Figure 4.

tRF5-GluCTC exhibits miRNA-/siRNA-like trans-silencing capacity. (a) Sequence alignment of Pp-anti_GluCTC_WT with three mutant luciferase sensor plasmids. Mutated nts are in bold and italic. The sequence of tRF5-GluCTC inhibitor and mimic are also present. (b) A549 cells in hexaplicate were co-transfected with a firefly (Pp) luciferase reporter plasmid (cognate “Pp-anti_GluCTC_WT ” plasmid or “Pp-vector ” plasmid, 0.1 µg/well of 24-well plate), a plasmid expressing renilla (Rr) luciferase, and 120 nmol/l antisense oligos (“anti-GluCTC ” or “anti-control”). After 2 hours post-transfection, cells were mock- or RSV-infected, then harvested at 6 hours post-infection to measure luciferase activities. In all experiments, Pp luciferase values were normalized to renilla (Rr) luciferase values. Values at y-axis (Pp/Rr) are a representative of three independent experiments and are expressed as mean ± SE. **On the second white bar (RSV-infected and Pp-anti_GluCTC transfected) denotes P value <0.01, relative to the second black bar (mock-infected and Pp-anti_GluCTC transfected). (c) A549 cells were treated as described in b and total RNA was subjected to northern hybridization as described in Figure 3b (left panel). Densitometric analysis of tRF5-GluCTC band intensity, quantified using VisionWorksLS image acquisition and analysis software from UVP (Upland, CA) is shown after normalization to 5S rRNA. Data are summarized from three independent experiments (right panel). **P < 0.01, relative to second white bar. (d) A549 cells in hexaplicate were transfected with indicated Pp luciferase reporter plasmids. At 24 hours post-transfection, cells were mock- or RSV-infected, then harvested at 15 hours post-infection to measure luciferase activities. All other descriptions are the same as in b. *P < 0.05, **P < 0.01, respectively, relative to second white bar. (e) A549 cells in hexaplicate were transfected with indicated tRF-mimic oligos. Luciferase plasmids, Pp-anti_GluCTC_WT or _Mut3 (0.1 µg/well), and a plasmid expressing Rr luciferase, were co-transfected. At 40 hours post-transfection, cells were lysed for luciferase assays. Pp values were first normalized by Rr values, and then the Pp/Rr values of “Pp-anti_GluCTC_WT ” were normalized to those of “Pp-anti_GluCTC_Mut3”, yielding relative Pp/Rr values (y-axis). **Denotes P value <0.01, relative to control-mimic (black bars) in respective concentration. All other descriptions are the same as in b. miRNA, microRNA; nts, nucleotides; ORF, open-reading frame; RSV, respiratory syncytial virus; siRNA, small-interfering RNA; tRF, tRNA-derived RNA fragment; tRNA, transfer RNA; WT, wild-type.