In the nematode Caenorhabditis elegans, different small RNA-dependent gene silencing mechanisms act in the germline to initiate transgenerational gene silencing. Piwi-interacting RNAs (piRNAs) can initiate transposon and gene silencing by acting upstream of endogenous short interfering RNAs (siRNAs), which engage a nuclear RNA interference (RNAi) pathway to trigger transcriptional gene silencing. Once gene silencing has been established, it can be stably maintained over multiple generations without the requirement of the initial trigger and is also referred to as RNAe or paramutation. This heritable silencing depends on the integrity of the nuclear RNAi pathway. However, the exact mechanism by which silencing is maintained across generations is not understood. Here we demonstrate that silencing of piRNA targets involves the production of two distinct classes of small RNAs with different genetic requirements. The first class, secondary siRNAs, are localized close to the direct target site for piRNAs. Nuclear import of the secondary siRNAs by the Argonaute HRDE-1 leads to the production of a distinct class of small RNAs that map throughout the transcript, which we term tertiary siRNAs. Both classes of small RNAs are necessary for full repression of the target gene and can be maintained independently of the initial piRNA trigger. Consistently, we observed a form of paramutation associated with tertiary siRNAs. Once paramutated, a tertiary siRNA generating allele confers dominant silencing in the progeny regardless of its own transmission, suggesting germline-transmitted siRNAs are sufficient for multigenerational silencing. This work uncovers a multi-step siRNA amplification pathway that promotes germline integrity via epigenetic silencing of endogenous and invading genetic elements. In addition, the same pathway can be engaged in environmentally induced heritable gene silencing and could therefore promote the inheritance of acquired traits.
Although excessive lipid accumulation is a hallmark of obesity-related pathologies, some lipids are beneficial. Oleic acid (OA), the most abundant monounsaturated fatty acid (FA), promotes health and longevity. Here, we show that OA benefits Caenorhabditis elegans by activating the endoplasmic reticulum (ER)-resident transcription factor SKN-1A (Nrf1/NFE2L1) in a lipid homeostasis response. SKN-1A/Nrf1 is cleared from the ER by the ER-associated degradation (ERAD) machinery and stabilized when proteasome activity is low and canonically maintains proteasome homeostasis. Unexpectedly, OA increases nuclear SKN-1A levels independently of proteasome activity, through lipid droplet-dependent enhancement of ERAD. In turn, SKN-1A reduces steatosis by reshaping the lipid metabolism transcriptome and mediates longevity from OA provided through endogenous accumulation, reduced H3K4 trimethylation, or dietary supplementation. Our findings reveal an unexpected mechanism of FA signal transduction, as well as a lipid homeostasis pathway that provides strategies for opposing steatosis and aging, and may mediate some benefits of the OA-rich Mediterranean diet.
Lipid droplet
Endoplasmic-reticulum-associated protein degradation
Regulation of gene expression by microRNAs (miRNAs) is essential for normal development, but the roles of miRNAs in the physiology of adult animals are poorly understood. We have isolated a conditional allele of DGCR8 / pash-1 , which allows reversible and rapid inactivation of miRNA synthesis in vivo in Caenorhabditis elegans . This is a powerful new tool that allows dissection of post-developmental miRNA functions. We demonstrate that continuous synthesis of miRNAs is dispensable for cellular viability but critical for the physiology of adult animals. Loss of miRNA synthesis in the adult reduces lifespan and results in rapid aging. The insulin/IGF-1 signaling pathway is a critical determinant of lifespan, and is modulated by miRNAs. We find that although miRNA expression is required for some mechanisms of lifespan extension, it is not essential for the longevity of animals lacking insulin/IGF-1 signaling. Further, misregulated insulin/IGF-1 signaling cannot account for the reduced lifespan caused by disruption of miRNA synthesis. We show that miRNAs act in parallel with insulin/IGF-1 signaling to regulate a shared set of downstream genes important for physiological processes that determine lifespan. We conclude that coordinated transcriptional and post-transcriptional regulation of gene expression promotes longevity.
Abstract Although excessive lipid accumulation is a hallmark of obesity-related pathologies, some lipids are beneficial. Oleic acid (OA), the most abundant monounsaturated fatty acid (FA), promotes health and longevity. Here we show that OA benefits C. elegans by activating the endoplasmic reticulum (ER)-resident transcription factor SKN-1A (Nrf1/NFE2L1) in a lipid homeostasis response. SKN-1A/Nrf1 is cleared from the ER by the ER-associated degradation (ERAD) machinery and stabilized when proteasome activity is low, and canonically maintains proteasome homeostasis. Unexpectedly, OA increases nuclear SKN-1A levels independently of proteasome activity, through lipid droplet (LD)-mediated enhancement of ERAD. In turn, SKN-1A reduces steatosis by reshaping the lipid metabolism transcriptome, and mediates longevity from OA provided through endogenous accumulation, reduced H3K4 trimethylation, or dietary supplementation. Our findings reveal a surprising mechanism of FA signal transduction, and a lipid homeostasis pathway that provides strategies for opposing steatosis and aging, and may mediate benefits of the OA-rich Mediterranean diet.
Endoplasmic-reticulum-associated protein degradation
Unfolded protein responses (UPRs) safeguard cellular function during proteotoxic stress and aging. In a previous paper (Lehrbach and Ruvkun, 2016) we showed that the ER-associated SKN-1A/Nrf1 transcription factor activates proteasome subunit expression in response to proteasome dysfunction, but it was not established whether SKN-1A/Nrf1 adjusts proteasome capacity in response to other proteotoxic insults. Here, we reveal that misfolded endogenous proteins and the human amyloid beta peptide trigger activation of proteasome subunit expression by SKN-1A/Nrf1. SKN-1A activation is protective against age-dependent defects caused by accumulation of misfolded and aggregation-prone proteins. In a C. elegans Alzheimer's disease model, SKN-1A/Nrf1 slows accumulation of the amyloid beta peptide and delays adult-onset cellular dysfunction. Our results indicate that SKN-1A surveys cellular protein folding and adjusts proteasome capacity to meet the demands of protein quality control pathways, revealing a new arm of the cytosolic UPR. This regulatory axis is critical for healthy aging and may be a target for therapeutic modulation of human aging and age-related disease.
NRF1
Proteostasis
Endoplasmic-reticulum-associated protein degradation
Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Proteasomes are essential for protein homeostasis in eukaryotes. To preserve cellular function, transcription of proteasome subunit genes is induced in response to proteasome dysfunction caused by pathogen attacks or proteasome inhibitor drugs. In Caenorhabditis elegans, this response requires SKN-1, a transcription factor related to mammalian Nrf1/2. Here, we use comprehensive genetic analyses to identify the pathway required for C. elegans to detect proteasome dysfunction and activate SKN-1. Genes required for SKN-1 activation encode regulators of ER traffic, a peptide N-glycanase, and DDI-1, a conserved aspartic protease. DDI-1 expression is induced by proteasome dysfunction, and we show that DDI-1 is required to cleave and activate an ER-associated isoform of SKN-1. Mammalian Nrf1 is also ER-associated and subject to proteolytic cleavage, suggesting a conserved mechanism of proteasome surveillance. Targeting mammalian DDI1 protease could mitigate effects of proteasome dysfunction in aging and protein aggregation disorders, or increase effectiveness of proteasome inhibitor cancer chemotherapies. https://doi.org/10.7554/eLife.17721.001 eLife digest Proteins perform many important roles in cells, but these molecules can become toxic if they are damaged or are no longer needed. A molecular machine called the proteasome destroys 'unwanted' proteins in animal and other eukaryotic cells. If the proteasome stops working properly, unwanted proteins start to accumulate and cells respond by increasing the activity of genes that make proteasomes. A protein called SKN-1 is involved in this response and activates the genes that encode proteasome proteins, but it is not understood how SKN-1 "senses" that proteasomes are not working properly. Here, Lehrbach and Ruvkun used a roundworm called Caenorhabditis elegans to search for new genes that activate SKN-1 when the proteasome's activity is impaired. The roundworms were genetically engineered to produce a fluorescent protein that indicates when a particular gene needed to make proteasomes is active. Lehrbach and Ruvkun identified some roundworms with mutations that cause the levels of fluorescence to be lower, indicating that SKN-1 was less active in these animals. Further experiments showed that some of these mutations are in genes that encode enzymes called DDI-1 and PNG-1. DDI-1 is able to cut certain proteins, while PNG-1 can remove sugars that are attached to proteins. Therefore, it is likely that these enzymes directly interact with SKN-1 and alter it to activate the genes that produce the proteasome. More work is now needed to understand the details of how modifying SKN-1 changes its activity in cells. In the future, drugs that target DDI-1 or PNG-1 might be used to treat diseases in which proteasome activity is too high or low, including certain cancers and neurodegenerative diseases. https://doi.org/10.7554/eLife.17721.002 Introduction The proteasome is a multi-protein complex responsible for the majority of protein degradation in eukaryotic cells (Tomko and Hochstrasser, 2013). The essential function of the proteasome, and its highly conserved structure and mechanism of proteolysis renders it an attractive target for bacteria and other competitors. Production of small molecule inhibitors and protein virulence factors that target the proteasome by some bacteria and fungi exploits this vulnerability to gain a growth advantage (Fenteany et al., 1995; Groll et al., 2008; Meng et al., 1999). In addition, environmental stresses antagonize the proteasome by causing accumulation of unfolded and aggregated proteins that can form a non-productive inhibitory interaction with proteasomes (Ayyadevara et al., 2015; Deriziotis et al., 2011; Kristiansen et al., 2007; Snyder et al., 2003). Human diseases in which proteasome dysfunction is implicated highlight the importance of maintaining proteasome function in the face of these challenges (Ciechanover and Kwon, 2015; Paul, 2008; Tomko and Hochstrasser, 2013), and it follows that animal cells possess mechanisms to monitor and defend proteasome function. A conserved response to proteasome disruption is the transcriptional up-regulation of proteasome subunit genes (Fleming, 2002; Meiners et al., 2003; Wójcik and DeMartino, 2002). In mammalian cells members of the Cap' n' Collar basic leucine zipper (CnC-bZip) family of stress responsive transcription factors mediate this transcriptional response. Two CnC-bZip franscription factors, Nrf1/NFE2L1 and Nrf2, have similar DNA-binding domains and may regulate an overlapping set of downstream targets. However, only Nrf1 is required for upregulation of proteasome subunits following proteasome disruption, whereas Nrf2 may activate proteasome expression under other circumstances (Arlt et al., 2009; Radhakrishnan et al., 2010; Steffen et al., 2010). The events leading to Nrf1 activation in response to proteasome disruption are complex. In vitro analyses in human and mouse cells indicate that Nrf1 is an endoplasmic reticulum (ER) membrane associated glycoprotein that is constitutively targeted for proteasomal degradation by the ER-associated degradation (ERAD) pathway. Upon proteasome inhibition Nrf1 is stabilized, undergoes deglycosylation and proteolytic cleavage, and localizes to the nucleus (Radhakrishnan et al., 2014; Sha and Goldberg, 2014; Wang, 2006; Zhang and Hayes, 2013; Zhang et al., 2015, 2007, 2014). How processing of Nrf1 is orchestrated, and its significance in responses to proteasome disruption in vivo are not understood. Upon proteasome disruption, C. elegans induces transcription of proteasome subunit, detoxification, and immune response genes, and animals alter their behavior to avoid their bacterial food source (Li et al., 2011; Melo and Ruvkun, 2012). The transcriptional response to proteasome disruption involves skn-1, which encodes multiple isoforms of a transcription factor with similarities to both Nrf1 and Nrf2 (Blackwell et al., 2015; Li et al., 2011). skn-1 was originally identified for its essential role in embryonic development (Bowerman et al., 1992), but is also required after these early stages for stress responses in a manner analogous to mammalian Nrf1/2 (An and Blackwell, 2003; Oliveira et al., 2009; Paek et al., 2012). SKN-1 binds to the promoters of proteasome subunit genes and mediates their upregulation in response to proteasome disruption, and is required for survival of a mutant with attenuated proteasome function (Keith et al., 2016; Li et al., 2011; Niu et al., 2011). The molecular mechanism that links SKN-1 activation to the detection of proteasome dysfunction has not been established. Here, we use genetic analysis to uncover the mechanism that couples detection of proteasome defects to these transcriptional responses in C. elegans. We find that an ER-associated isoform of SKN-1 (SKN-1A), is essential for this response. Our genetic data show that the ER-association of this transcription factor normally targets it for poteasomal degradation via ERAD, but is also required for its correct post-translational processing and activation during proteasome dysfunction. After ER-trafficking, our data argues that the PNG-1 peptide N-glycanase removes glycosylation modifications that occur in the ER, and then the DDI-1 aspartic protease cleaves SKN-1A. Each of these steps in SKN-1A processing is essential for the normal response to proteasomal dysfunction. This pathway is essential for compensation of proteasome function under conditions that partially disrupt the proteasome; when compensation is disabled, mild inhibition of the proteasome causes lethal arrest of development. Thus we reveal a vital mechanism of proteasome surveillance and homeostasis in animals. Results The aspartic protease DDI-1 and ERAD factors are required for transcriptional responses to proteasome disruption The proteasome subunit gene rpt-3 is upregulated in a skn-1-dependent manner in response to proteasome disruption (Li et al., 2011). We generated a chromosomally integrated transcriptional reporter in which the rpt-3 promoter drives expression of GFP (rpt-3::gfp). This reporter gene is upregulated in response to drugs such as bortezomib or mutations that cause proteasome dysfunction. To identify the genetic pathways that sense proteasome dysfunction and trigger the activation of SKN-1, we took advantage of a regulatory allele affecting the pbs-5 locus. pbs-5 encodes the C. elegans ortholog of the beta 5 subunit of the 20S proteasome. The pbs-5(mg502) mutation causes constitutive skn-1-dependent activation of rpt-3::gfp expression, but does not otherwise alter fertility or viability (Figure 1—figure supplement 1). Following EMS mutagenesis, we isolated a collection of recessive mutations that suppress the activation of rpt-3::gfp caused by pbs-5(mg502), and identified the causative mutations by whole genome sequencing (Table 1). The collection includes multiple alleles of genes encoding factors required for ERAD. In ERAD, misfolded glycoproteins are retrotranslocated from the ER lumen to the cytoplasm, where they are degraded by the proteasome (Smith et al., 2011). We isolated 3 alleles of sel-1, a gene that encodes the C. elegans orthologue of HRD3/SEL1. HRD3/SEL1 localizes to the ER membrane and recognizes ERAD substrates in the ER (Carvalho et al., 2006; Denic et al., 2006; Gauss et al., 2006), and a single allele of sel-9, which encodes the C. elegans orthologue of TMED2/EMP24, which is also ER-localized and implicated in ER quality control (Copic et al., 2009; Wen and Greenwald, 1999). We also found mutations in png-1, which encodes the C. elegans orthologue of PNG1/NGLY1. After ERAD substrates have been retrotranslocated to the cytoplasm, PNG1/NGLY1 removes N-linked glycans to allow their degradation by the proteasome (Kim et al., 2006; Suzuki et al., 2016). Most strikingly, we isolated six alleles of C01G5.6 (hereafter ddi-1), which encodes the C. elegans orthologue of DDI1 (DNA damage inducible 1). DDI-1 is an aspartic protease, highly conserved throughout eukaryotes (Sirkis et al., 2006). DD1's function is poorly understood, but it has been implicated in regulation of proteasome function and protein secretion (Kaplun et al., 2005; White et al., 2011). Table 1 EMS-induced mutations that disrupt rpt-3::gfp activation. https://doi.org/10.7554/eLife.17721.003 AlleleAffected geneEffectHomologuesmg563C01G5.6L245FDDI1. Aspartic protease.mg555C01G5.6C277Smg544C01G5.6G293Rmg542C01G5.6R350STOPmg543C01G5.6M244Img557C01G5.6L334Fmg565sel-1G594EHRD3/SEL1. ER membrane protein, required for ERAD substrate recognition.mg567sel-1A522Tmg547sel-1splice sitemg550sel-9S140FEMP24/TMED2. ER membrane protein.mg561png-1G498RPNG1/NJLY1. Peptide N-glycanase. Removes N-linked glycans during ERAD.mg564png-1splice site We examined activation of rpt-3::gfp in ERAD and ddi-1 mutant animals following disruption of proteasome function by RNAi of the essential proteasome subunit rpt-5. rpt-5(RNAi) caused larval arrest confirming that all genotypes are similarly susceptible to RNAi. While rpt-5(RNAi) causes robust activation of rpt-3::gfp in wild-type animals, mutants lacking ERAD factors or ddi-1 failed to fully activate rpt-3::gfp (Figure 1a). The requirement for sel-11, which encodes an ER-resident ubiquitin ligase required for ERAD (Smith et al., 2011), supports a general requirement for ERAD in activation of rpt-3::gfp expression. These genes are also required for upregulation of rpt-3::gfp following proteasome disruption by bortezomib (data not shown). Figure 1 with 1 supplement see all Download asset Open asset ER-associated degradation factors and the aspartic protease DDI-1 are required for responses to proteasome disruption. (a) rpt-3::gfp expression following disruption of proteasome function by rpt-5(RNAi) in various mutant backgrounds. Scale bars 100 µm. (b) Table showing growth vs. arrest phenotypes of various mutants in the presence of bortezomib or upon rpn-10 RNAi. For RNAi experiments, L1 animals were incubated for 3 days on indicated RNAi plates, and scored for developmental arrest (+; normal development, Lva; larval arrest). For bortezomib experiments, ~15 L1 animals were incubated for 4 days in liquid cultures containing varying concentrations of bortezomib, and scored for developmental progression. The number (n) of replicate bortezomib experiments performed for each genotype is shown on the right. Each colored rectangle is divided into equal parts to show results from each replicate. https://doi.org/10.7554/eLife.17721.004 Unlike the wild type, mutants defective in ERAD, or lacking DDI-1, arrested or delayed larval development in the presence of low doses of bortezomib (Figure 1b). We analyzed independently derived alleles of png-1 and ddi-1, indicating that hypersensitivity to proteasome inhibition is unlikely to be a consequence of linked background mutations. png-1 animals consistently showed the most severe defect, and were unable to grow in the presence of very low concentrations of bortezomib. Consistent with their drug sensitivity, mild disruption of proteasome function by RNAi-mediated depletion of the non-essential proteasome subunit RPN-10 causes a synthetic larval lethal phenotype in animals mutant for png-1. The bortezomib sensitivity of ddi-1; sel-11 double mutants was not enhanced compared to that of ddi-1 single mutants, suggesting that ddi-1 and ERAD factors act in the same genetic pathway. We conclude that ERAD and DDI-1 are required for transcriptional upregulation of proteasome subunits and survival during proteasome dysfunction. Given the defective activation of rpt-3::gfp, a direct target of SKN-1, it is likely that upon proteasome disruption, these factors are required to activate SKN-1. SKN-1A, an ER-associated isoform of SKN-1 mediates proteasome homeostasis The skn-1 gene generates 3 protein isoforms using alternative transcription start sites (SKN-1A, SKN-1B, and SKN-1C; Figure 2a). The isoforms share an identical C-terminal DNA binding domain, but differ in their N-termini. skn-1(RNAi) targets sequences common to all three transcripts. Both skn-1(RNAi), and the skn-1(zu67) mutation cause defective responses to proteasome dysfunction (NL unpublished). skn-1(zu67) is a nonsense allele affecting an exon shared by SKN-1A and SKN-1C, but that does not affect SKN-1B, suggesting SKN-1A and/or SKN-1C are required. SKN-1C encodes a 61 kD protein expressed specifically in the intestine, and SKN-1A encodes a 71 kD protein expressed in most tissues (An and Blackwell, 2003; Bishop and Guarente, 2007; Staab et al., 2014). SKN-1A differs from SKN-1C solely by the presence of 90 additional amino acids at the N-terminus that includes a predicted transmembrane domain (Figure 2b), and SKN-1A has been found to associate with the ER (Glover-Cutter et al., 2013). Figure 2 with 1 supplement see all Download asset Open asset SKN-1A, a transmembrane-domain-containing isoform of SKN-1 mediates transcriptional responses to proteasome disruption. (a,b) Schematic of the (a) skn-1 locus and (b) SKN-1A protein. In (a), the CRISPR-induced skn-1a-specific mutations are indicated. (c,d) rpt-3::gfp induction in wild type and isoform-specific skn-1 mutants in (c) the pbs-5(mg502) mutant, or (d) rpt-5(RNAi). Scale bars 100 µm. (e) Developmental arrest of isoform-specific skn-1 mutants exposed to mild proteasome disruption by rpn-10(RNAi) but not on control RNAi. Scale bar 1 mm. (f) Expression and localization of functional SKN-1A::GFP fusion protein after proteasome disruption by rpt-5(RNAi). Apparent GFP signal in control treated animals is background auto-fluorescence. Scale bar 10 µm. (g) No developmental arrest of skn-1 mutants carrying an isoform-specific skn-1a::gfp transgene, and exposed to mild proteasome disruption by rpn-10(RNAi). Scale bar 1 mm. (h) Table showing growth vs. arrest phenotypes of skn-1a mutants in in the presence of bortezomib. All skn-1a alleles are identical in their effect on skn-1a coding sequence (G2STOP). Experiments performed identically to those shown in Figure 1b, and data for the wild type from Figure 1 are shown for reference. https://doi.org/10.7554/eLife.17721.006 We used CRISPR/Cas9 to generate an isoform-specific genetic disruption of SKN-1A, by introducing premature stop codons to the skn-1a specific exons of the skn-1 locus (hereafter referred to as skn-1a mutants). Homozygous skn-1a mutant animals are viable, and under standard conditions show a growth rate and fertility indistinguishable from the wild type. However, skn-1a mutant animals fail to activate rpt-3::gfp in the pbs-5(mg502) mutant background, or upon RNAi of essential proteasome subunit genes, or exposure to bortezomib (Figure 2c,d, data not shown). We note that in these experiments skn-1a mutants failed to activate rpt-3::gfp in all tissues, including the intestine, where SKN-1C is expressed. Consistent with the failure to upregulate rpt-3::gfp, skn-1a mutants show larval lethality when proteasome dysfunction is induced by rpn-10(RNAi) or treatment with a low dose of bortezomib (Figure 2e,h). These skn-1a mutations specifically affect SKN-1A, but leave SKN-1B and SKN-1C unaltered, indicating that SKN-1A is essential for normal responses to proteasome disruption and in the absence of SKN-1A, the other isoforms are not sufficient. A number of stimuli that trigger stabilization and nuclear accumulation of a transgenic SKN-1C::GFP fusion protein are known, but relatively little is known about whether these stimuli also affect SKN-1A (Blackwell et al., 2015). We used miniMos transgenesis (Frøkjær-Jensen et al., 2014) to generate genomically integrated single-copy transgenes that expresses C-terminally GFP-tagged full length SKN-1A (SKN-1A::GFP), and a second C-terminally GFP tagged truncated SKN-1A that lacks the DNA binding domain (SKN-1A[∆DBD]::GFP). When driven by the ubiquitously active rpl-28 promoter, we did not observe accumulation of SKN-1A::GFP or SKN-1A[∆DBD]::GFP, consistent with constitutive degradation of these fusion proteins. Upon disruption of proteasome function, we observed stabilization and nuclear localization of SKN-1A::GFP and SKN-1A[∆DBD]::GFP in many tissues (Figure 2f, Figure 2—figure supplement 1a). For unknown reasons, the SKN-1A[∆DBD]::GFP transgene accumulated to higher levels than the full length transgene (Figure 2—figure supplement 1b). We generated similar transgenes to express tagged full length and truncated SKN-1C, but did not observe any effect of proteasome disruption (data not shown). These data suggest proteasome dysfunction triggers activation of SKN-1A, but not SKN-1C. We introduced the SKN-1A::GFP transgene into the skn-1a(mg570) and skn-1(zu67) mutant backgrounds. SKN-1A::GFP rescued the maternal effect lethal phenotype of skn-1(zu67). SKN-1A::GFP also restored wild-type resistance to proteasome disruption, as assayed by growth on rpn-10(RNAi) (Figure 3), or growth in the presence of low concentrations of bortezomib (data not shown). This indicates that the SKN-1A::GFP fusion protein is functional, and that SKN-1A::GFP is sufficient for normal responses to proteasome dysfunction even in the absence of SKN-1C (which is disrupted by the zu67 allele). As such, the transmembrane-domain-bearing SKN-1A isoform is necessary and sufficient for responses to proteasome dysfunction. Figure 3 Download asset Open asset ERAD is required for constitutive SKN-1A degradation, and for activation of SKN-1A upon proteasome disruption. (a) Western blot showing expression and post-translational processing of SKN-1A[∆DBD]::GFP in ERAD mutant animals, treated with either solvent control (DMSO) or 5 ug/ml bortezomib. SKN-1A[∆DBD]::GFP is only detected in wild-type animals upon bortezomib exposure; a major band at ~70 kD and a minor band at ~90 kD are detected. In ERAD defective mutants, the ~90 kD band is strongly detected under all conditions, and the ~70 kD band appears only following bortezomib treatment. Actin is used as a loading control. (b) Expression and localization of SKN-1A::GFP in wild type and sel-1 and sel-11 ERAD defective mutants after proteasome disruption by rpt-5(RNAi). In ERAD defective mutants, SKN-1A::GFP fails to localize to the nucleus. Scale bar 10 µm. (c) Expression and localization of SKN-1A::GFP in wild type and png-1 mutants after proteasome disruption by rpt-5(RNAi). In png-1 mutants, SKN-1A::GFP is able to localize to the nucleus, although at reduced levels compared to the wild-type. Scale bar 10 µm. https://doi.org/10.7554/eLife.17721.008 Mutation of ddi-1 does not enhance the sensitivity of skn-1a mutants to bortezomib, suggesting that DDI-1 acts through SKN-1A to promote resistance to proteasome inhibitors (Figure 2h). Additionally removing SEL-11 weakly enhanced the bortezomib sensitivity of ddi-1 skn-1a double mutants, and also caused occasional growth defects even in the absence of proteasome disruption, suggesting that ERAD promotes resistance to proteasome inhibitors largely, but not solely, through regulation of SKN-1A. We examined how ERAD factors regulate SKN-1A using the SKN-1A::GFP transgenes. sel-1 and sel-11 mutants accumulate high levels of SKN-1A[∆DBD]::GFP even in the absence of proteasome inhibitors, showing that SKN-1A is constitutively targeted for proteasomal degradation via ERAD (Figure 3a). Upon proteasome disruption, sel-1 and sel-11 mutants show defects in SKN-1A::GFP nuclear localization consistent with defective release from the ER (Figure 3b). Following proteasome disruption in png-1 mutants SKN-1A::GFP localizes to the nucleus, indicating PNG-1 acts downstream of release from the ER (Figure 3b). Lower levels of SKN-1A::GFP accumulate in the nuclei of png-1 mutants than in the wild type, but this mild effect is unlikely to fully account for the severely defective responses to proteasome inhibition in png-1 mutant animals, suggesting retention of glycosylation modifications normally removed by PNG-1 likely disrupts SKN-1A's nuclear function. These data suggest that activation of ER-associated and N-glycosylated SKN-1A is required for responses to proteasome dysfunction. DDI-1 aspartic protease localizes to both nucleus and cytoplasm, and is upregulated upon proteasome disruption To examine the expression and subcellular localization of the DDI-1 protease, we used miniMos to generate a single copy integrated transgene expressing full length DDI-1 fused to GFP at the N-terminus, under the control of the ddi-1 promoter. The GFP::DDI-1 fusion protein is expressed in most tissues and shows diffuse cytoplasmic and nuclear localization under control conditions, and can rescue a ddi-1 mutant (see below). Following disruption of proteasome function by rpt-5(RNAi), GFP::DDI-1 expression is dramatically induced, and GFP::DDI-1 is enriched in nuclei (Figure 4a). We used CRISPR/Cas9 to modify the ddi-1 locus to incorporate an HA epitope tag near the N-terminus of endogenous DDI-1. Following bortezomib treatment of ddi-1(mg573[HA::ddi-1]) animals, we observed strong upregulation (greater than 10-fold, based on blotting of diluted samples) of the HA-tagged endogenous DDI-1 (Figure 4b). The ddi-1 promoter contains a SKN-1 binding site (Niu et al., 2011). Upregulation of GFP::DDI-1 by rpt-5(RNAi) is greatly reduced in skn-1a(mg570) mutants (Figure 4c), suggesting that DDI-1 upregulation is mostly mediated by SKN-1A. The remaining weaker ddi-1 upregulation in the skn-1a mutant may represent a second skn-1a-independent mechanism that couples DDI-1 levels to proteasome function. Figure 4 Download asset Open asset DD1-1 is upregulated upon proteasome disruption. (a) A functional GFP::DDI-1 fusion protein is strongly induced and localizes to the nucleus upon proteasome disruption by rpt-5(RNAi). Scale bar 20 µm. (b) Western blot showing induction of HA-tagged endogenous DDI-1 upon proteasome disruption by bortezomib. Actin is used as a loading control. (c) Induction of GFP::DDI-1 upon proteasome disruption by rpt-5(RNAi) is lost in skn-1a mutants. Scale bar 20 µm. https://doi.org/10.7554/eLife.17721.009 DDI-1 is required for proteolytic cleavage of SKN-1A downstream of ER trafficking The EMS-induced ddi-1 missense alleles that cause failure to activate rpt-3::gfp are clustered within the aspartic protease domain of DDI-1, and affect conserved residues that are thought to form the substrate-binding pocket of the enzyme (Figure 5a,b), suggesting that the protease activity of DDI-1 is required (Sirkis et al., 2006). We used CRISPR/Cas9 mutagenesis to generate a protease dead mutant containing two amino acid substitutions at conserved residues of the catalytic motif, including the aspartic acid residue that forms the active site (D261N, G263A). We additionally isolated a CRISPR-induced deletion that deletes most of the aspartic protease domain and introduces a frameshift, which we presume to be a null allele. Both mutations cause a similar, strong defect in rpt-3::gfp activation by the pbs-5(mg502) mutant, or upon proteasome RNAi, and cause a similar sensitivity to bortezomib (Figure 5c, data not shown). Figure 5 with 1 supplement see all Download asset Open asset The DDI-1 aspartic protease is required for proteolytic activation of SKN-1A. (a) Schematic of the DDI-1 protein showing residues affected by EMS-induced loss of function alleles. (b) Multiple alignment of the aspartic protease domain of DDI-1. Red text indicates residues affected by mutations that disrupt rpt-3::gfp activation, blue text indicates the DS/TGAQ catalytic motif. (c) rpt-3::gfp induction in the pbs-5(mg502) mutant background (left panel), compared to animals carrying a ddi-1 deletion (middle panel), or a point mutation affecting the catalytic motif (right panel). Scale bars 100 µm. (d) Localization of SKN-1A::GFP in wild-type and ddi-1 mutant animals following disruption of proteasome function by rpt-5(RNAi). SKN-1A::GFP nuclear localization is intact in the ddi-1 mutant. In some gut cells of ddi-1 mutant animals SKN-1A::GFP localizes to puncta that are not detected in the wild type. Scale bars 10 µm. (e) Localization of SKN-1A[∆DBD]::GFP in gut nuclei of wild-type and ddi-1 mutant animals. Nuclear foci of SKN-1A[∆DBD]::GFP are found in ddi-1 mutants, but not wild type. (f) Western blot showing expression and processing of SKN-1A[∆DBD]::GFP in ddi-1 mutant animals, treated with either solvent control (DMSO) or 5 ug/ml bortezomib, and blotted for GFP. In the ddi-1 mutant animals, the major band detected is ~30 kD larger than in the wild type. (g) Western blot showing expression and processing of HA::SKN-1A:GFP in ddi-1 mutants animals, treated with either solvent control (DMSO) or 5 ug/ml bortezomib, and blotted for HA. In the wild type, a ~20 kD band is detected in animals exposed to bortezomib. In ddi-1 mutants this low molecular weight fragment is absent, and a ~110 kD band is detected. In (f) and (g) ddi-1 mutations were ddi-1(mg571)[deletion] or ddi-1(mg572)[active site] and actin is used as a loading control. https://doi.org/10.7554/eLife.17721.010 S. cervisiae Ddi1 contains an N-terminal ubiquitin-like (UBL) domain and a C-terminal ubiquitin-associated (UBA) domain, but these domains are not detected by standard protein sequence comparisons with C. elegans DDI-1. To address the possibility that UBL or UBA domains with highly divergent sequence may be present in DDI-1, we generated N-terminally truncated (∆N), and C-terminally truncated (∆C) gfp::ddi-1 transgenes. We tested their ability to rescue the bortezomib sensitivity phenotype of ddi-1(mg571) alongside wild-type gfp::ddi-1 and an aspartic protease active site (D261N) mutant. The active site mutation abolished rescue by the gfp::ddi-1 transgene, whereas the ∆N and ∆C truncated transgenes restored bortezomib sensitivity to near wild-type levels (Figure 5—figure supplement 1). These data are consistent with the lack of conservation of the UBL and UBA domains of DDI-1, and confirm the essential role of DDI-1 aspartic protease activity. In animals lacking DDI-1, SKN-1A::GFP localizes at normal levels to the nucleus upon proteasome disruption by rpt-5(RNAi), suggesting that DDI-1 regulates SKN-1A function after nuclear localization of the transcription factor (Figure 5d). We noticed that SKN-1A::GFP occasionally showed abnormal localization within gut nuclei of ddi-1 mutants, accumulating in highly fluorescent puncta. We observed this defect for both SKN-1A::GFP and SKN-1A[∆DBD]::GFP, indicating that the DBD of SKN-1A is not required for this mis-localization (Figure 5e). As in the wild type, SKN-1A[∆DBD]::GFP does not accumulate in the absence of proteasome disruption in ddi-1 mutants, indicating that the DDI-1 peptidase does not participate in constitutive degradation of SKN-1A by the proteasome (Figure 5f). SKN-1A[∆DBD]::GFP accumulates to similar levels upon proteasome disruption by bortezomib in wild-type and ddi-1 mutants, but in ddi-1 mutants is ~20 kD larger than in the wild type, and approximates the expected size of SKN-1A[∆DBD]::GFP. To test whether these differences reflect DDI-1-dependent proteolytic processing of SKN-1A, we generated a transgene that expresses full length SKN-1A with an N-terminal HA tag
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