Fig. 2: The Mirror reporters enable forward genetic identification of human factors required for MALAT1 processing at the ENE-mascRNA junction.

a Schematic of the effect of RNase P cleavage defect on fluorescence of the Mirror reporter. Low RNA levels of the RNase P-processed MALAT1(C8351G) Mirror reporter result from a stability-compromising mutation (highlighted in red) within its 3′ ENE, leading to low GFP fluorescence. Inhibition of mascRNA cleavage causes a switch to the downstream Bovine Growth Hormone (BGH) cleavage and polyadenylation site, increasing stability and thus GFP fluorescence of the Mirror reporter, enabling identification of human factors required for MALAT1 3′-end processing. b RT-qPCR quantification of RNase P processing of Mirror reporter variants shown in (d, e). Values are expressed relative to the RNase P-unprocessed Mirror-ENE(WT)-mascRNA(WT) transcript levels. ΔΔCt was used to quantify the relative expression levels. Data are presented as means of two biological × four technical replicates; error bars represent standard deviation. Statistically significant differences between knockout and control samples were determined by one-way ANOVA. Posthoc comparisons using Dunnett’s test were conducted to determine the overall difference between groups, and p values are labeled. Sequences of qPCR primers are listed in Supplementary Data S2. c RT-qPCR quantification of steady-state RNA levels of Mirror reporter variants (shown in (d, e)) transiently transfected in HEK293T cells. RNA levels were normalized to blasticidin S resistance (bsr)⁶⁹ mRNA expressed from a co-transfected control plasmid. All qPCR statistical tests for (c) were performed as described for (b); qPCR primers are listed in Supplementary Data S2. Source data are provided as a Source Data file. d Sequences and mutations in ENE-mascRNA regions of Mirror reporters shown in (a, b, c, e). Substitutions that destabilize ENE (C8351G)¹⁹ or disrupt RNase P cleavage (mut 8356-8370) at the ENE-mascRNA junction are highlighted in red. e Fluorescence of transiently transfected Mirror reporters visualized using Olympus IX70 fluorescence microscope and FACS analysis of the fluorescence of FRT site-incorporated Mirror reporters carrying 3′-end variants schematically shown to the right (nucleotide substitutions are detailed in (d)).