Figure 10.

Decapping enzyme participates in repression by the Pum N-terminus. (A) RNAi-mediated depletion of the Dcp2 mRNA after 5 days of dsRNA treatment was measured using RT-qPCR. Fold changes were calculated relative to non-targeting control (NTC). Mean log₂ fold change and 95% credible intervals for three biological replicates with three technical replicates each are reported in the graph. Data and statistics are reported in Supplementary Table S1. (B) The effect of inhibition of decapping on repression of Nluc 2× MS2 reporter protein and mRNA levels by the Pum N-terminus was measured using the tethered function dual luciferase assay. Decapping was inhibited by RNAi mediated depletion of Dcp2 and over-expression of the dominant negative mutant Dcp2 E361Q. Repression activity was calculated relative to tethered EGFP negative control within the same RNAi condition. Tethered Dcp1 served as a positive control. Mean log₂ fold change and 95% credible intervals are graphed from three biological replicates. Data and statistics are reported in Supplementary Table S1. For significance calling, a ‘*’ denotes a posterior probability >0.95 that the difference relative to the negative control is in the indicated direction. The ‘**' indicates a posterior probability of >0.95 that the indicated difference is at least 1.3-fold. An ‘x’ marks a posterior probability >0.95 that the indicated difference is no more than 1.3-fold in either direction. (C) Northern blot detection of Nluc 2× MS2 reporter and FFluc internal control in three biological replicate samples for tethered effectors analyzed in panel A. Each lane of the gel contains 5 μg of total RNA. Ethidium Bromide detection of rRNA was used to assess integrity and equivalent loading of the RNA samples. (D) Western blot of the V5-epitope tagged MS2 fusion effector proteins and Dcp2 E361Q used in panel B from three biological replicates. Equivalent mass of protein from each sample was probed with anti-V5 antibody.