RHA interaction with collagen 5′ SL RNA. (A) Pull-down of RHA with biotinylated 5′ SL RNA. Biotin-tagged 5′ SL RNA (5′ SL, lane 3) and inverted 5′ SL RNA (CON, lane 2) were used to pull down proteins from cytosolic extracts of HLF. Proteins were analyzed by SDS-PAGE and Coomassie staining. The protein bands identified by MALDI-TOF are indicated: RNA helicase A (RHA), ATP citrate lysate isoform 1 (ACLY1), ATP-dependent DNA helicase 2 subunit 1(XRCC6), eukaryotic initiation factor 4H (eIF4H). (B) Expression of proteins (immunoblot) used in gel mobility shift experiments. HA-tagged LARP6 and LARP6ΔC, His-tagged RHA and Flag-tagged control protein TRIM45 (CON) were transfected into HEK293 cells, and their expression in cytosolic extract was analyzed by Western blot. (–) Nontransfected cells. Actin (ACT) is shown as the loading control. (C) RHA does not bind 5′ SL in the gel mobility shift assay with high affinity. Gel mobility shift assay with wild-type (wt) 5′ SL RNA (lanes 1–7) and mutant 5′ SL RNA with a single point mutation that abolishes interaction with LARP6 (lanes 8–12) and extracts shown in B. Migration of RNA/protein complex and free probe is indicated. (Lane 1) The wt probe alone (P).
