To address this matter, we titrated the degree of cycloheximide so you can a sub-dangerous focus (0

Shape step three-source investigation dos

Source study to have Shape 3B, C, D–G, We and you may J: decay kinetics out-of chosen mRNAs from inside the structure addressed with translational inhibitors.

Figure 3-resource investigation 3

To begin to test these predictions, we directly perturbed the process of translation elongation and observed the effects on mRNA decay. Consistent with previous reports, we found that treating cells with a strong dose (50 ?g/mL) of the elongation inhibitor cycloheximide resulted in stabilized CIS3 and RPL25 transcripts and had no significant effect on ACT1 mRNA stability (Figure 3-figure supplement 1A) (Beelman and Parker, 1994). A potentially problematic aspect of these experiments is that high doses of cycloheximide completely halt translation thus shutting down a myriad of cellular processes, which in turn could lead to indirect effects. 2 ?g/mL) and assayed the effect of this level of elongation inhibition on transcript stability (Figure 3-figure supplement 2H). Even with this low concentration of cycloheximide, the CIS3 and RPL25 transcripts remained stabilized and the ACT1 mRNA again was not significantly affected (Figure 3B). The effects of cycloheximide on mRNA turnover were specific to elongation inhibition as mRNA half-lives were unchanged in a cycloheximide resistant mutant (rpl28-Q38K) (Figure 3-figure supplement 1B). We next tested an alternative translation elongation inhibitor, sordarin, which blocks the function of eukaryotic elongation factor 2 (Shastry et al., 2001). When cells were treated with a sub-lethal dose of sordarin, we again observed a stabilizing effect on mRNA half-lives (Figure 3C and Figure 3-figure supplement 1C). These stabilization effects were not due to an inability to incorporate 4TU into newly made transcripts as mRNA synthesis rates were not reduced upon treatment with either cycloheximide or sordarin (Figure 3-figure supplement 1D and E). Moreover, these effects were not specific to polyA selection. When these experiments were analyzed using total RNA, mRNAs were stabilized upon elongation inhibition and the half-life differences were unchanged in the case of low cycloheximide or further exaggerated as in the cases of high cycloheximide and sordarin (Figure 3-figure supplement 3A–C). The dramatic increase in mRNA stability in sordarin or high doses of cycloheximide is consistent with previous findings that decapping rather than deadenylation is blocked upon cycloheximide treatment (Figure 3-figure supplement 3A and C)(Beelman and Parker, 1994). Using a one-sided paired t-test, we find that 5 of the six measurements support the translation factor protection model (p(TP)<0.05) and none of the measurements support the stalled ribosome protection model (p(SR)<0.05). We conclude that inhibiting translation elongation stabilizes mRNAs.

While these results demonstrate that a stalled ribosome per se is not sufficient to induce decay, we could not exclude that cycloheximide or sordarin treatment might only poorly imitate slowed ribosomes on non-optimal codons since the acceptor-site of the ribosome remains occupied when these drugs are employed (Roy and Jacobson, 2013). To best mimic a non-optimal codon where the acceptor-site would be unoccupied, we treated cells with a sub-lethal dose (5 mM) of 3-amino-1,2,4-triazole (3AT), which results in histidine starvation thus lowering the concentration of histidyl-tRNAs (Figure 3-figure supplement 1F) (Klopotowski and Wiater, 1965). Indeed 3AT has previously been shown to stall ribosomes at histidine codons (Guydosh and Green, 2014). Histidine starvation also affects translation initiation by phosphorylation of eukaryotic initiation factor 2? via the Gcn2 kinase (Hinnebusch, 2005). In order to examine the effect on translation elongation by 3AT in isolation, all 3AT experiments were thus performed in gcn2? mutant cells. We examined the stability of 15 mRNAs with diverse spacing and position of histidine codons either untreated or treated with 3AT. Of these 15 paired measurements, we found that 11 are significantly stabilized and support the translation factor protection model (p(TP)<0.05). We find that none of the measurements support the stalled ribosome triggered decay model (p(SR)<0.05) (Figure 3D–G). This overall stabilization effect could not be explained by poor 4TU uptake as mRNA synthesis rates were not reduced upon 3AT treatment (Figure 3-figure supplement 1G). Interestingly, transcripts lacking histidine codons were also stabilized, which is consistent with the observation that 3AT limits glycine availability in addition to the depletion of histidine (Vital-Lopez et al., 2013). Again, we found that these results were recapitulated in the absence of polyA selection suggesting that the effect of 3AT is not limited to deadenylation but apply to steps downstream in mRNA decay as well (Figure 3-figure supplement 3D).