Deletion of the MALAT1 RNA 3' end Promotes Transcript Decay, TALAM1 downregulation, and Inhibits Proliferation in Gastric and Breast Cancer Cells

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 functions as a binary structural switch. Any perturbation (ranging from genomic deletions to a single-base insertion) triggers total transcript collapse and rapid exonucleolytic decay. This instability leads to locus-wide transcriptomic failure, characterized by the precipitous loss of the antisense transcript TALAM1, 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. Phenotypically, structural disruption of the 3 end significantly impairs the proliferative capacity of both cancer cellular models. These results identify the 3 triple-helix as an indispensable determinant of MALAT1 stability and provide endogenous validation for its role in cancer cells.