Figure 5.

(a) ANRIL was mainly localized in the nucleus of UCCs as determined by qRT-PCR for all transcripts (left panel) and overexpressed 3’-truncated variants in particular (right panel) subsequent to fractionated extraction of RNA. Purity of nuclear fractions was checked by measuring the antisense transcript LIT1 as a control (Figure S2d). (b) Abundance of the interaction partner CBX7 in UCCs was checked on the RNA (left panel) and protein level (right panel). (c,d) Direct interaction between lncRNA ANRIL and PcG proteins was determined by RNA immunoprecipitation (RIP) analysis. Although both proteins were sufficiently precipitated (d), only interaction of ANRIL with CBX7 was detectable by qRT-PCR ((c), left panel), but not with SUZ12 above background levels (c, right panel). Results from IgG isotype controls were set as 1. GAPDH served as another background control. Both full length (included in “all” assay, dark grey bar) and 3’-truncated ANRIL isoforms (light grey bar) interacted with CBX7 protein.