(A) UV crosslinking and RNA immunoprecipitation (RIP) was used to assess PUM1, PUM2, and RBMX interactions with GFP mRNA or the indicated NORAD constructs. After knock-in of the indicated constructs to the AAVS1 locus in HCT116 CRISPRi cells, endogenous NORAD was silenced with a lentivirally-expressed sgRNA. qRT-PCR was used to assess NORAD or GAPDH recovery in each RIP sample, expressed as fold-enrichment over pull-down with IgG. The data in the left graphs were generated with a primer pair in ND4 that does not amplify the 5′ fragment, while the right graphs used primers at the NORAD 5′ end. n = 2 biological replicates, each measured with three technical replicates. (B) Representative RNA FISH images of wild-type or mutant NORAD transcripts expressed from the AAVS1 locus in HCT116 CRISPRi cells after knockdown of endogenous NORAD. Probe 10 was used for full-length NORAD, PREmut, and 5′ del constructs; probe 1 was used for 5′ frag; and probe 6 was used for ND4.