NET1 3′ UTR localization elements are necessary and sufficient to target mRNA to the MB. (A) Schematic RT-qPCR-based reporter RNA approach for assaying MB RNA localization. Plasmids expressing Firefly and Renilla luciferases from a bidirectional reporter are integrated into the genome. Sequences to be tested for MB localization activity (NET1 full length 3′ UTR, LE and LE deletion) are fused to the 3′ UTR of Firefly luciferase. Using Taqman qPCR, the ratio of Firefly to Renilla luciferase mRNA is measured in MB and whole-cell samples. The ratio of these ratios (MB/WC) quantifies the MB enrichment of the Firefly luciferase transcript. By asking how this value changes upon appending additional sequences to the Firefly luciferase RNA, the effects of these additional sequences on MB RNA enrichment can be tested. (B) Diagram of reporter constructs used to interrogate NET1 localization activity in MBs. smFISH probes against the Firefly luciferase coding sequence are shown as red stars. (C) Localization of the reporter RNAs described in B to the MB as quantified using the strategy outlined in A. The value for the control reporter lacking additional sequence elements was set to one. P values were calculated using a t test. (D) smFISH visualizing the reporter RNAs described in B (yellow) in cells stably expressing Halo-MKLP1 (pink). Midbody is marked with an asterisk. The box indicates the area shown in a higher magnification inset. Scale bars: 10 μm. (E) Quantification of MB-localized Net1 UTR-containing reporter transcripts visualized in D. In all samples, reporter transcript counts in intercellular bridges and whole cells were quantified and the ratio of counts between the two locations is reported. These ratios were normalized by setting the value for the control reporter transcript lacking 3′ UTR additions to one. P values were calculated using a t test.