The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues , increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR. The human enzyme MTHFR links the folate and methionine cycles, which are essential for the biosynthesis of nucleotides and proteins. Here, the authors present the crystal structure and biochemical analysis of human MTHFR, providing molecular insights into its function and regulation in higher eukaryotes. Since the product CH3-THF is exclusively used by methionine synthase, and only the demethylated form (THF) may be recycled back to the folate cycle, MTHFR commits THF-bound one-carbon units to the methionine cycle.In humans, the folate and methionine cycles both generate products essential to cellular survival. Folate, the major cellular carrier of single carbon units, is required for the synthesis of purines and thymidine monophosphate. Within the methionine cycle, the methylation of homocysteine to methionine by methionine synthase (EC 2.1.1.13) produces an essential amino acid which may be used for protein synthesis or, crucially, be further converted to S-adenosylmethionine (SAM), a vitally important donor for the methylation of DNA, RNA and proteins as well as the creation of numerous methylated compounds. These two cycles intersect at the enzyme 5,10-methylenetetrahydrofolate reductase . MTHFR catalyzes the physiologically irreversible reduction of 5,10-methylene-tetrahydrofolate  and private2. Milder enzyme deficiencies, due to single nucleotide polymorphisms of the MTHFR gene, have been associated with various common disorders. The most studied of these is p.Ala222Val , identified as a risk factor for an overwhelming number of multifactorial disorders, including vascular diseases, neurological diseases, various cancers, diabetes and pregnancy loss (see e.g. review by Liew and Gupta3).In accordance with this essential role, major and minor deficiencies of human MTHFR are the direct or indirect causes of human disease. Severe MTHFR deficiency (MIM #607093) is inherited in an autosomal recessive manner and is the most common inborn error of folate deficiencyEscherichia (E.) coli7 and Thermus thermophilus8, in which the catalytic domain constitutes the entire sequence  barrel and have uncovered residues critical for binding the cofactor FAD4, the electron donor NADPH (NADH in bacteria7) and the product CH3-THF7. The bacterial structures, together with activity assay of trypsin cleaved porcine MTHFR9, indicate that the catalytic domain is sufficient for the entire catalytic cycle. Eukaryotic MTHFR orthologs additionally possess a C-terminal regulatory domain that is connected to the catalytic domain by a linker sequence 13, the demethylated form of SAM.Human MTHFR is a 656 amino acid multi-domain protein Fig. . The catnce Fig. , have bence Fig. . This C-14 and yeast15, or following immunoprecipitation from human cancer cell lines16. Phosphorylation has been associated with moderately decreased catalytic activity16 and increased total inhibition mediated by SAM14. Although phosphorylation mapping of this region has been thus far unsuccessful, scanning mutagenesis has revealed substitution of alanine for threonine at position 34 (p.Thr34Ala) to almost completely block phosphorylation15, suggesting Thr34 is the priming position. The cellular relevance of this modification remains unclear, although one group has suggested that phosphorylation at Thr34 can be accomplished by CDK1/cyclin B1 (ref. 16) and at Thr549 by polo-like kinase 1 (ref. 17) whereby they posit a role in histone methylation and replication.Human MTHFR further contains a 35 amino acid serine-rich region at the very N-terminus which is not found in MTHFR orthologs of bacteria, yeast or even lower animals Fig. . This reThe repertoire of bacterial MTHFR structures to date does not provide any mechanistic insight into the enzymatic regulation by phosphorylation and SAM binding, because both features are absent in prokaryotes. To this end, we have combined structural, biophysical and biochemical data of human MTHFR to provide a molecular view of MTHFR function and regulation in higher eukaryotes. We have identified specific phosphorylation sites and demonstrate a distinct relationship between phosphorylation and SAM inhibition. Further, using our 2.5 Å resolution crystal structure of the almost full-length human protein, we reveal that the regulatory domain utilizes a unique topology to bind SAH/SAM and transmit a catalytic inhibition signal, likely by long-range conformational change through the linker region.Hs)MTHFR, we generated full-length recombinant human MTHFR (HsMTHFR1–656) by baculovirus expression in Sf9 cells. Mass spectrometry-based phosphorylation mapping (with 92% coverage) identified 16 separate phosphorylation sites in HsMTHFR1–656 following purification from Sf9 cells   Fig. . All phoR HsMTHFR–656 by bed) Fig. . Additioed) Fig.  or 10 (Fed) Fig.  phosphat16. To investigate this more thoroughly, we used a very sensitive high-performance liquid chromatography (HPLC)-based activity assay which monitors the full enzymatic reaction in the physiological direction and allows determination of kinetic values18. Overall, we found similar kinetic values for HsMTHFR1–656 and HsMTHFR38–644  and kcat values (51.4 ± 4.2 vs. 40.7 ± 3.2 s−1), suggesting turnover was not meaningfully reduced. These values are comparable but higher than previous determinations from recombinant HsMTHFR (12.4 µmol min−1 mg−1 (ref. 13)) and purified porcine MTHFR (19.4 µmol min−1 mg−1 (ref. 19)). Importantly, CIP-treated HsMTHFR1–656 (dephosphorylated) retained nearly identical activity values to mock-treated HsMTHFR1–656  (Table KM) for the substrate CH2-THF (range: 21.3–25.5 µM) and electron donor NADPH (range: 23.5–35.5 µM) were very similar for all four proteins . Thus, we conclude that our assay is sensitive and specific, and that phosphorylation does not significantly alter MTHFR kinetic properties.Phosphorylation has been described to alter MTHFR kinetics, resulting in moderately decreased catalytic activity as measured by the NADPH-menadione oxidoreductase assayHsMTHFR1–656 which, in addition to phosphorylation, contained equivalent units of FAD , we were able to inhibit all recombinant HsMTHFR proteins by over 95%, regardless of the phosphorylation state  of ~3 µM, while CIP-treated HsMTHFR1–656 was approximately two-fold less sensitive to SAM inhibition, and HsMTHFR38–644 seven-fold less sensitive  derivatized protein , Pro (4), Glu (3) and Leu (3). Mediated by three β-turns, this linker makes multiple intricate contacts with both domains, and changes direction twice in traversing between the catalytic and regulatory domains.We determined the 2.5 Å resolution structure of in Table . The HsMins Fig.  to form ins Fig. , with itHsMTHFR38–644 structure allows the mapping of the 70 inherited missense mutations known to cause severe MTHFR deficiency, which lie on 64 different residues of the polypeptide  as the regulatory domain (20), with the remainder (6) found in the linker region. By proportion, however, the linker region has a higher density (24% of the sequence) of mutation sites than the catalytic (11%) and regulatory (7%) domains. Additionally, a number of sites in the catalytic and regulatory domains are in direct contact with the linker region. Further, the most severe mutations, those found either homozygously or in conjunction with a truncating mutation to result in enzymatic activity below 1.5% of control activity in patient fibroblasts20, cluster in the catalytic domain and the first two aa of the linker region, most of which are located where the linker meets the catalytic domain , strand β16, and the loop encompassing Asn386–Asn391  is ordered, while that of the other dimer subunit (chain B) is highly disordered, to the extent that only main chain atoms of the amino acid 40–58, 129–134 and 155–342 in chain B could be modelled.Within the homodimer, each of the two catalytic domains is presented away from the dimeric interface and their active sites are at opposite ends of the overall shape and face away from each other Fig. . In thisHsMTHFR38–644 crystal structure has captured the snapshot of an asymmetric dimer whereby the two catalytic domains have different orientations with respect to their own regulatory domains , and thus also permitting rigid body movement of these subunits in relative orientation to each other, we obtained a significantly improved fit  and treated with CIP (i.e. dephosphorylated and bound with SAH). The experimental scattering curves for as purified and CIP-treated HsMTHFR1–656 gave rise to slightly different profiles and derived parameters  to 1.56 Å resolution (Table HsMTHFR), low eukaryotic (ScMET12) and bacterial  orthologues. Consistent with their sequence conservation , ScMET12 and T. thermophilus MTHFR, this helix α1 is projected towards the interface between catalytic and regulator domains.The MTHFR catalytic domain adopts a TIM-barrel structure evolutionarily conserved across all kingdoms. In addition to HsMTHFR38–644, clear electron density for FAD was observed in the TIM barrel of chain A , the binding site in ScMET121–301 and subunit A of HsMTHFR38–644 is blocked by a crystal packing interaction from a nearby symmetry mate, making π–π stacking interactions with the FAD ligand H and substrate CHHsMTHFR38–644 structure with that of EcMTHFR bound with NADH   and the first strand β10 (T481INSQ485) of the central β-sheets, where a number of conserved residues are found. For example, Thr481  and Ser484 (98%) hydrogen-bond to the SAH adenine moiety, while Glu463 (99%) and Thr464 (62%) fixate the ribose hydroxyl groups. The strongest sequence conservation in the SAH binding site is found around the homocysteine moiety, including Pro348 (invariant) and Trp349 (99%) from the linker region, as well as Thr560 and Thr573 (both invariant) at the start and end of the β15-β16 turn. The SAH homocysteine sulphur atom is loosely contacted by Glu463 (3.8 Å) and Ala368 (3.7 Å). SAM is expected to bind to the same site in the regulatory domain, in a similar extended configuration as SAH and requiring the same set of binding residues. However, the additional methyl group in the sulphonium centre of SAM would create a steric clash to the Ala368 position of the structure  , Arg357 (HsMTHFR357–656) and Arg377 (HsMTHFR377–656) .Since there is no direct interface between the active site of the catalytic domain and the regulatory domain Fig. , SAM bin56) Fig. . All thr56) Fig. . This ag348–656 to either SAH or SAM resulted in shifts of elution volume (Ve) compared to as purified (apo-) protein  and SAH a rightward shift   as a means to study solution behaviour of the MTHFR regulatory domain in response to SAM/SAH binding. Exposure of MTHFRein Fig. , in contme) Fig. . By contand Fig. . A similand Fig. . Therefo348–656 resulted in protein that could no longer bind SAM  could lead to loss of SAH/SAM binding, and thus conformational change. Indeed, conservative mutation of Glu463 to either aspartate (p.E463D) or glutamine (p.E463Q) on MTHFRCatalytic regulation by phosphorylation and SAM binding distinguishes human MTHFR from its bacterial (which do not have phosphorylation or SAM binding regions) and lower eukaryotic (which do not have a phosphorylation region) counterparts. Until now, the molecular basis of how these two allosteric events modulate the catalytic machinery was entirely unknown, due to the absence of a structural context. Now, our structure-guided study has provided two major discoveries in this area: (1) identification of an extensive linker region that functionally connects SAM-binding in the regulatory domain with inhibition in the catalytic domain and (2) demonstration of the concerted effects of phosphorylation and SAM binding, individually mediated by regions more than 300 amino acids apart  as predicted from the sequence but also within the catalytic (3) and regulatory (2) domains. Many of the N-terminal phosphorylation sites identified are consistent with previous mutation analysis14, including Thr34 (refs. 16). The phosphorylated residues detected in the catalytic and regulatory domains were not reported before. Interestingly, two phosphorylated serines are located within the FAD binding site, although their physiological significance is currently unclear. Contrary to the recent observation of Li et al.17 we did not identify phosphorylation of Thr549.We mapped the entire phosphorylation landscape of 30 and in crystallo31, to avoid dis-inhibition by SAH. Secondly, phosphorylation could induce a conformational change to the protein that primes an inhibition ready state. The SAM Ki differences between phosphorylated and dephosphorylated protein, while relatively small (2–3 vs. 6–7 µM), are likely to be physiologically relevant. Intracellular SAM concentrations are reported to be 1–3 µM in human cells33, and the mTORC1 linked starvation sensor SAMTOR, which recognizes SAM for nutrient sensing, has a SAM dissociation constant of 7 µM34.An important finding with regard to MTHFR phosphorylation is that it does not directly alter the catalytic parameters of the enzyme, as determined by a sensitive HPLC-based activity assay. Perhaps this is not too surprising, since the first ordered residue of the structure, Glu40 (i.e. immediately following the phosphorylation region aa 1–37), is far removed from the catalytic site. Instead, MTHFR phosphorylation exerts a long-range influence on the SAM binding status at the regulatory domain some 300 amino acids away, by causing an increased sensitivity of the enzyme to SAM inhibition, but with no overall changes on total SAM inhibition. Phosphorylation likely enhances SAM sensitivity in two interdependent ways. Firstly, it enables protection of bound SAM from spontaneous degradation to SAH, a phenomenon widely observed for SAM-bound enzymes in vitroHsMTHFR are identified in this study, intact mass analysis shows that only 9–10 phosphorylations were present at a time, implying a high degree of heterogeneity. While multiply phosphorylated peptides were observed, they were not quantified using our methods. Identification of MTHFR proteoforms to assess whether phosphosites occur in combinations, perhaps using top-down MS35 would be very interesting, and may constitute the basis of future studies. Evolutionary conservation of the identified phosphorylation sites varies from absolute invariance through to yeast (e.g. Ser394), to poor conservation even among animals (e.g. Ser9 and Ser10) , whereby the additional metabolite-binding modules, not found in their bacterial counterparts, serve to fine-tune catalysis in response to the more intricate higher eukaryotic metabolic and signalling cues. We propose that MTHFR belongs to this class of allosteric enzymes that share a common mechanism—to regulate catalysis through steric sequestration of the catalytic site, in a ligand-dependent manner .We identified the MTHFR regulatory domain to constitute a novel SAM binding fold whose appendage to the well-conserved catalytic TIM-barrel is a relatively recent and contained evolutionary event. A similar phenomenon of domain organization is found in several eukaryotic enzymes, for example those involved in amino acid metabolism , the potential for inter-domain conformational changes is suggested by the following data. Firstly, the two chains in the crystal asymmetric unit show varying intrinsic order of the catalytic domain with respect to the regulatory domain. Secondly, SAXS and native mass spectrometry analysis between SAM-bound phosphorylated protein and SAH-bound dephosphorylated protein hint at subtle, but distinguishable changes to the protein dimensions. It remains to be determined from future studies whether MTHFR undergoes ligand-dependent conformational changes to rearrange domain orientation for catalysis, like PAH and CBS. If this should be the case, the MTHFR linker region will likely contribute to this role, as it makes extensive contacts with both the regulatory domain (e.g. SAM binding site) and catalytic domain (e.g. helices α3 and α4). This is also consistent with the concentration of deleterious disease mutations found in this region. Additional genetic data from our lab are in accord13, and suggestive of FAD being only loosely bound, as exemplified in chain B of our structure. Furthermore, a number of MTHFR mutations22 and polymorphisms13 are shown to affect FAD responsiveness. It is therefore possible that the inter-domain flexibility we observed, communicated by the SAM-bound signal, would alter the orientation of catalytic domains with respect to the rest of the protein, in a similar manner as the multi-domain enzymes CBS and PAH38. Such structural conformations are supported by overlays between chains A and B in our structure, and between apo- and holo-subunits in TtMTHFR8. In the case of MTHFR, the active site could be more sequestered (leading to FAD bound) or more exposed and mobile (leading to FAD loss) as a consequence.So in what aspects could the SAM-bound signal influence the catalytic domain, seeing that its kinetic parameters remain largely unaltered? One possibility is an effect on the stability or integrity of FAD, the essential cofactor. We observed that supplementation with FAD enabled rescue of activity to our recombinant MTHFR. This is indicative of cofactor loss, in agreement with previous findings3-THF to improve throughput of the methionine cycle and replenish SAM levels. Conversely, a high SAM/SAH ratio means abundant methylation capacity, in which case SAM-mediated allosteric inhibition of MTHFR turns off CH3-THF production, thereby lowering methionine cycle activity and concomitantly generation of SAM. This on/off switch is especially powerful at high SAM levels, as illustrated by almost complete inhibition of recombinant HsMTHFR at >200 µM SAM. Although these types of concentrations are unlikely to be seen inside the cell, HsMTHFR has been further outfitted with a dimmer switch, whereby protein phosphorylation increases sensitivity to SAM-mediated inhibition at normal (1–10 µM) cellular SAM levels. In this regard, phosphorylation allows linkage of the methionine cycle to other cellular pathways (e.g. cell cycle) through specific kinase activities .The SAM/SAH ratio is regarded as an indicator of a cell’s methylation potential and is a crucial indicator of the cells’ capacity to perform DNA methylation or create compounds which require methyl groups for assimilation. In the face of a low SAM/SAH ratio, meaning methyl donor deficiency, MTHFR is dis-inhibited, increasing the production of CHHsMTHFR homodimer is smartly tailor-made to facilitate this correlation. (1) The dimeric interface is entirely constituted by the regulatory domain to form a scaffold, while leaving each SAM binding site on a different face for its sensing and signal transmission functions. (2) The lack of contacts between inter-monomeric catalytic domains allows for the intrinsic intra-monomeric mobility with respect to the regulatory domains for signal propagation. (3) Importantly, this dimeric configuration brings the N- and C-termini of the polypeptide in proximity, projecting the phosphorylation region close to the regulatory domain dimer interface.The clear correlation we observed between phosphorylated MTHFR with SAM binding in solution (vs dephosphorylated MTHFR and SAH binding) leads us to interpret that the two regulatory properties act in concert. In fact, the architecture of the 40. Our work here constitutes a strong starting point for future, more precise investigation by structural, biochemical and cellular studies, for example towards: identification of the kinase(s) responsible for MTHFR phosphorylation in vivo; combining different structural methods to delineate conformational changes of the entire protein, and revealing the molecular basis of its specificity over NADPH.In summary, we provide a structural view of a eukaryotic MTHFR, pointing to the linker region playing a direct role in allosteric inhibition following SAM binding, and phosphorylation as a means to modulate SAM inhibition sensitivity. Modulating such finite control towards the level of a key metabolite may be of pharmacological interest, including in cancer metabolismE. coli (BL21(DE3)R3) expression, DNA fragments encoding human MTHFR (IMAGE: 6374885), mouse MTHFR (IMAGE: 6834886) and yeast MET12  harbouring different N- and C-terminal boundaries were amplified and subcloned in pNIC28-Bsa4 vector (accession number: EF198106) in-frame with a tobacco etch virus protease cleavable N-terminal His6-tag. For baculovirus expression, DNA fragments encoding human MTHFR (IMAGE: 6374885) harbouring different N- and C-terminal boundaries were cloned into the pFB-CT10HF-LIC vector (Addgene plasmid: 39191) in-frame with a tobacco etch virus protease cleavable C-terminal flag/His10-tag. Site-directed mutations were constructed using the QuikChange mutagenesis kit (Stratagene) and confirmed by sequencing. All primers are available upon request. Proteins expressed in E. coli were purified by affinity  and size exclusion  chromatography. Proteins expressed in insect cells in Sf9 media (ThermoFisher) were purified by affinity  and size exclusion (Superdex 200) chromatography, followed by cleavage of the C-terminal tag by His-tagged tobacco etch virus protease (1:20 mass ratio) overnight at 4 °C and re-passaged over Ni-NTA resin. Selenomethionine (SeMet)-derivatized proteins were expressed using SelenoMethionine Medium Complete (Molecular Dimensions) and purified as above.Numbering of the nucleotide changes follows the nomenclature of NM_005957.4, which places the A of the ATG initiation codon as the +1 nucleotide. The protein is numbered according to NP_005948.3. For ScMET121–302 as well as SeMet-derivatized and native HsMTHFR38–644 were concentrated to 15–20 mg mL−1, and crystals were grown by sitting drop vapour diffusion at 20 °C. The mother liquor conditions are summarized in Table 41). The HsMTHFR38–644 structure was solved by selenium multi-wavelength anomalous diffraction phasing using autoSHARP42, and subjected to automated building with BUCCANEER43. The SeMet model was used to solve the native structure of HsMTHFR38–644 by molecular replacement using PHASER44. This structure was refined using PHENIX45, followed by manual rebuilding in COOT46. Phases for ScMET121–302 were calculated by molecular replacement using 3APY as model. Atomic coordinates and structure factors for both ScMET121–302 (accession code: 6FNU) and HsMTHFR38–644 (accession code: 6CFX) have been deposited in the Protein Data Bank. Data collection and refinement statistics are summarized in Table HsMTHFR38–644 and ScMET121–302 contain respectively 95.6% and 98.6% in the favoured region, 4.3% and 1.1% in the allowed region, and 0.17% and 0.36% in the outlier region of the Ramachandran plot.Purified native 18 with modifications as described by Rummel et al.47 and Burda et al.22. Only minor adaptations were made for use with pure protein, including reducing the assay time to 7 min and the addition of bovine serum albumin (BSA) to keep purified proteins stable. Prior to assay, purified proteins were diluted from 15–20 to 1 mg mL−1 in 10 mM HEPES-buffer pH 7.4, 5% glycerol and 500 mM NaCl followed by successive dilutions of 1:100 and 1:32 in 10 mM potassium phosphate, pH 6.6 plus 5 mg mL−1 BSA, to a final MTHFR concentration of 312.5 ng mL−1. Specific activity was measured in 50 mM potassium phosphate buffer, pH 6.6, under saturating substrate concentrations  with and without the addition of FAD . The KM for NAPDH was determined by varying its concentration between 10 and 250 µM in the presence of 75 µM CH2-THF. The KM for CH2-THF was determined by varying its concentration between 2.5 and 100 µM in the presence of 75 µM NAPDH. All KM values were derived using a non-linear fit of Michaelis–Menten kinetics by GraphPad Prism (v6.07). For SAM inhibition, purified SAM48 was used. The Ki was estimated following a plot of log(inhibitor) vs. response and a four parameter curve fit as performed by GraphPad Prism (v6.07).All enzymatic assays, including SAM inhibition and thermolability, were performed using the physiological forward assay described by Suormala et al.−1), SYPRO-Orange (Invitrogen) diluted 1000× and buffer  in the presence of 0–250 µM SAM or SAH. Fluorescent intensities were measured from 20 to 78 °C with a ramp rate of 1 °C min−1. The temperature shifts, ΔTmobs, were calculated using a Boltzmann sigmoidal fit49, and half-maximal effective ligand concentrations, AC50, for each ligand was fit to a one-site total binding model, both using GraphPad Prism (v6.07).Analytical gel filtration was performed on a Superdex 200 HiLoad 10/30 column  pre-equilibrated with 10 mM HEPES pH 7.5, 150 mM NaCl and 5% glycerol in the presence or absence of 250 µM SAH or SAM (both Sigma-Aldrich). The column was calibrated using carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase (150 kDa) and apoferritin (443 kDa) as protein standards. Differential scanning fluorimetry was used to assay shifts in melting temperature caused by ligand binding in a 96-well PCR plate using an LC480 light cyler (Roche). Each well (20 µL) consisted of protein (0.1 mg mLHsMTHFR38–644 and HsMTHFR1–656 (phosphorylated and dephosphorylated) were performed at 0.99 Å wavelength Diamond Light Source at beamline B21 coupled to the Shodex KW403-4F size exclusion column  and equipped with Pilatus 2 M two-dimensional detector at 4.014 m distance from the sample, 0.005 < q < 0.4 Å−1 . The samples (20 mg mL−1) were in a buffer containing 20 mM Hepes-NaOH pH 7.5, 0.5 mM TCEP, 150 mM NaCl, 2% glycerol and 1% sucrose. SAXS measurements were performed at 20 °C, using an exposure time of 3 s frame−1. To perform dephosphorylation, as purified (phosphorylated) HsMTHFR1–656 was added with 10 mM Mg-acetate in the above buffer and incubated with 100U calf alkaline phosphatase at 37 °C for 2 h. SAXS data were processed and analysed using the ATSAS program package21 and Scatter (http://www.bioisis.net/scatter). The radius of gyration Rg and forward scattering I(0) were calculated by Guinier approximation. The maximum particle dimension Dmax and P(r) function were evaluated using the programme GNOM50. To demonstrate the absence of concentration-dependent aggregation and interparticle interference in the both SAXS experiments, we inspected Rg over the elution peaks and performed our analysis only on a selection of frames in which Rg was most stable. Overall, such stability of Rg over the range of concentrations observed in the SEC elution indicates that there were no concentration-dependent effects or interparticle interference. Ten runs of CORAL rigid body modelling were performed by defining residues 338–345 as a flexible linker and allowing the catalytic subunits to move while keeping the regulatory subunits fixed.SAXS experiments for the −1 in 0.1% formic acid and 50 µL was injected on to a 2.1 mm × 12.5 mm Agilent Zorbax 5um 300SB-C3 guard column housed in a column oven set at 40 °C. The solvent system used consisted of 0.1% formic acid in LC-MS grade water (solvent A) and 0.1% formic acid in LC-MS grade methanol (solvent B). Chromatography was performed as follows: initial conditions were 90% A and 10% B and a flow rate of 1.0 mL min−1. A linear gradient from 10% B to 80% B was applied over 35 s. Elution then proceeded isocratically at 95% B for 40 s followed by equilibration at initial conditions for a further 15 s. Protein intact mass was determined using a 6530 QTOF mass spectrometer (Agilent). The instrument was configured with the standard ESI source and operated in positive ion mode. The ion source was operated with the capillary voltage at 4000 V, nebulizer pressure at 60 psig, drying gas at 350 °C and drying gas flow rate at 12 L min−1. The instrument ion optic voltages were as follows: fragmentor 250 V, skimmer 60 V and octopole RF 250 V.Reversed-phase chromatography was performed in-line prior to mass spectrometry using an Agilent 1290 uHPLC system. Concentrated protein samples were diluted to 0.02 mg mL51. Briefly, 50 µg of protein was desalted and exchanged into 50 mM ammonium acetate pH 6.5 using three rounds of size exclusion spin column purification  following the manufacturer’s instructions. Approximately 50 µL was infused directly into the mass spectrometer via syringe pump at a rate of 6 µL min−1. The instrument was fitted with a standard source using a nebulizer pressure of 17 psi, drying gas flow rate 5 L min−1 and drying gas temperature 325 °C. The instrument was operated in positive ion, 1 GHz detector mode with fragmentor voltage 430 V, nebulizer pressure set to 17 psi, and collision gas pressure and energy at 0. Spectra were deconvoluted using the MaxEnt functionality within the Masshunter software package (Agilent) and multimeric states were determined manually using a charge table. The radius for native protein charge states was determined using the calculation z = R1.5 or R = z × 101/1.5 where z is the charge state and R is the radius in Ångstroms if the protein is assumed to be a sphere52. To allow comparison between spectra, ion intensities were normalized by dividing each value by the largest in that spectrum. Normalized ion intensity was plotted against charge radius for each ion.Mass spectrometry of MTHFR under native conditions was performed on the same MS instrument, and is described in detail elsewhere2 chromatography matrix (GL Sciences) and 2.5 mg ZrO2 powder. Non-phospopeptides were eluted using 80% ACN, 300 mg mL−1 DHB, 0.1% TFA. Phosphopeptides were eluted in 25% ammonium hydroxide, 40% ACN. Samples were dried-down by rotary evaporation and re-suspended in 5 µL of 2% ACN, 0.1% FA prior to LC-MSMS.Between 20 and 100 µg MTHFR was reduced in 100 µL of 100 mM ammonium bicarbonate buffer, pH 7.5 by addition of 1 µL of 1 M DTT and incubation at 56 °C for 40 min. Alkylation was performed by addition of 4 µL of saturated iodoacetamide solution and incubated at room temperature in the dark for 20 min. Endoprotease digestion was performed using either trypsin, Smart Digest trypsin (Thermo) or pepsin. Trypsin in 100 mM ammonium bicarbonate buffer, pH 7.5 was added in the ratio 20:1 w/w and incubated at 37 °C overnight. Smart Digest trypsin was incubated at 70 °C for 1 h following the manufacturer’s instructions. Pepsin digestion was performed in 0.1 M HCl in the ratio 20:1 w/w and incubated at 37 °C overnight. LC-MSMS analyses were performed using both whole endoprotease digests and metal oxide-affinity enriched samples. Metal oxide-affinity enrichment for phosphopeptides was performed using home made spin columns containing a mixed bed of 2.5 mg Titansphere TiO−1. Buffer A was 0.1% formic acid in LC-MS grade water: Buffer B was 0.1% formic acid, 80% LC-MS grade ACN. MSMS was performed in data-dependent mode with a scan rate of 26,000 m z−1 s−1 with three precursors per MS1 scan and active exclusion for 20 s. Charge state selection was +1, +2 and +3. Automated data analysis was performed using Data Analysis v 4.0 (Bruker). Database searching was performed using an in-house Mascot server, in which the MTHFR database was searched for variable modifications including oxidation (M) and phosphorylation (ST or Y) with an MS tolerance of 1.4 Da, MSMS tolerance of 0.5 Da, partials at 4, C13 at 1 and charge states of +1, +2, +3. All putative phosphopeptide assignments were evaluated manually with an assumed false discovery rate of zero. Composite MSMS for MTHFR coverage was 92%.Analysis was performed using a Dionex U3000 nanoHPLC coupled to a Bruker Esquire HCT ion trap mass spectrometer. Peptides were separated using a 200 µm × 5 cm Pepswift PS-DVB monolithic column . A gradient was developed from 2 to 17% B over 4 min, then 17–42% B over 2 min. The column was washed at 92% B for 1 min and finally equilibrated at 2% B for 6 min at a flow rate of 2.5 µL min6CFX and 6FNU, respectively. The full mass spectrometry phosphorylation dataset has been submitted to MassIVE with the accession number MSV000082179. All other data are available from the corresponding authors upon reasonable request.The crystal structures of HsMTHFR and ScMET12 have been deposited in the protein data bank (PDB) with the identifiers Supplementary InformationPeer Review File


Sub-zero temperatures put plants at risk of damage associated with the formation of ice crystals in the apoplast. Some freeze-tolerant plants mitigate this risk by expressing ice-binding proteins (IBPs), that adsorb to ice crystals and modify their growth. IBPs are found across several biological kingdoms, with their ice-binding activity and function uniquely suited to the lifestyle they have evolved to protect, be it in fishes, insects or plants. While IBPs from freeze-avoidant species significantly depress the freezing point, plant IBPs typically have a reduced ability to lower the freezing temperature. Nevertheless, they have a superior ability to inhibit the recrystallization of formed ice. This latter activity prevents ice crystals from growing larger at temperatures close to melting. Attempts to engineer frost-hardy plants by the controlled transfer of IBPs from freeze-avoiding fish and insects have been largely unsuccessful. In contrast, the expression of recombinant IBP sequences from freeze-tolerant plants significantly reduced electrolyte leakage and enhanced freezing survival in freeze-sensitive plants. These promising results have spurred additional investigations into plant IBP localization and post-translational modifications, as well as a re-evaluation of IBPs as part of the anti-stress and anti-pathogen axis of freeze-tolerant plants. Here we present an overview of plant freezing stress and adaptation mechanisms and discuss the potential utility of IBPs for the generation of freeze-tolerant crops. Plant growth is highly dependent on temperature, which dictates both the geographical range in which plants can be grown and potential yield. Apart from tropical zones, sub-zero temperatures can occur almost everywhere, making freezing damage a global concern. Plants vary in their “cold-hardiness” depending on the level of low temperature stress they can tolerate. Many temperate agricultural crops, including tobacco, tomatoes, potatoes, corn and apples, as well as certain ornamentals such as impatiens, begonias and phlox are considered “very tender” or “half-hardy” and sustain freezing damage between 0 and -4°C. As such, sub-zero temperatures in early spring and late autumn not only shorten the growing season but also result in substantial crop loss. For example, approximately 2 billion USD were lost in citrus crops due to a single frost event in California in 2013 . More recently, a spring frost in Austria resulted in a devastating 80% loss in the total fruit harvest resulting in more than 100 million Euro in damages (Statistics Austria).Figure 1A; Damage to plant cells invariably results following the growth of ice crystals in the extracellular space . Plant IBPs appear to be particularly efficient at adsorbing to ice crystals, thereby preventing the migration of water molecules at temperatures close to freezing or under freeze-thaw conditions. This property is known as ice recrystallization inhibition (IRI). Because of these functional properties, plant IBPs have also been variously known as antifreeze proteins (AFPs) or IRI proteins.Ice-binding proteins (IBPs) are one such family of low temperature-associated proteins found in certain cold-hardy animals and microbes as well as some freeze-tolerant plants. These proteins serve to control the growth of ice crystals and mitigate freezing damage . However, ice crystal formation occurs at higher temperatures by heterogeneous nucleation, catalyzed by the presence of dust, salts, organic molecules or ice-nucleation active (INA) bacteria . Apart fIn plants, liquids are generally contained in two compartments: the symplast, which encompasses water in the cytosol and central vacuole, and the apoplast consisting of water in the xylem-lumen space, the cell wall and the extracellular space surrounding cells . Since tPerennials, biannuals, and hardy annuals from temperate and polar regions have evolved means to adapt to low temperatures without sustaining significant damage in order to overwinter, produce seeds and/or survive seasonal frosts. Biochemical, metabolic, and physiological changes that allow plants to withstand low temperature stress are acquired through exposure to low temperatures and/or shortened day length prior to freeze events, a process known as cold acclimation . TypicalArabidopsis thaliana, has a maximal freezing tolerance of approximately -12°C, compared to -5°C observed in non-acclimated plants  with TH activities of ∼2°C , a property known as IRI . Of the six IBPs in winter rye, Secale cereale, two were identified as β-1,3-endoglucanases, two as class I and class II endochitinases, and two as thaumatin-like proteins, suggesting more than just a role in low temperature protection (Bromus inermis), wintersweet (Chimonanthus praecox), and spruce (Picea abies and P. peungens) (Triticum aestivum (A large number of plant IBP genes show sequence homology to PR genes (otection . These potection . This preungens) . A thaumaestivum . NotablyDaucus carota , and the closely related purple brome grass, B. distachyon (BdIRI), lowered the nucleation temperature of solutions containing P. syringae extracts by ∼1.9°C and ∼2.3°C, respectively . Some bacteria appear to promote ice crystal growth as a method of dispersal , howevererbicola . Such icerbicola . Plant Isyringae . Moreoveectively . Given tFigure 2A) results in ice morphologies that are indicative of their ice plane specificity. These morphologies can be readily examined under a microscope (∼ 40 X) on a cooled stage .The activity of IBPs results from their ability to adsorb to ice, a process that can be visualized by monitoring the “shaping” of ice in the presence of IBPs. Water or standard buffers will freeze as circular disks that expand quickly from the prism plane at temperatures close to the freezing point . In contLpAFP, direct ice crystal morphologies and burst patterns closer to that seen with hyperactive IBPs . These ice crystals shape as hexagonal bipyramids that instead burst from the a-axes resulting in expansion of the basal planes, clearly indicating high affinity for this site. At low protein concentrations, low TH IBPs take on a mild hexagonal shape , as seen in the cell extracts of cold-acclimated plants. This ice shaping is consistent for all plant IBPs identified to date with some minor differences in observed burst patterns, such as that seen with natively produced winter rye and purple brome IBPs, which burst from the tips of the prism plane, resulting in flower-shaped crystals . It is thought that similar to TH activity, IRI occurs by preventing the addition of water molecules from the quasi-liquid layer into the ice crystal lattice. However, IBPs likely also “pin” the surfaces of ice crystals, and as such, inhibit melting.As described by the Ostwald ripening effect, ice-recrystallization occurs because the growth of large ice crystals at the expense of smaller ones is a thermodynamically favorable reaction . This prThermal hysteresis and IRI are distinct properties with little correlation between measures of high IRI and high TH activity, as exemplified by plant AFPs which exhibit high IRI and low TH. However, the mechanisms that distinguish these ice-associating properties are currently unclear. It is worth noting that unlike TH, which requires a relatively high concentration of IBPs, IRI occurs at submicromolar concentrations , suggestThe mechanism underlying IBP adsorption to ice has been debated for decades. Early evidence suggested that hydroxyl groups on the surface of IBPs could generate hydrogen bonds between the protein and the ice crystal lattice . It was MpAFP and Maxi). These structures support the contention that IBFs organize surface waters with perfect complementarity to the primary prism plane . The properties of the IBF dictate how the water molecules will be organized and thus which plane of ice will be bound. This binding hypothesis has been considerably strengthened through protein crystallography which showed tightly bound water molecules on the proposed IBFs of crystallized bacterial and fish IBPs (sm plane . Since mTable 1) a crystal structure has only been obtained for LpAFP, although partial molecular dynamic models have been proposed for DcAFP and BdIRI . LpAFP has a right-handed beta-helix with 7 loops consisting of 14–15 residues per turn with the repeated ice-binding motif NXVXG/NXVXXG, where X is a solvent exposed-residue , that shares homology with dehydrins, is retained in the cytoplasm . An obvious exception is an IBP from L. perenne, LpIRI2, which has no sequence upstream of the ice-binding domain  pathway  lacking a signal peptide, and retained in the cytoplasm of transgenic plants, provided significantly less freeze protection compared to apoplastically localized LpIRI2 and LpIRI3  IBPs TaIRI1 and TaIRI2 by both cold-shock and gradual temperature decreases, whereas chitinase-, glucanase-, and thaumatin-like IBP transcripts were only induced by the latter regime  that more closely resemble autumn conditions, than after long days (16 h light) . It is pbove 4°C . LpAFP din vitro . Althougmostable .R. algida suspension cells following ABA induction also showed ice-binding activity . Glycosylated IBPs have been identified in disparate plants including L. perenne, S. dulcamara, D. carota, the Mongolian Menkhargana (Ammopiptanthus mongolicus), and H. rhamnoides . Initial efforts focused on the expression of fish IBPs with moderate levels of TH activity in tobacco (Nicotiana tobacum) and tomato (S. lycopersicum)  . Althougersicum) . The useobserved . Likely observed .Chloromonas sp., has recently been identified that has remarkably similar properties to that seen with plant IBPs, with a low TH (∼ 0.4°C at 5 mg/mL) and hexagonal ice-shaping (These successful experiments suggest that IBPs that have evolved from freeze-tolerant organisms could be better suited for transfer to other plants and the generation of transgenic crops. This may be true for a number of reasons. As mentioned earlier, ice formed in the presence of moderately active IBPs can produce sharp bipyrimidal ice crystals that result in spicular ice shards as they burst, presumably causing significant cellular damage. It is also likely that if freezing points are lowered significantly with the use of a high TH AFPs, explosive ice crystal growth could risk dangerous intracellular freezing. Interestingly, an IBP from the Antarctic microalga, -shaping . We sugg-shaping .A. thaliana (B. distachyon, and other perennial species of Brachypodium will be a valuable tool for the study of plant IBPs in the future. The prospect that these extraordinary proteins could not only contribute to future food security, but also the ornamental flower industry, will make these efforts well worthwhile.Recently, it has been demonstrated that expression of more than one IBP isoform, as would be expressed endogenously in plants, further enhanced freeze survival in transgenic thaliana . Thus, Ithaliana , indicatMB wrote this manuscript with revisions and editorial advice from VW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of dominant retinitis pigmentosa (RP), a type of inherited blindness caused by the dysfunction and death of photoreceptor cells. The P23H mutation, the most frequent single cause of RP in the USA, causes rhodopsin misfolding and induction of the unfolded protein response (UPR), an adaptive ER stress response and signalling network that aims to enhance the folding and degradation of misfolded proteins to restore proteostasis. Prolonged UPR activation, and in particular the PERK branch, can reduce protein synthesis and initiate cell death through induction of pro-apoptotic pathways. Here, we investigated the effect of pharmacological PERK inhibition on retinal disease process in the P23H-1 transgenic rat model of retinal degeneration. PERK inhibition with GSK2606414A led to an inhibition of eIF2α phosphorylation, which correlated with reduced ERG function and decreased photoreceptor survival at both high and low doses of PERK inhibitor. Additionally, PERK inhibition increased the incidence of inclusion formation in cultured cells overexpressing P23H rod opsin, and increased rhodopsin aggregation in the P23H-1 rat retina, suggesting enhanced P23H misfolding and aggregation. In contrast, treatment of P23H-1 rats with an inhibitor of eIF2α phosphatase, salubrinal, led to improved photoreceptor survival. Collectively, these data suggest the activation of PERK is part of a protective response to mutant rhodopsin that ultimately limits photoreceptor cell death. Retinitis pigmentosa (RP) is a group of inherited diseases that cause the progressive dysfunction and degeneration of rod and cone photoreceptor cells in the retina leading to blindness. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light sensing protein of rod cells, are the single most common cause of autosomal dominant RP, which primarily affect rod cells followed by the secondary loss of cone cells . The majDrosophila model of ALS  factors to enhance degradation. The UPR has three different signalling proteins which are kept in an inactive state by binding to the ER-chaperone binding immunoglobulin protein ; the inositol-requiring protein-1 (IRE-1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6) . The PERl of ALS .The involvement of ER stress and activation of UPR in retinal degeneration has been examined by several studies . ForTherefore, the role of ER stress in the pathogenesis of rhodopsin retinal degeneration is unresolved. For example, genetic ablation of CHOP does not protect against rhodopsin RP , whereasThe P23H-1 transgenic rat shows PERK activation  and undeRats were orally treated daily with a dose of 100 mg/kg PERKi, or vehicle, as this exceeds the dosing predicted to maximally inhibit PERK in the brain . SpecifiRetinal lysates of vehicle and PERKi treated P23H-1 rats were analysed by immunoblotting to assess whether oral administration with PERKi successfully inhibited PERK and PERK-mediated downstream signalling. The expression of mutant P23H rhodopsin in P23H-1 rat retina leads to the induction of the UPR ,23. SimiPhotoreceptor function was assessed by a full field scotopic electroretinogram (ERG) in dark-adapted rats at P35. Increasing light intensity correlates with a greater response of photoreceptor hyperpolarisation (a-wave), followed by the propagation of the signal through the retina with subsequent depolarisation (b-wave). Treatment with PERKi led to lower ERG responses as both a-wave and b-wave response amplitudes were reduced compared with vehicle-treated animals . This suT17M rhodopsin transgenic mice with genetically reduced levels of ATF4 were recently shown to have delayed photoreceptor degeneration . TherefoTo investigate if the inhibition of PERK affected the other branches of the UPR in the retina, the retinal lysates from the high and low dose treatments were immunoblotted for IRE1 and phospho-IRE1 (p-IRE1) and ATF6. The data revealed no significant changes in IRE1 level or phosphorylation , and no To investigate whether the reduction in visual function correlated with a reduction in photoreceptor numbers, optical coherence tomography (OCT) was used to measure the thickness of the outer nuclear layer (ONL), as a marker of photoreceptor survival. The measurement of the ONL along the temporal-nasal meridian is shown in Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was used to detect DNA fragmentation and measure apoptosis in the vehicle and PERKi treated P23H-1 retina. As expected with the active cell death associated with P23H expression, there was a high level of TUNEL reactivity in the vehicle-treated retina, which was further increased on PERKi treatment . ActivatAnalyses of the 10 mg/kg treated animals by OCT and histology , confirmAs inhibition of PERK activity was detrimental to P23H expressing photoreceptors, we wanted to test the hypothesis that prolonging PERK signalling could be protective. Salubrinal is a specific small molecule inhibitor of eIF2α dephosphorylation , and inhTo investigate the potential mechanisms underlying the accelerated retinal degeneration following PERK inhibition, immunohistochemistry was used to investigate rhodopsin traffic. Treatment with either high or low dose PERKi led to an increase in the co-localisation of rhodopsin with BiP in the ER as determined by co-localisation analyses . This suP23H rhodopsin can aggregate and form intracellular inclusions on heterologous expression in cell culture models . Therefoin vivo, a differential sedimentation assay ; increase ER exit (producing more factors to enable folding); and remove misfolded proteins through ERAD; however, sustained activation can have deleterious effects and lead to cell death. Although the role of ER stress and activation of the UPR in neurodegeneration has been extensively investigated, its role in disease progression remains controversial. The retina offers an excellent paradigm to study the role of cell stress in neurodegeneration, due to its accessibility, well-characterised function and the detailed understanding of genetic causes of retinal degeneration. Furthermore, it can be targeted with systemic, or local  drug delivery, or gene therapy. In this study, we used GSK2606414A, a selective inhibitor of PERK , to furtWe used the well-characterised P23H-1 transgenic rat model, which has well documented UPR induction ,23. ThisRecently, it was suggested that GSK2606414A, and the related compound GSK2656157, might not be specific to PERK, but can also inhibit RIP1 . FurtherInterestingly, prolonging eIF2α phosphorylation with salubrinal had a mild protective effect on the P23H transgenic rat retina. It was recently reported that salubrinal can reduce TUNEL reactivity in P23H transgenic mice and P23H knock-in mice without any WT allele (P23H/-), which both have PERK activation . It is nThe potential mechanisms of how PERK inhibition might affect photoreceptor viability were investigated and revealed that PERK inhibition led to an increase in rhodopsin aggregation in both cells and the P23H-1 rat retina. We also observed increased co-localisation of rhodopsin with the ER marker BiP, suggesting enhanced ER retention or increased expression. Previously, we have observed that increased PERK activation in the P23H-1 rat, stimulated by the heat shock protein coinducer arimoclomol, correlated with a decrease in photoreceptor cell death and reduced rhodopsin aggregation . The comDrosophila melanogaster, moderate ER stress caused by the loss of the fly rhodopsin specific chaperone, NinaA, protected photoreceptors from cell death  was from Sigma (UK).P23H rod opsin-GFP plasmid was as previously described . The mouad libitum.All procedures were conducted according to the Home Office (UK) regulations, under the Animals (Scientific Procedures) Act of 1986, and with the approval of local UCL-Institute of Ophthalmology, London, UK ethics committee. The P23H-1 line rats were kindly provided by Professor Matt LaVail,  and crossed with WT Sprague Dawley (SD) rats to generate hemizygous P23H-1 rats. SD rats were purchased from Harlan . All animals were housed under a 12: 12 light dark cycle, with food and water available P23H-1 rats were orally gavaged once daily from P21 to P35 with 10 mg/kg and 100 mg/kg GSK2606414A or vehicle at a volume of 0.2 ml per 50 g of rat or with 1 mg/kg Salubrinal dissolved in DMSO and then in milk and vehicle (milk). Rats were monitored daily for any adverse effects and for body weight gain.t test.At P35, rats were dark-adapted overnight in a ventilated light-tight box and anaesthetized with Ketamine/Xylazine at 0.2 ml/100 g intraperitoneally (i.p.). Full-field scotopic ERG was carried out using the Diagnosys system and Espion software  under red-light conditions and as previously described . Brieflyt test.Rat retinae were imaged using the Bioptigen Spectral-domain ophthalmic imaging system (SDOIS) as previously described . BrieflyRetinae were extracted and lysed with ice-cold RIPA buffer  containing 2% (v/v) mammalian protease inhibitor cocktail (PIC) 2% (v/v) mammalian phosphatase inhibitor cocktail. Retina lysates were sonicated for 2 × 30 s, centrifuged for 15 min at 12 000× g and at 4 °C and diluted in 5× sodium dodecyl sulphate (SDS) sample loading  to a final concentration of 1×. Samples for rod opsin were not denatured whereas samples for all the other antibodies were heated at 95 °C for 5 min before they were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Proteins were transferred to nitrocellulose with 25 mM Tris, 192 mM Glycine, 0.01% SDS (w/v) and 20% (v/v) Methanol. To prevent non-specific binding, membranes were blocked at 4 °C with either 5% (w/v) Marvel Milk in PBS with 0.1% (v/v) Tween buffer or and 5% (w/v) BSA in TBS with 0.1% (v/v) Tween buffer. Immunodetection of proteins of interest was carried out using the primary and secondary antibodies described in Materials section. Proteins were detected using the Luminata Forte and Luminata Crescendo HRP substrate kits. Rhodopsin sedimentation assay was performed as described previously ,23,28.in situ cell death detection kit (Fluorescein) following the manufacturer’s instructions.Eyes were dissected and fixed overnight in 4% paraformaldehyde at 4 °C following by cryoprotection with 30% sucrose in PBS. Eyes were then frozen and cryosectioned as described . Eye secSK-N-SH human neuroblastoma cells were maintained and transfected essentially as previously described . Three hImage acquisition of cells and retina was obtained using Carl Zeiss LSM700 laser-scanning confocal microscope. Images were exported from Zen 2009 software and prepared using Adobe Photoshop and Illustrator CS4. Cell morphology studies were scored as previously described . The PeaHMG online.Supplementary DataClick here for additional data file.


Symbiodinium types under stress, chemicals such as dichlorophenyl dimethylurea (DCMU) are widely used. Moreover, DCMU and recently menthol were used to generate aposymbiotic cnidarian hosts. While the effects of DCMU on Symbiodinium cells have been extensively studied, no studies have shown the mechanism behind menthol-induced coral bleaching. Moreover, no study has compared the effects of DCMU and menthol treatments on photosystem II (PSII) activity and generation of ROS in different Symbiodinium types. In this study, we utilized five freshly isolated Symbiodinium types (S. minutum (B1), S. goreaui (C1), C3, C15, and S. trenchii (D1a)) to compare the effects of DCMU and menthol treatments. Symbiodinium cells were exposed to DCMU and menthol at different concentrations for 4 h. Results showed that values of the 50% inhibitory concentration (IC50) for PSII inhibition were 0.72∼1.96 mM for menthol-treated cells compared to 29∼74 pM for DCMU-treated cells. Diverse responses of Symbiodinium types were displayed in terms of PSII tolerance to menthol (S. minutum > S. trenchii = C15 > C3 = S. goreaui), and also in the response curves. In contrast, responses were not so diverse when the different types were treated with DCMU. Three of five menthol-treated Symbiodinium types showed instant and significant ROS generation when PSII activity was inhibited, compared to no ROS being generated in DCMU-treated Symbiodinium types. Both results indicated that menthol inhibited Symbiodinium PSII activity through Symbiodinium type-dependent mechanisms, which were also distinct from those with DCMU treatment. This study further confirmed that photosynthetic functions Symbiodinium have diverse responses to stress even within the same clade.To understand the mechanism of photosynthetic inhibition and generation of reactive oxygen species (ROS) in  Symbiodinium spp. are associated with marine invertebrate hosts, including Protista, Porifera, Cnidaria, and Mollusca  treatment)   or streseatment) . Disrupty metals . Howeveriodinium .Symbiodinium cells have extensively been studied . A sample collection permit (no. 488-100-01) was obtained from the Kenting National Park Authority as part of the long-term environmental monitoring project. Coral colonies were transferred to the laboratory within 3 h in an aerated plastic box, and maintained in an aquarium tank under conditions described in −2 s−1 light) for 1 week before initiating experiments. The glass sea anemone, Exaiptasia pulchella , coral fragments with 5∼10 cm2 of live tissue and excised tentacles from sea anemones were homogenized and washed with artificial seawater (ASW) prepared from Instant Ocean  as described by Symbiodinium ITS2 types C1 (S. goreaui), C3, C15, D1a (S. trenchii), and B1 (S. minutum) from S. pistillata, A. humilis, P. lutea, G. fasicularis and E. pulchella, respectively, based on denaturation gradient gel electrophoresis (DGGE) according to To prepare freshly isolated Fv∕Fm), and the minimum (F0) and maximum (Fm) fluorescence levels were measured to calculate the variable fluorescence  using a DIVING-PAM fluorometer  at a setting of 8 for the measuring light and saturating flash of actinic light.PSII activities of FIS were determined by the maximum quantum yield (50), triplicate samples with 5 ml of FIS (around 106 cells ml−1) of each type showing Fv∕Fm values of >0.6 . 50. Fv∕Fm values of FIS in menthol or DCMU were directly determined with the DIVING-PAM fluorometer without dark adaptation at 4 h of incubation, and were converted to PSII inhibition by comparing to values in fresh isolates as below: Fv∕Fm)0h and (Fv∕Fm)4h values were respectively measured at 0 and after 4 h of incubation. After plotting PSII inhibition against the logarithm-transformed concentration of menthol (or DCMU), the equation for calculating the IC50 was obtained from the best curve-fit-model provided in SigmaPlot 10.0 software.When determining values of the half-maximal inhibitory concentration (IC2DCFDA) probe. FIS  or 130 pM DCMU as the positive control, for 4 h in which the PSII activity of FIS would be completely inhibited. The mentioned concentration used to examine ROS generation refers to the highest concentration causing maximum inhibition of PSII activity. Also, during the trials of menthol-induced bleaching of the coral hosts, the effective concentration varied between different hosts associated with different Symbiodinium types, but all displayed significant photosystem II (PSII) breakdown in the endosymbiotic Symbiodinium  for 30 min in the dark, followed by fluorescence microscopic examination  of menthol and DCMU on PSII inhibition of FIS were calculated from samples of each treatment. The curve equations for IC50 calculation were determined by the best-fit regression curve of PSII inhibition (%) against the logarithm-transformed reagent concentration, which are described in Dose effects  followed by Fisher’s least significance difference (LSD) test, with a significance level of 0.05. The coefficient of variance (CV) was used to evaluate the variation in IC50 measurements between replicates.Comparisons of 4-h ICSymbiodinium algae were incubated in menthol-supplemented ASW for 4 h, all five Symbiodinium types  displayed typical dose–response curves under menthol concentrations of 0.19∼2.43 mM  and low slope factors (8.7∼9.5 for S. goreaui and S. trenchii). But those for DCMU-treated FIS displayed only one group of slope factors that ranged 4.3∼6.3. The 4-h IC50 values for menthol and DCMU treatments on five Symbiodinium types were calculated, and results are listed in 50 values for menthol ranged 0.72∼1.96 mM with CV values ranging 1.1%∼12.3%, and those for DCMU ranged 29∼74 pM with CV values of 2.3%∼14.1% , and S. minutum was found to be the most tolerant type to both menthol and DCMU irritation (p < 0.05). In contrast to the comparable tolerability to DCMU among different Symbiodinium types, S. trenchii and type C15 were significantly more tolerant to menthol than were S. goreaui and C3 (p < 0.05).Regression coefficients for curve fitting ranged 0.989∼0.999 for menthol treatments and 0.995∼0.999 for DCMU treatments. When converting parameter “3%∼14.1% . Two setSymbiodinium varied among types. As shown in S. minutum, consistent with the ASW control in S. trenchii, there were only mild ROS signals detected  of that of S. trenchii (35 ± 3 fluorescence units cell−1), and more than three times that of S. minutum (18 ± 2 fluorescence units cell−1) , even though Fv∕Fm values were reduced to <0.1. In order to monitor the times needed for Symbiodinium types to respond to menthol irritation, Fv∕Fm values and ROS levels of the C3 type were examined every 5 or 10 min after suspending the alga in menthol-supplemented ASW. As shown in Fv∕Fm value was significantly reduced from 0.685 to <0.2 within 5 min of incubation, and to nearly 0 after 10 min of incubation. As with the decrease in Fv∕Fm value, ROS generation in the menthol treated Symbiodinium reached a maximum within 5 min of incubation  . ROS flucubation .Symbiodinium types. Loss of photosynthetic activity in Symbiodinium might explain the coral bleaching phenomenon of menthol-treated corals and sea anemones  and non-ROS generating (S. minutum and S. trenchii). The reason for such a distinction needs to be addressed in future studies.This study indicated that millimolar levels of menthol significantly inhibited PSII activities of five freshly isolated anemones . Howeverolerance , thermalolerance , and expolerance . Therefo menthol , Table 12+ stimulated exocytosis. This has been demonstrated in previous results from transcriptomic studies suggesting the disruption of calcium homeostasis in both corals and sea anemones during stress-induced bleaching M8, menthol might also be inducing symbiophagy  have tried to understand the mechanisms involved in loss of Symbiodinium cells when treated with menthol, the clarity as to what really happens is still lacking and needs to be explored in more detail in future studies.In conclusion, activity of (TRP)M8 might not be a general phenomenon. We observed diverse responses based on biophagy  in the h10.7717/peerj.3843/supp-1Data S1Click here for additional data file.10.7717/peerj.3843/supp-2Figure S1Symbiodinium in Isopora palifera during menthol bleaching. Bleaching treatment was conducted with a repeat incubation of 8 h in 0.58 mM menthol/artificial seawater (ASW) following with 16 h in ASW. Measurement was stopped at the third day of incubation due to low Symbiodinium population. Data are presented with mean ± S.D (n = 8).An example study on maximum quantum yield measurements of Click here for additional data file.10.7717/peerj.3843/supp-3Figure S2Symbiodinium types when treated with artificial seawater (ASW) containing DCMU. Freshly isolated Symbiodinium (FIS) was incubated in DCMU-supplemented ASW for 4 h, followed by 2′, 7′-dichlorofluorescin diacetate labeling and microscopic examination of fluorescence. DCMU concentrations used in FIS incubation were 0.13 mM for all Symbiodinium tested, which would cause complete breakdown of PSII activity in the algae. ROS signals in type C3 treated with the ASW control (A) and DCMU-treated Symbiodinium types B1, D1a, C15, C3, and C1 (B F) are presented with a representative photo. Scale bar in the photo represents 50 mm.Diverse reactive oxygen species (ROS) generation levels among Click here for additional data file.


RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA (pA) site in sense gene RNASEH2C, which generated longer 3′ untranslated region (3′UTR) and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene’s protein level in RNASEH2C-KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.Natural antisense transcripts (NAT) and alternative polyadenylation (APA) of messenger RNA (mRNA) are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense (S-AS) gene pairs, and finally focused on The online version of this article (10.1007/s13238-017-0497-0) contains supplementary material, which is available to authorized users. Cis-encoded NATs are driven by promoters at the opposite strand of the so-called sense gene, usually partially reverse complementary to its sense partner. The sense-antisense (S-AS) pairs were found to express synergistically rather than by chance , defined as the polyadenylation of precursor messenger RNA (pre-mRNA) at multiple sites, is another layer of gene regulation that contributes to transcriptome complexity at the last step of mRNA maturation . Then, tail-to-tail S-AS gene pairs were chosen for further study since they overlapped in the polyadenylation sites and more likely to have mechanistic interaction between antisense transcription and APA. Compared to non-overlapped genes, tail-to-tail S-AS gene pairs were found more enriched with APA genes , implying intrinsic relevance between antisense transcription and APA.To explore whether antisense transcripts and APA have possible connections genome-widely, we analyzed our previously published PA-seq datasets from 13 human tissues  site of one gene in tail-to-tail S-AS gene pair always stayed on the way of the transcription of the other gene, we next examined the correlation between change of NATs expression and the distal pA site usage of the sense gene by PA-seq, which can quantify both the distal/proximal pA site usage and the relative gene expression level , resulting in a transcript variant with shorter 3′UTR and not overlaps with KAT5 . Second, 3′ RACE (rapid-amplification of 3′ cDNA ends) showed two bands corresponding to distal  and proximal pA sites , which was further validated in 6 additional human cell lines . To investigate further the effect of NAT in controlling pA site usage of the sense gene, we perturbed the expression of NAT gene (KAT5) and then measured the pA site usage in RNASEH2C. Interestingly, we found that ectopic (or in trans) overexpression of KAT5 did not affect the pA site usage of RNASEH2C  using CRISPR/Cas9 approach led to a significant lower usage of distal pA site of RNASEH2C   . In contrast, in trans overexpression of KAT5 did not affect the protein level of RNASEH2C  and CCND1 , were also sharply declined .The cellular and molecular phenotypes of reduced RNASEH2C level were next investigated. Cell growth ability dramatically decreased in lls Fig. A, and thned Fig. B and 4C.in cis over-expression of KAT5, we then examined the cell growth behavior in cells over-expressing KAT5 in cis. Interestingly, these cells also exhibited reduced cell growth rate , which has been demonstrated to play regulatory roles in alternative polyadenylation  compared to control cells , thus the cis-regulatory elements of polyadenylation might be not as strong as the conserved proximal one, and the usage of distal pA site possibly needs more trans-acting factors recruited by Pol II to help.To explain the higher usage of distal pA site upon elevation of Pol II occupancy, we hypothesize that distal pA site is more sensitive to local Pol II concentration compared to the proximal one. Two lines of evidence support this hypothesis. First, distal pA site has much lower usage than proximal one in most of the human tissues , while other mammalians such as mouse and rat did not have the distal polyA signal, resulting the absence of isoform with long 3′UTR   Figs. B and S14lls Fig. C and 6D.ERBB2, an important regulator of breast cancer, turns out to be not successful because of the co-amplification and over-expression of its neighbor genes  caused a distal pA site usage shift in sense gene (RNASEH2C), which reduced the production of corresponding protein. However, in mouse cells, due to the absence of distal pA site, in cis overexpression of KAT5 cannot trigger APA, and result in the unchanged expression of RNASEH2C.S-AS gene pair can either be coding or non-coding. In the case of present study, both ion Fig. A, thus iRNASEH2C-KAT5 gene pair, distal pA site is far away from RNASEH2C promoter, but is much closer to KAT5 promoter , consistent with the conclusion drawn by Ji et al. .Pol II has been reported to regulate APA Ji, . Moreoveter Fig. A. Thus ii et al. . Previoui et al. . IntriguKAT5 in cis did not downregulate RNASEH2C protein level in mouse cells, suggesting the newly evolved distal pA site in human was critical to mediate APA in RNASEH2C and ultimately reduced the protein level. We speculated that such function of pA site usage might depend on the interplay between the cis-regulatory elements and trans-acting factors. Although the ideal experimental design to prove the importance of APA in down-regulating sense gene’s protein level was to completely disrupt the usage of distal pA site in RNASEH2C, it was technical challenging to delete all the cis-elements near the distal pA site completely. Actually, we tried multiple gRNAs and still can not obtain cell clones with mutated polyA signal upstream of the distal pA site, possibly due to the inefficiency of the related gRNA . Nevertheless, we successfully deleted polyadenylation-related cis-elements downstream of the distal pA site and observed the decreased expression of the long isoform of RNASEH2C , suggesting that these elements were helpful for the recognition and cleavage of the distal pA site. To circumvent this technical limitation, we introduce mouse cells that lack of distal pA site for RNASEH2C to serve as an indirect evidence instead, wherein, in cis upregulation of KAT5 could not affect the expression of RNASEH2C  on the second day. Cells were harvested for RNA and protein extraction 48 h after transfection.All cell lines  used in this study were cultured in DMEM medium supplemented with 10% FBS at 37°C with 5% COKAT5 knockdown in cis, cells were transfected with 2 μg plasmids (pBC2-gRNA). 24 h after transfection, cells were screened by 300 μg/mL hygromycin for two days, then the survival cells were trypsinized, diluted and seeded into a 96-well plate for single cell clone selection. For KAT5 overexpression in cis, cells were co-transfected with 2 μg pBC2-gRNA plasmid and puroR-CMV-T-easy vector, with pBC2-gRNA plasmid and puroR-T-easy vector serving as control, respectively. 24 h after transfection, cells were screened by 300 μg/mL hygromycin for two days, and the survival cells were cultured continuously in medium with 2 μg/mL puromycin for ~5 days, during which period, medium was replaced every day to remove the dead cells. The final survival cells were cultured for an additional 7–10 days for single cell clone selection. For distal pA site mutation in human RNASEH2C, we adopted an episomal vector-based CRISPR/Cas9 system for higher gene knockout efficiency, and screened for stable single cells according to the published methods . After one day selection, the survival cells were trypsinized and split into two parts, one for cell proliferation assay, the other part was cultured for additional two days in the medium with puromycin (2 μg/mL). Then the cells were harvested for RNA and protein extraction to validate RNASEH2C knockdown efficiency.For knockdown RNASEH2C shRNA plasmid construction, the DNA oligonucleotides were annealed and then cloned into the pLKO.1 plasmid by EcoRI and AgeI restriction site. Sequences corresponding to short or long 3′UTR of RNASEH2C were PCR amplified by Q5 high-fidelity DNA polymerase (NEB), and then inserted into the Renilla luciferase vector (psiCHECK-2 vector). Meanwhile, a mutated long 3′UTR was constructed by replacing the proximal polyadenylation signal AATAAA with CAATTG. As to the vector for in trans overexpression of KAT5, the complete coding sequences (CDS) were acquired by PCR amplification from human 293T cDNA, then the fragments were inserted into the pcDH_EF1_MCS_T2A_Puro plasmid by EcoRI and BamHI restriction site.For KAT5 in cis were constructed following the description below. For gRNA vectors construction, gRNAs were designed using online CRISPR Design (http://crispr.mit.edu/), and the synthesized oligos were annealed with sticky terminus of HpaI and XbaI restriction site, and then integrated downstream U6 promoter in a middle vector PUG3 (U6-gRNA). The U6-gRNA fragments were then inserted into the final plasmid PBC2 by SalI and AfII restriction site. When two or more gRNAs used, one U6-gRNA was first PCR amplified from gRNA-PUG3 by primers with XbaI and XhoI restriction site and cloned into downstream of another gRNA-PUG3 vector, finally, the tandem U6-gRNA fragments were inserted into the final plasmid PBC2.The vectors for knock-out and knock-in of puro (puroR) gene and homologous arms (HA) upstream and downstream puroR. The other plasmid is HA-puroR-CMV-HA-T-easy vector, among of which, HA sequences were acquired by PCR amplification from 293T genomic DNA, CMV and puroR gene were PCR amplified from the templates of pCDNA3.1 and PT2B vector, respectively. Then they were ligated successively by overlap-PCR and finally inserted into T-easy vector. Q5 high-fidelity DNA polymerase was used for all PCR reactions to minimize the introduction of possible error bases, and Sanger sequencing was applied for fidelity confirmation.For knock-in CRISPR/Cas9, two donor plasmids were constructed based on the skeleton of T-easy vector. One was the control plasmid with insertion of fragments containing anti-Total RNA and protein was extracted with TRIzol reagent (Sigma) according to the manufacturer’s instruction. For cDNA synthesis, 1 μg total RNA was reverse-transcribed into the cDNA by FastQuant RT Kit (TIANGEN) and oligo (dT) primer. Then quantitative PCR was performed using 2× SYBR mix (KAPA) on Bio-Rad CFX manager machine.For Western blot, the primary antibody for detecting RNASEH2C , SRSF3 , C-terminal domain (CTD) of Pol II  and GAPDH  was 1:1000 diluted and incubated for 2 h at room temperature. The second antibody  was 1:5,000 diluted and incubated for 1 h at room temperature.RNASEH2C according to the manufacturer’s handbook with some modifications . Briefly, denatured RNA was resolved by 1% agarose gel, and then was transfered to NC membrane  overnight at 4°C. Following UV cross-link, the membrane was pre-hybridized in hybridization solutions at 65°C incubator for 2 h and then incubated with Dig-labelled-probe for hybridization overnight. Then the membrane was washed and blocked in blocking solutions for 30 min at room temperature. Membrane was next incubated with antibody solutions (anti-Dig-AP antibody in Blocking solutions) for 30 min and was washed twice for 15 min by wash solutions. Finally, immunological signal was detected after the membrane was equilibrated in detection buffer.We performed Northern blot to detect the short and long isoforms of The interaction of SRSF3 with the C-terminal domain (CTD) of Pol II was detected by Co-IP experiment. Firstly, 10 μg of vectors  was individually transfected into 293T cells by Lipofectamine 2000. Cells were harvested in lysis buffer 24 h post transfection. We removed 20 μL cell lysate as input and the rest was incubated with anti-flag agarose beads  at 4°C for 6 h. The agarose beads mixture was then washed twice according to the vendor’s handbook. Western blot loading buffer was next added to the beads and incubated at 95°C for 10 min to denature protein. Finally, Western blot was performed to detect SRSF3-flag and the CTD of Pol II by anti-flag  and anti-CTD antibody, respectively.RNASEH2C was measured by adding actinomycin D at a final concentration of 5 μg/mL into 293T cells to block transcription. Cells were harvested at the time point of 0 h, 4 h, 8 h, 12 h, 24 h, 32 h, respectively, and total RNA was extracted with the methods described above. The difference in stability was then evaluated by qRT-PCR. The long isoform RNASEH2C_L was quantified using primer specific to long 3′UTR, while the overall expression of RNASEH2C was quantified using primers shared by both short and long 3′UTRs (RNASEH2C_S).The stability for short and long isoforms of Firstly, total RNA was treated with DNase I to eliminate DNA contamination. Then 500 ng DNA-free RNA was reverse-transcribed into cDNA with SMARTScribe reverse trascriptase . The primer used for reverse transcription is composed of 19 known nucleotides plus a downstream 30 dTs and two-anchoring nucleotides  from 5′ to 3′ end . The cDNA product was diluted, and PCR was performed with one primer targeting on the gene and the other primer partially same as the primer mentioned above . Then PCR products were monitored by agarose gel, and validated by Sanger sequencing.293T cells were transfected with psiCHECK-2 vectors containing short-3′UTR, long-3′UTR and mutated-3′UTR, respectively, using Lipofectamine 2000 in 24-well plate. After transfection for 24 h, the firefly and renilla luciferase activities were measured one by one according to the manufacture’s instruction (Promega). The final renilla luciferase activity was normalized to the firefly luciferase signal.DOJINDO) was then added according to the manufacturer’s protocol to each well every 24 h post seeding, lasting for four days. After each treatment, cells were incubated for two hours at 37°C, then the optical density (OD) at 450 nm and 600 nm were measured respectively for each well by microplate reader (Tecan i-control).After selection by puromycin, the survival cells were trypsinized and diluted, then subcultured in 96-well plate, with 2000 cells per well and three replicates for each time point. CCK-8 reagent , 1× protease inhibitor). Cell lysate was incubated on ice for 10 min and then centrifuged at 500 ×g for 5 min, the supernatant was carefully pipetted into a new tube. Then wash the pellet once with ice-cold lysis buffer, and centrifuge again at 500 ×g for 5 min. The supernatants harvested from these two steps were combined to make the cytoplasmic fraction, and the pellet left was the nuclear fraction. Finally, RNA was prepared from each fraction by using TRIzol reagent.To separate the nuclear and cytoplasmic components, cellular fractionation was performed following the description below. Cells were trypsinized, washed twice with ice-cold 1× PBS, and resuspended in ice-cold lysis buffer CFB (20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L KCl, 100 mmol/L NaF, 1 mmol/L DTT, 1% NP40, 1.5 mmol/L MgClThe nascent RNA assay was performed with two methods, the Click-iT method . Then the 60 nt at the 3′ end of each read was cut off to remove the potential contamination of adapters introduced in library preparation. The clean reads were then aligned to human hg38 genome using BWA (v0.7.10)  ChIP-seq reads were gained after sequencing on Illumina HiSeq 4000. The sequencing quality was first evaluated using FastQC Below is the link to the electronic supplementary material.