Abstract
Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1u (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound ribosomes as WT RTA, did not inhibit HAC1u mRNA splicing. A ribosome-binding mutant, which showed reduced binding to ribosomes but retained depurination activity, inhibited HAC1u mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of HAC1u mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that ribosomes play a role in splicing of HAC1u mRNA. We show that HAC1u mRNA is associated with ribosomes and does not get processed on depurinated ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1u mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1u mRNA that is associated with ribosomes.
Highlights
Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER
We previously showed that precursor form of WT RTA (preRTA) with its own signal sequence translocated into the ER and inhibited activation of the unfolded protein response (UPR), whereas an inactive form that translocated into the ER induced the UPR [17]
Because activation of the UPR would induce transcription of ER-associated degradation (ERAD) components, which normally translocate misfolded proteins from the ER to the cytosol for degradation, we proposed that inhibition of the UPR may allow ricin to enter the cytosol and avoid degradation [17]
Summary
To determine whether slowing the rate of ribosome depurination affects RTA-mediated inhibition of the UPR, we transformed yeast with R193A/R235A, which has an intact active site but shows greatly reduced ribosome binding in vitro and delayed ribosome depurination in vivo; G212E, which binds ribosomes like WT mRTA in vitro but has very low depurination activity due to a mutation near the active site [19]; and the mature form of WT RTA (mRTA) (Table S1). Yeast expressing G212E showed little effect on HAC1-HA level and appeared similar to VC (Fig. S2) These results indicate that the reduction in HAC1i mRNA and protein levels correlates with the depurination activity of RTA mutants on the ribosome. B, -fold change in GFP mRNA expressed from the UPRE-GFP reporter quantified by qRT-PCR in yeast carrying WT or mutant RTA expression plasmids compared with VC using total RNA from cells grown in dextrose or galactose. C, -fold change in HAC1i mRNA expressed from the UPRE-GFP reporter quantified by qRT-PCR in yeast carrying mRTA or mutant RTA expression plasmids compared with VC using total RNA from cells grown in Dex and Gal. The y axis shows the average -fold change in HAC1i mRNA, with error bars representing the range of expression from two biological replicates using three technical replicates for each.
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