PUM1&2-mediated repression is directed via the decapping-dependent pathway. Mean log2 fold change values ±SEM are plotted and listed in Supplemental Table S1. (*) P-value <0.05 relative to the negative control MS2-HT (above x-axis) or between the indicated conditions (below the x-axis). (A) The Nluc 4xMS2 MALAT1 reporter gene used in the tethered function assays. The 3′ end of this reporter terminates with the MALAT1 triple helix structure. (B) Repressive activity of MS2-fused PUM1 N, PUM2 N, or CNOT7 effector proteins measured by the tethered function assay with either Nluc 4xMS2 pA (black bars) or MALAT1 (blue) reporters, relative to MS2-HT negative control. n = 9. (C) Western blot of MS2-tagged protein from a representative experimental replicate for B. GAPDH served as a loading control. (D) The tethered function assay was used to determine the effect of overexpressed, dominant negative DCP2 mutant (DCP2 mt) on repression activity of MS2-tethered PUM1 N, PUM2 N, or CNOT7 effector proteins, relative to MS2-HT negative control. Repression of Nluc 4xMS2 pA and Nluc 4xMS2 MALAT1 reporters was measured without (−) or with (+) overexpressed DCP2 mt. n = 9. (E) Western blot of V5-tagged proteins and myc-tagged DCP2 mt from a representative experimental replicate for D.
