Supplementary Figure 5: Firre interacts with hnRNPU in mESc and adipose contexts.
From: Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre
(a) The RNA pull-down technique was developed and optimized by testing known lncRNA-protein interactions. For optimization, telomerase RNA TERC was chosen as well as others, such as SRP and 7SK (data not shown). The binding partners of TERC, Dyskerin and TERT, were recovered with high efficiency and specificity. (b) The optimized RNA pull-down technique was used to find the binding partners of Firre. The analysis of mass spectrometry data is outlined. (c) Based on the highest unique peptide counts across different isoforms and lysate conditions, we identified 8 candidate proteins that physically associate with Firre in an RRD-dependent manner. The top candidate was hnRNPU. (d) Firre interacts with hnRNPU in mouse adipose tissue as well as in mESC lysate. Western blots are shown for the pull-downs using in vitro biotinylated isoforms of Firre: five with RRD and one without. We compared end labeling (EL) and body labeling (BL) to exclude the possibility of a bias in binding and used TERC antisense (AS) and sense (S) as negative controls. hnRNPK and A1 are shown for RRD-specific interactions. 20% of the input protein lysate was loaded in the first lane for each Western blot. Input protein lysate and input RNA are included as loading controls.
