The yeast Rab GTPase Ypt1 modulates unfolded protein response dynamics by regulating the stability of HAC1 RNA

Abstract

The unfolded protein response (UPR) is a conserved mechanism that mitigates accumulation of unfolded proteins in the ER. The yeast UPR is subject to intricate post-transcriptional regulation, involving recruitment of the RNA encoding the Hac1 transcription factor to the ER and its unconventional splicing. To investigate the mechanisms underlying regulation of the UPR, we screened the yeast proteome for proteins that specifically interact with HAC1 RNA. Protein microarray experiments revealed that HAC1 interacts specifically with small ras GTPases of the Ypt family. We characterized the interaction of HAC1 RNA with one of these proteins, the yeast Rab1 homolog Ypt1. We found that Ypt1 protein specifically associated in vivo with unspliced HAC1 RNA. This association was disrupted by conditions that impaired protein folding in the ER and induced the UPR. Also, the Ypt1-HAC1 interaction depended on IRE1 and ADA5, the two genes critical for UPR activation. Decreasing expression of the Ypt1 protein resulted in a reduced rate of HAC1 RNA decay, leading to significantly increased levels of both unspliced and spliced HAC1 RNA, and delayed attenuation of the UPR, when ER stress was relieved. Our findings establish that Ypt1 contributes to regulation of UPR signaling dynamics by promoting the decay of HAC1 RNA, suggesting a potential regulatory mechanism for linking vesicle trafficking to the UPR and ER homeostasis.

Figure 1. Ypt1 associates in vivo with unspliced HAC1 in a UPR–dependent manner.

GST-tagged Ypt1 was purified from yeast cells grown under normal (A) or UPR-induced conditions (B) using anti-GST conjugated beads. UPR was induced by addition of 10 mM DTT for 50 min. Co-purifying RNAs were labeled and bound to a DNA microarray. “Mock-corrected IP enrichment” values were calculated by subtracting Mock IP signal from Ypt1 IP signal on a gene-by-gene basis (see “Materials and Methods”). Arrows indicate enrichment values for HAC1 probes (“JXNs” = junctions, 5 probes total present on the arrays- 2 unspliced junctions, intron, ORF, and spliced junction).

Figure 2. Ypt1 knockdown affects HAC1 splicing and expression.

(A) Gene expression of ypt1 (A) or sec12 (B) knockdown strain compared to isogenic parental wild type strain. In black, the signal distribution of (Log₂(Knockdown/WT)) values for all genes is shown. In red, the distribution of (Log₂(Knockdown/WT)) values for annotated UPR target genes is shown. Red arrows point to values for canonical UPR target genes (HAC1i, KAR2, ERO1, PDI1) and black arrows show values obtained from HAC1u probes. Data presented are averages of two replicate experiments.

Figure 3. Ypt1 knockdown effect on HAC1 RNA transcription and decay.

(A) Knocking down YPT1 does not affect HAC1 promoter activity as evaluated by assaying RNA levels of a transcriptional reporter gene containing the HAC1 promoter sequence fused to a GFP ORF sequence and the ACT1 3′UTR. Normalized expression, reported as a fraction of actin RNA levels, was determined using quantitative RT-PCR. Data represent the average of three replicate experiments. (B-C) Quantitative RT-PCR measurements of unspliced (B) and spliced (C) HAC1 RNA expression 30 min post addition of 3 ug/ml thiolutin (“Treated”) relative to HAC1 RNA levels in untreated cells. Values are normalized to GAPDH levels and data are averages of 3–4 replicates. Asterisks indicate significant differences. * = p<0.05 by t-test.

Figure 4. Ypt1 levels affect UPR attenuation.

ypt1-DAmP or its isogenic parental wild type strain were treated with 10 mM DTT for 1 hr, then washed, and re-suspended in fresh YPD media. A recovery assay was performed (up to one hour after wash) and expression levels of spliced HAC1 (A) and the canonical UPR target gene KAR2 (B) were measured by quantitative RT-PCR. Data represent average of four replicates per strain. All values are normalized to actin levels. Asterisks indicate significant differences. * = p<0.05, ** = p<0.005 by t-test.

Figure 5. Ypt1 negatively regulates HAC1u RNA expression in the absence of ER stress.

Under normal growth conditions (left panel). Ypt1 regulates HAC1 RNA expression by destabilizing the transcript. This is likely accomplished by recruiting HAC1 away from the ER and in proximity to decay factors. Ire1 and Ada5 are necessary for the interaction and could play an active role in recruiting HAC1 to the ER, thus facilitating the Ypt1-HAC1 association. Under UPR-induced conditions (right panel). The GTPase no longer interacts with HAC1. Instead, HAC1u is spliced by Ire1 and the mature HAC1i mRNA is translated to activate the ER stress response cascade. Dotted line denotes putative recruitment of HAC1u by Ire1; solid lines show established interactions. HAC1u = unspliced HAC1; HAC1i = spliced HAC1; ER = endoplasmic reticulum; UPR = unfolded protein response.