Deletion of the MALAT1 RNA 3' end Promotes Transcript Decay and Inhibits Proliferation in Gastric and Breast Cancer Cells

Abstract

The long non-coding RNA MALAT1 is a conserved oncogenic driver whose function relies on a 3 triple-helix motif. While its biochemistry is well-characterized in vitro, the endogenous requirement for this motif in regulating the stability of the transcript and other genes residing in its locus remains unclear. In this study, we employed a dual-sgRNA CRISPR-Cas9 approach to systematically excise triple-helix-forming sequences from the native MALAT1 locus in gastric (AGS) and breast (MCF7) cancer cells. Our findings demonstrate that the 3 end strongly contributes to MALAT1 stability. Perturbations ranging from genomic deletions to a single-base changes trigger transcript collapse and rapid exonucleolytic decay, while the biogenesis of the small RNA mascRNA (a byproduct of MALAT1, also involved in cancer) remains decoupled and unaffected. In cellulo, DMS probing reveals that edited transcripts retain structural complexity in the 3 region. Phenotypically, structural disruption of the 3 end significantly impairs proliferation of both cancer cellular models. These results identify the 3 triple-helix as a determinant of MALAT1 stability and provide endogenous validation for its role in the analyzed AGS and MCF7 cells.