Fig. 1: Schematics of the pathways acting on the 3′ end of MALAT1 and the Mirror approach to identify them.
a Crystal structure (PDB ID: 4PLX) of MALAT1 triple-helical ENE stability element1,19 that protects the 3′ end of MALAT1 against nuclear degradation pathways. Position of the ENE-destabilizing mutation C8351G19 used in this study is indicated by the red arrow. The image is made using Mol* Viewer68. b Schematics of the MALAT1 3′-end formation by RNase P cleavage of the tRNA-like mascRNA structure and formation of the triple-helical ENE structure1,18,19 that protects the 3′ end of MALAT1 against nuclear degradation pathways. c The stability-compromised ENE(C8351G) structure provides only partial protection against nuclear degradation pathways. d Schematics of the Mirror concept for identifying nuclear pathways acting on nuclear long non-coding RNAs using reporters utilizing cytoplasmic fluorescence. The schematic GFP protein was created in BioRender. Che, J. (2025) https://BioRender.com/ht3ohze. e The “Red” Mirror reporter cell line for simultaneous identification of two nuclear pathways acting on the MALAT1 3′ end: the first, a degradation pathway targeting the 3′ end of MALAT1 but prevented from degrading MALAT1 by its intact triple-helical ENE; and the second, the pathway required for MALAT1 3′-end processing. Major sensitivity-enhancing features of the Fireworks fluorescence amplification system employed by Mirror are described35. The 3′ end of the MALAT1 ENE Mirror reporters is formed by RNase P cleavage upstream of mascRNA, as indicated by the orange arrow. Steady-state levels of the MALAT1 ENE Mirror reporters depend on the (1) integrity of their 3′-end triple-helical ENE stability element, which is inserted after the β-globin 3′ UTR and can be disrupted by a single nucleotide substitution C8351G (shown in red). In addition to the “Red” Mirror cell line shown here, an orthogonal “Green” reporter cell line (shown schematically in Figs. S1a and S2) provides controls for cell line- and fluorescent protein-specific effects during genome-wide forward genetic screening and candidate gene validation. f Anticipated effects of inhibiting pathways of nuclear degradation and processing on the fluorescence of “Red” Mirror cell line.
