The unfolded protein response (UPR) maintains homeostasis of the endoplasmic reticulum (ER). Residing in the ER membrane, the UPR mediator Ire1 deploys its cytoplasmic kinase-endoribonuclease domain to activate the key UPR transcription factor Xbp1 through non-conventional splicing of Xbp1 mRNA. Ire1 also degrades diverse ER-targeted mRNAs through regulated Ire1-dependent decay (RIDD), but how it spares Xbp1 mRNA from this decay is unknown. Here, we identify binding sites for the RNA-binding protein Pumilio in the 3′UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio binds both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced is stabilized by Pumilio. Furthermore, Pumilio displays Ire1 kinase-dependent phosphorylation during ER stress, which is required for its stabilization of Xbp1spliced. hIRE1 can phosphorylate Pumilio directly, and phosphorylated Pumilio protects Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an unexpected regulatory link between an RNA-binding protein and the UPR.
Metazoan cells respond to endoplasmic reticulum (ER) stress by activating an intracellular network of signaling pathways, known as the unfolded protein response (UPR)1,2. In higher eukaryotes, the UPR involves three ER transmembrane transducers: inositol-requiring enzyme 1 (IRE1), pancreatic ER kinase (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6). When misfolded proteins accumulate in the ER, IRE1 activates the downstream transcription factor X-box binding protein 1 (XBP1), via the non-conventional splicing of Xbp1 mRNA3,4,5,6,7. The cytoplasmic kinase- endoribonuclease domain of IRE1 mediates the splicing of a 26-nucleotides long intron from the Xbp1 mRNA, causing a frame-shift during translation that introduces a new carboxyl domain in the XBP1 protein. The resulting spliced form of Xbp1, XBP1spliced, is a functionally active transcription factor that upregulates the expression of ER chaperones and other UPR target genes8.
The intracellular localization, stability and translation of mRNAs is regulated by interaction of RNA-binding proteins (RBPs) or microRNAs with specific sequences present in the 3′ untranslated regions (3′UTR) of the mRNA9,10,11. In budding yeast, the 3′UTR of the Xbp1-orthologue Hac1 targets Hac1 mRNA to foci of activated IRE1 at the ER membrane, which enables IRE1-mediated splicing of Hac1 mRNA12. In contrast, in mammalian cells the 3′UTR of Xbp1 seems to be dispensable for the targeting of Xbp1 mRNA to activated IRE113. Instead, the targeting occurs through the tethering of 2 hydrophobic regions (HR1 and HR2) present in the XBP1unspliced protein to the cytosolic side of the ER membrane13,14. This latter process involves a tripartite complex comprising Xbp1 mRNA, a ribosome, and the nascent XBP1 protein, and additionally requires translational pausing of the Xbp1unspliced mRNA14.
Besides Xbp1 mRNA splicing, the endoribonuclease (RNase) domain of Ire1 also performs a function known as regulated Ire1-dependent decay (RIDD), which mediates the depletion of specific mRNAs15 and/or microRNAs16. While in Drosophila cells RIDD degrades ER-targeted mRNA relatively promiscuously, in mammalian cells it is thought to depend on a specific Xbp1-like mRNA sequence endomotif within a stem-loop structure, and on translational state of the mRNA target17. The phosphorylation and oligomerization state of IRE1 plays an additional role in controlling IRE1′s RNase activity18. RIDD can act as a post-transcriptional mechanism to deplete mRNAs, thereby affecting both ER homeostasis and cell fate19,20,21,22.
RNA-binding proteins (RBPs) constitute an important class of post-transcriptional regulators. RBPs are involved in multiple critical biological processes, relevant to cancer initiation, progression, and drug resistance23. Several recent studies validate the role of the Pumilio family of RBPs in diverse biological processes in several organisms. Specific mRNA targets of two human Pumilio isoforms (PUM1 and PUM2) include oncogenes, tumor suppressors, and other factors implicated in oncogenic and cell death pathways24,25,26. In several organisms, PUM proteins also play a conserved role in stem cell proliferation and self- renewal27,28,29,30. PUM proteins are essential for development and growth, and their dysfunction has been associated with neurological diseases, infertility, movement disorders, and cancer31,32,33,34,35,36.
Drosophila Pumilio–the founding member of the PUM family—is characterized as a translational repressor and is involved in embryo patterning, fertility and the regulation of neuronal homeostasis31,37,38,39,40. PUM proteins act as post-transcriptional regulators, by interacting with consensus sequences called Pumilio regulatory elements (PRE) in the 3′UTRs of target mRNAs9 to modulate their translation and/or degradation41,42. Recent findings support a direct role of PUM proteins in the activation/protection of specific RNAs26. PUM proteins are composed of distinct functional domains: N-terminal repressor domains (N), which are unique to different PUM orthologues, and a Pumilio homology domain (PumHD), which recognizes the PRE. These domains mediate the normal repressive role of PUM proteins by antagonizing the translational activity of Poly(A) binding protein (PABP)42,43. The PUM N terminus is required to fully rescue developmental defects of a pum mutant44,45.
Although much progress has been made toward understanding the biological roles of PUM proteins, it remains to be determined how they regulate their target mRNAs particularly under diverse biological conditions such as cellular stress. Here, we uncover an unexpected functional link between Pumilio and the Ire1-Xbp1 pathway. We show that Pumilio protects Drosophila Xbp1 against RIDD, without perturbing canonical Ire1-driven Xbp1 splicing. Furthermore, we provide evidence that Ire1 phosphorylates Pumilio under ER stress and that this is essential for Pumilio-mediated protection of Xbp1 mRNA. These results identify an important regulatory mechanism connecting RBPs and the UPR.
The 3′UTR of Xbp1 contains cis elements that regulate mRNA stability
The 3′UTRs of Drosophila Xbp1unspliced and Xbp1spliced differ in the length, the former being 600 bp longer than the latter (Fig. 1a). We conducted a search on the 3′UTR of Drosophila Xbp1 mRNA and identified two putative Pumilio regulatory elements (UGUAXAUA) (Fig. 1a, b). To test experimentally if these putative PREs can regulate mRNA, we constructed reporters containing GFP, expressed under the control of the metalotheionin promoter46,47, together with the 3′UTR of Xbp1 in wild type or a mutated version of each of the two PREs (Fig. 1b). To examine the impact of these PREs on mRNA stability, we mutated the sequence encoding the PRE (UGUAXAUA) to the non-functional element ACAAXAUA (Fig. 1b).
The mRNA decay of the resulting reporters was analyzed in transiently transfected Drosophila S2 cells, which were treated with actinomycin D to stop transcription. For each mutated version of the PREs the stability of the GFP reporter decreased as compared to the wild-type 3′UTR, suggesting a role for these regulatory regions in the post- transcriptional regulation of Xbp1. We also compared the effect of the 3′UTRs of Xbp1spliced and Xbp1unspliced forms by quantitative RT-PCR, which showed a greater effect of the former 3′UTR on stability of the GFP reporter mRNA (Supplementary Fig. 1a). This result suggests that other, yet unidentified regulatory regions of RNA stability may be present in the 3′UTR of Xbp1unspliced.
Pumilio binds Xbp1 mRNA in vitro and in the developing Drosophila eye
To confirm the interaction between Pumilio and Xbp1 mRNA, we tested the binding affinity of Pumilio to one of the PRE elements present in the Xbp1 3′UTR by electrophoretic mobility shift assays (EMSA - Fig. 1c). For this experiment we used purified protein of human PUM1, containing the Pum-HD domain (Supplementary Fig. 2a), which shares 80% of sequence conservation to Drosophila Pum-HD. Interaction between hPUM1-HD and the PRE1 in the Xbp1 3′UTR was tested using a range of protein concentration (100-300 nM) and a biotinylated RNA oligo containing the WT PRE1 sequence (5′-N5-UGUACAUA-N12-3′BioTEG). hPUM1-HD binds the RNA oligo, as seen by appearance of a slower migrating band in the native gel, corresponding to the RNA-protein complexes. The binding selectivity of hPUM1-HD to the RNA oligo was examined by the presence of increasing concentrations of an unlabeled RNA oligo competitor, containing the WT or the mutated (UGUA to ACAA) target sequence for Pumilio binding. Although the shifted band corresponding to the RNA-PUM complex disappeared following the addition of the WT competitor oligo, the addition of the mutant competitor oligo yielded no effect on the binding of hPUM1-HD to the labeled WT Xbp1 oligo.
To analyze if Pumilio could act as a regulator of Xbp1 in fly tissues, we expressed a transgene encoding a TAP-tagged48 Pumilio RNA-binding motif (UAS-TAP-PumHD), under control of the eye-specific GMR-GAL4 driver. We conducted TAP pull-down RNA affinity purifications from heads of transgenic adult flies with the genotype GMR– GAL4>UAS-TAP-PumHD, and performed RT-PCR to determine whether the endogenous Xbp1 mRNA was enriched in flies over-expressing TAP-Pum-HD (Fig. 1d, Supplementary Fig. 1b). We found that PumHD could bind equally well to the 3′UTR of Xbp1unspliced and Xbp1spliced (Fig. 1d). These results suggest that endogenous Xbp1 transcripts in the Drosophila eye can be regulated by Pumilio.
Pumilio regulates the stability of Xbp1spliced mRNA
The activity of Ire1 in mediating Xbp1 splicing can be assayed with an Xbp1-GFP reporter49, wherein Xbp1spliced is tagged with GFP. We constructed a new Xbp1 reporter (Xbp1-HA-GFP) (Fig. 2a), which distinguishes the translation product of Xbp1unspliced, fused to an HA tag, from that of Xbp1spliced, fused to GFP. We first tested the expression of the reporter translation products by western blot under non stress and stress conditions in the presence of dithiothreitol (DTT), an ER stress inducing chemical (Fig. 2b). In this context, overexpression of full-length Pumilio50 led to an increase in Xbp1spliced, but Xbp1unspliced protein levels remained unchanged in comparison with controls (transfected cells with Xbp1-HA-GFP only). Overexpression of PumR750, a Pumilio mutant defective for RNA binding, did not cause a change in Xbp1spliced or Xbp1unspliced levels, in comparison with controls.
Subsequently, we determined the stability of the Xbp1-HA-GFP reporter mRNA in transiently transfected S2 cells, either with Pumilio RNAi or control LacZ RNAi depletion (Fig. 2c, Supplementary Fig. 1c). Depletion of Pumilio decreased the stability of the Xbp1-HA-GFP reporter transcript as compared with controls (Fig. 2c). When Pumilio levels were restored by co-transfecting S2 cells depleted of Pumilio with a plasmid expressing either the full-length Pumilio protein (Pum-FL)50 or the Pumilio RNA binding domain (Pum-HD), the mRNA stability of Xbp1-HA-GFP reporter returned to the levels of the control experiment (LacZ RNAi treatment), but it did so only when the full-length protein was present (Fig. 2c). These results indicate that Pumilio has a protective role against degradation of the Xbp1 mRNA, and that although the HD region confers specific RNA interaction with the Xbp1 transcripts, the full-length Pumilio protein is required for its protective role.
Pumilio regulates Xbp1spliced during photoreceptor differentiation
Next we tested the regulation of Xbp1 mRNA by Pumilio in a physiological, developmental context. The Ire1 signaling pathway is activated during the pupal stages in the photoreceptors51, where it is required for photoreceptor differentiation and morphogenesis of the rhabdomere – the light-sensing organelle of photoreceptors. We generated transgenic flies with the Xbp1-HA-GFP reporter, and performed immunofluorescence analysis with antibodies against GFP and HA, to assess the expression levels of Xbp1spliced-GFP and Xbp1unspliced-HA in eyes containing clones of the Ire1f02170 null mutation51. As expected, and validating the Xbp1-HA-GFP reporter, Xbp1spliced-GFP expression was absent from Ire1 f02170 homozygous cells (labeled by the absence of DsRed), but Xbp1unspliced-HA expression was unaffected in these Ire1 homozygous mutant cells (Fig. 2d). We next examined the expression of Xbp1-HA-GFP in eyes containing clones of the Pumilio null mutation pumET152. In pupal eyes (50 hr), in the absence of Pumilio (labeled by the absence of DsRed), the expression of Xbp1spliced-GFP was reduced as compared to the wild-type cells (Fig. 2e, f). However, expression of Xbp1unspliced-HA remained unchanged (Fig. 2f), indicating that Pumilio exerts its regulatory role on the mRNA of Xbp1spliced-GFP but not Xbp1unspliced-HA.
Pumilio undergoes IRE1 kinase-dependent phosphorylation during ER stress
It is known that Pumilio proteins may become activated to regulate target mRNAs upon phosphorylation53. Furthermore, the observation that the stability of the Xbp1spliced transcript was dependent on the full-length Pumilio protein (Fig. 2c) indicated that other domains of Pumilio, besides the RNA-binding domain, might also be important for Xbp1 mRNA stability. Recent studies36,42,50 indicate that additional regions in the N terminus of Pumilio act as repressor domains, with two specific segments – designated Pumilio conserved motif (PCM) a and b – being found in both Drosophila and human PUMs (Fig. 3a and Fig. S2a). We screened the Pumilio protein sequence for potential serine/threonine phosphorylation sites using online bioinformatic tools54 combined with phosphorylation sites reported in other screens (http://www.phosphosite.org/). We found potential phosphorylation sites across the different domains of Pumilio (Fig. 3a). To investigate if Pumilio is phosphorylated during ER stress, we conducted Phostag analysis using Pumilio proteins tagged with a C-terminal V5 epitope (Fig. 3a). It was difficult to analyze phosphorylation of full-length Pumilio50, due to its relatively large size of ~180 kDa (data not shown). Therefore, to facilitate the analysis, we constructed 3 truncated versions of V5-tagged Pumilio (D1 = aa 1 to 547; D3 = aa 777 to 1091 and D1D3 = aa 1 to 1091), which contained the predicted phosphorylation sites.
Phostag analysis revealed that, during ER stress (4 hr DTT), Pumilio D1 displayed a 6-fold increase in phosphorylated forms, relative to non-ER stress conditions (0 hr DTT) (Fig. 3b, c). This phosphorylation pattern was confirmed by treatment with phosphatases (+CIP or + λPP) and the use of a triple phospho-mutant (M3 = T537A, S540A, S544A), which reduced the phosphorylated forms of Pumilio D1 (Fig. 3b, d).
During ER stress and after IRE1 trans-autophosphorylation, the cytosolic kinase-endoribonuclease domain of IRE1 rotates to a back-to-back configuration2,18, possibly allowing the kinase active site to access other potential substrates. We reasoned that through binding to the 3′UTR of Xbp1, Pumilio might localize to the vicinity of the Ire1 kinase domain. Therefore, we tested whether Ire1 could phosphorylate Pumilio during ER stress, using the inhibitors of Ire1 kinase activity (Apy29)55 and compound #1856 or an inhibitor of IRE1 RNase activity (4μ8C)57. Upon DTT exposure (4 h), in S2 cells transfected with PUM-D1, Apy 29 attenuated the generation of PUM-D1 hyper-phosphorylated forms (Fig. 3d). In contrast, 4μ8C did not change the pattern of PUM-D1 phosphorylation, which is expected, as 4μ8C is specific for the RNase activity and does not inhibit IRE1 kinase activity57. We also observed ER stress induced phosphorylation of PUM-D3, and by constructing phosphorylation site mutant versions of PUM-D3 we could identify S902 as a site that is phosphorylated under ER stress conditions (Fig. 3e).
hIRE1 phosphorylates Pumilio and hPUM1
To confirm that IRE1 kinase activity could directly phosphorylate Pumilio, we performed in vitro kinase reactions using the hIRE1α kinase-endoribonuclease (KR) domain57 and purified versions of Pumilio domains D3 and D1D3. Phostag immunoblot analysis (Fig. 3f, g) showed specific bands indicating phosphorylation of PUM- D3 and PUM-D1D3 upon incubation with hIRE1 KR. Pumilio phosphorylation was abolished upon Apy 29 or λ-PP treatment. To complement these results, we performed in vitro radioactive kinase assays using hIRE1α KR and purified versions of Pumilio and hPum1 protein domains (Fig. 4a, b and Supplementary Fig. 2b). The hIRE1α KR protein phosphorylated both Pumilio and hPum1 proteins. For negative controls, we used a kinase-dead hIRE1α KR as enzyme, or BIP (a luminal ER protein that should not be a direct target for IRE1 phosphorylation) as substrate (Supplementary Fig. 3a). Furthermore, to assure that the phosphorylation pattern observed for the PUM proteins in the radioactive assays did not simply reflect IRE1 auto-phosphorylation, we used an antibody specific for phosphorylated IRE158. While we observed phosphorylated monomers, dimers and oligomers of hIRE1α KR as expected, we could not detect any signal in the 40 KDa region (Supplementary Fig. 3b), which corresponds to the phosphorylated forms of Pumilio that were detected with the V5 antibody (Fig. 4b). These results show that hIRE1α KR can directly phosphorylate Pumilio.
Pumilio protects Xbp1 mRNA from regulated IRE1-dependent decay
We have shown that Pumilio has a protective role in the stability of Xbp1 mRNA (Fig. 2c). Ire1 can also mediate RIDD, the selective decay of ER-bound mRNAs, thereby reducing the load of nascent proteins entering the ER in order to be folded59. RIDD plays an import role during persistent ER stress19 and in Drosophila cells it can lead to the complete degradation of even ectopic mRNAs, such as GFP15,60. Furthermore, Le Thomas et al.58 identified a variety of mRNAs in human cells that do not contain the consensus stem-loop endomotif present in Xbp1 and in many canonical mammalian RIDD targets (dubbed RIDDLE – RIDD lacking endomotif), but are nonetheless bona fide targets of IRE1-dependent RNA decay. Indeed, Le Thomas et al showed that in its purified, fully phosphorylated oligomeric form hIRE1α KR (KR-3P) degraded not only RIDD and RIDDLE mRNA targets, but also hXBP1 mRNA. We therefore hypothesized that Pumilio may protect Xbp1 mRNA from decay by Ire1 under ER stress conditions. To test this possibility, we performed mRNA stability assays with the Xbp1-HA-GFP reporter in S2 cells treated with DTT, in the absence or presence of the Ire1 RNase inhibitor 4μ8C. The destabilization of Xbp1-HA-GFP mRNA in cells depleted of Pumilio was reverted upon 4μ8C treatment (Fig. 5a), indicating that Pumilio stabilizes Xbp1 mRNA against the RNase activity of Ire1. We also conducted mRNA stability assays using in vitro transcribed Drosophila Xbp1 and human XBP1 mRNAs incubated with non-phosphorylated (0P) or fully phosphorylated (3P) hIRE1α KR. Since we could not produce purified recombinant full-length Pumilio, we used different forms of hPUM1, which were pre-incubated with the Xbp1 transcript in order to assess their protection against degradation by IRE1. Full-length hPUM (hPUM1-FL) protected the Drosophila Xbp1 (Fig. 5b) and hXBP1 (Supplementary Fig. 4a) mRNAs from degradation by hIRE1α KR-3P, but did not block or even diminish the hairpin motif-dependent splicing of Xbp1 and hXBP1 mRNAs by KR-0P or KR-3P. By contrast, hPUM1-B (2-827) did not protect Xbp1 mRNA from decay, consistent with the absence of the HD region necessary for binding to Xbp1 mRNA in this hPUM variant. Likewise hPUM1-C (828-1186) is incapable of such protection. Only the full-length hPUM1 protein contains all the requisite domains for protection of Xbp1 mRNA, in keeping with the above results (Fig. 2c).
Pumilio’s protection of Xbp1 mRNA is dependent on its phosphorylation state
Having identified T537, S540, S544 and S902 as sites for Pumilio phosphorylation under ER stress (Fig. 3), we next asked if the phosphoryation status of Pumilio could regulate the effect on the stability of Xbp1spliced. To examine this, we compared the half-life of Xbp1 upon overexpression of wt Pumilio or Pumilio phosphomutants (S902A and a quadruple mutant:T537A, S540A, S544A and S902A) in RNAi Pumilio-depleted S2 cells (Fig. 5c). As compared to wild-type Pumilio, overexpression of the phosphomutants lead to a decrease in the stability of Xbp1spliced, demonstrating that Pumilio phosphorylation is required to promote its protective effect on Xbp1 transcripts under ER stress.
Pumilio regulation of Xbp1 is important for the cellular response to ER stress
Finally, we investigated the importance of this regulatory mechanism of Pumilio on Xbp1 mRNA for the response of cells under ER stress. We treated Drosophila larvae from the Pumilio mutation PumET1 with/without tunicamycin (Tm) yeast paste food. After 20 hours of Tm treatments, around 1000 L1-L2 stage PumET1 homozygous or control heterozygous PumET1/TTG larva were collected and processed for RT-qPCR. The results (Fig. 5d) show that, upon tunicamycin treatment, the levels of Xbp1spliced and the induction of the Xbp1 targets Acat2 and Hsc3 are diminished in PumET1 homozygous larva, in comparison with control heterozygous PumET1/TTG larva. Similarly, we did experiments using human MDA-MB-231 cells treated with Thapsigargin (Tg) or vehicle control (DMSO). In this case the RT-qPCRs results (Supplementary Fig. 4b) reveal diminished levels for human XBP1s and the XBP1s target SYVN1, upon treatments with siRNAs for PUM1 and PUM2 (siPUM1+2).
Our results uncover a novel protective effect of Pumilio on Xbp1 mRNA during ER stress. This protective effect depends on the phosphorylation status of Pumilio, which can be mediated by Ire1 kinase activity in response to ER stress. We propose a model depicted in Fig. 6, which involves 3 steps.
In the absence of ER stress, Ire1 is not active and Xbp1 mRNA is not spliced, but Pumilio can bind the 3′UTR of Xbp1unspliced mRNA (Fig. 6–1). Indeed, our RNA pull down experiment (Fig. 1d) showed that Pumilio can bind equally well to the 3′UTR of Xbp1unspliced and Xbp1spliced. Accumulation of misfolded protein in the lumen of the ER causes ER stress and activates Ire1. At this stage, in order to be spliced, Xbp1 mRNA must localize in the vicinity of the cytoplasmic domain of Ire1, which harbors both kinase and endoribonuclease activities. Upon ER stress, Pumilio is phosphorylated via a mechanism that requires Ire1 kinase activity (Fig. 3). As demonstrated by our in vitro assays (Fig. 4), the kinase domain of Ire1 can directly phosphorylate Pumilio; however, we cannot exclude at this stage the possibility that other kinases also may contribute to Pumilio phosphorylation. It is known that phosphorylation of Pum family proteins contributes to their activation and function53. We suggest that in this case, Ire1- dependent phosphorylation of Pumilio causes a conformational change in the mode of interaction between Pumilio and Xbp1 mRNA, such that much of the transcript, though not the canonical stem-loop structures, is inaccessible to the more promiscuous endoribonuclease activity of Ire1 (Fig. 6: step 2). This allows Xbp1 splicing to occur and furthermore permits Ire1 to carry out endomotif-directed RIDD as well as endomotif-lacking RIDDLE while sparing Xbp1spliced mRNA and allowing its more efficient translation (Fig. 6–3).
We do not know if Pumilio also protects Xbp1 mRNA from other ribonucleases or for how long the protective effect against RIDD/LE lasts. Other studies61 have shown, using cultured mouse cells, an increase in the stability of Xbp1spliced mRNA at a time of translational repression during “early” phases of ER stress (4 h treatment with DTT). These authors suggested that Xbp1spliced mRNA could be protected from degradation by an unidentified protein factor. Our results suggest that Pumilio is such a factor, while Ire1 RIDD/LE activity is the degradation-causing agent.
From our model, we also predict that upon overcoming ER stress conditions, Pumilio phosphorylation levels will be reduced, either due to the activity of phosphatases and/or due to Ire1 dephosphorylation and attenuation62. At this stage, Pumilio’s protective role over Xbp1 mRNA may subside, presumably making the transcript more vulnerable to the action of the mRNA decay machinery and the ribosome-associated quality control, as previously shown for Hac163. This regulatory step may avoid an accumulation of Xbp1spliced under non-stress conditions, which could be detrimental for cellular homeostasis.
Previous studies have shown that Pumilio proteins play a translational repressive role or promote degradation of their target mRNAs, which is in contrast with our results. However, another study has shown that, contrary to the canonical repressive activity, PUM1/2 rather promote FOXP1 expression through direct binding to two consensus PREs present in the FOXP1-3′UTR64. Furthermore, an additional report has identified RNAs that are positively regulated by human PUM1 and PUM265.
Our results demonstrate that Ire1 is responsible for the phosphorylation status of Pumilio during ER stress and that this phosphorylation has implications for Xbp1spliced protein levels. Recent data in zebrafish implicate Pum1 phosphorylation as an initial key step for the sequential activation of cyclinB1 mRNA translation during oocyte maturation, although the kinase involved remains unidentified66. In Xenopus, Nemo-like kinase (NLK)—a typical mitogen-activated protein kinase that is activated during an early phase of oocyte maturation67—was shown to directly phosphorylate Pum1 as well Pum2 in vitro. It is possible that other kinases may be involved in Pumilio phosphorylation under non-ER stress conditions or specific developmental stages.
In conclusion, our present work uncovers an unanticipated mechanism that regulates one of the key branches of the UPR through the action of an RNA binding protein. This involves Ire1-kinase-driven phosphorylation of Pumilio, which in turn protects Xbp1spliced mRNA from Ire1-RNase-driven RIDD and RIDDLE, thereby coordinating the two major endoribonuclease outputs of Ire1 to enable a more efficient intracellular response and ER stress mitigation.
Drosophila S2 cells (Schneider, 1972) were cultured at 25 °C in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (FBS, Invitrogen), 2 mM glutamine (Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen). MDA-MB-231 cells were all obtained from ATCC, authenticated by short tandem repeat (STR) profiles, and tested to ensure mycoplasma free within 3 months of use. All cell lines were cultured in RPMI1640 media supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma), 2 mM glutaMAX (Gibco) and 100 U/ml penicillin plus 100 μg/ml streptomycin (Gibco).
Transgenic lines were generated using pUAST-AttB and phiC31 integrase-mediated DNA integration (BestGene) that allows the insertion of the transgenes in a specific site of the acceptor fly genome. Clones of mutant eye tissue were generated by the Flp/FRT technique68, with Flipase expression under the control of the eyeless promoter. Drosophila stocks obtained from the Bloomington Stock Center (Indiana University, Bloomington, IN, USA): GMR-Gal4 (active in the eye, under the control of the glass multiple reporter); eye-flip GMR-Gal4 (promotes recombination in the eye) and Actin5c- Gal4 (ubiquitous expression). Drosophila stocks were maintained at 25 °C on standard cornmeal media in an incubator with a 12 h light/dark cycle. TAP-PUM stocks were a kind gift from André Gerber. The stock FRT, pumET1 was a kind gift from Yuh Nung Jan. Tunicamycin feeding experiments with Drosophila larva was done as previously described69. Briefly, cages with apple juice plates (6 cm) were set with PumET1/TM3-TTG (TM3 balancer with twiGAL4 > UAS-GFP) flies for 24 h and aged for 24 h more. At 48 h after egg laying, larva in the plates were exposed to 500 μL of yeast paste food with/without tunicamycin (5 mg/mL) for around 20 h, after which GFP positive (Control heterozygous) and GFP negative (PumET1 homozygous) larva were manually collected under Leica fluorescent scope and processed for RNA extraction.
The 3′UTRs of Xbp1spliced and Xbp1unspliced forms were amplified from the cDNA clone GH09250 (Flybase) using specific primers (Supplementary Table 1) and cloned downstream of the green fluorescent protein (GFP) coding sequence in the vector pRmHa, containing the metallothionein promoter. The mutations in the Pumilio binding sites were introduced using oligonucleotide-mediated site-directed mutagenesis and inverse PCR. The UAS-Xbp1-HA-GFP construct was made by PCR cloning. All cloning was performed with the Phusion High-Fidelity PCR Master Mix with HF Buffer or GH Buffer according to the manufacturer’s protocol. All clones were confirmed by sequencing (Stabvida). The full-length Pumilio protein with a C-terminal V5 tag50 was a gift from Aaron Goldstrohm, from which the different truncated dPUM-D1, dPUM-D3 and dPUM-D1D3 were constructed.
Transfection and stable cell line establishment
Cells were co-transfected with the different plasmids using Effectene reagent according to manufacture indications (Qiagen). For stable transfection, cells were selected by replacing Schneider’s complete Drosophila media with fresh medium supplemented with the appropriate antibiotics (zeocin, puromycin) according to the resistance gene present in the transfected plasmids and maintained under selective media until the formation of resistant clones. The reagents are described in Supplementary Table 2.
RNAi was performed as described previously70. Primer pairs tailed with the T7 RNA polymerase promoter (Supplementary Table 1) were used to amplify PCR fragments obtained from cDNA clones. PCR products with an average size of 600 bp were then used as templates for dsRNA production with the T7 RiboMAX system (Promega). For transfection, 15 μg/ml dsRNA against Drosophila Pumilio or LacZ control were added to S2 cells in 12-well plates, during 9 days. mRNA depletion was confirmed by RT-PCR before further analysis.
Total RNA and protein extraction
For RNA stability and Western blot experiments, 4,5 × 105 S2 cells were seeded in 12-well plates the day before treatments, transfections and protein or RNA extraction. Transfection was performed with Effectene (Qiagen) according to the manufacturer’s instructions. Expression of the GFP reporter under the metallothionein promoter was induced by adding 7 mM CuSO4 to the cell culture media for 3 h. For the UAS dependent Xbp1 reporters, transcription was induced by cotransfection of Actin-Gal4 plasmid. For mRNA half-life measurements, transcription was blocked with actinomycin D (5 μg/ml; Sigma-Aldrich) and the cells were harvested at the indicated time points. Total RNA was then extracted using Zymo Research quick-RNA miniprep Kit.
qPCR and RT-PCR
Primers for qRT–PCR (Supplementary Table 1) were designed according to MIQE guidelines (Minimum Information for Publication of qRT-PCR Experiments - Supplementary Table 3) using NCBI primer blast, choosing a melting temperature of 62 °C. By using three serial dilutions of cDNA, primer efficiencies were determined, only primers with efficiencies varying around 100% were used for analysis.
Equal amounts of total RNA was retro-transcribed using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo/Fermentas). Each PCR reaction was performed on 1/40 of the cDNA obtained, using SSoFast EvaGreen Supermix (Bio-Rad) according to the manufacturer’s instructions and Bio-Rad CFX-96 as detection system. All samples were analyzed in triplicates and from 3 independent biological RNA samples. For each sample, the levels of mRNAs were normalized using rp49 as a loading control. Normalized data then were used to quantify the relative levels of mRNA using the ΔΔCT method. qPCR was carried out using a CFX-96 Biorad instrument. Biological replicates represent independently grown and processed cells. Technical replicates represent multiple measurements of the same biological sample.
For experiments with human MDAMB231 cells, siRNAs treatments used either a non-targeting control (siNTC) (Dharmacon #D-001810-10-05) or PUM1 (#L-014179-00-0005) and PUM2 (#L-014031-02-0005) combined (siPUM1+2), following the RNAiMAX protocol from Invitrogen (#13778075). 72 h later, cells were treated for 6 h with DMSO or Tg, and then proceed to RNA extraction using the RNeasy Plus kit (Qiagen #74134). Equal amounts of RNA were reverse transcribed and amplified using the TaqMan™ RNA-to-CT™ 1-Step Kit (Applied Biosystems #4392938) on the ABI QuantStudio 7 Flex Real-Time PCR System. The delta-delta CT values were calculated by relating each individual CT value to its internal GAPDH control. Taqman primers for XBP1s (#Hs03929085_g1), SYVN1 (#Hs00381211_m1), PUM1 (#Hs00472881_m1), PUM2 (#Hs00209692_m1), and GAPDH (#Hs02758991_g1) were from Life Technology.
Total protein lysates were prepared in lysis buffer containing protease inhibitors. Proteins were size-separated by SDS-PAGE and transferred onto nitrocellulose or PDVF membranes (Biorad). For Western blot analysis, primary antibodies (Supplementary Table 2) were rat anti-GFP (3H9) (1:1000, Chromotek), mouse anti-V5 (1:5000, Invitrogen), mouse anti-HA (1:5000, Covance) and mouse anti-α-tubulin (AA4.3) (1:1000, Developmental Studies Hybridoma Bank).
Drosophila Pumilio domains (PUM-D1, PUM-D3, Pum-D1D3) fused to V5-6xHis were subcloned into pET 28a and pET26a vectors for protein expression in bacteria. Plasmids were transformed into BL21(DE3) competent cells and recombinant proteins were induced with 1 mM IPTG at 25 °C. Bacteria were lysed in lysis buffer (500 mM NaCl, 50 mM Tris-Cl, pH8.0, 0,1% Triton, Protease inhibitors). Samples were lysed by sonication, centrifuged twice at 14,000 × g for 15 min, and the cleared supernatant was bound to Ni-NTA Superflow beads (Qiagen) by gravity filtration. Unbound proteins were washed with lysis buffer at pH 8.0 supplemented with increasing amounts of imidazole (5 mM, 40 mM, 60 mM). Recombinant proteins were eluted from beads in lysis buffer with 100–250 mM imidazole at pH 8.0. For recombinant proteins retained in inclusion bodies (PUM-D3), solubilization was done by including 6M urea in lysis buffer. All purified Pumilio recombinant proteins were dialized overnigth at 4 °C, against a final buffer (20 mM Hepes, 200 mM NaCl, 5% Glicerol, 2 mM DTT) and concentrated with appropriate MW cut-off Vivaspin columns (Merck). The concentration of purified proteins was determined by colorimetric assay (Bio-Rad DC Protein Assay) and verified by electrophoresis alongside of BSA standards with Coomassie staining.
Immunoprecipitation of TAP-PUM from flies
Extracts from adult flies heads were prepared as described in48. After immunoprecipitation of TAP-PUM, total RNA was extract with Trizol and Xbp1 mRNA levels detected by RT-PCR using specific primers for each Xbp1 transcript.
Electrophoretic mobility shift assay
EMSA was performed according to Chemiluminescent RNA EMSA Kit (Thermo scientific). RNA oligos (Supplementary Table 1) were designed to cover 30 bases of the Xbp1 3′UTR, including the Pumilio PRE, UGUACAUA sequence. The oligo pum1site-wt-3′BioTEG was labeled with biotin at the 3′ end, and the identical oligo without biotin labeling served as competitor probe. Mutant competitor oligo pum1site-mut contained the mutations UGUA to ACAA. For this experiment we used purified protein of human PUM1, containing the Pum-HD domain, which shares 80% of sequence conservation to Drosophila Pum-HD and both have similar affinity for Nanos response sequences71.
Binding reactions were in a binding buffer containing 10 mM Hepes/KOH pH 7.4, 50 mM KCl, 3 mM MgCl2, 1 mM EDTA, 0.1 mg/ml yeast tRNA, 2 mM dithiothreitol, 0.01% (w/v) Tween-20, 0.2 U rRNAsin (Promega), and 5% (v/v) glycerol in a total volume of 20 µl (Chen et al., 2008). In the competition assay, the reaction mixture was supplemented and incubated with 5x, 10x and 50-fold molar excess of unlabeled competitor oligos before adding the biotin-labeled probe (10 nM). The protein-RNA complexes were allowed to form for 1 h on ice, followed by electrophoresis through 6% non-denaturing polyacrylamide gels in 0.5 X TBE at 100 V at room T °C. The samples were transferred to a Hybon N+ Membrane at 350 mA for 45′ at 4 °C, using Trans-Blot (Bio-Rad). The membrane was cross-linked for 3 min at 120.000 joules/cm2, followed by blocking for 1 hour in 5% milk in TBST (Tris-buffered saline, 0.1% Tween 20). Blots were incubated with HRP-conjugated anti-biotin antibody (1:1000, Cell Signaling, 7075P5) for 2 h at room temperature. After four times 5 min washing in TBST, the membrane was developed with ECL western blotting detection system (Amersham) and imaged using the ChemiDoc Imaging System (Bio-Rad).
In vitro phosphorylation assays
IRE1 phosphorylation assays were performed by incubation of purified hIRE1α KR (3,3 μg/μl) with purified Pumilio protein (dPumD1, dPumD3, hPumFl) in IRE1 kinase buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM DTT, 5% glicerol), containing either 10 μCi of γ-ATP[32P] for radioactive assays or cold ATP [2 mM] for phostag immunoblot assays (in a total volume of reaction of 20 μl). Inhibitors of IRE1 Kinase (Apy29 [2,5 μM/μl] and compound #18 [2,5 μM/μl]) were pre-incubated with hIRE1α KR and all reactions were assembled on ice, prior to addition of ATP and incubation for 2 h at 25 °C. Phosphatase treatment with λPP (NEB) or CIP (NEB) were performed after phosphorylation reactions, for 40 min at 30 °C. Each reaction was stopped by addition of SDS-PAGE loading buffer and run on pre-cast SDS-PAGE gels (Biorad). The autophosphorylation of hIRE1α KR was confirmed by western blot using a pSer phospho-specific antibody (Genentech) and phosphorylated Pumilio proteins were detected using anti-V5 antibody (Invitrogen). In the case of kinase radioactive assays, gels were dried and kinase activity visualized by autoradiography.
S2 cells were lysed in CIP buffer (100 mM NaCl, 50 mM Tris- HCl pH 7.9, 10 mM MgCl, 1 mM DTT), 1 mM PMSF, 0.1% NP40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Calbiochem). For CIP treatment phosphatase inhibitor cocktail was omitted, and lysate was incubated at 37 °C, for 60 min in 2 units CIP (NEB) per 50 μL reaction containing 50μg of total protein. For λ-phosphatase (NEB) treatment, lysates were incubated with 1 unit of phosphatase for 30 min at 30 °C in λ−phosphatase buffer. Lysates were cleared by centrifugation and subjected to SDS-PAGE. To detect phosphorylated Pumilio in SDS-PAGE, we used Phos-tag AAL-107 (Wako Chemicals GmbH) according to the manufacturer’s instruction72,73. Western blotting was performed using mouse anti-V5 (1:5000, Invitrogen), followed by the corresponding Horseradish Peroxidase (HRP) conjugated secondary antibodies (1:5000, GE Healthcare) and visualized using the ECL Plus Western Blotting detection system (GE Healthcare).
Immunofluorescence and confocal microscopy
For Drosophila pupal dissections, white pre-pupae (0h pupa) were collected and maintained at 25 °C until the required stage. Larval, pupal and adult eyes were dissected in 1xPBS, fixed in 1xPBS + 4% Formaldehyde for 40 min at room temperature and washed 3 times with 1xPBS 0.3% Triton X- 100. Primary antibodies (Supplementary Table 2) were incubated in 1xPBS, 1% BSA, 0.1% Tween 20, 250 mM NaCl overnight at 4 °C. Samples were washed 3 times with 1xPBS + 0.3% Triton X-100 and incubated with appropriate secondary antibodies (from Jackson Immuno- Research Laboratories) for 2 h at room temperature. Samples were mounted in 80% glycerol in a bridge formed by two cover slips to prevent the samples from being crushed while analyzed on the confocal microscope (Leica TCS SP5, 63X magnification oil immersion lens).
Xbp1 RNA cleavage assay
T7 RNA was generated from pOT2-Xbp1, containing the ORF and UTR of Drosophila Xbp1. 1 μg of RNA was digested at room temperature by 1 μg of human IRE1α KR 0P or 3P recombinant protein (~0.8 μM) for 45 min in RNA cleavage buffer (HEPES pH7.5 20 mM; K acetate 50 mM; Mg acetate 1 mM; TritonX-100 0.05% (v/v)). The total volume of the reaction was 25 μl. The digestion was then complemented by an equal volume of formamide and heated up at 70 °C for 10 min to denature the RNA. The mixture was immediately placed on ice for 5 min, and then 20 μl was run on 3% agarose gel at 160 V for 1 h at 4 °C. The PUM proteins were incubated with the RNA for 40 min on ice prior to RNA digestion.
All panels data are represented as mean ± SD, from at least three independent biological replicates experiments. All statistical comparisons for significance between control and experimental groups was calculated using a significance cut off p < 0.05. and denoted by *p < 0.05,**p < 0.01, and ***p < 0.001, based on two-tailed unpaired t-Student’s test or one-way ANOVA followed by an appropriate post-hoc analysis. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc) and online resources (https://astatsa.com/OneWay_Anova_with_TukeyHSD).
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
The data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data for each figure are provided with this paper as a source data file. Source data are provided with this paper.
Ron, D. & Walter, P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol. 8, 519–529 (2007).
Walter, P. & Ron, D. The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081–1086 (2011).
Cox, J. S. & Walter, P. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87, 391–404 (1996).
Yoshida, H., Matsui, T., Yamamoto, A., Okada, T. & Mori, K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891 (2001).
Shen, X. et al. Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development. Cell 107, 893–903 (2001).
Calfon, M. et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415, 92–96 (2002).
Uemura, A., Oku, M., Mori, K. & Yoshida, H. Unconventional splicing of XBP1 mRNA occurs in the cytoplasm during the mammalian unfolded protein response. J. Cell. Sci. 122, 2877–2886 (2009).
Acosta-Alvear, D. et al. XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks. Mol. Cell 27, 53–66 (2007).
Quenault, T., Lithgow, T. & Traven, A. PUF proteins: repression, activation and mRNA localization. Trends Cell Biol. 21, 104–112 (2011).
Vazquez-Pianzola, P., Urlaub, H. & Suter, B. Pabp binds to the osk 3′UTR and specifically contributes to osk mRNA stability and oocyte accumulation. Dev. Biol. 357, 404–418 (2011).
Webster, M. W., Stowell, J. A. & Passmore, L. A. RNA-binding proteins distinguish between similar sequence motifs to promote targeted deadenylation by Ccr4-Not. Elife 8, W39 (2019).
Aragón, T. et al. Messenger RNA targeting to endoplasmic reticulum stress signalling sites. Nature 457, 736–740 (2009).
Yanagitani, K. et al. Cotranslational targeting of XBP1 protein to the membrane promotes cytoplasmic splicing of its own mRNA. Mol. Cell 34, 191–200 (2009).
Yanagitani, K., Kimata, Y., Kadokura, H. & Kohno, K. Translational pausing ensures membrane targeting and cytoplasmic splicing of XBP1u mRNA. Science 331, 586–589 (2011).
Hollien, J. et al. Regulated Ire1-dependent decay of messenger RNAs in mammalian cells. J. Cell Biol. 186, 323–331 (2009).
Upton, J.-P. et al. IRE1α cleaves select microRNAs during ER stress to derepress translation of proapoptotic Caspase-2. Science 338, 818–822 (2012).
Moore, K. & Hollien, J. Ire1-mediated decay in mammalian cells relies on mRNA sequence, structure, and translational status. Mol. Biol. Cell (2015). https://doi.org/10.1091/mbc.E15-02-0074
Gardner, B. M. & Walter, P. Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response. Science 333, 1891–1894 (2011).
Ghosh, R. et al. Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress. Cell 158, 534–548 (2014).
Lu, M. et al. Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis. Science 345, 98–101 (2014).
Coelho, D. S. & Domingos, P. M. Physiological roles of regulated Ire1 dependent decay. Front. Genet. 5, 1–6 (2014).
Huang, H.-W., Zeng, X., Rhim, T., Ron, D. & Ryoo, H. D. The requirement of IRE1-XBP1 in resolving physiological stress during Drosophila development. J. Cell. Sci. (2017) https://doi.org/10.1242/jcs.203612
Perron, G. et al. A general framework for interrogation of mRNA stability programs identifies RNA-binding proteins that govern cancer transcriptomes. CellReports 23, 1639–1650 (2018).
Guan, X. et al. PUM1 promotes ovarian cancer proliferation, migration and invasion. Biochem. Biophys. Res. Commun. 497, 313–318 (2018).
Dai, H. et al. PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells. Cell Death Dis. 10, 595 (2019).
Bohn, J. A. et al. Identification of diverse target RNAs that are functionally regulated by human Pumilio proteins. Nucleic Acids Res. 46, 362–386 (2018).
Forbes, A. & Lehmann, R. Nanos and Pumilio have critical roles in the development and function of Drosophila germline stem cells. Development 125, 679–690 (1998).
Naudin, C., Hattabi, A., Michelet, F., Blood, A. M.- N.2017. PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells. Am. Soc. Hematology https://doi.org/10.1182/blood-2016-10-747436
Shigunov, P. et al. PUMILIO-2 is involved in the positive regulation of cellular proliferation in human adipose-derived stem cells. Stem Cells Dev. 21, 217–227 (2012).
Mak, W., Fang, C., Holden, T., Dratver, M. B. & Lin, H. An important role of Pumilio 1 in regulating the development of the mammalian female germline. Biol. Reprod. 94, 134 (2016).
Vessey, J. P. et al. Mammalian Pumilio 2 regulates dendrite morphogenesis and synaptic function. Proc. Natl Acad. Sci. U. S. A. 107, 3222–3227 (2010).
Miles, W. O., Tschöp, K., Herr, A., Ji, J.-Y. & Dyson, N. J. Pumilio facilitates miRNA regulation of the E2F3 oncogene. Genes Dev. 26, 356–368 (2012).
Driscoll, H. E., Muraro, N. I., He, M. & Baines, R. A. Pumilio-2 regulates translation of Nav1.6 to mediate homeostasis of membrane excitability. J. Neurosci. 33, 9644–9654 (2013).
Gennarino, V. A. et al. Pumilio1 haploinsufficiency leads to SCA1-like neurodegeneration by increasing wild-type Ataxin1 levels. Cell 160, 1087–1098 (2015).
Gehrke, S. et al. PINK1 and parkin control localized translation of respiratory chain component mRNAs on mitochondria outer membrane. Cell Metab. 21, 95–108 (2015).
Goldstrohm, A. C., Hall, T. M. T. & McKenney, K. M. Post-transcriptional regulatory functions of mammalian pumilio proteins. Trends Genet. 34, 972–990 (2018).
Lehmann, R. & Nüsslein-Volhard, C. Involvement of the Pumilio gene in the transport of an abdominal signal in the Drosophila embryo. Nature 329, 167–170 (1987).
Wharton, R. P., Sonoda, J., Lee, T., Patterson, M. & Murata, Y. The Pumilio RNA-binding domain is also a translational regulator. Mol. Cell 1, 863–872 (1998).
Menon, K. P. et al. The translational repressor Pumilio regulates presynaptic morphology and controls postsynaptic accumulation of translation factor eIF-4E. Neuron 44, 663–676 (2004).
De Jesus-Olmo, L. A. et al. Pumilio regulates sleep homeostasis in response to chronic sleep. Deprivation in Drosophila melanogaster. Front. Neurosci. 14, 1–15 (2020).
Wharton, R. P. & Aggarwal, A. K. mRNA regulation by Puf domain proteins. Sci. STKE 2006, pe37–pe37 (2006).
Arvola, R. M. et al. Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs. Nucleic Acids Res. 48, 1843–1871 (2020).
Weidmann, C. A., Raynard, N. A., Blewett, N. H., Van Etten, J. & Goldstrohm, A. C. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation. RNA 20, 1298–1319 (2014).
Salazar, A. M., Silverman, E. J., Menon, K. P. & Zinn, K. Regulation of synaptic pumilio function by an aggregation-prone domain. J. Neurosci. 30, 515 (2010).
Miller, M. A. & Olivas, W. M. Roles of Puf proteins in mRNA degradation and translation. Wiley Interdiscip. Rev. RNA 2, 471–492 (2011).
Bunch, T. A., Grinblat, Y. & Goldstein, L. S. Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 16, 1043–1061 (1988).
Cairrao, F., Halees, A. S., Khabar, K. S. A., Morello, D. & Vanzo, N. AU-rich elements regulate Drosophila gene expression. Mol. Cell. Biol. 29, 2636–2643 (2009).
Gerber, A. P., Luschnig, S., Krasnow, M. A., Brown, P. O. & Herschlag, D. Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc. Natl Acad. Sci. 103, 4487–4492 (2006).
Ryoo, H. D., Domingos, P. M., Kang, M. J. & Steller, H. Unfolded protein response in a Drosophila model for retinal degeneration. EMBO J. 26, 242–252 (2007).
Weidmann, C. A. & Goldstrohm, A. C. Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor. Mol. Cell. Biol. 32, 527–540 (2012).
Coelho, D. S. et al. Xbp1-independent Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila. Cell Rep. 5, 791–801 (2013).
Ye, B. et al. Nanos and Pumilio are essential for dendrite morphogenesis in Drosophila peripheral neurons. Curr. Biol. 14, 314–321 (2004).
Kedde, M. et al. A Pumilio-induced RNA structure switch in p27-3′ UTR controls miR-221 and miR-222 accessibility. Nat. Cell Biol. 12, 1014–1020 (2010).
Gnad, F., Gunawardena, J. & Mann, M. PHOSIDA 2011: the posttranslational modification database. Nucleic Acids Res. 39, D253–D260 (2011).
Korennykh, A. V. et al. The unfolded protein responsesignals through high-order assembly of Ire1. Nature 457, 687–693 (2008).
Harrington, P. E. et al. Unfolded Protein Response in Cancer: IRE1α Inhibition by Selective Kinase Ligands Does Not Impair Tumor Cell Viability. ACS Med. Chem. Lett. 6, 68–72 (2014).
Cross, B. et al. The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule. PNAS 109, E869–E878 (2012).
Le Thomas, A. et al. Decoding non-canonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α. Nat. Comm. 12, 7310 (2021).
Hollien, J. & Weissman, J. S. Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response. Science 313, 104–107 (2006).
Gaddam, D., Stevens, N. & Hollien, J. Comparison of mRNA localization and regulation during endoplasmic reticulum stress in Drosophila cells. Mol. Biol. Cell 24, 14–20 (2013).
Majumder, M. et al. A novel feedback loop regulates the response to endoplasmic reticulum stress via the cooperation of cytoplasmic splicing and mRNA translation. Mol. Cell. Biol. 32, 992–1003 (2012).
Chang, T.-K. et al. Coordination between Two Branches of the Unfolded Protein Response Determines Apoptotic Cell Fate. Mol. Cell 71, 629–636.e5 (2018).
Peschek, J. & Walter, P. tRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay. Elife 8, 213 (2019).
Naudin, C. et al. PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells. Blood–2016–10–747436–35 (2017). https://doi.org/10.1182/blood-2016-10-747436
Uyhazi, K. E. et al. Pumilio proteins utilize distinct regulatory mechanisms to achieve complementary functions required for pluripotency and embryogenesis. Proc. Natl Acad. Sci. U. S. A. 3, 201916471 (2020).
Saitoh, A., Takada, Y., Horie, M. & Kotani, T. Pumilio1 phosphorylation precedes translational activation of its target mRNA in zebrafish oocytes. Zygote 26, 372–380 (2018).
Ota, R., Kotani, T. & Yamashita, M. Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes. J. Biol. Chem. 286, 2853–2863 (2011).
Golic, K. G. Site-specific recombination between homologous chromosomes in Drosophila. Science 252, 958–961 (1991).
Coelho, D. S., Gaspar, C. J. & Domingos, P. M. Ire1 mediated mRNA splicing in a C-Terminus deletion mutant of Drosophila Xbp1. Plos One 9, e105588 (2014). 8.
Clemens, J. C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl Acad. Sci. 97, 6499–6503 (2000).
Zamore, P. D., Williamson, J. R. & Lehmann, R. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins. RNA 3, 1421–1433 (1997).
Kinoshita-Kikuta, E., Kinoshita, E., Matsuda, A. & Koike, T. Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS PAGE gel. Proteomics 14, 2437–2442 (2014).
Kinoshita, E., Kinoshita-Kikuta, E. & Koike, T. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat. Protoc. 4, 1513–1521 (2009).
We thank Aaron Goldstrohm for plasmids. We thank Yuh Nung Jan, Stefan Luschnig and André Gerber for Pumilio fly stocks. We thank David Ron and Heather Harding for providing the purified hIRE1〈 KEN, 4μ8C and helpful discussions. This work received funding by grants from the Fundação para a Ciencia e Tecnologia (PTDC/BIA-BCM/105217/2008; PTDC/SAU- OBD/104399/2008; PTDC/BEX-BCM/1217/2012; FCT-ANR/ NEU-NMC/0006/2013; PTDC/NEU-NMC/2459/2014; SFRH/ BPD/93893/2013 and DL57/2016 to FC and SFRH/BD/ 130817/2017 to CS). The project leading to these results has also received funding from ‘la Caixa’ Foundation (ID 100010434), under the agreement <LCF/PR/HR17/52150018>.
A.LeT., S.M., and A.A. were employees of Genentech, Inc., a member of the Roche Group, during the performance of these studies. All other authors declare no competing interests.
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Cairrão, F., Santos, C.C., Le Thomas, A. et al. Pumilio protects Xbp1 mRNA from regulated Ire1-dependent decay. Nat Commun 13, 1587 (2022). https://doi.org/10.1038/s41467-022-29105-x