A functional UPR in fission yeast
UPR induction in all eukaryotic cells analyzed to date involves the Ire1-mediated, non-conventional splicing of Hac1/XBP1
mRNA. The splice sites at which Ire1 cleaves the mRNA to initiate splicing lie in well-conserved stem/loop structures that are readily identified (Gonzalez et al., 1999
). We and others were therefore perplexed when bioinformatic analyses failed to identify Hac1/XBP1 orthologs in S. pombe
and other yeasts of the same genus () (Hooks and Griffiths-Jones, 2011
; Frost et al., 2012
). The Hac1/XBP1 transcription factors are well conserved between species and are easily recognized by sequence alignment among the superfamily of bZIP transcription factors (). By contrast, Ire1 is well conserved in S. pombe
, with all of the functionally important hallmarks identified in other eukaryotes, including its ER lumenal unfolded protein sensing domain and its cytosolic kinase and RNase domains. Moreover, Ire1 was essential for S. pombe
growth on tunicamycin (Tm) (), which induces ER stress by blocking N
-linked glycosylation, indicating that Ire1 serves an essential function in allowing cells to cope with ER stress. This function required Ire1's RNase activity, as Ire1(H1018N)
carrying a single amino acid substitution of a catalytic residue in Ire1's RNase active site failed to support cell growth on tunicamycin ().
The UPR in fission selectively down-regulates ER-targeted mRNAs.
ER stress dependent mRNA down-regulation
To address the conundrum posed by the missing Ire1 splicing substrate in S. pombe, we first explored the scope of UPR-dependent changes in gene expression. To this end, we isolated polyA+ RNA from wild type and Ire1Δ cells, in which the UPR was induced with the reducing agent dithiothreitol (DTT). DTT causes ER stress by impairing disulfide bond formation in the ER. The purified mRNA population was reverse-transcribed and subjected to deep-sequencing. Unexpectedly, we observed widespread Ire1-dependent mRNA down-regulation, but virtually no mRNA up-regulation (). Thirty-nine mRNA species were reduced by more than twofold in a DTT- and Ire1-dependent manner (, bottom left grayed area). Most members of this set of down-regulated mRNAs were abundantly expressed, as depicted by the size of the plotted circles. Down-regulation, however, did not correlate with mRNA abundance (). Intriguingly, the set of down-regulated genes exclusively encoded proteins targeted to the ER (identified by signal sequences and/or transmembrane segments) (, red circles). As shown in , the genome-wide profile of Ire1- and ER stress-dependent mRNA changes of genes encoding ER-targeted proteins is skewed to a significantly greater extent toward down-regulation than that of other mRNAs (p<1×10−20). More than half of the most down-regulated mRNAs encode proteins with annotated functions in the secretory pathway, in particular proteins involved in lipid metabolism, trafficking, and ER functions ().
As the reduction in mRNA abundance was ER stress- and Ire1-dependent, we next explored if Ire1 could be directly involved in destabilizing ER-bound mRNAs. To this end, we sought to trap any putative primary Ire1-cleavage products prior to degradation by deleting Ski2, which encodes a helicase component of the cytosolic Ski complex (cytosolic exosome) that mediates 3′ → 5′ RNA decay. Northern blot analysis of Ski2Δ cells revealed that Gas2 mRNA (which is down-regulated 2.5-fold in an ER stress and Ire1-dependent manner) yielded two discrete cleavage products upon ER stress (). Gas2 mRNA cleavage was dependent on Ire1, as no mRNA reduction and no cleavage products were observed in Ire1Δ Ski2Δ double deletion cells (). Another target, Yop1, behaved similarly (). In time-course experiments, reduction of Gas2 mRNA and accumulation of the cleavage products peaked at 30 min after UPR induction (); at later time points the abundance of intact full-length mRNA increased, suggesting that newly transcribed mRNA is not cleaved if the Ire1-dependent cleavage products are not further degraded. Indeed, Ski2Δ cells failed to grow on plates containing tunicamycin (), indicating that an intact mRNA decay is important for S. pombe cells to cope with ER stress.
Ire1 cleaves down-regulated mRNAs at specific sequences.
To determine the mRNA cleavage sites genome-wide, we prepared total RNA fractions from Ski2Δ
and from Ire1Δ Ski2Δ
cells. We used tRNA ligase to attach linker sequences specifically to those RNA fragments terminating in a 2′,3′-cyclic phosphate, which is the expected product of Ire1-catalyzed RNA cleavage (Schutz et al., 2010
). We then amplified the cleavage products in 3′ RACE reactions priming at the linker sequence. Alignment of the sequencing data to the S. pombe
genome identified the 3′ ends of Ire1-dependent fragments. In particular, we identified 39 Ire1-dependent fragments mapping to 24 of the most down-regulated genes, as shown in for Gas2
mRNA (left panel). By size estimation, the major Ire1-dependent peak corresponded to the smaller, more abundant Gas2
mRNA cleavage product (labeled ▲ in ). A second, less abundant short fragment was also observed in the sequencing data (labeled × in ). (Fragment × was absent or below the detection limit on the Northern blot unless the primary cleavage site was mutated (see , discussed below).) Spliceosomal U6 RNA normally terminates in a 2′,3′-cyclic phosphate and thus provided a valuable control for the ligation reaction (, right panel).
Alignment of the experimentally determined Ire1-dependent cleavage sites revealed a core motif with a signature of three conserved nucleotides (UG\C) that flank the Ire1-dependent cleavage sites at positions −2, −1, and +1 with an additional strong bias against G in position +2 (). Most mapped mRNA cleavage sites (34 of 39), including those in Gas2 mRNA, localized within the open reading frames. Indeed, a Gas2 reporter construct transcribed off a heterologous alpha-tubulin (Nda2) promoter and containing only the Gas2 ORF flanked by heterologous 5′ and 3′ tubulin untranslated regions (UTRs), was down-regulated upon ER stress in an Ire1-dependent manner (). This degradation was quantitatively comparable to that of the native Gas2 transcript (), indicating that the information contained within the Gas2 ORF is sufficient to confer susceptibility to Ire1-dependent cleavage.
To assess the functional importance of the identified Gas2 mRNA cleavage site experimentally, we mutated the UGC-residues of the ▲-site (UG\CU). As expected, ER stress-dependent cleavage of the Gas2 reporter mRNA at the mapped site was abolished (). In its place, however, we observed two new Ire1-dependent fragments (labeled × and *). Scanning gel densitometry revealed that fragment × is distinctly smaller than fragment ▲, and hence represents a cryptic site that is only utilized when site ▲ is mutated. Fragment * likely corresponds to the lower abundance cleavage product observed in , which becomes more prominent in the mutant construct. Taken together, we conclude that Ire1-dependent mRNA cleavage in S. pombe is sequence dependent.
The data presented so far suggest that homeostatic control of ER protein folding is regulated differently in S. pombe than S. cerevisiae. Rather than relying on a transcriptional program to upregulate genes that enhance ER protein folding capacity as in S. cerevisiae, S. pombe cells reduce the amount of specific proteins entering the organelle by decreasing the level of ER-targeted mRNAs using Ire1-dependent mRNA degradation.
Bip1 mRNA processing and stabilization in response to ER stress
In all species analyzed to date, Bip1 is a major UPR target gene that is upregulated when cells experience ER stress. Paradoxically, we found S. pombe Bip1 mRNA among the 39 down-regulated mRNAs identified by the analyses shown in . Analysis by Northern blotting yielded seemingly conflicting results: by this analysis, Bip1 mRNA was fourfold more abundant in ER stressed cells (). Intriguingly, the appearance of a faster migrating mRNA species (‘tBip1 mRNA’) indicates that Bip1 mRNA changes size in cells experiencing ER stress (, lanes 3–4). Appearance of the tBip1 mRNA species was Ire1-dependent and in wild type cells accounted for the increase in overall mRNA abundance. The increase did not result from augmented transcription. We measured the activity of a heterologous reporter in which the Bip1 promoter was fused to GFP and showed no Ire1-dependent change in mRNA abundance with ER stress (). In agreement with this result, we found that the stability of an mRNA bearing the Bip1 ORF and 3′ UTR showed a more than threefold increase in half-life from T1/2=20 min for the unprocessed form present in unstressed cells to T1/2=70 min for the processed form present in ER-stressed cells (). Furthermore, the Bip1 3′ UTR and the presence of a signal sequence were sufficient to a heterologous mRNA construct to confer Ire1-dependent processing ().
Ire1 truncates Bip1 mRNA within the 3′ UTR.
Sequencing of the expressed genome in UPR-induced and uninduced cells revealed the molecular difference between Bip1 and tBip1 mRNA (). For these experiments, we extracted total RNA and then, without selecting for polyA+ RNA, removed rRNA by subtractive hybridization. After reverse transcription, deep-sequencing of the cDNA pool from uninduced cells revealed good coverage of reads spanning the entire Bip1 mRNA including its 5′ and 3′ UTR (, left, blue profile). By contrast, cDNA isolated from DTT-treated cells revealed a precipitous drop in reads mapping to the 3′ end (, left, red profile and ). We also performed 3′-RACE to determine the 3′ end of tBip1 mRNA. The sequence of the amplified DNA confirmed that tBip1 mRNA lacks a polyA tail and terminates at G373 in the 3′ UTR (, right panel). In seven independently isolated clones, we found no sequence variations in the tBip1 linker junction. The sequences flanking G373 align with the UG\CU motif (), suggesting that tBip1 mRNA is produced by truncation of Bip1 mRNA in an Ire1-dependent RNA cleavage reaction that resembles those of the Ire1-dependently down-regulated mRNAs described above.
Mutational analysis of the cleavage site confirmed that specific sequences are required. Mutation of G373 to C or U and its deletion together with preceding nucleotides abolished Ire1-dependent Bip1 mRNA processing (). By contrast, a mutation of the preceding G370 to C diminished cleavage only marginally (less than twofold). In all analyzed mutants of Bip1 mRNA, UPR-induction increased abundance of the transcript approximately twofold (a level comparable to that observed in Ire1Δ cells) (, right panel), whether processing took place or not, perhaps due to compensatory transcriptional regulation that is independent of Ire1. For all mutants, however, the increased abundance stayed shy of the fourfold increase observed for wild type Bip1 mRNA.
As Bip1 mRNA truncation resulted in a loss of the polyA tail, this result resolves the paradox of why Bip1 mRNA appeared to be down-regulated in the polyA+ mRNA pool analyzed in . Indeed, directly comparing the UPR-dependent fold-change in mRNA abundance of polyA+ RNA and rRNA-depleted total RNA uniquely positioned Bip1 sequences as an anti-correlated outlier, whereas all other mRNAs were well correlated between the samples (). From these data we conclude that, remarkably, Bip1 mRNA is the only stable mRNA in the cell that loses its polyA tail upon UPR induction.
It was unexpected to find an mRNA that had lost its polyA tail to be more stable in cells.
To determine the translation proficiency of tBip1 mRNA, we subjected UPR-induced cells to polysome profiling. These experiments confirmed that despite lacking its polyA tail, tBip1 mRNA sedimented in the polyribosome fractions in sucrose gradients (). Moreover, ribosome footprinting demonstrated that Bip1 mRNA in uninduced cells and tBip1 mRNA in UPR-induced cells were engaged with actively translating ribosomes, mapping throughout the Bip1 ORF (). The larger number of reads obtained upon UPR induction correlated with the higher abundance of tBip1 mRNA. We note a significant ribosome occupancy preceding the translation start site in both Bip1 and tBip1 mRNA most likely presenting previously unrecognized small uORFs (see for a zoomed-in view). The relative ribosome occupancy of these putative uORFs did not change with UPR induction. Translation of the processed mRNA resulted in an enhanced steady-state concentration of Bip1 protein, as shown by quantitative Western blotting ().
tBip1 mRNA is translated and is important for fitness during ER stress.
To assess the physiological consequences of this unique regulatory mechanism of Bip1 expression, we explored the growth of strains carrying a mutation of the Bip1 mRNA processing site (ΔTTAACTGGTG\C). Liquid cultures of Bip1 mRNA mutant, that were exposed to a pulse of ER stress (tunicamycin) and allowed to recover after washout of the drug showed a marked growth delay in early log phase () and enhanced cell death (), indicating that Bip1 mRNA processing is important for maintaining cell fitness in the face of ER stress. By contrast to cell growth in liquid culture, Bip1 mutant cells grew on UPR-inducing tunicamycin plates only marginally worse that wild type cells (). The importance of Bip1 mRNA processing, therefore, varies with growth conditions.