bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint
Pumilio protects Xbp1 mRNA from regulated Ire1-dependent decay
Fatima Cairrao1, Cristiana C Santos1, Adrien Le Thomas2, Scot Marsters2, Avi Ashkenazi2 and Pedro M. Domingos1
1 - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal 2 - Cancer Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA Correspondence should be sent to [email protected] or [email protected]
SUMMARY
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. We identified binding sites for the RNA-binding protein Pumilio in the 3’UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio bound both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced was stabilized by Pumilio. Furthermore, Pumilio displayed Ire1 kinase-dependent phosphorylation during ER stress, which was required for its stabilization of Xbp1spliced. Human IRE1 could directly phosphorylate Pumilio, and phosphorylated Pumilio protected Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an important and unexpected regulatory link between an RNA-binding protein and the UPR.
INTRODUCTION targeting of Xbp1 mRNA to activated IRE113. Instead, the targeting occurs through the tethering Metazoan cells respond to endoplasmic reticulum of 2 hydrophobic regions (HR1 and HR2) present in (ER) stress by activating an intracellular network of the XBP1unspliced protein to the cytosolic side of the signaling pathways, known as the unfolded protein ER membrane13,14. This latter process involves a response (UPR)1,2. In higher eukaryotes, the UPR tripartite complex comprising Xbp1 mRNA, a involves three ER transmembrane transducers: ribosome, and the nascent XBP1 protein, and inositol-requiring enzyme 1 (IRE1), pancreatic ER additionally requires translational pausing of kinase (PKR)-like ER kinase (PERK), and the Xbp1unspliced mRNA14. activating transcription factor 6 (ATF6). When Besides Xbp1 mRNA splicing, the misfolded proteins accumulate in the ER, IRE1 endoribonuclease (RNase) domain of Ire1 also activates the downstream transcription factor X-box performs a function known as regulated IRE1- binding protein 1 (XBP1), via the non-conventional dependent decay (RIDD), which mediates the splicing of Xbp1 mRNA3-7. The cytoplasmic kinase- depletion of specific mRNAs15 and/or microRNAs16. endoribonuclease domain of IRE1 mediates the While in Drosophila cells RIDD degrades ER- splicing of a 26-nucleotides long intron from the targeted mRNA relatively promiscuously, in Xbp1 mRNA, causing a frame-shift during mammalian cells it is thought to depend on a translation that introduces a new carboxyl domain specific Xbp1-like mRNA sequence endomotif in the XBP1 protein. The resulting spliced form of within a stem-loop structure, and on translational Xbp1, XBP1spliced, is a functionally active state of the mRNA target17. The phosphorylation transcription factor that upregulates the expression and oligomerization state of IRE1 plays an of ER chaperones and other UPR target genes8. additional role in controlling IRE1’s RNase The intracellular localization, stability and activity18. RIDD can act as a post-transcriptional translation of mRNAs is regulated by interaction of mechanism to deplete mRNAs, thereby affecting RNA-binding proteins (RBPs) or microRNAs with both ER homeostasis and cell fate19,20,21,22. specific sequences present in the 3’ untranslated RNA-binding proteins (RBPs) constitute an regions (3’UTR) of the mRNA9-11. In budding yeast, important class of post-transcriptional regulators. the 3’UTR of the Xbp1-orthologue Hac1 targets RBPs are involved in multiple critical biological Hac1 mRNA to foci of activated IRE1 at the ER processes, relevant to cancer initiation, membrane, which enables IRE1-mediated splicing progression, and drug resistance23. Several recent of Hac1 mRNA12. In contrast, in mammalian cells studies validate the role of the Pumilio family of the 3’UTR of Xbp1 seems to be dispensable for the RBPs in diverse biological processes in several Cairrão et al, 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint organisms. Specific mRNA targets of two human test experimentally if these regions can regulate Pumilio isoforms (PUM1 and PUM2) include mRNA, we constructed reporters containing GFP, oncogenes, tumor suppressors, and other factors expressed under the control of the metalotheionin implicated in oncogenic and cell death pathways24- promoter46,47, together with the 3'UTR of Xbp1 in 26. In several organisms, PUM proteins also play a wild type or a mutated version of each of the two conserved role in stem cell proliferation and self- PREs (Fig. 1b). To examine the impact of these renewal27-30. PUM proteins are essential for PREs on mRNA stability, we mutated the sequence development and growth, and their dysfunction has encoding the PRE (UGUAXAUA) to the non- been associated with neurological diseases, functional element ACAAXAUA (Fig. 1b). infertility, movement disorders, and cancer31-36 . The mRNA decay of the resulting reporters Drosophila Pumilio - the founding member was analyzed in transiently transfected Drosophila of the PUM family – Is characterized as a S2 cells, which were treated with actinomycin D to translational repressor and is involved in embryo stop transcription. For each mutated version of the patterning, fertility and the regulation of neuronal PREs the stability of the GFP reporter decreased homeostasis31,37-40. PUM proteins act as post- as compared to the wild-type 3'UTR, suggesting a transcriptional regulators, by interacting with role for these regulatory regions in the post- consensus sequences called Pumilio regulatory transcriptional regulation of Xbp1. We also elements (PRE) in the 3’UTRs of target mRNAs9 to compared the effect of the 3'UTRs of Xbp1spliced and modulate their translation and/or degradation41-42. Xbp1unspliced forms by quantitative RT-PCR, which Recent findings support a direct role of PUM showed a greater effect of the former 3’UTR on proteins in the activation/protection of specific stability of the GFP reporter mRNA (Fig. S1). This RNAs26. PUM proteins are composed of distinct result suggests that other, yet unidentified functional domains: N-terminal repressor domains regulatory regions of RNA stability may be present (N), which are unique to different PUM orthologues, in the 3'UTR of Xbp1unspliced. and a Pumilio homology domain (PumHD), which recognizes the PRE. These domains mediate the Pumilio binds Xbp1 mRNA in the developing normal repressive role of PUM proteins by Drosophila eye antagonizing the translational activity of Poly(A) To analyze if Pumilio could act as a regulator of binding protein (PABP)42,43. The PUM N terminus is Xbp1 in fly tissues, we expressed a transgene required to fully rescue developmental defects of a encoding a TAP-tagged48 Pumilio RNA-binding pum mutant44,45. motif (UAS-TAP-PumHD), under control of the eye- Although much progress has been made specific GMR-GAL4 driver. We conducted TAP pull- toward understanding the biological roles of PUM down RNA affinity purificationsP (R As) from heads proteins, it remains to be determined how they of transgenic adult flies with the genotype GMR– regulate their target mRNAs particularly under Gal4>UAS-TAP-PumHD, and performed RT-PCR diverse biological conditions such as cellular stress. to determine whether the endogenous Xbp1 mRNA Here, we uncover an unexpected functional link was enriched in flies over-expressing TAP-Pum- between Pumilio and the Ire1-Xbp1 pathway. We HD. Xbp1 transcripts were strongly enriched in show that Pumilio protects Drosophila Xbp1 against GMR-Gal4>UAS-TAP-PumHD flies as compared to RIDD, without perturbing canonical Ire1-driven UAS-TAP- PumHD controls (Fig. 1c,d). We found Xbp1 splicing. Furthermore, we provide evidence that Pumilio could bind equally well to the 3’UTR of that Ire1 phosphorylates Pumilio under ER stress Xbp1unspliced and Xbp1spliced (Fig. 1d). These results and that this is essential for Pumilio-mediated suggest that endogenous Xbp1 transcripts in protection of Xbp1 mRNA. These results identify an the Drosophila eye are subject to regulation by important regulatory mechanism connecting RBPs Pumilio. and the UPR. Pumilio regulates the stability of Xbp1spliced RESULTS mRNA The activity of Ire1 in mediating Xbp1 splicing can The 3'UTR of Xbp1 contains cis elements that be assayed with an Xbp1-GFP reporter49, wherein regulate mRNA stability Xbp1spliced is tagged with GFP. We constructed a The 3'UTRs of Drosophila Xbp1unspliced and new Xbp1 reporter (Xbp1-HA-GFP) (Fig. 2a), Xbp1spliced differ in the length, the former being which distinguishes the translation product of 600bps longer than the latter (Fig. 1a). We Xbp1unspliced, fused to an HA tag, from that of conducted a search on the 3'UTR of Drosophila Xbp1spliced, fused to GFP. We first tested the Xbp1 mRNA and identified two putative Pumilio expression of the reporter translation products by regulatory elements (UGUAXAUA) (Fig. 1a,b). To western blot under non stress and stress conditions
Cairrão et al, 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
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Figure 1- The 3’UTR of Xbp1 contains ATG TAA1 cis elements of mRNA stability a Intron PRE1 PRE2 DBD Pum Pum a) Schematic representation of Non stress Drosophila Xbp1 transcripts (unspliced 5’UTR 3’UTR (900 bp) Xbp1 unspliced and spliced). ATG (start codon), TAA (stop codon), DBD (DNA binding domain), Intron (23-nt hairpin structure ATG TAA2 DBD PumPRE1 PumPRE2 recognized by IRE1 RNase domain. Two Transactivation domain Stress Pumilio regulatory elements (PRE1 and 5’UTR 3’UTR (300 bp) PRE2) are present in the 3’ UTR. Xbp1 spliced b) Xbp1 3’UTR pRM GFP reporters bearing the WT and mutant PREs from the Xbp1 3’UTR. The consensus binding sites UGUA for Pumilio were b pRM GFP–Xbp1s 3'UTR mutagenized into ACAA for PRE1mut and Pumilio binding sites PRE2mut; pMT, metallothionein promoter. wt 100 Xbp1s 3’UTR Half-life: 106.2 min PRE1 PRE2 Reporters were transiently transfected in pMT S2 cells. Total RNA was isolated at 80 Xbp1s 3’UTR PRE1 Mut GFP Half-life: 65.3 min different points following the addition of pMT PRE1Mut PRE2 60 Xbp1s 3’UTR PRE2 Mut GFP actinomycin D (5 µg/ml). Stability of the Half-life: 60.7 min GFP reporters was assessed by 40 pMT PRE1 PRE2Mut quantitative RT-PCR (qRT-PCR), using GFP remaining primers specific for GFP and rp49 20 PRE1Mut: acaACAUA mRNAs (control). Levels of mRNA 0 reporter were normalized to those of rp49 PRE2Mut: acaAUGUAU Normalized % of gfp mRNA 0 60 120 180 mRNA, and averages and standard deviations from three independent Time after ActD (min) experiments are plotted. c) TAP tag RNA affinity purification (AFP) c d Xbp1 transcripts AFP Tap-PUM HD from adult heads of transgenic flies 14 expressing the Pumilio RNA-binding Total RNA input Affinity purified RNA 12 motif (GMR GAL4>UAS-TAP-PumHD) 10 under the GMR driver (expression in 8 eyes). Xbp1 transcripts are enriched in ve to control
Ď 6 Pumilio RNA affinity purifications * 4 compared with controls (UAS TAP- 2 PumHD, but no GMR GAL4). 1-UAS-TAP-PUM HD A.U rela 0 2-GMR GAl4>UAS-TAP-PUM HD d) Quantitative RT-PCR using primers Xbp1Total totalXbp1Intron UnsplicedSpliced Spliced specific for Xbp1Spliced, Xbp1Unspliced and UAS-TAP-PUM HD total Xbp1 transcripts, from GMR IP L339 GAL4>UAS-TAP-PumHD RNA pull IPGMRTap Pum GAL4> UAS-TAP-PUM HD downs, normalized against levels in the UAS-TAP-PumHD control pull downs. Results show a specific interaction in the presence of dithiothreitol (DTT), an ER stress between Xbp1Spliced and Xbp1Unspliced inducing chemical (Fig. 2b). In this context, PUM transcripts and PumHD. overexpression led to an increase in Xbp1spliced, but not Xbp1unspliced protein levels. Pumilio regulates Xbp1spliced during Subsequently, we determined the stability of photoreceptor differentiation the Xbp1-HA-GFP reporter mRNA in transiently Next we tested the regulation of Xbp1 mRNA by transfected S2 cells, either with or without Pumilio Pumilio in a physiological, developmental context. RNAi depletion (Fig. 2c). Depletion of Pumilio The Ire1 signaling pathway is activated during the decreased the stability of the Xbp1-HA-GFP pupal stages in the photoreceptors51, where it is reporter transcript as compared with controls (Fig. required for photoreceptor differentiation and 2c). When Pumilio levels were restored by co- morphogenesis of the rhabdomere – the light- transfecting S2 cells depleted of Pumilio with a sensing organelle of photoreceptors. We plasmid expressing either the full-length Pumilio generated transgenic flies with the Xbp1-HA-GFP protein50 or the Pumilio RNA binding domain (Pum- reporter, and performed immunofluorescence HD), the mRNA stability of Xbp1-HA-GFP reporter analysis with antibodies against GFP and HA, to returned to the levels of the control experiment (no assess the expression levels of Xbp1spliced-GFP and RNAi treatment), but it did so only when the full- Xbp1unspliced-HA in eyes containing clones for an Ire1 length protein was present (Fig. 2c). These results null mutation51. As expected, and validating the indicate that Pumilio has a protective role against Xbp1-HA-GFP reporter, Xbp1spliced-GFP expression degradation of the Xbp1 mRNA, and that although was absent from cells homozygous for Ire1 the HD region confers specific RNA interaction with mutation (labeled by the absence of DsRed), but the Xbp1 transcripts, the full-length Pumilio protein Xbp1unspliced-HA expression was unaffected in Ire1 is required for its protective role. mutant cells (Fig. 2d). We next examined the expression of Xbp1-HA-GPF in eyes containing
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XBP1-HA-GFP-3’UTR mRNA
a XBP1 HR2 HA GFP 3’UTR
SPLICING UPON STRESS NON STRESS CONDITIONS
XBP1 GFP XBP1 IN HR2 HA XBP1Spl: GFP XBP1Uns: HA b c wt PumF Xbp1-HA-GFP : 3'UTRspliced DTT - RNAi 0h 8h 0h 8h (5mM) kDa kDa + RNAi Pum 245- 100 245- Pum-V5 + RNAi +PumHD 180- 80 180- + RNAi +FullPum 60 Xbp1spl:GFP 40
63- mRNA remaining 63- (56 kDa)
Normalized % of 20 48- gfp 48- 0 Xbp1uns:HA 0 60 120 180 35- (29,8kDa) 35- Time after ActD (min) Figure 2- Pumilio regulates the stability of Xbp1spliced mRNA PIre1f02170 /DsRED Xbp1spl-GFP Xbp1Uns-HA a) Schematic representation of the Xbp1-HA-GFP reporter d construct. This reporter allows the detection of XBP1Spl (Spliced) and Xbp1Uns(Unspliced) forms. The pUAST-Xbp1-HA-GFP reporter, contains an HA tag fused in frame with the coding sequence of Xbp1Unspliced. GFP only gets in frame with Xbp1Spliced after Ire1 mediated splicing of the 23bp intron. b) Expression of Xbp1-HA-GFP reporter in S2 cells under non stress (0h) and stress conditions (8h DTT treatment). Xbp1spl GFP (56kDa) is detected under stress conditions. Overexpression of Pumilio (Pum-V5) increases the levels of pumET1/ DsRED Xbp1spl-GFP ELAV Xbp1spl-GFP , but not of Xbp1uns-HA (29,8 kDa). e c) Stability of the reporter was compared between non-treated S2 cells (-RNAi) and RNAi treated cells (+RNAi) against Pumilio. Inactivation of Pumilio by RNAi destabilizes Xbp1spl- GFP reporter (black squares). Reposition of Pumilio levels by transfection with full length Pumilio (Full Pum, green squares) restores the stability of the GFP reporter in S2 cells, in contrast to the RNA binding domain of Pumilio (PumHD, blue triangules). Total RNA was extracted after induction of stress pumET1/ DsRED Xbp1spl-GFP Xbp1uns-HA with DTT (4h at 5µM) and mRNA stability was analysed by f quantitative RT-PCR at defined periods after actinomycinD treatment (5µg/ml). d) The Xbp1-HA-GFP signaling reporter is activated in the Drosophila photoreceptors during midpupal stages (50 hrs). Xbp1spl-GFP is observed in WT photoreceptors (Red) but not in photoreceptors that are homozygous for an Ire1 null mutation (PIre1f02170), labeled by the absence of DsRed. Xbp1uns-HA (blue) is observed in all cells. e) The expression of Xbp1spl-GFP (green) is reduced in cells homozygous for the Pum null mutation (PumET1), labeled by the absence of DsRed expression, in comparison with wildtype cells (DsRed positive). Elav (blue) was used as a marker of the 52 photoreceptors. clones of the Pumilio null mutation pumET1 . In f) The expression Xbp1spl-GFP (green) is reduced but Xbp1uns- pupal eyes (50 hr), in the absence of Pumilio HA (blue) is unaltered in cells homozygous for PumET1, (labeled by the absence of DsRed), the expression labeled by the absence of DsRed expression, in comparison of Xbp1spliced-GFP was reduced as compared to the with wildtype cells (DsRed positive), indicating that the regulatory role of Pumilio is only on the Xbp1Spl form. Anti-HA wild type cells (Fig. 2e,f). However, expression of was used to label Xbp1Uns-HA protein. Xbp1unspliced-HA remained unchanged (Fig. 2f), indicating that Pumilio exerts its regulatory role on the mRNA of Xbp1spliced-GFP but not Xbp1unspliced-HA .
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Figure 3 – Pumilio undergoes Ire1 kinase- PUM HD
Pum FL 379 547 777 847 1091 1426 1523 dependent phosphorylation during ER stress a 1 PCMa 2 PCMb 3 V5-H6 a) Diagram of Drosophila Pumilio domains, with S453 S477 T803 S805 S902 possible phosphorylation sites identified in D1D3 Phosphosite (www.phosphosite.org and Phosida 1 a 2 b 3 V5-H6 (http://www.phosida.com). Indicate below in red for S453S477 T803S805 S902 each amino acid residue. Truncated version of the D1 D3 1 a V5-H6 b 3 V5-H6 full-length Pumilio protein were constructed carrying
S453 S477 T803 S805 S902 different domains of Pumilio (Pum FL: Full length protein, D1D3: domain 1 to 3, D1: domain 1, D3: domain 3, PUM-HD: RNA binding Pumilio homology b 0h 2h M 4h 8h c domain. Constructs are tagged with V5 and 6xHis at CIP - + - + - + - + PUM-D1 their C-terminal end. The motifs represented by P 10.0 D1 letters a and b refer to conserved motifs (PCMa and 8.0 ** PCMb) in Drosophila and human PUM proteins. * b) Drosophila S2 stable cell lines expressing the V5 6.0 tagged Pumilio D1 were submitted to ER stress D1 4.0 (DTT 5mM) and samples collected at defined times α-V5 before (0h) and after stress induction (2h, 4h 8h). 2.0
Fold normalized to Phosphorylation of Pumilio domains was α-tubulin Unphosphorylated PUM-D1 0.0 determined based on changes in their 0h 2h 4h electrophoretic mobility through a standard SDS- DTT (5mM) PAGE gel using the Phostag compound [50mM]. Pumilio proteins were detected by immunoblot (IB) d e with a mouse monoclonal V5 antibody. The mobility dPUM-D1 + 4h DTT dPUM-D3 + 4h DTT shift of Pumilio is specific for phosphorylation as it is Apy29 4µC8 λPP S902A reversed by calf intestinal phosphatase treatment CIP 0µM 80µM 0µM CIP 100µM (+CIP). Total protein levels were monitored by P P P D1 D3 tubulin levels. D1 : hyper-phosphorylated forms, D1:
D3 non phosphorylated form of D1. M: protein size D1 markers. α-V5 α-V5 c) Quantification of fold increase of PUM D1P during ER stress, normalized to non phosphorylated form α-tubulin α-tubulin (D1). The statistical significance was calculated by one-way ANOVA coupled with Tukey’s post hoc test (* p<0.05 ; ** p<0,01) f g d) An inhibitor of Ire1 kinase activity impairs the phosphorylation of dPUM-D1. Immunoblot (IB) with MW MW (kDa) (kDa) Phostag phosphorylation analysis of Pumilio D1 P P after ER stress (4h DTT treatment). Cells were D3 D1D3 48- 135- incubated with an inhibitor of Ire1 kinase activity D1D3 (APY29, 80µM) or an inhibitor of IRE1 RNase
100- activity (4µ8c, 100µM). The phosphorylation pattern D3 WB:α-V5 WB -V5 of dPUM-D1 was monitored by IB with anti-V5 35- α antibody. Total protein loading was monitored by in vitro dPUM-D1D3 Phostag in vitro dPUM-D3 Phostag levels of tubulin. CIP (alkaline phosphatase treatment of cell extracts after 4h of DTT treatment. e) Phostag immunoblot (IB) for phosphorylation analysis of Pumilio Domain 3 (dPUM-D3) after ER Pumilio undergoes IRE1 kinase-dependent stress (4h DTT treatment). dPUM-D3 was detected phosphorylation during ER stress by IB using anti-V5 antibody. Total protein loading It is known that Pumilio proteins may change their was monitored by levels of Tubulin. Mutation of site activity according to their phosphorylation state53. Ser902 to Ala (S902A) prevents the DTT induced Furthermore, the observation that the stability of the phosphorylation of dPUM-D3. λPP: λ phosphatase spliced treatment of cell extracts after 4h of DTT treatment. Xbp1 transcript was dependent of the full- f, g) Phostag immunoblot (IB) after in vitro length Pumilio protein (Fig. 2c) indicated that other phosphorylation assay with purified domains of domains of Pumilio, besides the RNA-binding Pumilio (D3 and D1D3) incubated with human domain, are also important for Xbp1 mRNA stability. IRE1-alpha [464-977] in kinase buffer (2mM ATP). 36,42,50 Controls: IRE1 alone, PUM alone (D3 or D1D3); Recent studies indicate that additional regions Inhibition of phosphorylation was observed upon in the N terminus of Pumilio act as repressor treatment with λ-phosphatase or the Apy 29 Ire1 domains, with two specific segments – designated kinase inhibitor. Pumilio proteins were detected with Pumilio conserved motif (PCM) a and b – being anti-mouse V5 antibody . found in both Drosophila and human PUMs (Fig. 3a and Fig. S2a). We screened the Pumilio protein of Pumilio (Fig. 3a). To investigate if Pumilio is sequence for potential serine/threonine phosphorylated during ER stress, we conducted phosphorylation sites using online bioinformatic Phostag analysis using Pumilio proteins tagged tools54 combined with phosphorylation sites with a C-terminal V5 epitope (Fig. 3a). It was reported in other screens (http:// difficult to analyze phosphorylation of full-length www.phosphosite.org/). We found potential Pumilio50, due to its relatively large size of ~180 kDa phosphorylation sites across the different domains Cairrão et al, 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
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(data not shown). Therefore, to facilitate the analysis, we constructed 3 truncated versions of V5-tagged Pumilio (D1= aa 1 to 547; D3= aa 77 to 1091 and D1D3= aa 1 to 1091), which contained a the predicted phosphorylation sites. MW Phostag analysis revealed that, during ER (kDa) stress (4 hr DTT), Pumilio D1 displayed a 6-fold increase in phosphorylated forms, relative to non- 135- 32P ER stress conditions (0 hr DTT) (Fig. 3b). This dPUM-D1D3- phosphorylation pattern was confirmed by 100- treatment with phosphatases (+CIP or + λPP), 75- which reverted Pumilio to a non-phosphorylated hIRE1α- 32P state (Fig. 3b). 63- [464-977] During ER stress and after Ire1 trans- autophosphorylation, the cytosolic kinase- 48- endoribonuclease domain of IRE1 rotates to a dPUM-D1D3 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 may localize to the vicinity of the Ire1 kinase domain. Therefore, we b tested whether Ire1 could phosphorylate Pumilio MW during ER stress, using the inhibitors of Ire1 kinase (kDa) activity (Apy 29)55 and compound #1856 or an inhibitor of IRE1 RNase activity (4µ8C)57. Upon 180- DTT exposure (4 hr), in S2 cells transfected with 135- PUM-D1, Apy 29 attenuated the generation of 100- hIRE1α-3232PP PUM-D1 hyper-phosphorylated forms (Fig. 3d). In 63- [464-977] contrast, 4µ8C did not change the pattern of PUM- 48- D1 phosphorylation, which is expected as 4µ8C is dPUM-D3-3232PP specific for the RNase activity and does not inhibit 35- 57 IRE1 kinase activity . We also observed ER stress 25- induced phosphorylation of PUM-D3, and by dPUM-D3 constructing phosphorylation site mutant versions of PUM-D3 we could identify S902 as a site that is phosphorylated under ER stress conditions (Fig. 3e). Figure 4- hIRE1 KR phosphorylates Pumilio in vitro a and b) In vitro radioactive phosphorylation assays of purified hIRE1 phosphorylates Pumilio and hPUM1 Pumilio dPUM-D1D3 (a) and dPUM-D3 (b) with hIRE1 KR To confirm that IRE1 kinase activity could directly (464-977). Purified proteins were incubated 2h in IRE1 kinase 32 phosphorylate Pumilio, we performed in vitro buffer containing γ-ATP[ P]. Detection of radioactive dPUM- D1D3, dPUM-D3 and auto-phosphorylation of hIRE1 KR are kinase reactions using the hIRE1alpha kinase- denoted by a yellow bullet 32P. Negative controls: BIP as a endoribonuclease (KR) domain57 and purified luminal ER protein non-phosphorylated by IRE1, and proteins versions of Pumilio domains D3 and D1D3. without incubation with IRE1. Specificity of phosphorylation Phostag immunoblot analysis (Fig. 3f,g) showed was monitored by treatment with lambdaPP or incubation with specific inhibitors of IRE1 kinase activity (Apy 29 and specific bands indicating phosphorylation of PUM- compound #18). MW: protein molecular weights in kDa. 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 reflect IRE1 auto-phosphorylation, we used an assays using hIRE1 KR and purified versions of antibody specific for phosphorylated IRE1. While Pumilio and hPum1 protein domains (Fig. 4a,b and we observed phosphorylated monomers, dimers Fig. S2b). The hIRE1 KR protein phosphorylated and oligomers of hIRE1 KR as expected, we could both Pumilio and hPum1 proteins. For negative not detect any signal in the 40 KDa region (Fig. controls, we used a kinase-dead hIRE1 KR as S3b), which corresponds to the phosphorylated enzyme, or BIP (a luminal ER protein that should forms of Pumilio that were detected with the V5 not be a direct target for IRE1 phosphorylation) as antibody (Fig. 4b). These results show that hIRE1 substrate (Fig. S3a). Furthermore, to assure that KR can directly phosphorylate Pumilio. the phosphorylation pattern observed for the PUM proteins in the radioactive assays did not simply
Cairrão et al, 6 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
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a b IRE1 KR wt 100 1/2t: 138,62 min pumRNAi 80 1/2t: 77,01 min pumRNAi+4µ8c DMSO alone + PUM1 B + PUM1 C + PUM1 A
remaining 60 1/2t: 138,6 min RNA ladder 40 Kb ** 20 1.0 - ** 0.5 - % of mRNA ** 0 0.3 - 0 60 120 180 0.15 - Time after ActD (min) dXbp1
PUM1 B= 2-827 fragment Human PUM PUM1 C= 828-1186 fragment Human PUM c PUM1 A= 2-1186 Full human PUM dPUM-FL vs Phosphomutants Figure 5 - Pumilio protects Xbp1spliced mRNA from regulated Ire1-dependent decay FL a) WT S2 cells were compared to cells treated with RNAi 100 1/2t= 138,62 min against Pumilio (pumRNAi) and with or without 4µ8c [100 µM], S902A an inhibitor of IRE1 RNase activity. Following ER stress 80 1/2t= 86,6 min induction (5mM DTT), and in the presence ActinomycinD
remaining 60 ** TetraMut [5µg/ml], total RNA was isolated from cell extracts at different ** spliced 1/2t= 69,3 min periods. The levels Xbp1 -GFP mRNA were normalized to 40 rp49 mRNA levels, and the half-lives calculated by regression analysis of amount of %mRNA remaining against time (min). 20 qRT-PCR analysis was used to determine mRNA levels using
% of mRNA 0 specific primers for gfp and rp49. Averages of % mRNA 0 60 120 180 remaining and corresponding standard deviations were calculated from at least 3 independent experiments. The Time after ActD (min) statistical significance was calculated by one-way ANOVA coupled with Tukey’s post hoc test (* p<0.05 ; ** p<0,01). b) In vitro transcripts of dXbp1 were incubated with purified hIRE1 KR. Human PUM1 FL protects Xbp1 RNA from Ire1 Pumilio protects Xbp1 mRNA from regulated dependent decay, but does not impair Ire1 dependent Xbp1 IRE1-dependent decay splicing. dXBP1 = Drosophila XBP1 T7 transcript, hPUM1 B = We have shown that Pumilio has a protective role [2-827] fragment Human PUM; hPUM1 C=[828-1186] fragment in the stability of Xbp1 mRNA (Fig. 2c). Ire1 can Human PUM, hPUM1 A= [2-1186]; 3P-IRE1 = phosphorylated also mediate RIDD, the selective decay of ER- IRE1. c) qRT-PCR results from Pumilio RNai treated S2 cells bound mRNAs, thereby reducing the load of transfected with plasmid expressing dPUM-FL, a single or a nascent proteins entering the ER in order to be quadruple phosphomutant of dPUM-FL (full length Pumilio folded58. RIDD plays an import role during containing the single mutation S902A; and a quadruple mutant: persistent ER stress19 and in Drosophila cells it can T537A, S540A, S544A, S902A). The Pumilio protective role upon Xbp1spliced-GFP is diminished in the dPUM-FL lead to the complete degradation of even ectopic phosphomutants. The statistical significance was calculated by mRNAs, such as GFP15,59. We therefore one-way ANOVA coupled with Tukey’s post hoc test (* p<0.05 hypothesized that Pumilio may protect Xbp1 mRNA ; ** p<0,01). 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 hPUM (hPUM1-FL) protected the Xbp1 mRNA from treated with DTT, in the absence or presence of the degradation by hIRE1 KR, but did not block or even Ire1 RNase inhibitor 4µ8C. The destabilization of diminish the hairpin motif-dependent splicing of Xbp1-HA-GFP mRNA in cells depleted of Pumilio Xbp1 mRNA. By contrast, hPUM1-B (2-827) did not was reverted upon 4µ8C treatment (Fig. 5a), protect Xbp1 mRNA from decay, consistent with the indicating that Pumilio stabilizes Xbp1 mRNA absence of the HD region necessary for binding to against the RNase activity of Ire1. Xbp1 mRNA in this hPUM variant. Likewise We also conducted mRNA stability assays using in hPUM1-C (828-1186) is incapable of such vitro transcribed Drosophila Xbp1 mRNA incubated protection. Only the full-length hPUM1 protein with hIRE1 KR. Since we could not produce purified contains all the requisite domains for protection of recombinant full-length Pumilio, we used different Xbp1 mRNA, in keeping with the above results (Fig. forms of hPUM1, which were pre-incubated with the 2c). Xbp1 transcript in order to assess their protection against degradation by IRE1 (Fig. 5b). Full-length
Cairrão et al, 7 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint
Pumilio’s protection of Xbp1 mRNA is dependent on its phosphorylation state Having identified S902 as a site of Pumilio phosphorylation under ER stress (Fig. 3e), 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: S902A,T537A, S540A, S544A) in RNAi Pumilio- depleted S2 cells (Fig. 5d). As compared to wild type Pumilio, overexpression of the Xbp1 splicing phosphomutants lead to a decrease in the stability of Xbp1spliced, demonstrating that Pumilio phosphorylation is required to promote its Xbp1unspliced protective effect on Xbp1 transcripts under ER stress. Xbp1spliced DISCUSSION Translation
Our results uncover a novel protective effect of Figure 6- Model for Ire1-controlled post-transcriptional Pumilio on Xbp1 mRNA during ER stress. This regulation of Xbp1 by Pumilio. protective effect depends on the phosphorylation Step 1- In non ER stress conditions, Xbp1unspliced is present in status of Pumilio, which can be mediated by Ire1 the cytosol and PUM may bind to the PRE elements in the Xbp1 3’UTR; Step 2- ER stress conditions lead to Ire1 kinase activity in response to ER stress. We autophosphorylation and Pumilio phosphorylation. propose a model depicted in Fig. 6, which involves Phosphorylated Pumilio protects Xbp1spliced mRNA from Ire1 3 steps. dependent decay, but does not impair Ire1 dependent Xbp1 splicing. Step 3 - Xbp1spliced transcripts can be efficiently In the absence of ER stress, Ire1 is not spliced active and Xbp1 mRNA is not spliced, but Pumilio translated into the Xbp1 transcription factor. unspliced can bind the 3’UTR of Xbp1 mRNA (Fig. 6 - long the protective effect against RIDD lasts. Other 1). Indeed, our RNA pull down experiment (Fig. 1d) studies have shown60, using cultured mouse cells, showed that Pumilio can bind equally well to the an increase in the stability of mRNA Xbp1spliced at a 3’UTR of Xbp1unspliced and Xbp1spliced. Accumulation of misfolded protein in the lumen of the ER causes time of translational repression during “early” phases of ER stress (4 hr treatment with DTT). ER stress and activates Ire1. At this stage, in order spliced to be spliced, Xbp1 mRNA must localize in the These authors suggested that Xbp1 mRNA vicinity of the cytoplasmic domain of Ire1, which could be protected from degradation by an harbors both kinase and endoribonuclease activity. unidentified protein factor. Our results suggest that Upon ER stress, Pumilio is phosphorylated via a Pumilio is such factor, while Ire1 RIDD activity is the mechanism that requires Ire1 kinase activity (Fig. degradation-causing agent. 3). As demonstrated by our in vitro assays (Fig. 4), From our model, we also predict that upon the kinase domain of Ire1 can directly overcoming ER stress conditions, Pumilio phosphorylate Pumilio; however, we cannot phosphorylation levels will be reduced, either due to the activity of phosphatases and/or due to Ire1 exclude at this stage the possibility that other 61 kinases also may contribute to Pumilio dephosphorylation and attenuation . At this stage, phosphorylation. It is known that phosphorylation of Pumilio’s protective role over Xbp1 mRNA may Pum family proteins contributes to their activation subside, presumably making the transcript more and function53. We suggest that in this case, Ire1- vulnerable to the action of the mRNA decay machinery and the ribosome-associated quality dependent phosphorylation of Pumilio causes a 62 conformational change in the mode of interaction control, as previously shown for Hac1 . This regulatory step may avoid an accumulation of between Pumilio and Xbp1 mRNA, such that much spliced of the transcript, though not the canonical stem- Xbp1 under non stress conditions, which could loop structures, is inaccessible to the more be detrimental for cellular homeostasis. promiscuous endoribonuclease activity of Ire1 (Fig. Previous studies have shown that Pumilio 6: step 2). This allows Xbp1 splicing to occur and proteins play a translational repressive role or furthermore permits Ire1 to carry out RIDD while promote degradation of their target mRNAs, which sparing Xbp1spliced mRNA and allowing its more is in contrast with our results. However, another efficient translation (Fig. 6 - 3). study has shown that, contrary to the canonical We do not know if Pumilio also protects repressive activity, PUM1/2 rather promote FOXP1 Xbp1 mRNA from other ribonucleases and for how expression through direct binding to two consensus PREs present in the FOXP1-3'UTR63. Furthermore, Cairrão et al, 8 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint an additional report has identified RNAs that are with HF Buffer or GH Buffer according to the manufacturer's positively regulated by human PUM1 and PUM264 . protocol. All clones were confirmed by sequencing (Stabvida). The full-length Pumilio protein with a C-terminal V5 tag50 was a Our results demonstrate that Ire1 is gift from Aaron Goldstrohm, from which the different truncated responsible for the phosphorylation status of dPUM-D1 , dPUM-D3 and dPUM-D1D3 were constructed. Pumilio during ER stress and that this phosphorylation has implications for Xbp1spliced Transfection and stable cell line establishment protein levels. Recent data in zebrafish implicate Cells were co-transfected with the different plasmids using Pum1 phosphorylation as an initial key step for the Effectene reagent according to manufacture indications sequential activation of cyclinB1 mRNA translation (Qiagen). For stable transfection, cells were selected by during oocyte maturation, although the kinase replacing Schneider's complete Drosophila media with fresh involved remains unidentified 65. In Xenopus, medium supplemented with the appropriate antibiotics (zeocin, puromycin) according to the resistance gene present in the Nemo-like kinase (NLK) – a typical mitogen- transfected plasmids and maintained under selective media activated protein kinase that is activated during an until the formation of resistant clones. early phase of oocyte maturation66 – was shown to directly phosphorylate Pum1 as well Pum2 in vitro. Total RNA and protein extraction It is possible that other kinases may be involved in For RNA stability and Western blot experiments, 4,5×105 S2 Pumilio phosphorylation under non-ER stress cells were seeded in 12-well plates the day before treatments, conditions or specific developmental stages. transfections and protein or RNA extraction. Transfection was In conclusion, our present work uncovers an performed with Effectene (Qiagen) according to the unanticipated mechanism that regulates one of the manufacturer's instructions. Expression of the GFP reporter key branches of the UPR through the action of an under the metallothionein promoter was induced by adding 7 mM CuSO4 to the cell culture media for 3hrs. For the UAS RBP. This involves Ire1-kinase-driven dependent Xbp1 reporters, transcription was induced by co- phosphorylation of Pumilio, which in turn protects transfection of Actin-Gal4 plasmid. For mRNA half-life Ire1-RNase-driven Xbp1spliced against RIDD, thereby measurements, transcription was blocked with actinomycin D coordinating the two major endoribonuclease (5 µg/ml; Sigma-Aldrich) and the cells were harvested at the outputs of IRE1 to enable a more efficient indicated time points. Total RNA was then extracted using intracellular response and ER stress mitigation. Zymo Research quick-RNA miniprep Kit. qPCR and RT-PCR METHODS Primers for qRT–PCR analysis were designed according to Cell culture MIQE guidelines (Minimum Information for Publication of qRT- PCR Experiments) using NCBI primer blast, choosing a Drosophila S2 cells (Schneider, 1972) were cultured at 25 °C melting temperature of 62°C. By using three serial dilutions of in Schneider's Drosophila medium (Invitrogen) supplemented cDNA, primer efficiencies were determined, only primers with with 10% heat inactivated fetal bovine serum (FBS, Invitrogen), efficiencies varying around 100% were used for analysis. 2 mM glutamine (Invitrogen), 100 U/mL penicillin and 100 0,5 μg of total RNA was retro-transcribed using RevertAid H μg/mL streptomycin (Invitrogen). Minus First Strand cDNA Synthesis Kit (Thermo/Fermentas). Each PCR reaction was performed on 1/40 of the cDNA Transgenic flies obtained, using SSoFast EvaGreen Supermix (Bio-Rad) according to the manufacturer's instructions and Bio-Rad Transgenic lines were generated using pUAST-AttB and CFX-96 as detection system. All samples were analyzed in phiC31 integrase-mediated DNA integration (BestGene) that triplicates and from 3 independent biological RNA samples. For allows the insertion of the transgenes in a specific site of the each sample, the levels of mRNAs were normalized using rp49 acceptor fly genome. Clones of mutant eye tissue were as a loading control. Normalized data then were used to generated by the Flp/FRT technique67, with Flipase expression quantify the relative levels of mRNA using the ΔΔCT method. under the control of the eyeless promoter. Drosophila stocks qPCR was carried out using a CFX-96 Biorad instrument. obtained from the Bloomington Stock Center (Indiana Biological replicates represent independently grown and University, Bloomington, IN, USA): GMR-Gal4 (active in the processed cells. Technical replicates represent multiple eye, under the control of the glass multiple reporter); eye-flip measurements of the same biological sample. GMR-Gal4 (promotes recombination in the eye) and Actin5c- Gal4 (ubiquitous expression). Drosophila stocks were Protein analysis maintained at 25°C on standard cornmeal media in an incubator with a 12h light/dark cycle. TAP-PUM stocks were a Total protein lysates were prepared in lysis buffer containing kind gift from André Gerber Laboratory. protease inhibitors. Proteins were size-separated by SDS- PAGE and transferred onto nitrocellulose or PDVF membranes Plasmid construction (Biorad). For Western blot analysis, primary antibodies were anti-GFP (3H9) (1:1000, Chromotek), anti-mouse V5 The 3′UTRs of Xbp1spliced and Xbp1unspliced forms were amplified (1:5000,Invitrogen), anti-mouse HA (1:5000, Covance) and from the cDNA clone GH09250 (Flybase) using specific primer anti-α-tubulin (AA4.3) (1:1000, Developmental Studies pairs, and cloned downstream of the green fluorescent protein Hybridoma Bank). (GFP) coding sequence in the vector pRmHa, containing the metallothionein promoter. The mutations in the Pumilio binding Protein Purifications sites were introduced using oligonucleotide-mediated site- directed mutagenesis and inverse PCR. The UAS-Xbp1-HA- Drosophila Pumilio domains (PUM-D1, PUM-D3, Pum-D1D3) GFP-3'UTR constructs were made by PCR cloning. All cloning fused to V5-6xHis were subcloned into pET 28a and pET26a was performed with the Phusion High-Fidelity PCR Master Mix vectors for protein expression in bacteria. Plasmids were transformed into BL21(DE3) competent cells and recombinant
Cairrão et al, 9 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint proteins were induced with 1mM IPTG at 25ºC. Bacteria were RNAi was performed as described previously 70. Primer pairs lysed in lysis buffer (500 mM NaCl, 50 mM Tris-Cl, pH8.0, tailed with the T7 RNA polymerase promoter were used to 0,1% Triton, Protease inhibitors). Samples were lysed by amplify PCR fragments obtained from cDNA clones. PCR sonication, centrifuged twice at 12,000rpm for 15min, and the products with an average size of 600 bp were then used as cleared supernatant was bound to Ni-NTA Superflow beads templates for dsRNA production with the T7 RiboMAX system (Qiagen) by gravity filtration. Unbound proteins were washed (Promega). For transfection, 15 μg/ml dsRNA was added to S2 with lysis buffer at pH 8.0 supplemented with increasing cells in 12-well plates, during 9 days to silence Drosophila amounts of imidazole (5mM, 40mM, 60mM). Recombinant Pumilio. mRNA depletion was confirmed by RT-PCR before proteins were eluted from beads in lysis buffer with 100-250 further transfections. mM imidazole at pH 8.0. For recombinant proteins retained in inclusion bodies (PUM-D3), solubilization was done by Immunofluorescence and confocal microscopy including 6M urea in lysis buffer. All purified Pumilio recombinant proteins were dialized overnigth at 4°C, against a For Drosophila pupal dissections, white pre-pupae (0h pupa) final buffer (20mM Hepes, 200 mM NaCl, 5% Glicerol, 2mM were collected and maintained at 25°C until the required stage. DTT) and concentrated with appropriate MW cut-off Vivaspin Larval, pupal and adult eyes were dissected in 1xPBS, fixed in columns (Merck). The concentration of purified proteins was 1xPBS + 4% Formaldehyde for 40 minutes at room determined by colorimetric assay (Bio-Rad DC Protein Assay) temperature and washed 3 times with 1xPBS + 0.3% Triton X- and verified by electrophoresis alongside of BSA standards 100. Primary antibodies were incubated in 1xPBS, 1% BSA, with Coomassie staining. 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 Immunoprecipitation of TAP-PUM from flies with appropriate secondary antibodies (from Jackson Immuno- Research Laboratories) for 2 hours at room temperature. Extracts from adult flies heads were prepared as described in Samples were mounted in 80% glycerol in a bridge formed by 48. After immunoprecipitation of TAP-PUM, total RNA was two cover slips to prevent the samples from being crushed extract with Trizol and Xbp1 mRNA levels detected by RT-PCR while analyzed on the confocal microscope (Leica TCS SP5, using specific primers for each Xbp1 transcript. 63X magnification oil immersion lens).
In vitro phosphorylation assays Xbp1 RNA cleavage assay
Ire1 phosphorylation assays were performed by incubation of T7 RNA was generated from pOT2-Xbp1, containing the ORF purified hIre1 KR (3,3µg/µl) with purified Pumilio protein and UTR of Drosophila Xbp1. 1 μg of RNA was digested at (dPumD1, dPumD3, hPumFl) in IRE1 kinase buffer (25mM room temperature by 1 μg of human IRE1α KR recombinant HEPES, pH 7.5, 150mM NaCl, 5mM DTT, 5% glicerol), protein (~0.8 μM) for 45 min in RNA cleavage buffer (HEPES containing either 10µCi of γ-ATP[32P] for radioactive assays or pH7.5 20 mM; K acetate 50 mM; Mg acetate 1 mM; TritonX-100 cold ATP [2mM] for phostag immunoblot assays (in a total 0.05% (v/v)). The total volume of the reaction is 25 μl. The volume of reaction of 20µl). Inhibitors of IRE1 Kinase (Apy 29 digestion was then complemented by an equal volume of formamide and heated up at 70°C for 10 min to denature the [2,5µM/µl] and compound #18 [2,5µM/µl]) were pre-incubated RNA. The mixture was immediately placed on ice for 5 min, and with hIRE1 and all reactions were assembled on ice, prior to then 20 μl was run on 3% agarose gel at 160V for 1 hour at 4°C. addition of ATP and incubation for 2h at 25ºC. Phosphatase The PUM proteins were incubated with the RNA for 40 min on treatment with λPP (NEB) or CIP (NEB) were performed after ice prior to RNA digestion. phosphorylation reactions, for 40 min at 30ºC. Each reaction was stopped by addition of SDS-PAGE loading buffer and run Statistical Methods on pre-maid SDS-PAGE gels (Biorad). The autophosphorylation of hIRE1 KR was confirmed by western All panels data are represented as mean ± SEM, from at least blot using a pSer/24 phospho-specific antibody (Genentech) three independent biological replicates experiments. All and phosphorylated Pumilio proteins were detected using anti- statistical comparisons for significance between control and mouse-V5 antibody (Invitrogen). In the case of kinase experimental groups was calculated using a significance cut off radioactive assays, gels were dried and kinase activity p < 0.05. and denoted by *p< 0.05,**p< 0.01, and ***p< 0.001, visualized by autoradiography. based on two-tailed unpaired t-Student’s test or one-way ANOVA followed by an appropriate post-hoc analysis. Phostag-gels Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc) and online resources (https:// S2 cells were lysed in CIP buffer (100 mM NaCl, 50 mM Tris- astatsa.com/OneWay_Anova_with_TukeyHSD). HCl pH 7.9, 10 mM MgCl2, 1 mM DTT), 1 mM PMSF, 0.1% NP40, protease inhibitor cocktail (Roche), and phosphatase AUTHOR CONTRIBUTIONS inhibitor cocktail (Calbiochem). For CIP treatment phosphatase inhibitor cocktail was omitted, and lysate was incubated 37°C FC designed the study, performed the experiments, analyzed 60 min in 2 units CIP (NEB) per 50 μL reaction containing 50µg data and wrote the manuscript. CS constructed reagents for of total protein. For λ-phosphatase (NEB) treatment, lysates the Phostag assays and contributed to the RNA IP assays. were incubated with 1 unit of phosphatase for 30 min at 30ºC ALT conducted the Xbp1 mRNA in vitro cleavage assays. SM in λ phosphatase buffer. Lysates were cleared by centrifugation provided the purified versions of hPUM1. PD performed the IF and subjected to SDS-PAGE. To detect phosphorylated staining in Fig.2. PD and AA designed and supervised the Pumilio in SDS-PAGE, we used Phos-tag AAL-107 (Wako study, analyzed data and wrote the manuscript. All authors Chemicals GmbH) according to the manufacturer's instruction read and edited the manuscript. 68,69. Western blotting was performed using mouse anti-V5 (1:5000, Invitrogen), followed by the corresponding ACKNOWLEDGMENTS Horseradish Peroxidase (HRP) conjugated secondary antibodies (1:5000, GE Healthcare) and visualized using the We thank Aaron Goldstrohm for plasmids. We thank Yuh Nung ECL Plus Western Blotting detection system (GE Healthcare). Jan, Stefan Luschnig and André Gerber for Pumilio fly stocks. We thank David Ron and Heather Harding for providing the dsRNA treatments purified hIRE1α KEN, 4µ8C and helpful discussions. This work
Cairrão et al, 10 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint received funding by grants from the Fundação para a Ciencia mbc.E15–02–0074 (2015). doi:10.1091/mbc.E15-02- e Tecnologia (PTDC/BIA-BCM/105217/2008; PTDC/SAU- 0074 OBD/104399/2008; PTDC/BEX-BCM/1217/2012; FCT-ANR/ 18. Gardner,B. M. & Walter, P. Unfolded proteins are Ire1- NEU-NMC/0006/2013; PTDC/NEU-NMC/2459/2014; SFRH/ activating ligands that directly induce the unfolded BPD/93893/2013 and DL57/2016 to FC and SFRH/BD/ protein response. Science 333, 1891–1894 (2011). 130817/2017 to CS). The project leading to these results has 19. Ghosh, R. et al. Allosteric Inhibition of the IRE1α also received funding from ‘la Caixa’ Foundation (ID RNase Preserves Cell Viability and Function during 100010434), under the agreement
Cairrão et al, 11 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint
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Cairrão et al, 12 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
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Xbp1-GFP- 3'UTR
110 y = 100e-0.005x R² = 0.94753 90 Uns half-life: 138.6 min y = 100e-0.012x 70 Spl R² = 0.96816
remaining half-life: 57.76 min 50
% mRNA 30
10 0 60 120 180 240
Time (min) after ActD treatment
Figure S1- Xbp1 3’UTR Unspliced and 3’UTRSpliced differ in their stability effect Stability of the GFP reporters fused with Xbp1 3’UTRspliced or 3’UTRunspliced was assessed by quantitative RT-PCR (qRT-PCR), using primers specific for GFP and rp49 mRNAs (control). The levels of mRNA reporter were normalized to those of rp49 mRNA, and averages and standard deviations from three independent experiments are plotted. 3’UTRunspliced shows a higher stability effect of the reporter half-life (2 fold stabilization) relatively to the spliced form.
Cairrão et al, 13 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint
Y717 Y83 S124 S229 T711 T1150 a S19 S75 T112 S209 T514 S709 S806 Y1123 1 PCMa 2 PCMb 3 PUF PUF PUF PUF PUF PUF PUF PUF
Hu PUM1 A (S2-I 1186) 118 kDa
Hu PUM1 B (S2-S827) 82,5kDa
Hu PUM1 C (G828-I 1186) 35,8kDa
b
Ire1α + + - + + + + hPUM1 A - - + + + + + λPP - - - - + - - Ire1a + - + + + + Apy29 - - - - - + - hPUM1 B - + + + + + #18 ------+ λPP - - - - - + BIP + ------Apy29 - - - + - - #18 - - - - + - MW MW (kDa) (kDa) 135- 245-
32P 180- 100- hPUM1 A 135- 32P 75- 100- hPUM1 B
32P 75- 32 63- hIRE1α hIRE1α P [464-977] 63- [464-977]
48- 48- hPUM1 A hPUM1 B
Ire1α + + - + + + + hPUM1 C - - + + + + + λPP - - - - + - - Apy29 - - - - - + - #18 ------+ BIP + ------
MW (kDa)
180-
135-
100- 32P 75- hIRE1α 63- [464-977]
48- 32P hPUM1C 35- [828-1186]
hPUM1 C Figure S2 hIRE1 KR domain phosphorylates hPUM1 in vitro a) Schematic representation of domains and putative phosphorylation sites present in Human PUM1. b) in vitro radioactive kinase assay with γ-[32P]ATP of purified hPUM1 forms incubated with hIRE1 KR domain. Specificity of phosphorylation was monitored by treatment with lambdaPP or incubation with specific inhibitors of IRE1 kinase activity (Apy 29 and #18). Arrows express hPUM1 phosphorylated forms corresponding to the expected MW of each purified protein. Treatment with kinase inhibitors decreased the amount of detected radioactive protein bands.
Cairrão et al, 14 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430300; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Preprint
a hPUM1 C dPUM D3 hIre1α + - - + - - + - - + hIRE1KD - + - - + - - - + - hPUM1 C - - + + + + - - - - dPumD3 ------+ + + + λPP - - - - - + - - - +
MW 245- (kDa) 180- 135- 32P 100- hIRE1α [464-977] 75- 63- 32P 48- hPUM1 C [828-1186]
35- 32 dPUM D3 P 25- [777-1091]
hPUM1 C hPUM1 C b + +
hIRE1α + + + - + + + + ATP - + + + + + + + λPP - + - - - + - - Apy ------+ - #18 ------+ M
MW (kDa) Oligomers
100 – Dimers
75 – monomers 32P 63 – hIRE1α [464-977]
35 –
hPUM1 C (G828-I 1186) 35,8kDa
Figure S3 Validation of in vitro phosphorylation hPUM1-C and dPUM-D3 a) In vitro phosphorylation hPUM1-C domain and dPUM-D3 domain by hIRE1 KR using the control kinase-dead Ire1 (hIRE1KD, D688N). Assays were conducted as described before. Arrows denote phosphorylated forms of hPUM-C (Blue arrow) and dPUM- D3 purified proteins (red arrow). Autophosphorylation of hIRE KR is denoted by a black arrow. b) Phostag immunoblot of phosphorylation of hPUM-C with an antibody specific for phosphorylated IRE1(anti-rabbit Phospho- Ire1). Specificity of phosphorylation was monitored by treatment with λPP or incubation with specific inhibitors of IRE1 kinase activity (Apy 29 and compound #18) and a reaction lacking ATP (first lane). The phosphorylation state of hIRE1 KR is decreased in control reactions as expected comparatively to the reactions containing hIRE1+ATP or hIRE1 + hPUM-C + ATP. An additional control was made by conducting a kinase assay of hPUM-C without incubation with hIRE1 KR domain. M – Protein molecular weight marker lane.
Cairrão et al, 15