However, their biological functions and therapeutic potential remain unknown.
To address this, we performed a high-throughput CRISPR interference screen in NALM6 cells targeting 1855 transcription start sites of leukemia-specific lncRNAs to identify those that are important for cancer cell survival. 5 lncRNAs were subsequently selected for further functional characterization based on their subcellular co-localization with P-bodies, the spliceosome, and nuclear bodies by smiFISH and immunofluorescence. These candidates were then subjected to CRISPR-Cas13 knock-down followed by direct RNA nanopore sequencing using the latest RNA004 chemistry with replicates. This yielded over 280M single molecule reads from both poly(A) enriched and total RNA libraries, a major improvement over the previous RNA002 chemistry.
De novo transcriptome assembly identified over 8k novel intergenic transcripts in these samples (including a previously unidentified lncRNA directly upstream of the well-studied KRAS proto-oncogene) and an order of magnitude more new isoforms of known genes, dozens of which are differentially expressed following CRISPR knock-down. Two of the selected lncRNAs presented major transcriptome-wide perturbations, exposing their role as important regulators of gene expression in biological processes, includin protein sumoylation, cell proliferation, as well as cell cycle regulation and myelin formation, respectively.
In addition to gene expression differences, the impact of lncRNA perturbation on RNA modifications was assessed, focusing on the detection of m6A with the recently updated m6Anet deep learning models, the results of which will be presented herein. This work supports the importance of designing functional studies that include lncRNAs, which can govern a broad range of biological processes and provide effective targets for novel therapeutic strategies.