FIG 5.

hnRNP A1 interacted directly with G-rich RNA of LANA and enhanced translation. (A) Affinity pulldown assay of wild-type (WT) LANA RNA. Wild-type LANA RNA oligonucleotide was biotinylated and incubated with lysate from KSHV-positive BCBL-1 cells, and streptavidin beads were added to pull down RNA-bound proteins, followed by resolution of the samples on an SDS-PAGE gel and detection by hnRNP A1. Scrambled (Scrmb) RNA oligonucleotide was used as a negative control. WT LANA and Scrmb LANA RNA lanes were imaged at high exposure to detect binding. WT LANA RNA showed relatively higher binding with LANA than with the scrmb LANA RNA. The input lane was imaged at a lower exposure for comparable band intensities. (B) RNA CLIP assay confirming the direct interaction between hnRNP A1 and G-rich RNA of LANA. HEK293T cells transfected with pA3F-G4 wild type and pA3F-G4 disrupted plasmids were harvested and fixed 24 h posttransfection. The cells were lysed and then incubated with hnRNP A1 antibody and protein A/G beads. Bead-bound RNA was purified and quantified using an iTaq Universal SYBR green one-step kit with G4 wild-type and G4 disrupted clone-specific primers (a) and 7sk gene-specific primers (b), which served as a positive control for hnRNP A1 RNA CLIP. (C) RNA CLIP assay confirming the direct interaction between endogenous hnRNP A1 and G-rich RNA of LANA in KSHV-positive cells. BCBL-1 cells were treated with DMSO and TMPyP4 for 24 h, following which the cells were lysed and incubated with hnRNP A1 antibody and protein A/G beads. Bead-bound RNA was purified and quantified using an iTaq Universal SYBR green one-step kit with primers specific to the G-rich region (amino acids 857 to 916) (a) and specific to the region from amino acids 1 to 32, which does not form any G-quadruplexes. (D) Luciferase assay showing an increase in translation of LANA with increasing concentrations of hnRNP A1. HEK293L cells were transfected with pGL3-LANA and pA3F-hnRNA1 (at various concentrations). Cell lysates were used for the luciferase levels using a dual-luciferase assay. (E) Effects of hnRNP A1 on translation of the G4 wild type. HEK293T cells were transfected with pA3F-G4 wild type and pA3F-G4 disrupted plasmids along with pA3F-hnRNP A1 at increasing concentrations. Twenty-four hours posttransfection, the cell lysates were resolved by SDS-PAGE and proteins were detected using anti-Flag antibody. Anti-GAPDH antibody was used to ensure equal loading of the proteins.