a, Schematic representation of an inducible system to visualize expression of the Men ε/β reporter ncRNAs and recruitment of paraspeckle proteins. A LacO array, a tetracycline response element (TRE) array, and 24 MS2 stem loop repeats are placed upstream of the Men ε/β gene allowing visualization of the locus by expression of ECFP-LacI. Transcription is initiated by the addition of DOX and nascent transcripts are visualized through binding of EYFP-MS2 to the MS2 stem loops. Not drawn to scale. b, Live-cell imaging shows that transcriptional induction of Men ε/β ncRNAs initiated de novo formation of paraspeckles labeled by PSP1 (arrow). Scale bars, 5 μm. c, De novo formed paraspeckles (arrow) can retain inverted repeat-containing mRNAs, similar to the endogenous paraspeckles (arrowhead), confirming that they are bona fide functional paraspeckles. d, Quantification shows that Men ε/β transcription initiated the recruitment of different paraspeckle proteins, but not SC35 and SF2/ASF, nuclear speckle RNA binding proteins; and resulted in the retention of different inverted repeat containing mRNAs, but not mCat2 mRNA (n>50 cells in each condition).