RFLAT-1 5′-UTR inhibits translation in vitro and in vivo. (a) Absorbance profile (254 nm) of sucrose gradients from lysates of resting (D0) or PHA-activated (D1 and D5) PBLs. (b) Nine fractions were collected from each gradient, and equal volumes of each fraction were separated on an agarose gel and analyzed by Northern hybridization with an RFLAT-1 probe. 18S and 28S rRNA in each fraction were visualized by ethidium bromide staining. (c) RFLAT-1 expression constructs. (full-length [RF] or lacking the 5′-UTR [ΔU]) were subjected to in vitro transcription-translation in the presence of ³⁵S methionine. The products were detected by autoradiography. (d–g) pcDNA3.1 Luc (Luc) or pcDNA 3.1 5′-UTR Luc (5′-UTR Luc) were subjected to in vitro transcription-translation (d) or transiently transfected into Jurkat T cells (e and g) or HEK293 cells (f) and assayed for luciferase activity. The data are presented as relative luciferase activity where the activity of the 5′-UTR Luc construct is arbitrarily set to 1. *P < 0.05 (d, e, and f). Decreasing amounts of lysates from transfected Jurkat cells were subjected to an RPA (g) using luciferase (upper panel) and actin riboprobes (bottom panel). Lanes 2 and 5, 30 μl; 3 and 6, 15 μl; 4 and 7, 7.5 μl lysate.