Figure 2. Reaper and hid reporter mRNAs containing wild type MREs are present on actively translating ribosomes.

(A) Representative UV absorbance (254 nm) traces of polysome gradients on which either cytoplasmic extracts from emetine (E) treated or harringtonine (H) treated cells were fractionated. (B) Northern blots and quantitation of signal from fractions of polysome gradients as in (A); Blots from cells expressing reaper and hid reporter mRNAs containing wild type MREs are on the left. Sedimentation profiles, as assessed by splinted ligation, and signal quantitation of endogenous mir-13b and bantam miRNAs are shown on the right. (C) Northern blot and quantitation of signal from fractions of polysome gradients in which extract of cells treated with emetine (E) or harringtonine (H) were sedimented. The probes were to either endogenous reaper or to polyA binding protein (PABP) mRNAs.

DOI: http://dx.doi.org/10.7554/eLife.12880.006

Figure 2—figure supplement 1. Reporter mRNAs containing mutant MREs are present on translating ribosomes.

(A) (B) and (C) are exactly as described for (A) (B) and (C) in Figure 2 except that reporters containing mutant MREs were analyzed.

DOI: http://dx.doi.org/10.7554/eLife.12880.007

Figure 2—figure supplement 2. First indication of cotranslational decay of reaper reporter mRNA containing a wild type MRE.

Norther blot as shown in Figure 2 (A) was exposed for a longer time. Note fragments smaller than full length.

DOI: http://dx.doi.org/10.7554/eLife.12880.008

Figure 2—figure supplement 3. Most mRNAs are accounted for in polysome gradients.

We did an accounting experiment analogous to that shown in Maroney et al., 2006. Equal numbers of cell from wt or mutant reporter cell lines were processed as described in methods to prepare cytoplasmic (cyto) extract or to lyse with Trizol (total). RNA was then prepared from half of each sample. The remaining half of cytoplasmic extract was centrifuged through the same sucrose gradients used for polysome analysis. Fractions were pooled (gradient) and pellets (pellet) resuspended and RNA was prepared from both. After EtOH precipitation all samples were resuspended in the same volume. Equal aliquots of each were used in Northern blots and probed for both reporter and control (Rp49) mRNAs.

DOI: http://dx.doi.org/10.7554/eLife.12880.009

Figure 2—figure supplement 4. Reporter mRNAs containing wild type MREs are associated with lighter polysomes than reporter mRNAs containing mutant MREs.

(A) Direct comparison of sedimentation reporter mRNAs containing wild type WT or mutant MT MREs. These are the same blots and quantitations as those shown in a different context in Figure 2 and Figure 2—figure supplement 1. (B) Same as in (A) but hid reporter mRNAs were analyzed here.

DOI: http://dx.doi.org/10.7554/eLife.12880.010