Supplementary Figure 6: Raly binds CCR5AS and the 3’UTR of CCR5 mRNA.

a, Western blot analysis was performed to confirm the presence of Raly protein in the (AS) pulldown as compared to the SC pulldown. b, In silico prediction of the interaction between the long (left) and short (right) forms of the CCR5AS transcript and Raly protein was done using a freely available algorithm (http://service.tartaglialab.com/newsubmission/globalscore), which integrates properties of protein and RNA structures into overall binding propensity. Global score predicts global and local interactions between RBP and lncRNA. A high interaction score (≈1) predicts a strong interaction between Raly protein and CCR5AS transcript. c, A Myc-tagged Raly protein sequence was transfected in Hut-78 cells. The c-Myc tagged protein was immunoprecipitated using anti-Myc antibody coated magnetic beads, but not with nonspecific IgG, as confirmed by Western blot. d, The Hut-78 cell line was transfected with either control siRNA (siCon) or Raly siRNA (siRaly). Cells were harvested at 48h and 72h post-transfection. Western blot analysis showed downregulation of Raly after 72h. Alpha tubulin was used as the housekeeping control. e CCR5 3’UTR was cloned downstream of Renilla luciferase into a dual luciferase reporter psicheck2 vector. The Raly binding site in the CCR5 3’UTR sequence is indicated by a red bar. f, Hut-78 cells were transfected with a vector encoding Myc-tagged Raly protein (Raly-Myc) along with either Control siRNA (siCon1) or CCR5AS siRNA (siLnc1). Cells were lysed and RNA immunoprecipitation was carried out using anti-Myc antibody (Raly-IP) or control IgG (IgG-IP). Precipitation of Raly protein by anti-Myc antibody, but not control IgG, was confirmed by Western blot. Input represents straight lysate (that is no immunoprecipitation). g, Fold enrichment of CCR5AS in the pulldown was determined using qPCR. CCR5AS was enriched in the Raly pulldown (Raly-IP) as compared to IgG in the siCon1 treated cells. No enrichment of CCR5AS was observed in the Raly pulldown of cells transfected with siLnc1. h, CCR5 mRNA decay was determined by 5-Ethylene uridine (EU) pulse-labeling of RNA using the Click-iT Nascent RNA Capture Kit. Hut-78 cells were transfected with siRaly or siCon and pulsed with EU after 60 hours. Eighteen hours after EU-pulsing, the cells were washed, supplemented with fresh growth medium and harvested at one hour intervals for 4 hours. Total RNA was isolated from cells and quantitated. The EU-labeled RNA was biotinylated, precipitated, and captured using streptavidin coated magnetic beads as per the manufacturer’s protocol. The RNA captured on beads was used for cDNA synthesis and qPCR analysis. Data are represented as relative expression levels of CCR5 mRNA in the siCon or siRaly transfected cells. The mRNA half-life (50% mRNA remaining, t_1/2) for the CCR5 mRNA in siRaly (4h) was higher as compared to the siCon treated cells (2.9h).