TNRC6A–TNRC6C silence the expression of bound transcripts independently of the Argonaute proteins. (A–C) Human HEK293 cells were transfected with a mixture of three plasmids: the R-Luc-5BoxB reporter or the R-Luc control lacking the BoxB sequences, a plasmid expressing firefly luciferase as a transfection control, and vectors expressing the λN-HA-peptide or λN-HA-TNRC6A–TNRC6C. Renilla luciferase activity and mRNA levels were normalized to those of the firefly luciferase and set to 100 in cells expressing the λN-HA-peptide alone. Mean values ± standard deviations from three to four independent experiments are shown for the R-Luc-5BoxB reporter. Data corresponding to the R-Luc control (lacking the BoxB sequences) are not shown. In B, the expression levels of the λN-HA-TNRC6A–TNRC6C proteins and fragments were analyzed by Western blot. HA-MBP served as a transfection control. C shows a representative Northern blot. (D–F) Human HEK293 cells were transfected with a mixture of three plasmids: the R-Luc-5BoxB reporter or the R-Luc control reporter lacking the BoxB sequences, a plasmid expressing firefly luciferase as a transfection control, and vectors expressing the λN-HA-peptide or N- and C-terminal fragments of λN-HA-TNRC6A–TNRC6C. Renilla luciferase activity and mRNA levels were normalized to those of the firefly luciferase and analyzed as described in A. Representative Northern blots are shown in the lower panels. (G) Silencing activity of TNRC6C silencing domain (SD) measured as described in A.
