C. elegans neurons triggers the Ire1-Xbp1-mediated stress response in an age-dependent and cell-autonomous manner. The p38 MAPK PMK-3 regulates the chronic stress through IRE-1 phosphorylation. Overexpressing gap junction protein also activates autophagy. The insulin pathway functions through autophagy, but not the transcription of genes encoding ER chaperones, to counteract the p38-Ire1-Xbp1-mediated stress response. Together, these results reveal an intricate cellular regulatory network in response to chronic stress in a subset of cells in multicellular organism.ER stress occurs in many physiological and pathological conditions. However, how chronic ER stress is alleviated in specific cells in an intact organism is an outstanding question. Here, overexpressing the gap junction protein UNC-9 (Uncoordinated) in  in vivo. Here, we designed a genetic system in the nematode C. elegans, which allows us to induce ER stress in specific cells, without drug treatment or any other external stimuli, and then to monitor the stress response. The p38 MAPK directly acts on the phosphorylation of IRE-1 to promote the stress response. Meanwhile, the insulin receptor function through autophagy activation to counteract the p38-IRE-1-XBP-1 pathway. Together, these results reveal an intricate cellular regulatory network in response to chronic stress in multicellular organism.The accumulation of unfolded proteins triggers the ER stress response (UPR), which allows cells to fight against fluctuations in protein expression under both physiological and pathological conditions. Severe acute ER stress responses can be induced by drug treatment. However, such intense ER stress rarely occurs ubiquitously in every cell type  Proteins destined for secretion or insertion into membranes enter the endoplasmic reticulum (ER) in an unfolded form and generally leave only after they have reached their native states. During normal function, a secretory cell may experience dramatic variations in the flux of new proteins through its ER in response to changes in demand. Disruption of the balance between secretory protein synthesis and the folding capacity of the ER activates a signaling network called UPR (the Unfolded Protein Response) in an attempt to maintain homeostasis . The UPRxbp1 mRNA produces a short, activated form of XBP1, called XBP1s, which activates expression of genes encoding chaperones and other ER-associated degradation proteins that expand the folding capacity of the ER and increase the breakdown of misfolded proteins -like ER kinase), and ATF6 (activating transcription factor 6) . In respproteins . In variproteins ,12. In vesponses .Macroautophagy (hereafter referred to as autophagy) is a cellular degradation process initiated in response to stress. It attempts to restore metabolic homeostasis through lysosomal degradation of cytoplasmic organelles or cytosolic components . AutophaC. elegans is transparent throughout its life cycle. We reasoned that if we could induce the ER stress response by overloading the cell with a non-secreted plasma membrane protein, we would then be able to detect the occurrence of UPR by directly observing the sub-cellular localization of this protein in vivo. Gap junction channels direct cell-to-cell communication and defects in gap junction formation and/or function are the cause of at least 10 human pathological conditions [C. elegans neurons activates the IRE-1-XBP-1-mediated UPR in a cell-autonomous manner. Using this model, we further identified that the p38 MAP kinase PMK-3 functions through IRE-1 phosphorylation to regulate UPR. Loss of function of daf-2 (encoding the insulin-like receptor) suppresses the mis-localization defect of overexpressed UNC-9 in pmk-3, ire-1 or xbp-1 mutants. Intriguingly, although the reduced transcription of ER chaperone Bip/hsp-4 (immunoglobulin heavy chain-binding protein/heat shock protein 4) is not rescued by daf-2 mutation, autophagy induction is largely restored. This study reveals the intricate molecular interactions underlying the ER stress response in a limited subset of cells in an intact organism, and provides insights into the pathophysiology of many human degenerative diseases.The nematode nditions . Moreovenditions –24. HereC. elegans to establish the in vivo ER stress model. The D-type motor neurons, including 6 DDs and 13 VDs, are a set of GABAergic neurons with cell bodies distributed along the ventral nerve cord and neuronal processes projecting both along the ventral cord and to the dorsal cord [unc-25 encodes the GABA biosynthetic enzyme glutamic acid decarboxylase [unc-25 promoter, the GFP (green fluorescent protein)-fused gap junction protein UNC-9 could be specifically overexpressed in DD/VD neurons. We found that the ectopically expressed UNC-9::GFP is distributed in a punctate pattern along the neuronal processes and in the cell body region of DD/VD neurons  animals, the UNC-9::GFP signal no longer takes the characteristic punctate form. Instead, it is diffusely distributed in the cell body region of DD/VD neurons. Except for a few small residual dots, there is no GFP signal on the neurites  or ATF6 (atf-6 mutant) did not affect the punctate pattern of UNC-9::GFP in DD/VDs , we found that the majority of UNC-9::GFP signal in ire-1 mutants displays a punctate pattern similar to wild type , the unbiased genetic screen also identified a pmk-3 allele, xd74. In xd74 animals, the UNC-9::GFP signal is diffusely distributed in the cell body region of DD/VDs and is greatly reduced in DD/VD neuronal processes  and pmk-3(tm745), display similar UNC-9::GFP localization defects in DD/VD neurons to pmk-3(xd74) animals ;ire-1(v33) and pmk-3(tm745);xbp-1(zc12) double mutants, the UNC-9::GFP distribution resembles the ire-1 or xbp-1 null (pmk-3(tm745);ire-1(v33); xbp-1(zc12) triple mutant also mimics ire-1 or xbp-1 single null mutants  lost its capability to rescue the UNC-9::GFP localization defect in pmk-3 mutants  (We showed above that the kinase-defective (KD) mutant form of PMK-3  . We furtt PMK-3) . Fourth,t PMK-3) . Furthert PMK-3) . Togethexbp-1(xd131) strain and performed a further genetic suppressor screen to explore whether the malfunctioning ER stress response can be repressed. From the screen, we identified the xd211 mutation. In the presence of xd211, the punctate pattern of UNC-9::GFP was restored along the neurites and in the cell body region of DD/VD neurons (xbp-1(xd131) or xbp-1(zc12) was also suppressed by the daf-2(e1370) allele (daf-2 function is responsible for the genetic suppression of xbp-1. DAF-2 negatively regulates the FOXO transcription factor DAF-16. In xbp-1(xd131) daf-2(xd211);daf-16(mu86) triple mutant animals, the suppression of xbp-1 by daf-2 is completely blocked by daf-16 mutation  allele   driven by the unc-25 promoter, we examined the autophagy activity in DD/VD neurons. In the absence of UNC-9::GFP, mCherry::LGG-1 is diffusely distributed along the neuronal processes and there are typically less than 4 mCherry::LGG-1 puncta in the cell body region  contain diffusely distributed UNC-9::GFP signal. After starvation, the UNC-9::GFP adopted a punctate distribution pattern in a significant proportion (about 58%) of DD/VD cells . Although the detailed mechanisms underlying this restricted effect remain elusive, it is possible that the broadness or/and the severity of ER stress in the nervous system determines whether the stress response can spread to neighboring tissues.In response to ER stress, transcription of the ER chaperone gene is activated by the IRE-1/XBP-1 pathway . Indeed,nd cells ,40. Pan-podermis . In contire-1 or xbp-1 mutants, a diffuse UNC-9::GFP signal accumulates in the ER. Evidently, with no need for drug treatment or any other external stimuli, the distinct UNC-9::GFP subcellular localization offers a dual marker to monitor functional versus defective UPR at single-cell resolution in an intact animal. Notably, the subcellular localization of endogenous UNC-9 and of ectopic muscle-expressed UNC-9::GFP is unaffected by ire-1 or xbp-1. Thus, the IRE-1/XBP-1 UPR is not involved in the UNC-9 protein localization under normal growth condition. Previous studies demonstrated that that the UPR is also function in absence of external ER stress and the UPR genes are critical for maintaining secretory protein metabolism[The cell-autonomous stress response triggered by overloading of UNC-9::GFP protein ensures that UNC-9::GFP adopts its distinctive punctate pattern in DD/VD neurons. In contrast, when UPR fails, such as in etabolism,42. Herepmk-3/p38 MAPK is involved in the ER stress response triggered by excess UNC-9::GFP. Among the three p38 MAPKs in C. elegans [ire-1-xbp-1 pathway protects animals from Pseudomonas aeruginosa infection, inactivation of the PMK-1-mediated immune response suppresses the lethal effect of P. aeruginosa infection in xbp-1 mutants [pmk-3 functions similarly to ire-1 and xbp-1. In contrast, neither PMK-1 nor the upstream MAPKK (MAPK kinase) SEK-1 nor MAPKKK (MAPK kinase kinase) NSY-1 [ire-1 and xbp-1) are shared, other regulatory components are employed in a tissue- or/and intensity-dependent manner. How does PMK-3/p38 participate in this cell-autonomous chronic ER stress response? A previous study showed that p38 MAPK directly phosphorylates the activated form of XBP1 and enhances its nuclear migration [xbp-1 mutants in which the putative phosphorylation sites of p38 MAPK (T191 and S283) were disrupted could fully rescue the UNC-9::GFP mis-localization defect in xbp-1 (the percentage of DD/VD cell bodies showing defective UNC-9::GFP distribution was dropped to 8.7% and 11.3% respectively). In addition, overexpressing ire-1 suppresses the pmk-3 mutant phenotype, while upregulation of pmk-3 does not bypass the functional requirement for ire-1. This suggests that pmk-3 functions upstream of ire-1. Consistent with this idea, PMK-3 binds to IRE-1 and the phosphorylation status of IRE-1 is affected by the presence of functional PMK-3. Among three putative p38 MAPK phosphorylation sites in IRE-1, T949 is located in the C-terminal RNase domain. Phosphorylation of the corresponding site does not have a direct impact on the splicing activity in vitro, but has a noticeable effect in vivo [xbp-1 mRNA in DD/VD neurons is partially affected by pmk-3 and mutation of this T949 site significantly decreased the rescue activity of IRE-1. Given that phosphorylation on the RNase domain may be involved in the recruitment of unknown cofactors to maximize the RNase activity of IRE-1 [Aided by unbiased genetic screens, we further identified that  elegans , PMK-1 i elegans –49. Whil mutants . Here, iigration . Here, h in vivo . Here, wof IRE-1 , it is fdaf-2 are long-lived, and both ire-1 and xbp-1 make a large contribution to the long lifespans of daf-2 mutants and also increase their ER stress resistance [daf-2 could restore protein secretion in ire-1 and xbp-1 mutants in a daf-16 dependent manner [daf-2 mutation also improved the ER stress resistance of xbp-1 and ire-1 mutant animals when they were treated with low but not high tunicamycin concentrations, and DAF-16 acts through ER-associated degradation systems independently of ire-1 [ire-1, and further reducing insulin signaling restored the normal PVD neuronal morphology [xbp-1 is not involved in PVD morphogenesis at all, which suggests that the antagonistic role of insulin signaling may not act through the non-canonical splicing of xbp-1 mRNA as in the UPR process [daf-2 mutation suppresses the defective ER stress response in both ire-1 and xbp-1 animals. Because the cell autonomous reduction of hsp-4 expression in ire-1 or xbp-1 mutants is not restored by daf-2, we suspected that daf-2 may act on alternative path to regulate the stress response. Intriguingly, autophagy induction was restored in ire-1 or xbp-1 mutant cells by loss of daf-2 function. It has been shown that the daf-2 mutants exhibit increased levels of autophagy, and autophagy is required for their extended lifespan [daf-2 mutation in both wild-type and ire-1 mutant animals [daf-2 mutation, the reduction of autophagy in ire-1 or xbp-1 mutants was indeed suppressed by daf-2. The FOXO forkhead transcription factors are key transcriptional regulators of autophagy [daf-16 mutation .Therefore, we suspected that in ire-1 or xbp-1 single mutants, the DAF-16/FOXO remains inactive due to the presence of DAF-2. Since neither transcription of ER chaperone genes (for instance hsp-4/Bip) nor autophagy induction could occur in ire-1 or xbp-1 single mutants, the proper localization of overexpressed protein could not be achieved. In contrast, when both ire-1/xbp-1 and daf-2 are removed, the DAF-16 is activated due to the absence of DAF-2. Hence, although the transcriptional upregulation of ER chaperone genes (such as hsp-4/Bip) is missing-because of the lack of ire-1/xbp-1 pathway, the active DAF-16 could act through autophagy or other protein degradation process or ER homeostasis to facilitate the proper localization of overexpressed protein. Intriguingly, the accumulation of misfolded proteins in the ER appears not to directly activate DAF-16 [ire-1-xbp-1 branch of the UPR are removed. Nevertheless, the availability of two parallel systems , dlk-1(tm4024), daf-16(mu86), mom-4(ne1539), mtk-1(ok1382). LGII: ire-1(v33), nsy-1(ok593), xdIs15. LGIII: xbp-1(zc12), xbp-1(xd131), xbp-1(tm2457), xbp-1(tm2482), daf-2(e1370 ts), daf-2(xd211), kin-18(pk71), xbp-1(zc12). LGIV: pmk-3(tm745), pmk-3(ok169), pmk-3(xd74), jnk-1(gk7), kgb-1(um3), pmk-1(km25), kgb-2(gk361), unc-30(ju54), mak-2(tm2927), wnk-1(ok266), lgg-1(bp500), xdIs13. LGV: rpm-1(ju44), mlk-1(ok247). LGX: atf-6(ok551), atf-6(tm1153), pek-1(ok275), pek-1(ok275), mkk-4 (ok1545), jkk-1(km2), mek-1(ks54), sek-3(tm1344), sek-6(tm4305 tm4136), sek-1(km4). The genetic screen was performed according to the previous report [xdIs15 or xbp-1(xd131);xdIs15 worms were treated with EMS (ethylmethane sulfonate) solution for 4 hours. F2 worms were examined for phenotype alteration at the young adult stage under a fluorescence microscope. Mutant animals were recovered to fresh plates for maintenance. A total of 10,000 and 5,000 mutagenized haploid genomes were screened respectively for xdIs15 and xbp-1(xd131);xdIs15 worms. 10 mutants were identified from the genetic screen using xdIs15 as a start strain, which could be classified to 4 categories: diffused UNC-9::GFP signal in the cell body region of DD/VD neurons (xd74 and xd131), enlarged UNC-9::GFP puncta accumulated in the cell body region of DD/VD neurons (2 mutants), reduced UNC-9::GFP puncta number along ventral nerve cord (3 mutants) and decreased UNC-9::GFP expression (3 mutants). In the xbp-1(xd131) mutant genome, a point mutation at W318 creates a stop codon in the active form of xbp-1. Only the daf-2(xd211) mutant was isolated from the xbp-1(xd131);xdIs15 suppressor screen. To isolate daf-2(xd211) alone mutant, the classical recombination method was used. Total 999 F2 worms were sequenced and finally a single daf-2(xd211)/ daf-2(xd211) xbp-1(xd131) hetezygote was identified. All isolated mutants were outcrossed with wild type at least four times. The strains containing daf-2(e1370 ts) were cultured at 16°C until L2/L3 stage, then shifted to 25°C to reach adulthood before phenotype characterization.Culture and manipulation of  methods . Mutantss report . Brieflyunc-25 promoter was inserted between the BamHI and SphI sites of the pSM-GFP vector (a generous gift from Dr. Kang Shen) and the UNC-9 cDNA was inserted into the BamHI site. xbp-1, ire-1 and pmk-3 cDNAs were amplified by reverse transcription. The ER stress reporter plasmid Punc-25::XBP-1us::mCherry was modified from XB3 plasmid (a generous gift from Dr. Christopher Rongo). Punc-25::cytb-5.1::mCherry was kindly provided by Dr. Yingchuan Qi. Punc-25:: mCherry::MANS, Punc-25:: mCherry::RAB-7, Punc-25:: mCherry::RAB-5, and Punc-25::mCherry::LGG-1 were constructed by the recombination method. Transgenic animals were produced as previously described [pmk-3 and the kinase dead form of pmk-3(pmk-3KD) were amplified by PCR and then inserted between the EcoRI and XbaI sites of pFLAG-CMV-2 plasmid. The cytosolic C-terminus of IRE-1 was amplified by PCR and inserted between the NheI and KpnI sites of pcDNA 3.1/myc-His vector. Integrated strains were obtained by UV irradiation. The strain BCN1071 which expresses Phsp-4::mCherry was kindly provided by Ben Lehner. All integrated transgenic animals were out-crossed at least 3 times. The corresponding transgenes are listed in The escribed . The wilunc-9 gene. This UNC-9::GFP knock in strain (PHX727) was generated by SunyBiotech and was verified with DNA sequencing. This strain was outcrossed with N2 for 4 times before use.The UNC-9::GFP knock-in strain was created using sg1:CGTATGGTTGCAACTCACGCCGG and sg2: CCGGAGAACTACCCTGTTACGAG. The GFP sequence was inserted before the stop codon TGA of unc-25::mCherry marker was used. Phsp-4::mCherry was used to detect induction of the chaperone gene hsp-4 in the ER stress response. The average Phsp-4::mCherry fluorescence intensity in the anterior and posterior intestine regions was measured and normalized with the wild type. To quantify the xbp-1 splicing activity, the Punc-25::XBP-1us::mCherry plasmid was used and DD/VD cell bodies with mCherry signal were counted. To characterize the distribution of UNC-9::GFP under starvation conditions, well-fed L4 ire-1(v33);xdIs15 worms were collected and washed 8 times with M9 buffer. After sedimentation, worms were plated on NGM plates without food. After 24 hours, the starved adult worms were examined using Leica TCS SP8 confocal microscopy. All graphical data are presented as mean ± s.e.m. Two-tailed unpaired and paired Student’s t-tests were performed for comparison between two groups of samples. To compare multiple groups, one-way ANOVA was used with an appropriate multiple comparisons post hoc test . *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.Animals were mounted on 2% agar pads in M9 buffer containing 1% 1-phenoxy-2-propanol and examined by fluorescence microscopy unless indicated otherwise. Fluorescence photographs were taken using a Zeiss Axioimager A1 with an AxioCam digital camera and Axiovision rel. 4.6 software (Carl Zeiss) or an IX81 Olympus inverted confocal microscope. All images were taken at the young adult stage unless specifically indicated. The UNC-9::GFP distribution defect in the cell body region was quantified by counting the percentage of cell bodies with diffusely localized UNC-9::GFP. To quantify the UNC-9::GFP puncta distribution in DD/VD cell body region, the size of UNC-9::GFP co-localized with either myr::mCherry, mCherry::RAB-5, mCherry::RAB-7 or mCherry::LGG-1 was measured using ImageJ. The ratio of the co-labeled UNC-9::GFP to the total UNC-9::GFP was calculated. Signals from 5 DD/VD cells were scored for each genotype. To quantify the number of DD/VD neurons, the Punc-25::mCherry::LGG-1 was constructed. Images at the young adult stage were collected and the numbers of mCherry::LGG-1 foci in DD/VD cell body regions were counted. To characterize mCherry::LGG-1 puncta in strains carrying daf-2((e1370 ts), embryos were collected and raised at 25°C until L2/L3 stage before phenotypic examination.To analysis autophagy activity in DD/VD neurons, PHEK293T (human embryonic kidney) cells were maintained in Dulbecco’s modified Eagle’s medium containing 12% FBS. For cell transfection, polyethylenimine (Polysciences) was used according to the manufacturer’s instructions. The clones Myc-tagged cytoplasmic region of IRE-1 and the Flag-tagged full-length wild-type or kinase inactive form of PMK-3 were co-transfected into HEK293T cells. 24 hours after transfection, cells were harvested and lysed for 30 min at 4°C. After centrifugation, the supernatants were incubated with anti-FLAG M2 affinity gel beads at 4°C overnight and then washed three times with washing buffer and incubated with SDS-sample buffer. Samples were resolved by standard immunoblotting techniques. All the co-immunoprecipitation experiments were repeated at least three times.The Myc-tagged cytoplasmic region of IRE-1 and the Flag-tagged full-length wild-type or kinase inactive form of PMK-3 were co-expressed in HEK293T cells and then the IRE-1 protein was immunoprecipitated with Myc antibodies. The phosphorylation level of IRE-1 was examined with P-Thr-Pro-101antibody , which detects the conserved phosphorylation site (S/TP) for p38 MAPK. The signal intensity of individual protein bands was analyzed with ImageJ. The relative protein level was determined by subtracting the respective FLAG control. All the co-immunoprecipitation experiments were repeated at least three times.The pGEX-4T-1-GST::IRE-1 plasmid was transformed into BL21(DE3) competent cells. The fusion protein was induced by 1mM IPTG at 18°C for 24hr and then purified by Glutathione Sepharose 4B. The Flag::PMK-3 and myc::SEK-1 plasmids were transfected into HEK293T cells. 24 hours after transfection, cells were lysed and purified by anti-FLAG M2 affinity beads. Subsequently, the protein samples were concentrated and replaced with the kinase buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl, 1 mM DTT). The phosphorylation of IRE-1 by active PMK-3 was monitored using a Universal Kinase Activity Kit . Purified IRE-1 and 200μM ATP were mixed with purified PMK-3 and coupling phosphatase CD39L2 in phosphatase buffer . As a control group, PMK-3 was replaced by 1x kinase buffer. The reaction is carried out in 50 μL for 10 minutes at room temperature. The coupling phosphatase CD39L2 can generate inorganic phosphate from ADP resulting from the kinase reaction. Malachite Green Reagent was added to terminate the reaction. After incubation for 20 minutes at room temperature to stabilize the color development. The amount of phosphorylated protein was determined using a microplate reader set to 620 nm. The experiments were repeated three times independently. To further determine the phosphorylation of IRE-1, anti-P-Thr-Pro-101 antibody was used to detect the phosphorylation site (S/TP) for p38 MAPK. The GST::IRE-1 was incubated with Flag::PMK-3 in kinase buffer  at 25°C for 30 minutes. As a control group, the Flag::PMK-3 was replaced by 1x kinase buffer. The samples were boiled with loading buffer to terminate the reaction. The experiments were repeated three times independently.unc-9 sequence and the reverse primer located in gfp sequence. Primer1: Forward: 5-CTACCCTGTTACGAGCTTGC-3 Reverse: 5-TTCTACCGGTACCCTCCAAG-3 Primer2: Forward: 5-CTACCCTGTTACGAGCTTGC-3; Reverse: 5-TGCCCATTAACATCACCATC-3. Relative levels of unc-9::gfp expression were normalized to act-1 in the same sample.Total RNA was isolated from different genotype worms using Trizol reagent (Invitrogen). cDNA was generated using a Superscript II reverse transcriptase kit (Invitrogen). qRT-PCR experiments were performed using Transstart Green qPCR superMix (Transgen Biotech) with a Stratagene Mx3000P system (Agilent). The following two pair primers were used and the forward primer located in ire-1(v33); daf-2(e1370);xdIs15, xbp-1(zc12); daf-2(e1370); xdIs15, pmk-3(tm745); daf-2(e1370); xdIs15 worms were cultured at 25°C to reach adulthood and then transferred onto NGM plates containing 5 mM chloroquine. After 4 hours incubation, the UNC-9::GFP distribution defect in the cell body region of DD/VD neurons was quantified by counting the percentage of cell bodies with diffusely localized UNC-9::GFP signal.The chloroquine treatment was performed following a modified protocol from previous report . BrieflyS1 FigN = 5. (B) A worm-scale image of UNC-9::GFP knock in (KI) (green). NR, nerve ring. VC, ventral cord. (C) Quantification of Phsp-4::mCherry signal in the anterior intestine region. (D) Quantification of Phsp-4::mCherry signal in the posterior intestine region. NS, not significant; **P < 0.01, *P < 0.05. One-way ANOVA with Dunnett’s test. (E) Expression of Phsp-4::mCherry (red) in wild-type (wt), ire-1 and xbp-1 animals with or without UNC-9::GFP. Scale bar represents 25 μm. (F) Phsp-4::mCherry distribution in wild type with no UNC-9::GFP overexpression in DD/VD cells. F’ is enlarged from F (boxed region). (F) Phsp-4::mCherry (red) is widely and weakly expressed in multiple tissues. F’ is enlarged from F (boxed region) showing a part of ventral cord region. (G) Phsp-4::mCherry (red) is induced in UNC-9::GFP (green) expressing DD/VD neurons (white arrows). G’ is enlarged from G (boxed region) showing a DD/VD cell (red). Scale bar represents 50 μm(A) Quantification of the UNC-9::GFP co-distribution with Myr::mCher, RAB-5:mChery or RAB-7::mCherry. (TIF)Click here for additional data file.S2 Figxbp-1 gene. The molecular lesions are indicated for the zc12, tm2482, tm2457, and xd131 alleles. Green boxes indicate exons. (B) Quantification of the UNC-9::GFP localization defect in various genotypes. N = 20 for each genotype; ***P < 0.001; **P < 0.01. One-way ANOVA with Dunnett’s test. (C and D) Quantification of the mRNA expression of Punc-25::UNC-9::GFP in various genotypes. Three independent biological repeats.(A) The structure of the (TIF)Click here for additional data file.S3 Figwt), ire-1(v33), xbp-1(zc12), and pmk-3(tm745) animals carrying the Pmyo-3::UNC-9::GFP transgene. White lines highlight the muscle cells. Dashed lines highlight the regions from which the UNC-9::GFP puncta are absent. Scale bar represents 25 μm. (B) UNC-9::GFP expressed at the endogenous level from a knock-in allele (UNC-9::GFP KI) is distributed on the neuronal processes and cell surface of DD/VD neurons (asterisks) in ire-1 (A) and xbp-1 (B)mutants. Scale bar represents 5 μm.(A) The localization of UNC-9::GFP (green) in muscle cells in wild-type ((TIF)Click here for additional data file.S4 Figunc-53 expressing neurons (green) in wild-type (wt), ire-1(v33), and xbp-1(zc12) animals carrying the Punc-53::UNC-9::GFP transgene. White arrows indicate UNC-9::GFP puncta on neurites. Dashed lines encircle the cell bodies. Scale bars represent 5 μm.(A) The localization of UNC-9::GFP in P(TIF)Click here for additional data file.S5 Figpmk-3 gene. The molecular lesions are indicated for the ok169, tm745 and xd74 alleles. The kinase domain is indicated. Green boxes indicate exons. (B) The RT-PCR results for pmk-3 in wild type and pmk-3(xd74).(A) The structure of the (TIF)Click here for additional data file.S6 Figxbp-1 represented by mCherry signal in wild-type (wt) and pmk-3 animals. N is indicated for each genotype. (C) The in vitro kinase assay. ***P < 0.001. Student’s t-test. Three independent biological repeats.(A) The localization of UNC-10::GFP (green) in DD/VDs in various genotypes. Dashed lines encircle the cell bodies. Scale bars represent 5 μm. (B) Alternative splicing of (TIF)Click here for additional data file.S7 Fighsp-4::mCherry signal in the anterior intestine region. (C) Quantification of Phsp-4::mCherry signal in the posterior intestine region. ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant. One-way ANOVA with Dunnett’s test. (D) The overexpression of hsp-4 does not suppress the mutant phenotype of ire-1(v33) or xbp-1(zc12) animals.(A) The PMK-3-IRE-1-XBP-mediated UPR acts in parallel with insulin-inhibited autophagy to alleviate chronic stress induced by excess UNC-9::GFP proteins. (B) Quantification of P(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex). Ribonucleoprotein complexes (RNPs), composed of both RNA molecule(s) and one or more RNA-binding proteins (RBPs), are known to play key roles in cell homeostasis and cell fate by controlling post-transcriptional gene regulation and RNA processing. This can involve gene expression, RNA storage, RNA stability and RNA transport . Until rOver the last two decades, several techniques have been developed to study the interplay between RNA and their cognate RBPs. These methods use an RNA-centric or protein-centric approach and target a specific RNP or an entire group of RNPs. For such a rapidly evolving field, it can be challenging to maintain an overview of the available methods and more importantly how they can be applied. Additionally, standard ways of analyzing, presenting and visualizing the resulting data have yet to emerge.It has become clear that RNA is more than solely a blueprint to be translated into functional proteins, as it can also function as a scaffold to coordinate and organize protein networks and vice versa. Earlier, protein-centric approaches, such as Cross-Linking and ImmunoPrecipitation (CLIP), were applied to describe these interactions ,20. AlthDespite the recent availability of multiple RNA-centric methodologies discussed in As mentioned in Urdaneta et al. (2019) and Beckmann (2017), we have to highlight that the term RBP has a historical role in functional RNA biology. Since cross-linking an RNP is based on the physical proximity of the RNA and the protein rather than on the physiological role of the protein, the term RBP becomes broader or a new term has to be introduced, as suggested by Urdaneta et al. Because of this proximity binding, the question of whether the identified binding proteins are functional binders or just spatially close to a given RNA molecule during the time of cross-linking can arise. Together with the fact that a lot is still unknown about the role and importance of numerous transient RNA–protein interactions, it is advisable to experimentally validate identified targets, no matter how stringent the purification procedure ,24.A first step when embarking on a study of the ribonome is to choose whether or not a covalent bond will be introduced between the RNA molecule and its interacting proteins. Depending on the sample type and goal of the set-up, no cross-linking, chemical- and UV-induced cross-linking could be used. An overview of the strategies is provided in Isolating RNPs in vivo in their native state under physiological conditions is as close as it gets to the true nature of these complexes and therefore, if successful, harbors the most accurate and desirable information. Nevertheless, these native conditions have several practical challenges. The native RNP is as strong as its weakest non-covalent interaction, and therefore mild lysis conditions and washing procedures have to be used to maintain these interactions. These washing conditions do not discriminate between protein–RNA and protein–protein interactions, leaving secondary binding proteins in the RNP complex and leaving non-RNA binding protein contaminants to be co-purified. Additionally, aspecific RNPs can re-associate after cell lysis, making the discrimination between specific and contaminating interactions more difficult . Under nChemical cross-linking is a widespread technique used successfully for multiple protein- and RNA-centric approaches . Often tUltraviolet light (UV) promotes the formation of an irreversible covalent bond between a single-stranded RNA molecule (ssRNA) and its interacting RBPs . To prodConventional UV cross-linking uses a wavelength of 254 nm, and both laser light and UV lamps can be utilized to introduce enough energy to form the covalent link . SpecifiAmino acids differ in structure and therefore also in preference to form a covalent link with their interacting nucleotides. A study by Kramer et al., 2014 found that all amino acids, with the exception of Asp, Asn, Glu and Gln, could cross-link with RNA. Cys, Lys, Phe, Trp and Tyr are cross-linked with a greater frequency than other amino acids . Asp andHomo sapiens cell lines . T. T74]. TInspired by chromatin immunoprecipitation (ChIP), where protein-DNA interactions are investigated, Simon et al. developed in 2011 the Capture Hybridization Analysis of RNA Targets (CHART) . SimultaO-CH2-4′ bridge, increasing the probes’ melting temperature up to 10 °C per LNA nucleotide in the mixmer sequence . P. P78]. Pn nature . In a naWhereas UV cross-linking is a very specific cross-linking technique, it lacks efficiency, which is its main drawback. It is estimated that only 1–5% of the RNPs are actually cross-linked, leaving 95–99% free. Nevertheless, we believe that because of its specificity and ease of use, UV cross-linking is still the most suitable and general strategy to use in RNA-centric approaches. Therefore, we will only elaborate on methodologies that make use of UV cross-linking. This does not mean that our rationale cannot be applied to chemical cross-linking or native experiments.Peptides, unlike DNA/RNA, cannot be amplified. Until a technique is found to do so, this means that what you isolate is what you have to detect. Depending on the goal, overcoming the detection limit of mass spectrometry can be challenging. Rogell et al., making use of LNA/DNA mixmer probes, estimated that the amount of a captured single RNA target should be around 2 µg to reach the mass spectrometry detection limit . These nAt first sight, non-cross-linked (free) RNA is not perceived as a problematic contaminant in RNP isolation studies. Indeed, in theory, it harbors no protein contaminants and thus will not affect the mass spectrometry analysis. However, we believe that removing free RNA is one of the major protocol improvements to make. Free RNA not only contaminates the isolate by dragging in transiently bound unlinked proteins but also competes with RNPs to be isolated. Knowing that most of the RNA is not cross-linked, this generates strong competition and makes methods such as (e)RIC, RAP-MS, specific capture with antisense LNA/DNA mixmer probes, RICK and CARIC less efficient A. These As described, organic phase separation methods such as XRNAX and PTex isolate RNPs at the interphase. Importantly, besides isolating the whole RBPome, these techniques also separate the RNPs from free RNA and proteins, leaving a purer RNP mixture, and are comparable with the strategy of the Tandem RNA Isolation Procedure (TRIP) . Using tMcHugh et al., 2015 successfully used RAP-MS to identify interacting proteins of the lncRNA Xist. Although already proven to work on high-copy-number RNPs, such as ribosomal and spliceosomal complexes, they were the first to successfully use this technique on a low-copy-number RNA molecule, highlighting its potential. After such success, one would expect an explosion of similar studies. To the best of our knowledge, only a few papers appeared that specifically identify interacting proteins to a single specific RNA target ,108,109.10 cells per sample  and 76.8 mL of MyOne streptavidin C1-coated dynabeads representing a value of approximately 24,000 euros (at the time of writing) were used. Half of the amount of dynabeads was used to pre-clear the lysate from native biotin-linked molecules. This scale and cost exceed the costs of an appealing, routinely usable procedure for wide adoption. As discussed above, an organic phase separation pre-step already eliminates most of the free RNA, which will theoretically increase the RAP-MS methods’ yield by ~20 times . In addition, PTex needs 100 times less starting material in comparison with RIC to achieve a similar goal. Along with the higher RNP capture efficiency, biotin-linked molecules are also removed. This makes a biotin pre-clearing step unnecessary, reducing the experimental time and amounts of expensive materials. If the above assumptions are correct, a similar RAP-MS experiment based on an organic phase separation fraction would be possible for the assumed cost of 600 euro in streptavidin beads, which makes it much more appealing to be routinely used. Additionally, other techniques which do not discriminate between free RNA and their RNP target can benefit from this organic phase separation pre-purification step such as (e)RIC, PPR protein, HyPR-MS, CHART-MS and ChiRP-MS, but this still needs to be established in practice.This suggests the lack of a scalable, cost-efficient and easy to execute protocol. To give an idea of the used scale and cost, 8 × 10RICK and CARIC enable the highly interesting feature of making a distinction between RNPs comprising newly transcribed RNA relative to older complexes. This characteristic could enable the dynamic mapping of RNPs upon different biological/chemical conditions. A challenging part of the click-based approach is to perform an efficient click reaction to link biotin to the 5-Ethynyl-uridin (EU) analogs without causing RNA degradation due to formed Cu(I) ligands and lysate debris. Although Huang et al. (2018) already use ultrafiltration tubes to pre-purify the RNP fraction, we think that using an organic phase pre-separation could minimize RNP degradation during the click reaction. This is due to the increased probability of optimal reaction conditions and the lack of RNase/proteinase and other interfering lysate components. Along with the possibility to control the reaction conditions, naturally present biotin-linked proteins and metabolites are removed. Therefore, the lysate will not have to be pre-cleared before performing the streptavidin-coupled bead capture, reducing experimental time and expensive chemicals.Together, the above-discussed feature of removing free RNA results in a more efficient capture experiment on the same amount of input material. When scaling up an experiment, the required volumes could exceed practical sizes. The organic phase pre-separation can easily handle big volumes to process down to a much smaller RNP fraction. This efficiently tackles potential scaling problems on the condition that starting material is not limited. We believe that this property, together with separating RNPs from free RNA and free proteins, will make organic phase pre-purification a gold standard in all facets of future RNP research.While this review is focused on RNA-centric methods, starting from a clean RNP fraction can also be very helpful to enhance downstream protocols to elucidate ribonome transcriptomics, RBP mapping ,43,110, The study of the ribonome is a rapidly evolving field. With the new organic phase separation methods, the field is pushed even further. Multiple methodologies are being developed in parallel which make use of different experimental conditions and choices. Experimental differences occur at all stages, starting with the choice of cell or tissue type, cross-linking methods and doses, use of different control sampling, mass spectrometry equipment and bioinformatic proteomic approaches. Lacking a clear consensus on the experimental set-up and on how to analyze and visualize data makes comparisons between methods difficult.Both ChiRP-MS and RAP-MS were independently performed to study the interacting proteins of Xist lncRNA. Due to the similar research question, these techniques are a good example to illustrate that differences in experimental set-up may greatly influence outputs. For ChiRP-MS, 81 interacting proteins were identified, whereas only 10 were identified for RAP-MS. The identified proteins for both techniques show an overlap of eight. ChiRP-MS makes use of human HeLa cell lines, whereas RAP-MS uses a mouse pSM33 ES cell line. Differences in cell type may contribute to the different identified RBPs . In addiAnother example is the comparison of three mRNA interactome capture experiments conducted in Arabidopsis, which only share an overlapping set of 79 proteins of the total 1815 proteins detected. As stated by Köster et al., apart from the differential mass spectrometry data analysis, the diverse developmental stages of the plants examined in each of these studies may contribute to the different outputs . Other dFurthermore, when analyzing mass spectrometry data, different false discovery rates, peptides required for protein identification, number of biological replicates, protein enrichment fold cut-offs, mass spectrometry equipment, quantitative proteomics approaches, etc., also contribute to a more difficult comparison of the outputs of each experiment and method.A final important aspect is the evaluation of the efficacy of the experiment. This is often presented as the relative abundance of the target RNA compared to a reference RNA after the experiment in contrast to its ratio in the input sample, meaning that the levels of the target RNA after purification are normalized to the levels in the input sample. This is known as the fold-enrichment of the target RNA. By comparing to the input sample, the target RNA may be enriched several-fold; however, it is possible that in absolute numbers, the target RNA, when capturing a low-copy-number RNA for example, may not exceed the abundance of the reference and other contaminating RNA molecules. In quantitative proteomics approaches, contamination will be indeed identified as being contamination. However, it is possible, especially for low-copy-number RNA, that protein contamination, if too abundant, complicates the detection of the low-abundant target proteins due to the occupation of the ion trap sampling space by these contaminants. Additionally, the often-used semiquantitative approaches based on spectral counting have troubles in reliably identifying low-abundance proteins due to inherent variations . As a reThe complex life of RNPs is still far from being a fully understood system. In the last decade, a wealth of methods has been developed to address pieces of this puzzle. Each method, however, has its own characteristics and strengths, but also there are clear technical limitations holding back the possibility of studying RNPs with a clear and general approach. We believe that the arrival of the phase separation methods, such as XRNAX, OOPS and PTex, will catalyze the former techniques into more effective tools to study RNPs. Due to both experimental and bioinformatics differences in set-up, it is often difficult to compare and evaluate techniques, often resulting in different outputs for similar research questions. Therefore, these methods can be considered often rather complementary than equal, rendering it more important to choose the technique tailored to the desired output.


We find that τc can more reliably predict high-magnitude events during a short P-wave time window (PTW) but produces greater uncertainty in the low-magnitude range, whereas Pd is highly correlated with the event magnitude depending on the selection of an appropriate PTW. Here, we propose a threshold-based evolutionary magnitude estimation method based on a specific combination of τc and Pd that both offers more robust advance magnitude estimates for large earthquakes and ensures the estimation accuracy for low-magnitude events. The advantages of the proposed approach are validated using data from 2016–2017 and the Ms 8.0 Wenchuan earthquake in an offline simulation. The proposed concept provides a useful basis for the future implementation of an EEW system in the Sichuan–Yunnan region.The Sichuan–Yunnan region is one of the most seismically vulnerable areas in China. Accordingly, an earthquake early warning (EEW) system for the region is essential to reduce future earthquake hazards. This research analyses the utility of two early warning parameters (τ Following several decades of development, EEW systems are currently operational and provide alerts to the public in several countries or regions worldwide2, including Japan3, Taiwan4, Mexico5 and South Korea6; in contrast, some EEW systems operating in India7, Romania8, Turkey8 and the West Coast of the United States9 issue alerts only to selected users. In addition, other EEW systems are currently under real-time testing or development in locations including Italy10, Chile11, Coast Rica2, EI Salvador2, Nicaragua2, Switzerland2, Israel12, and the Beijing capital region13 and Fujian region14 of China.Earthquake early warning (EEW) systems offer promise as a practical tool in seismic hazard mitigation that could help reduce the losses caused by destructive earthquakes. Using data from a dense instrument array around the source region or near the target of concern, EEW systems aim to broadcast advance warning of potential damage from an ongoing earthquake to target areas before the arrival of destructive seismic wavesTwo main approaches are adopted for EEW systems: regional warning (network-based) and onsite warning . In the onsite warning approach, the initial P-wave motion of a single station or local array is used to predict the subsequent peak ground shaking at the same site. In contrast, to issue a regional warning, data from seismic networks deployed in the potential source area are used to estimate the source parameters (location and magnitude) and predict the ground motion for the concerned region. The advantage of onsite warning is that it provides faster warnings of future ground motion to sites at short distances from the epicenter, commonly through setting a P-wave amplitude threshold without necessarily determining the location and magnitude of the event, whereas the regional warning approach provides a more robust ground motion prediction for a wider region with continuously available network data and source parameters updated in real time. The earliest EEW systems generally adopted the onsite warning method due to the limited network density. At present, following increases in network density, the onsite algorithm is employed in most network-based warning systems to integrate the advantages of the regional and onsite warning approaches, thereby providing more information and the quickest warnings.15 on 3 June 2007, the Ms 7.0 Lushan earthquake17 on 20 April 2013, and the Ms 6.5 Ludian earthquake18 on 3 August 2014. Of particular note is the Ms 8.0 Wenchuan earthquake, which occurred on 12 May 2008 and resulted in more than 69,000 deaths and financial losses reaching hundreds of billions of RMB19. As the effects of potential earthquake hazards will continue to increase with the rising population and economy in this area, scientific research on diminishing future earthquake hazards is essential for the Sichuan–Yunnan region. In this context, to strengthen seismic prevention and disaster mitigation measures therein, the Sichuan–Yunnan region has been designated the key monitoring area of the Seismic Intensity Rapid Reporting and Earthquake Early Warning (SIRR & EEW) project, and developing an EEW system for this region is of great concern.The Sichuan–Yunnan region, located at the eastern edge of the Qinghai–Tibet Plateau, coincides with the middle to southern section of the north–south seismic zone that extends across the central part of the Chinese mainland. The high level of seismicity in this region is associated with its complex tectonic setting. The Sichuan–Yunnan region has experienced numerous destructive earthquakes that have caused extensive casualties and property losses; remarkable examples include the Ms 6.4 Ning’er earthquakec), the most commonly used parameter for estimating the magnitude with regard to the P-wave frequency, is correlated with the earthquake magnitude20. In addition, the peak amplitude of the vertical displacement (Pd) in the first few seconds of the P-wave has been shown to be related to the event magnitude and is used mainly as information about the amplitude content of early P-waves to predict the earthquake magnitude22. Models based on τc and Pd have been proposed for the EEW systems in China to estimate the earthquake magnitude using only sequence data for large events that occurred in the Sichuan–Yunnan region24. Fortunately, with the operation of the China Strong Motion Network Center (CSMNC), large quantities of high-quality digital strong motion data have been recorded, constituting a reliable database from which the relationships between the event magnitude and the EEW parameters τc and Pd can be established for the Sichuan–Yunnan region.Rapidly estimating the magnitude of an earthquake event is the most critical obstacle to the widespread development of EEW systems and is typically achieved based on empirical scaling relationships between the magnitude and early warning parameters from initial P-waves. Research has confirmed that the average period 27. For the reliable and early assessment of destructive earthquakes, Wu and Kanamori20 originally proposed the integrated use of τc and Pd to establish a threshold-based EEW system, which was subsequently applied in various areas28. However, low signal-to-noise ratio (SNR) records from low-magnitude events may produce an unexpectedly large τc value29, in which case the direct combination of these two parameters with such an unexpected error may overestimate a small event, resulting in a false alarm.Previous studies have shown that τc and Pd. We proposed first judging whether τc should be combined with Pd to improve the magnitude estimation stability, and then we further explored the evolutionary magnitude estimation of the proposed approach. Here, we selected 1596 vertical components from 273 events that occurred within the Sichuan–Yunnan region during 2007–2015 as the fitting dataset for linear regression; furthermore, data from 13 events that occurred in the same region during 2016–2017 are employed as the validation dataset to evaluate the reliability of the proposed approach  reliably represents low-SNR data and is closely related to the displacement drift during the computation of τc. Accordingly, we set a threshold of Pv = 0.05 cm/s to identify low-SNR waveforms and found that Pv < 0.05 cm/s typically corresponds to low-magnitude events with abnormally high τc values  to the integrated displacement. Only one earthquake record with a magnitude larger than 6 in our dataset features Pv < 0.05 cm/s, as highlighted by the arrow in Fig. v = 0.05 cm/s) is small enough that the stronger filter (0.15 Hz high-pass filter) will rarely be applied to large events, thereby avoiding the loss of considerable low-frequency components.We calculated τc value of each event for PTW = 3 s was established, and the result is illustrated in Fig. After applying this criterion to restrict low-SNR data, the best-fit relationship between the magnitude and mean τThe magnitude is estimated as follows.c values and resulted in scaling τc well with the magnitude up to M = 8. In addition, the correlation coefficient of this relationship increased from 0.412 to 0.683. According to our magnitude estimation equation ; otherwise, our method extends the PTW [situations (1)-(3)].As more information is needed for situations (2) and (3) to discriminate large events and the PTW needs to be extended to estimate larger-magnitude events by Pc to calculate the average magnitude only when both Pd10km and τc exceed their respective thresholds [situation (1)] and otherwise take the magnitude estimation of Pd10km as the magnitude estimate under any other set of conditions [situations (2)–(4)]. For situation (1), we introduced weights to compute the final magnitude estimate by combining the magnitude estimates from Pd10km and τc. Unlike the typical weights established based on the standard error of magnitude relationships under the overall magnitude range, here, considering that the main purpose of situation (1) is to reduce the magnitude saturation of large events, we decided to compute the weights based on the underestimation error for M = 8 events according to the relationship established above under each fixed PTW, as presented in Supplementary Table d10km and τc for PTW = i s and the magnitude estimate of a single station based on these weights was obtained as follows.c(Pd10km) for PTW = i s, respectively.With a certain PTW, our approach implements different strategies to estimate the event magnitude from the observation of a single station by analyzing the four potential situations stated above. We jointly use τi and PTWi are the predicted magnitude and PTW, respectively, for the i-th triggered station.The final magnitude was computed based on the results of multiple triggered stations in a seismic network. Since numerous distant stations with short PTWs will tend to lower the estimates of the average magnitude, we obtained average values based on the length of the PTW for each station as follows.For clarity, we have summarized the steps of our evolutionary magnitude estimation process in a flow chart Fig. .Figure 6To further investigate the reliability of the established relationships and our threshold-based approach, we applied our method to the validation dataset to evaluate the magnitude estimates, and we demonstrated the evolutionary magnitude estimation algorithm for the largest earthquake considered herein: the Ms 8.0 Wenchuan earthquake.d10km and τc only when data were available for PTW = 2 s. After comparisons with the corresponding thresholds (dashed lines), magnitude estimation was undertaken by following the threshold-based magnitude estimation steps presented in Fig. d can provide robust magnitude estimates with a low standard deviation for all small events. In addition, the inclusion of τc allows the magnitude for large events to be more precisely estimated than using Pd alone: our method produced estimates of 6.72 and 6.33 for the M = 7 Jiuzhaigou earthquake with PTW = 2 s for the estimation with and without τc, respectively. These results indicate that our method is stable and that it performed well for all 13 earthquakes considered with an average magnitude estimation error of 0.22 magnitude units for both PTW = 2 s and PTW = 3 s.Figure c value did not reach the corresponding threshold, only Pd10km was used, and the proposed method yielded a magnitude estimate of 6.7. Approximately 8.5 s after the OT, both Pd10km and τc exceeded their respective thresholds at the second triggered station (051PXZ), yielding a magnitude estimate of 8.13 at this station when PTW = 2 s. After averaging the estimates from the first two stations, a magnitude estimate of 7.41 was obtained just 8.5 s after the OT, reflecting a smaller underestimation error of 0.59 compared to the result (1.47) of using Pd10km only (dashed blue line). The third station (051PXZ) exhibited small displacement amplitudes at 2 and 3 s; these displacements were insufficient to reach Pd10km threshold. Accordingly, the estimates using Pd10km only for this station lowered the average magnitude for the three stations 10.3–12.3 s after the OT. We included the average estimates without considering the PTW length for all three stations (black circles) in Fig. Figure c and Pd) based on CSMNC data during 2007–2015 for the Sichuan–Yunnan region. In particular, we established a τc-magnitude relationship with a fixed PTW of 3 s and a Pd10km-magnitude relationship with increasing PTW (2–10 s). Based on the derived relationships, we proposed a threshold-based evolutionary approach for estimating the event magnitude within seconds of the P-wave arrival.We investigated two earthquake magnitude estimation parameters ; this selective criterion reduces such errors considerably.Our study highlights the shortcomings of existing methods, for which the applications of Pd results and setting a threshold to ensure that τc participates in the magnitude estimation process only for large earthquakes. Previous studies have proposed using τc and Pd together to improve reliability, but the occurrence of abnormally high τc values for small events will introduce errors when combining these two parameters. Our approach applies an advance judgment (compared with a predefined threshold) and implements reasonable strategies based on the levels of the observed parameters. Specifically, situation (1) indicates that an event is most likely a large event, so we jointly use the estimates of τc and Pd10km. Situation (2) indicates that an event may be a large earthquake with a small Pd10km or a small-moderate earthquake with an unexpectedly large τc. To avoid the potentially serious consequences of failing to trigger an alarm for a large earthquake, we suggest extending the PTW and using only Pd10km for the magnitude estimation in this scenario. Although a small Pd10km will underestimate a potential large event, this underestimation can be improved by extending PTW, which helps mitigate the magnitude overestimation for potential small-moderate earthquakes caused by combining unexpectedly large τc values. Situation (3) indicates that an event may be a large earthquake with an unexpectedly small τc or a small-moderate earthquake with a large Pd10km. Considering the small error of Pd10km found in the small-moderate magnitude range and the underestimation of potential large earthquakes caused by jointly using small τc values, we also suggest extending the PTW to avoid excluding large earthquakes and using only Pd10km for the magnitude estimation in this case. Situation (4) indicates that an event is most likely a small-moderate event, so we use the estimates of only Pd10km and cease extending PTW beyond 3 s. Accordingly, this approach integrates the timeliness of estimating large events based on τc and the robustness of general magnitude estimation based on Pd; moreover, the proposed method reduces the impacts of the uncertainty in τc and the magnitude saturation of Pd. We believe this approach provides the most accurate estimate in the shortest time.Our proposed threshold-based methodology is based primarily on Pd10km alone. Moreover, we show that the pull-down effects of distant stations with short PTWs can be mitigated by averaging the magnitude estimates based on the lengths of the records available at those stations, thereby reducing the weights of stations with short PTWs. Since the underestimation caused by the third station (051PXZ) was so obvious, for future real-time application, we suggest retaining the previous magnitude estimation results without introducing low-amplitude stations if the event is judged most likely to be a large earthquake based on the near-source stations. It is important to note that the distances used in this study were based on the post-location. Accordingly, the real performance will be affected by the error in locating earthquakes in real-time.Offline applications to 13 events that occurred during 2016–2017 and the Ms 8.0 Wenchuan earthquake demonstrated the robustness and feasibility of our proposed methodology. According to the simulated results, the average magnitude estimation error of 0.2 magnitude units can be achieved using a PTW of 2 s for earthquakes with M ≤ 7. For the largest event considered herein, namely, the Ms 8.0 Wenchuan earthquake, a relatively stable magnitude estimate of 7.74 was obtained 12.3 s after the OT. Our approach offers clear advantages over the prediction approach using P10, and our approach will provide a robust magnitude estimate for M < 6.5 earthquakes within 8 s after the OT. Therefore, assuming Vs = 3.5 km/s, the lead time corresponding to a specific hypocentral distance (R) between the epicenter and target will be given by (R/3.5–8) s. A lead time of 6 s is available for a hypocentral distance of 50 km, and 20 s is available at 100 km. Specifically, for the Ms 8.0 Wenchuan earthquake, a relatively stable magnitude estimate of 7.74 can be obtained 12.3 s after the OT by our approach. Note that the region that was severely damaged by the Wenchuan earthquake (Beichuan) was almost 90 km away from the epicenter31; our method would provide the people in Beichuan a lead time of 13.4 s. Indeed, expanding the PTW of near-source stations cannot avoid the inclusion of S-waves; however, stable magnitude estimates were provided by our approach without severe overestimation caused by the contamination of S waves.In addition to robustly declaring the event magnitude, it is equally important for an EEW system to send an alert for the largest lead time . With regard to the timeliness, the proposed method needs only to obtain the initial information after P-wave arrivals, following which the method can automatically choose a specific strategy based on pre-established thresholds and magnitude relationships without complex computations; hence, this magnitude estimation method requires little execution time. Typically, the lead time is controlled by the difference in the P-S-wave traveltimes. Assuming a mean station spacing of 20 km within a network ensures that 3 stations are triggered within 5 s of the OTc and Pd10km thresholds to increase the reliability of the preliminary determination on whether the event is large, and based on the judgment for selectively applying period and amplitude parameters to predict the magnitude at the early stage of earthquake occurrence. When continuous waveform data from multiple stations are streamed to the network processing center, our approach combines the information from all available stations and chooses whether to extend the PTW to update the source parameters to provide the most robust warning, similar to a regional (network-based) warning. We believe this method can be widely used in other geographical regions to improve the robustness and timeliness of magnitude estimation. Accurate magnitude estimations closely related to the determination of a potential damage area will help those areas that will not receive alarms due to an underestimated magnitude; this will be conducive to reducing earthquake hazards and providing guiding to the emergency response a few seconds in advance.The proposed method was conceived to be applied to a regional EEW system and to provide robust source parameters (magnitude) with continuous information from a dense network. Our approach begins to provide a magnitude estimate after the first station is triggered, similar to the single-station, threshold-based onsite approach, which quickly provides approximate warnings on whether the following ground motion will be damaged. Similarly, our approach uses a four-entry decision table composed of τ32 was used to detect P-waves from vertical acceleration records, and the waveforms were checked manually to correct the P-wave arrivals as necessary. The acceleration signals were integrated once (twice) with respect to the velocity (displacement). A four-pole 0.075 Hz high-pass filter was used to remove low-frequency drift in the velocity and displacement. The peak amplitude Pd is the maximum absolute value of the vertical displacement in the first several seconds after the P-wave arrival. We applied an extended P-wave time window (2–10 s) to obtain the coefficients for the Pd attenuation relationship through least-squares multi-regression analysis:d, M and R are the peak displacement (cm), magnitude and hypocentral distance (km), respectively.An automatic P-wave picker described by Allend, we normalized Pd to a reference distance of 10 km to obtain Pd10km and calibrated the linear regression relationship between the event-averaged Pd10km and magnitude.To retrieve the magnitude dependence of Pc is defined as follows.0 is the time window over which τc is computed, starting at the first P-wave arrival and typically equaling 3 s. We averaged the τc values for all records for each event and used the least-squares method to fit an event-averaged τc value; this reduced scatter induced by site conditions and radiation patterns.The parameter τSupplementary Information 1.Supplementary Information 2.


Pancreatic and biliary tract cancers often present with non-specific symptoms, resulting in diagnosis at a late stage. This may be too late for curative surgery. Earlier detection and characterisation may guide treatment options and increase survival. Natural “circles” of RNA (circRNAs) are shown to regulate cancer-related genes, and act as cancer “biomarkers”. Recent research has shown that circRNAs are both abundant and stable, both of which are desirable characteristics for clinically useful biomarkers. In this systematic review, we describe the roles of circRNAs in pancreatic and biliary tract cancers, summarise the current published research and explore their utility as a biomarker. A total of 32 articles were included: 22 considering Pancreatic Cancer, 7 for Bile Duct Cancer and 3 for Gallbladder Cancer. CircRNA proved an exciting prospect as a biomarker for these cancers and future work should continue to develop and expand this field of research.Pancreatic Ductal Adenocarcinoma (PDAC) and biliary-tract cancers (BTC) often present at a late stage, and consequently patients have poor survival-outcomes. Circular RNAs (circRNAs) are non-coding RNA molecules whose role in tumourigenesis has recently been realised. They are stable, conserved and abundant, with tissue-specific expression profiles. Therefore, significant interest has arisen in their use as potential biomarkers for PDAC and BTC. High-throughput methods and more advanced bioinformatic techniques have enabled better profiling and progressed our understanding of how circRNAs may function in the competing endogenous RNA (ceRNA) network to influence the transcriptome in these cancers. Therefore, the aim of this systematic review was to describe the roles of circRNAs in PDAC and BTC, their potential as biomarkers, and their function in the wider ceRNA network in regulating microRNAs and the transcriptome. Medline, Embase, Scopus and PubMed were systematically reviewed to identify all the studies addressing circRNAs in PDAC and BTC. A total of 32 articles were included: 22 considering PDAC, 7 for Cholangiocarcinoma (CCA) and 3 for Gallbladder Cancer (GBC). There were no studies investigating Ampullary Cancer. Dysregulated circRNA expression was associated with features of malignancy in vitro, in vivo, and ex vivo. Overall, there have been very few PDAC and BTC tissues profiled for circRNA signatures. Therefore, whilst the current studies have demonstrated some of their functions in these cancers, further work is required to elucidate their potential role as cancer biomarkers in tissue, biofluids and biopsies. The incidence of pancreatic ductal adenocarcinoma (PDAC) has been increasing over the past three decades. Currently, almost 10,000 new cases are diagnosed each year in the UK, which is almost equal to the number of deaths related to PDAC . This isThe role of circRNAs has been demonstrated in various conditions, for example, diabetes  and AlzhIn this narrative and systematic review, we describe the characteristics, biogenesis and biological mechanisms of action of circRNAs. We then perform a systematic review aiming to describe original research into candidate circRNA molecules: all studies quantifying and considering the clinical implications of circRNAs dysregulation in PDAC and other biliary malignancies will be included. This will be discussed in the context of the wider literature, with a focus on their demonstrated molecular mechanisms and potential clinical utilisation.The vast majority of RNA molecules do not take part in protein translation and are therefore termed “non-coding” RNAs; this family accounts for 95% of the RNA pool ,9. This The inherent structure of circRNA results in a deficiency of 5′ to 3′ polarity and a lack of a polyadenylated tail . This afAs circRNAs typically do not code for a protein molecule and initially had no clear function they were thought to be a by-product of typical post-transcriptional mRNA modification: formed by “mis-splicing” or indistinguishable from RNA lariats ,24,25. DCircRNAs are formed during the post-transcriptional process of splicing pre-messenger RNA (pre-mRNA), where the canonical pathway typically excludes introns to form mature messenger RNA molecules as covalently linked exons. Back-splicing of pre-mRNA transcripts occurs with the formation of covalent bonds between a downstream 5′ splice donor site and an upstream 3′ acceptor site  19,27].,27.19,27quaking (QKI) [muscleblind (MBL/MBNL1) [The predominant mechanism for spliceosomal circRNA biogenesis relies on the presence of a reverse-complementary RNA sequence within introns flanking a potential circRNA . Exons ang (QKI)  and muscL/MBNL1)  or the iL/MBNL1) .6A motifs after specific RBPs recruitment [As circRNAs lack 3′ and 5′ ends degradation, requires an initiating step of internal cleavage, for which a number of pathways have been identified. Primary sequence-dependent degradation can be Ago2/miR-671 mediated, as demonstrated in ciRS-7 (Cdr1as) , or mediruitment . Howeverruitment . Additioruitment . This coruitment .One of the principal mechanisms through which circRNAs have been proposed to regulate biological mechanisms is labelled “miRNA sponging”. This describes a regulatory axis between non-coding RNA molecules and mRNA termed the competing endogenous RNA (ceRNA) network ,14,43. MAs mentioned, circRNAs can function as a direct miRNA sponge assisted by AGO2, one of the RBPs, but circRNAs can also act in an indirect way on mRNA transcription through RBPs, or compete with mRNA to combine with RBPs, to influence mRNA translation. Examples include circANRIL (circular isoform of the Antisense Non-coding RNA In the INK4 Locus), which was shown in vitro to control ribosomal RNA maturation by binding to a domain of Pescadillo Ribosomal Biogenesis Factor 1 (PES1), thereby preventing pre-rRNA binding and exonuclease-mediated rRNA maturation . Other eIn the post-transcriptional setting, circRNAs share exons with linear isoforms. Spliceosomal mediated generation actively competes with canonical linear splicing. This may limit the production of linear homologs and has the potential to promote alternate “exon lacking” variants which may have attenuated or absent biological function . For exa6A motifs are widely prevalent in circRNAs and likely to have a role in regulating protein synthesis, demonstrating the ability to enable and accelerate this process in vitro [The direct translation of circRNAs into proteins, however, remains controversial . It has in vitro .CircRNAs have been proposed as a source of non-colinear pseudogenes  . Retro-tOne of the biggest challenges in the study of circRNAs has been identification and differentiation from other non-coding RNA molecules. Early recognition has been driven through identification of the “back-spliced” sequences . One limTM) investigates for 13,617 circRNAs and was utilised by two studies comparing circRNAs expression in PDAC and para-cancerous tissue. These two projects identified 351 (fold change of >1.5) and 289 (fold change of 2) dysregulated circRNAs expression profiles respectively as potential targets for future investigation. The results were submitted to the Gene Expression Omnibus under numbers GSE69362 (6 paired samples) [CircRNA profiling can be broadly divided into two techniques: tissue microarray or RNA sequencing. Tissue microarray is a high throughput technique in which specific circRNA junction sequences are identified to quantify expression. The Arraystar human CircRNA array (Arraystarsamples)  and GSE7samples) . Other psamples) , but is samples) .After identification of specific candidate circRNAs, the expression can be quantified via Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). To achieve this, outward-facing primers are utilised with a complementary nucleotide sequence to the “back-splice” . This isRNA research techniques can be limited by RNA degradation, although meticulous handling in an RNase-free environment and minimisation of pre-analytical time from sample to bench can limit this to an extent. This impact may be further attenuated when working with circRNAs, due to their increased stability, and resistance to enzymatic degradation. Currently, the majority of circRNAs research utilises fresh frozen tissue samples, although some researchers have considered the role of alternate tissue sources. Indeed, one study considered the potential of fresh frozen paraffin-embedded (FFPE) tissue samples . This sthttps://circinteractome.nia.nih.gov) is one web tool that has been developed to explore circRNA structure and potential interaction with RNA binding proteins (RBPs) and miRNA within the ceRNA network [The ability for researchers to identify potential ceRNA networks through complementary binding sites has advanced significantly with the expansion of publicly available sequence databases and bioinformatic technology. CircInteractome  and Arraystar’s homemade miRNA prediction software, miRanda and StarBase (for miRNA and mRNA interactions) . IdentifPublicly available datasets such as Gene Ontology (GO) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) allow evaluation of potential interplay between ceRNA networks and known genetic and molecular pathways ,74,75. GThe defined search strategy identified 270 articles of which 129 duplicates were excluded. At title/abstract screening 62 articles were excluded as clearly unrelated to the study question or excluded article type. On full-text review, a further 47 articles were excluded that did not present unique data with adequate candidate evaluation. No additional studies were identified through the reference review. Therefore, 32 articles were included in this systematic review: 22 PDAC, 7 CCA and 3 GBC. This is summarised in Twenty studies validating circRNAs expression in PDAC were included, all of which utilised real-time quantitative polymerase chain reaction (RT-qPCR) to evaluate . A totalBioinformatic techniques were utilised by 20 groups to evaluate the relationship of circRNAs molecules in the ceRNA network, demonstrating a number of complementary miRNA binding sites to hypothesis regulatory roles over molecules and molecular pathways .CircRNA expression in PDAC cell lines generally correlates with the differential expression demonstrated in tissue samples. Furthermore, cell studies have allowed quantification of the impact of differentially expressed circRNAs with cellular features of malignancy  . In thosAnimal studies have demonstrated that PDAC cells in which implicated circRNAs are overexpressed are associated with increased tumour size and evidence of metastatic disease, while silencing results in a reduction of tumour size and growth .Given the potential mechanisms and cellular effects that are described, it is not surprising to find that the dysregulation of implicated circRNAs in PDAC is associated with poor prognostic features and ultimately shortened survival time . No assoLess work has been undertaken to explore the role of circRNAs in other biliary malignancies, although studies investigating both CCA and GBC have been published. A total of 7 studies considering CCA, and 3 considering GBC were identified .Within CCA, the circRNA hsa_circ_0005230 has been demonstrated to be upregulated in cancer tissue compared to control, and correlated with increased tumour size, lymphatic spread and disease stage . Its actWith regards to GBC, the circRNA hsa_circ_0008234, or circFOXP1, demonstrated increased expression in cancer compared to controls . Higher Molecular pathways and proteins that are associated with the development of the malignant features in PDAC are demonstrated to be part of dysregulated circRNAs ceRNA networks . Various3K/AKT, with evidence that these can be influenced by circRNAs. EGFR is an upstream regulator and activator of a number of pathways associated with the development of malignancy, including the Signal Transducers and Activators of Transcription (STATs). Activated STAT may then stimulate specific oncogenes, leading to the development of malignancy. CircRNAs are shown to have a regulatory role, for example, ciRS-7 (Cdr1as) is able to promote the EGFR/STAT3 pathway through suppression of miR-7 in the ceRNA network [3K/AKT pathway, PI3K is activated through ligand binding, which phosphorylates AKT, again promoting cellular proliferation. This pathway has been implicated in the growth and progression of PDAC with emerging evidence that circRNAs play a role in its regulation [Enhanced proliferation can be achieved through a number of mechanisms, including resistance to apoptosis. A number of molecular pathways regulate the cell cycle including EGFR/STAT and PI network . In the gulation . HOXC6, gulation .The integrity of the cellular micro-environment, including cell–cell interactions and extracellular matrix, is key to homeostasis. Both interruption of the endothelial barrier and disruption of the extracellular membrane permit cellular dissemination, synonymous with the features of invasion and metastasis in malignancy. Circ-IRAS has been implicated in the development of tumour invasion and metastasis through attenuating the cell tight junction barrier. Attenuation of miR-122 within the ceRNA network results in increased RhoA activity with subsequent increase in F-actin and decrease ZO-1 tight membrane proteins. This results in increased endothelial monolayer permeability .In addition to acting through the “miRNA sponge” studies have associated circRNA protein binding with the progression of malignancy, for example between circFOXK2 and both YBX1 and hnRNPK . The forAngiogenesis is critical in both physiological and pathological states . It is aLymph node dissemination, enhanced by lymphangiogenesis, is one pathway though to be responsible for rapid spread and metastasis in PDAC . CurrentBoth CircUBAP and hsa_circ_0000977 have been hypothesised to promote malignancy in PDAC through attenuation of the host immune system ,110. CirCurrent research has demonstrated a relationship between circRNAs and the process of epithelial to mesenchymal transition (EMT). Definable, and typically increased, regulation of many circRNAs has been demonstrated in cells that have been induced to go through this process . EMT is Identification of cancer stem cells (CSCs) can be achieved by evaluation for surface markers, such as CD44 and CD133, via immunostaining, or by performing sphere formation assays and in vivo tumour initiation assays. CircRNAs have been shown to maintain CSCs through a variety of mechanisms, such as WNT and Notch signalling regulation . CircFOXGemcitabine resistance is one of the principal challenges in providing effecting chemotherapy for advanced PDAC. Although the underlying mechanisms remain unclear, previous work has demonstrated that overexpression of some members of the ceRNA network is associated with chemotherapy resistance in PDAC, driving the loss of miR-410-3p . More reThese studies clearly demonstrate significant differential expression of certain circRNAs molecules in pancreatic and biliary tract tumours compared to normal tissues. Furthermore, this differential expression can persist in both serum and circulating exosomes. Both in vivo and in vitro, specific circRNA expression has been associated with features of malignancy. These data, along with increasing evidence that these molecules are abundant and stable, demonstrate the significant potential for utilisation of circRNAs as biomarkers in PDAC and BTC as diagnostics and prognostics, with the potential for developing future predictive and therapeutic applications. Therapeutic applications would be related to their individual biological roles, including their relationship with the features of malignancy, and any association with the development of chemotherapeutic resistance.Consideration of circRNAs as biomarkers has been undertaken in a variety of gastrointestinal cancers. One meta-analysis included 13 cancer studies: 7 gastric, 5 hepatocellular and 1 colorectal, demonstrating an area under the Receiver Operating Curve (AUC) of 0.81 for single circRNA candidates, with a corresponding sensitivity of 0.72, and specificity of 0.77 . Its posOne limitation when using tissue for diagnostics is the invasive nature of sample collection, and this is typically obtained at the time of surgery when the diagnosis is already clear. This has led to the consideration of other potential sites of interest, including saliva, shown to be relevant in other malignancies , both cyBoth plasma and serum are easy to isolate from peripheral blood samples, with defined standard operating procedures . Two stuExtracellular vesicles (EVs) are a compartment that is received increasing attention. Exosomes are 40–150nm sized EVs generated from the plasma membrane of cells as part of the endosome pathway . These sThe profile of exosomal circRNAs expression has been described in one study that sequenced RNA in 14 serum samples (GSE100232) . This stFurther to acting as a diagnostic biomarkers, plasma/serum circRNAs level may allow prognostication of disease and prediction of response to treatments. Thus, dysregulated peripheral blood circRNA expression has been strongly correlated with tumour factors and survival outcomes in PDAC, and unlike pathological evaluation, can allow a non-invasive, real-time, assessment of the tumour transcriptome. This “liquid biopsy” has the potential to allow proactive monitoring of the disease process, permitting improved recognition of treatment success or failure, and act as a step towards tailor-made treatments . RegularWith curative surgical intervention possible in only a limited number of PDAC patients , primarySome circRNAs, such as hsa_circ_0001649, have been demonstrated to generate tumour suppressive effects, and these offer potential as therapeutic targets . StimulaExosomes and other EVs have been suggested as a possible delivery vehicle for anti-cancer therapy. In animal models, exosomes have been used to increase the therapeutic index of doxorubicin , and theAt present, the circRNA component of the transcriptome has only been assessed in a limited number of PDAC tissue samples. PDAC is a heterogeneous disease characterised by multiple genetic abnormalities, often present in many core signalling pathways , and so Researchers have also considered the differential circRNA expression in other clinically useful samples other than tissue , however these avenues require further exploration. Blood sampling in particular may offer a simple, and non-invasive approach to investigating PDAC, potentially allowing real-time assessment of response to cancer treatment. However, only a limited number of circRNAs have been investigated in PDAC as blood-based biomarkers to date ,116,127.https://www.arraystar.com/), the latter implementing its own circRNA naming conventions. Arraystar does not offer the complete annotations for their circRNA probe set that would be needed to systematically compare sequencing data and microarray data, which effectively promotes a division of the circRNA field into microarray studies and sequencing studies.The circRNA field is currently suffering from a lack of naming convention. In the early days of whole-transcriptome-based circRNA studies (from 2013 to 2015), circBase was the primary database supporting circRNA research , and as Finally, the utility of circRNAs for liquid biopsy requires consideration in the context of other relevant biomarkers, including circulating tumour cells (CTCs), cell-free DNA, and other RNA molecules ,141,142.This systematic review was performed in keeping with the PRISMA guidelines. This review is based on previous study results and so no ethical approval or consent was required. This review was registered on the National Institute for Health Research (NIHR) PROSPERO database, identification number CRD42019156889.Medline, Embase, Scopus and PubMed were interrogated without limit on the time period with the following search strategy on the 16 April 2020. Both Title/Abstract and Medical Subject Heading (MeSH) were utilised (where available) for article identification, search strategy outlined below. Non-English and duplicated articles were excluded at this stage. Authors CL and AF performed title and abstract screening and for all identified articles, followed by full-text review. In addition, a manual search of identified reviews and referencing was performed to identify any additional relevant studies.(Circular RNA OR circRNA) AND . For each term, common synonyms and MeSH headings were incorporated.Articles types excluded at screening were those retracted, reviews, editorials, book chapters and conference abstracts. Additionally, those clearly not in the context biliary cancer or unrelated to the subject of interest were excluded.On full text review inclusion required quantification of circRNA expression in the context of biliary tract cancer and evaluation against the feature of malignancy in vitro, in vivo or in the context of clinical outcomes.Authors CL and AF performed data collection independently against a predefined database. After initial data collection, both authors undertook further data collection. For each article, the circRNA of interest was recorded along with related miRNA, mRNA, technique through which expression and relationships were validated; in vitro study outcomes; in vivo study outcomes; along with associated clinical tumour and patient evaluation.Meta-analysis was not performed. Data are presented in the subgroups of Pancreatic Ductal Adenocarcinoma and then other biliary tract malignancies.Of all the non-coding RNA molecules, the abundance, stability and tissue specific expression patterns of circRNAs lend them to use in diagnostics, prognostics and molecular-targeted therapies for various diseases including PDAC. These features have only recently been realised, and so it is unsurprising that circRNAs have received increased attention over the past few years. The differential expression profile of circRNAs in malignancies such as PDAC has now been demonstrated, with validation of several molecules of interest. The majority of investigated circRNAs in PDAC are upregulated and are associated with malignant cellular processes such as proliferation, invasion and metastasis. The proposed oncogenic mechanism is predominantly through competitive inhibition of miRNAs, frequently termed “miRNA sponge”, through the ceRNA network. Other proposed mechanisms include interaction with RBPs, protein and peptide regulation, and pseudogene generation. It is ultimately likely to be through a range of actions dependant on the specific circRNA molecules in question, with further work required to expand this understanding. Bioinformatic analysis has enhanced our ability to understand the interaction of circRNAs within genetic and molecular pathways, but has also served to illustrate the complexity of these networks.Like any investigation, the consideration and implementation of circRNAs as biomarkers for PDAC must proceed with caution. Despite the demonstrated significance in diagnostics and prognostics, there is still large room for error. The role of circRNAs must be considered in the wider context of each disease stage, and the treatments given. Ultimately, the future of PDAC diagnosis, prognosis and treatment is likely to be planning individualised care to each patient, and although research is starting to granulate the role of circRNAs and the ceRNA network in PDAC, it is clear that there is still a long way to go. Future research should continue to map out the differential circRNA expression profiles in PDAC, and establish how deregulated circRNAs are associated with tumour and clinical features. This should be expanded to include clinically useful samples such as blood, biopsies and biofluids. Evaluation of specific circRNAs that may have a role in diagnosis and prognostication should continue to be evaluated alone, and as a panel, as well as in combination with other diagnostic tests, including established tumour markers.