Figure 3. Cotranslational decay of reaper reporter mRNA.
(A) Schematic representation of probes used for detection of reaper reporter mRNA. (B) Representative UV absorbance trace (254 nm) of a polysome gradient fractionating cytoplasmic extract prepared from cells treated with actinomycin D for ten minutes. Actinomycin treatment for this time had no effect on overall protein synthesis as assessed by polysome profiling (Figure 3—figure supplement 1). (C) Northern blot analyses of fractions from polysome gradients in which cytoplasmic extracts from cells expressing reaper reporter mRNAs containing either a wild type (WT) or mutant (MT) MRE were sedimented. Northern blots were probed with a fragment that hybridized to the 3’ region of the firefly reporter open reading frame, see (A). (C) The same as in (D) except that the blots were probed with a fragment that hybridized to the 5’ region of the firefly luciferase open reading frame, see (A).
Figure 3—figure supplement 1. 10 min actinomycin D treatment does not affect overall protein synthesis.
Figure 3—figure supplement 2. Evidence for cotranslational mRNA degradation of hid reporter mRNA containing wild type MREs.
