Disrupting C2A/C2A homodimerization in UNC-13L-rescued animals had no effect on synaptic transmission, but fully restored the evoked release and the release probability of unc-10/RIM mutants deficient for C2A/RIM heterodimerization. Thus, our results support the model that RIM binding C2A releases UNC-13L from an autoinhibitory homodimeric complex to become fusion-competent by functioning as a switch only.UNC-13 proteins play an essential role in synaptic transmission by recruiting synaptic vesicles (SVs) to become available for release, which is termed SV priming. Here we show that the C2A domain of UNC-13L, like the corresponding domain in mammalian Munc13-1, displays two conserved binding modes: forming C2A/C2A homodimers, or forming a heterodimer with the zinc finger domain of UNC-10/RIM (C2A/RIM). Functional analysis revealed that UNC-13L’s C2A promotes synaptic transmission by regulating a post-priming process. Stimulus-evoked release but not SV priming, was impaired in  We found that tonic neurotransmitter release at a low Ca2+ concentration and stimulus-evoked release were significantly reduced in the unc-10(nu487 K77/79E) mutants, whereas SV release was normal in the UNC-13L(K49E)-rescued unc-13 mutants. Moreover, the hypertonic sucrose-evoked responses were unaltered following disruption of homodimerization and heterodimerization, suggesting that SV priming is not determined by the state of the C2A domain. Interestingly, simultaneously disrupting C2A/C2A homodimerization in the unc-10(nu487 K77/79E) mutant fully restored both tonic and evoked release. These results suggest that the synaptic transmission defect in the unc-10(nu487 K77/79E) mutant is most likely caused by formation of more C2A/C2A homodimers, and that the role of UNC-10/RIM binding UNC-13 C2A domain is to release the C2A from an autoinhibitory state, allowing it to become a fusion-competent monomeric form. Finally, we show that UNC-13’s C2A domain increases release probability but has little effect on SV priming.Here, we examined the functional importance of the C2A domain by generating a transgenic line of UNC-13L with a point mutation in the C2A domain (K49E) that blocks formation of C2A/C2A homodimers, and a mutant carrying two mutations in the The UNC-13L C2A domain and the UNC-10/RIM ZF domain share high sequence identity with the corresponding domains of mouse Munc13-1 (50%) and RIM (48%) , suggest2+, as we have previously found that the mIPSCs are almost arrested in 0 mM Ca2+ but are normal 1 mM Ca2+ in some mutants  null mutant, and were fully restored by neuronal expression of full-length UNC-13L  at the neuromuscular junction. Both the mEPSCs and the mIPSCs were recorded in the presence of 0 mM and 1 mM Ca mutants , suggest UNC-13L . Deletin-13L . Compared to wild-type animals, unc-10(nu487 K77/79E) mutants display slightly uncoordinated behaviour (data not shown). Analysis of tonic release showed that the mEPSCs and mIPSCs were markedly reduced in unc-10(nu487 K77/79E) mutants in 0 mM Ca2+, whereas both were unaltered in 1 mM Ca2+ . As shown in 2+ concentration from 0 mM to 0.05 mM and 0.1 mM caused robust enhancement of the mEPSC and mIPSC frequencies in wild-type animals. In unc-10(nu487 K77/79E) mutants, a significantly lower mIPSC frequency was observed in 0.05 mM Ca2+ but not in 0.1 mM Ca2+, whereas mEPSCs were unaltered in both 0.05 mM and 0.1 mM Ca2+ ; unc-10(nu487 K77/79E) double mutant by crossing the unc-10(nu487 K77/79E) into the UNC-13L(K49E) rescue strain. In 0 mM Ca2+ recordings, the UNC-13L(K49E); unc-10(nu487 K77/79E) double mutant mIPSC rate was significantly higher than that observed in unc-10(nu487 K77/79E) single mutants, suggesting that blocking homodimer formation was sufficient to prevent the decreased tonic GABA release caused by the unc-10(nu487 K77/79E) mutation (2+ in unc-10(nu487 K77/79E) mutants (unc-10(nu487 K77/79E) double mutant in 1 mM Ca2+, similar to those in the UNC-13L(K49E) rescue and the unc-10(nu487 K77/79E) mutant  mutant . Taken tunc-10(nu487 K77/79E) mutants, exhibiting decreased amplitude and total charge transfer compared to wild-type animals (unc-10(nu487 K77/79E) mutant (44%) was similar to that observed in the UNC-13LΔC2A rescue animals (46%), suggesting that C2A/RIM heterodimerization accounts for the role of the C2A domain in evoked release. Thus, our results revealed that C2A/RIM heterodimerization is required for evoked neurotransmitter release. It should be noted that the evoked EPSCs were recorded in 1 mM Ca2+ solution in which, however, the mEPSCs were unchanged  double mutants. We found that preventing C2A/C2A homodimerization in the unc-10(nu487 K77/79E) mutant fully restored the evoked EPSC to a wild-type level  were indistinguishable from those rescued by wild-type UNC-13L, indicating that C2A homodimers are not required for evoked responses . To detepe level . These rpe level . These rpe level , Table 1unc-13 null mutants, and was fully restored by expressing a wild-type UNC-13L transgene in all neurons (A decrease in evoked EPSCs may arise from a priming defect or a change in release probability. We therefore examined whether SV priming is regulated by the C2A domain and its two different states (homodimer and heterodimer). SV priming was measured by the pulsed application of hypertonic sucrose solution  onto the ventral nerve cord, and the size of the RRP was calculated by integrating the sucrose-evoked current. The current was almost eliminated in the  neurons . Strikin neurons , Table 1 neurons . Despite neurons , supportunc-10(K77/79E) mutant by puffing hypertonic sucrose . Our data showed that the sucrose-evoked currents in these animals were comparable to those observed in UNC-13L rescue and wild-type animals (unc-10(K77/79E) mutant was also confirmed by puffing a lower concentration of sucrose . Consequently, the probability of synaptic vesicle release (Pvr) calculated by the ratio of the charge transfer of the evoked EPSCs to the charge transfer of the sucrose-evoked currents was significantly reduced in the ΔC2A rescue and the unc-10(K77/79E) mutant  rescue animals and the  animals , demonst) mutant . It shou) mutant . Togetheunc-10(K77/79E) mutant could arise from a change in the expression level of UNC-13, or a change in sub-localization of the UNC-13 proteins at the nerve terminals. We next analyzed the UNC-13 protein levels from different transgenic rescue lines. A fluorescence protein (mApple) was fused to the C terminus of the various UNC-13L constructs , and expressed in unc-13 null mutants. Our prior study showed that c-terminal mCherry tags do not interfere with UNC-13’s function in promoting SV exocytosis  mutant was caused by a change in the sub-localization of UNC-13L. To determine this, we compared colocalization of UNC-13L and an active zone marker (ELKS-1) in wild-type controls and in unc-10(K77/79E) mutants. UNC-13L and ELKS-1 were strongly colocalized in wild-type (unc-10(K77/79E) mutants  mutant, we asked whether the synaptic depression and recovery would be altered by disruption of C2A/RIM heterodimerization. An integrated line expressing ChIEF, a variant of channelrhodopsin-2 (ChR2) in the cholinergic motor neurons was used to record the EPSCs evoked by a train or a paired light stimulus (unc-10(K77/79E) mutants exhibited synaptic depression following a 1 Hz or 5 Hz train stimulus (i) to the amplitude of the first EPSC (EPSC1). The unc-10(K77/79E) mutants displayed a similar depression rate to that of the control animals (2) to the first EPSC amplitude (EPSC1). As shown in unc-10(K77/79E) mutants. This indicates that disrupting C2A/RIM binding does not affect refilling of the SVs at synapses.Because of the reduced Pstimulus . Both thstimulus . The dep animals , indicat animals . The recC. elegans, for SV priming and release probability. Prior studies in mouse and C. elegans have reported contradictory results regarding the role of the C2A domain in priming  exhibit a normal RRP, arguing that the C2A domain is not required for priming (unc-13 mutant (n2609) with a point mutation in the C2A domain of UNC-13L exhibits a similar reduction in evoked EPSCs to UNC-13LΔC2A, whereas docked SV numbers are normal in the unc-13(n2609) mutant. This suggests that the C2A domain is not required for docking , consistent with previous studies (unc-10(K77/79E) mutant also exhibits an impairment in evoked EPSCs but a normal RRP, thereby leading to a decreased Pvr  mutant was blocked by disrupting C2A/C2A homodimerization (unc-10(nu487 K77/79E) animals  is comparable to that in the wild-type animals (data not shown), demonstrating that UNC-10/RIM is not required for SV priming. These results suggest that UNC-10/RIM primarily regulates SV fusion by increasing release probability and not by promoting SV priming. UNC-10/RIM increases release probability via its ability to bind UNC-13 but also through its requirement for recruiting CaV2 calcium channels to presynaptic terminals  double mutants, RIM binding to the UNC-13L C2A domain only appears to be playing a switch role. The C2A domain must therefore have additional functions for regulation of SV release. This is confirmed by our observations in relation to tonic release, which has been proposed to have a different regulatory mechanism from evoked release  mutant, and UNC-13L(K49E); unc-10(nu487 K77/79E) double mutants in 1 mM Ca2+. We only observed a change in the Ca2+ sensitivity in very low Ca2+ at GABAergic synapses, and this phenotype disappeared when the Ca2+ levels were increased ;  release . The mEPfindings , revealincreased . Our obsStrain maintenance and genetic manipulation were performed as previously described . AnimalsWild-type, N2 bristolunc-13(s69) IKP6901 nu487 unc-10(nu487 K77/79E), XKP7503 snb-1::UNC-13L];unc-13(s69)KP6893 nuEx1515 ZTH6 unc-13(s69);hztEx91 [Psnb-1::UNC-13L(K49E)]ZTH454 unc-10(nu487 K77/79E);hztEx91 ZTH7 Punc-17::ChR2(H134R)::YFP +lin-15(+)] VZX460 zxIs6 ZTH605 unc-13(s69);hztEx77 [Psnb-1::UNC-13LΔC2A::mApple]ZTH606 unc-13(s69);hztEx78 [Psnb-1::UNC-13L(K49E)::mApple]ZTH607 [Punc-129::ELKS-1::Cerulean::Cerulean LG]JSD0025 tauIs12 (9x) oxSi91 IIEG5793 unc-10(nu487 K77/79E);oxSi91, MosSCIZTH608 ‘Ex’ in the strain name indicates that the rescue experiments use extrachromosomal array.snb-1 promoter (3 kb) was inserted into MCSI between the SphI and BamHI sites. The C2A domain deletion removed the first 150 amino acids. The 5’ primer (GCTGCAGACTGTAGAATCAACAACTGT) and 3’ primer (ACAGTTGTTGATTCTACAGTCTGCAGC) were used to create the Psnb-1::UNC-13L(K49E) construct.A 5.451 kb cDNA corresponding to UNC-13L was amplified by PCR from the N2 cDNA library and inserted into MCSII of the JB6 vector between the KpnI and NotI sites. The Transgenic strains were isolated by microinjection of various plasmids using either Pmyo-2::NLS-GFP (KP#1106) or Pmyo-2::NLS-mCherry (KP#1480) as the co-injection marker. Integrated transgenes were obtained by UV irradiation of strains carrying extrachromosomal arrays. All integrated transgenes were outcrossed at least seven times.C. elegans as previously described  near the ventral nerve cord (one muscle distance from the recording pipette) and applying a 0.4 ms, 85 μA square pulse using a stimulus current generator (WPI). A pulsed application of sucrose  was applied onto the ventral nerve cord using a Picospritzer III (Parker). The glass pipette containing sucrose was placed at the end of the patched muscle .Electrophysiology was conducted on dissected escribed . Worms wAnimals were immobilized on 2% agarose pads with 30 mM levamisole. Fluorescence imaging was performed on a spinning-disk confocal system (3i Yokogawa W1 SDC) controlled by Slidebook 6.0 software. Animals were imaged with an Olympus 100 × 1.4 NA Plan-Apochromat objective. Z series of optical sections were acquired at 0.11 μm steps. Images were deconvolved with Huygens Professional version 16.10  and then processed to yield maximum intensity projections using ImageJ 1.51 n .unc-10 62–121 K77/79E.ITC experiments were performed using a Nano ITC (TA Instruments). ITC titrations were performed in degassed 20 mM HEPES pH 7.5, 150 mM NaCl at 25°C. After baseline subtraction, the area under each peak was integrated and data were fit to a one site independent binding model usingNanoAnalyze software (TA Instruments) to determine the binding affinity (K), enthalpy (dH) and stoichiometry (n); binding was not observed for Protein expression and purification (for ITC experiments): C. elegans UNC-13 long isoform residues 1–258 bearing the K49E mutation (which abolishes homodimer formation), C. elegans UNC-10 residues 62–121 WT or C. elegans UNC-10 K77/79E were expressed from pGEX6p1 as a GST fusion in Rosetta2 (DE3) cells. Cells were grown at 37°C in 6L 2xyt with carbenicillin (100 µg/mL), chloramphenicol (35 µg/mL) and ZnSO4 , to O.D.600 ~ 0.5, then allowed to cool down to 18°C for an hour before induction with 1 mM IPTG overnight at 18°C. The cells were harvested the next day and lysed in 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM PMSF, 1% Triton X-100, 1 mM DTT, 200 µM ZnSO4 (UNC-10 constructs only); the cells were sonicated 3 × 40 s using a midi tip, and lysates clarified 45 mins @ 40,000 rpm, 4°C in a Sorvall Type 45 Ti rotor. Clarified lysates were bound to 1.5 mL (bed volume) glutathione sepharose beads tumbling overnight at 4°C. The next day, the beads were washed extensively, and HRV3C protease added for on-beads cleavage overnight at 4°C. The eluted soluble constructs were concentrated and subjected to gel filtration on a Superdex 75 10/300 against 20 mM HEPES pH 7.5 150 mM NaCl at 4°C. Single peaks corresponding to the monomeric proteins were concentrated to 21 µM (UNC-13L residues 1–258), to 140 µM (UNC-10 62–121 WT) and to 116 µM (UNC-10 62–121 K77/79E mutant), and used in the ITC experiment.All recordings were obtained using a HEKA EPC10 double amplifier (HEKA Elektronik) filtered at 2 kHz, and analyzed by built-in programs in Igor seven software (WaveMetrics). Each set of data represents the mean ± SEM of an indicated number (n) of animals. Statistical significance was determined using student’s-t test or one-way ANOVA followed by Dunnett’s test to control for multiple comparisons. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Josep Rizo as the Reviewing Editor, and the evaluation has been overseen by Randy Schekman as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Nils Brose (Reviewer #2) and Janet Richmond (reviewer #3).Thank you for submitting your article "Heterodimerization of UNC-13/RIM regulates synaptic vesicle release probability but not priming" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Liu et al. present a very interesting story on a very important aspect of synapse biology: the mechanism by which the interplay between UNC-13 and UNC-10 determines presynaptic function. UNC-13/Munc13 proteins are essential regulators of synaptic vesicle priming. Their loss causes a complete arrest of transmitter release, and their regulation by protein-interactors or second messengers determines several key aspects of synapse function, most notably short-term synaptic plasticity. In this regard the focus on the UNC-13/UNC-10 interaction is important and of very substantial interest because the accounts in the literature are confusing, with evidence for (and against) roles in synapse targeting and sub-synaptic localization of UNC-13/Munc13 and in the regulation of UNC-13/Munc13 dimerization and UNC-13/Munc13 activity. Liu et al. show that the UNC-13L C2A domain homodimerizes and also heterodimerizes with UNC-10, in correlation with previous results obtained with the mammalian proteins. Their data suggest that the UNC-13L C2A domain and its interaction with UNC-10 are important to increase the release probability but do not control priming. The latter result contrasts with data described in Camacho et al., 2017. The reasons for this apparent discrepancy are not clear, but the reviewers believe that the present results will be of substantial interest to the field of synapse biology and, overall, are supportive of the manuscript. However, they also have a number of concerns that are summarized below.Essential revisions:C. elegans showed altered distributions of docked vesicles away from the active zone in the presence of UNC-13 constructs lacking the C2A domain as well as UNC-10 mutants, and this positional effect likely accounts for the reduced release probability observed in the present study. Liu et al. did not analyze sub-cellular localization and did not present expression data to determine the levels of the protein constructs reintroduced in the nerve cord. This is problematic given that all of the UNC-13 constructs are extrachromasomal arrays and thus could be highly or differentially expressed. Furthermore, the present study would have benefited from using CRISPR or some other form of homologous recombination throughout, as was done for their UNC-10 (K77/79E) construct so that expression level effects do not confound the interpretation of the results. Of particular concern is the possibility that overexpression of UNC-13 K49E in the rescue of release in the UNC-10 (K77/79E) background may not be physiologically relevant.1) Earlier studies showed that a truncation of mammalian Munc13-1 that abolishes RIM1 binding leads to a priming defect that can be  overcome by prolonged strong overexpression of the truncated Munc13-1 variant . This indicated that the Munc13/RIM interaction might play a role in the synaptic trafficking and sub-synaptic positioning and anchoring of Munc13s. This notion is supported by the fact that Munc13-1 and ubMunc13-2 levels are massively reduced in RIM1 KO mice. Subsequent analyses in RIM1 KOs showed that synaptic targeting and active zone anchoring of Munc13-1  are perturbed in the absence of RIM1 . In a parallel study, it was shown that perturbation of the Munc13-RIM interaction by infusion of the isolated RIM2 ZnF domain strongly inhibits transmitter release from the calyx of Held while a Munc13-binding-deficient ZnF domain variant does not , which corroborates the earlier/parallel work cited above. Since the first study on the functional effects of the RIM-Munc13 interaction on Munc13-dimerization , the whole field focused almost exclusively on this issue, without addressing the effects of perturbing RIM/Munc13 or UNC-13/UNC-10 interactions on Munc13/UNC-13 localization . This is unfortunate because we know that the levels of Munc13/UNC-13 at synapses and its sub-synaptic positioning affect key synapse features. In addition, previous studies in For these reasons, the present study and its interpretation would profit tremendously from a comparative analysis of the expression levels and sub-cellular localization of the different UNC-13 variants at synapses (as well as of UNC-13 levels and positioning in the presence of the different UNC-10 variants). If analyzing sub-synaptic UNC-13 localization cannot be performed within a reasonable timeframe for this communication, the authors should at least quantify the synaptic UNC-13 levels. The authors should also discuss these various issues and tone down their conclusions accordingly (including the definitive nature of the title).2) Crucial differences observed between the results described by Liu et al. and by Camacho et al., 2017 correspond to the measurements of the readily-releasable pool (RRP) of vesicles by treatment with hypertonic sucrose. It is unclear whether these are indeed real differences between the mammalian and worm systems, or the different results arise from differences in experimental protocols. For instance, the authors use 1 M sucrose instead of 500 mM sucrose, which is more common and was used by Camacho et al. It is also plausible that differences might arise from the method of application of sucrose. The authors should perform experiments with 500 mM sucrose to examine whether their results stand at this lower concentration, provide more experimental details on these experiments and discuss how experimental details might influence the results obtained. Raw traces for both concentrations should be provided and, when collecting the 500 mOsm data, the leak current for each trace used in the analysis should be noted.C. elegans" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Randy Schekman as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Nils Brose (Reviewer #2); Janet E Richmond (Reviewer #3).Thank you for submitting your article "Heterodimerization of UNC-13/RIM regulates synaptic vesicle release probability but not priming in The reviewers have discussed the reviews with one another and with the Reviewing Editor. They found most revisions satisfactory, but there are still some concerns that are summarized below to help you prepare a revised submission.1) The authors produced new extrachromasomal lines for the UNC-13 constructs tagged with C-terminal Apple, which allowed them to measure comparable expression levels, but these were not the arrays used to do the functional electrophysiological analysis in the paper. It is the case that arrays can vary in expression levels from one injection to the next. The authors only provide assurances that the behavior was rescued but did not show the data or the extent of the rescue. More concerning, they did not repeat the electrophysiological analyses on these strains. Without this, how can we be certain that these are similar to the original array-expressing strains and that the same results would be found.unc-13 null and the UNC-13 rescuing arrays tagged with Apple, this would have greatly improved confidence in the conclusions of the paper. The leak currents should be provided and the data should be interpreted considering these issues.2) As for the hyperosmotic traces, only those for the unc-10(k77/79E) were reexamined in 0.5M sucrose and the requested leak currents for the two data sets (i.e. 0.5 and 1M) were not provided. The concern is that at 1M sucrose, if there were substantial leak, the large inward currents observed would arise not from sucrose-induced release but poor seals. This problem has arisen in the past in analyses of fly embryos in which large inward currents were observed in mutants that were subsequently shown to have no primed vesicles; the reason was leaky recordings. If the authors could have produced these data for the 3) In the decision letter a critical discussion of the use of extrachromosomal-array-based expression was provided. Clearly, overexpression via this approach could mask effects that would become detectable with WT-like expression levels. This issue should be discussed briefly as a possible caveat. Essential revisions:1) Earlier studies showed that a truncation of mammalian Munc13-1 that abolishes RIM1 binding leads to a priming defect that can be  overcome by prolonged strong overexpression of the truncated Munc13-1 variant .[…] Furthermore, the present study would have benefited from using CRISPR or some other form of homologous recombination throughout, as was done for their UNC-10 (K77/79E) construct so that expression level effects do not confound the interpretation of the results. Of particular concern is the possibility that overexpression of UNC-13 K49E in the rescue of release in the UNC-10(K77/79E) background may not be physiologically relevant.For these reasons, the present study and its interpretation would profit tremendously from a comparative analysis of the expression levels and sub-cellular localization of the different UNC-13 variants at synapses (as well as of UNC-13 levels and positioning in the presence of the different UNC-10 variants). If analyzing sub-synaptic UNC-13 localization cannot be performed within a reasonable timeframe for this communication, the authors should at least quantify the synaptic UNC-13 levels. The authors should also discuss these various issues and tone down their conclusions accordingly (including the definitive nature of the title).We agree that the synaptic levels and localization of Munc13/UNC-13 proteins at the nerve terminals are essential for SV exocytosis. The use of extrachromosomal arrays in this study raised the question of whether the UNC-13 protein levels from distinct transgenic lines  are similar or not.unc-13 null mutants rescued the locomotion defects (data not shown). Indeed, the UNC-13 with a C-terminal tag is fully functional in mediating SV exocytosis . By analyzing the fluorescence intensity of mApple, we showed that the average proteins levels of UNC-13L, UNC13LK49E, and UNC-13LDC2A are not significantly different . These results exclude the possibility that the decreased SV release in the UNC-13LDC2A rescue is due to a change in the protein level, and that disrupting the C2A/C2A homodimerization has no effect on protein level. It should be noted that we didn’t compare release in these new mApple transgenic strains, as prior studies have shown that a fluorescence tag does not affect UNC-13 protein’s function .To determine this, we made all the rescue constructs with a fluorescence protein tag (mApple). Re-introducing these constructs into the -1 (tagged with Cerulean) exhibit an obvious colocalization . This colocalization was still observed in unc-10(K77/79E) mutants . These results suggest that disrupting the UNC-13L/RIM interaction does not dramatically alter UNC-13L synaptic localization and that UNC-13L likely has other binding partners that promote its active zone localization.To examine whether the sub-localization of UNC-13L is altered when heterodimerization with RIM is disrupted, we analyzed UNC-13L co-localization with ELKS1, another active zone protein. In wild-type background, UNC-13L (tagged with mCherry) and ELKSAll the above results have been incorporated into the figures and text.unc-10(K77/79E) mutant could arise from a change of the expression level of UNC-13, or a change of the sublocalization of the UNC-13 proteins at the nerve terminals. […] These results suggest that disrupting UNC-13L binding to RIM does not prevent synaptic localization of UNC-13L.”“The decreased release probability in the UNC-13LDC2A rescue and the The new generated imaging lines have been added into the “Strain names” in the Materials and methods section.2) Crucial differences observed between the results described by Liu et al. and by Camacho et al., 2017 correspond to the measurements of the readily-releasable pool (RRP) of vesicles by treatment with hypertonic sucrose. It is unclear whether these are indeed real differences between the mammalian and worm systems, or the different results arise from differences in experimental protocols. For instance, the authors use 1 M sucrose instead of 500 mM sucrose, which is more common and was used by Camacho et al. It is also plausible that differences might arise from the method of application of sucrose. The authors should perform experiments with 500 mM sucrose to examine whether their results stand at this lower concentration, provide more experimental details on these experiments and discuss how experimental details might influence the results obtained. Raw traces for both concentrations should be provided and, when collecting the 500 mOsm data, the leak current for each trace used in the analysis should be noted.unc-10(K77/79E) arise from the experimental protocols we used, as the large priming defect in the unc-13 mutants has been consistently observed in our lab. Nevertheless, to address the reviewer’s question, we compared the synaptic currents elicited by 0.5M sucrose between wild type and the unc10(K77/79E) mutants. To record a relatively stable sucrose response under this condition, a 5s puffing duration was used (unc-10(K77/79E) mutants were comparable to those in wild-type worms (Since Rosemund and colleagues first established the assay of using hypertonic sucrose-evoked synaptic current to estimate the size of the readily releasable vesicular pool (RRP), this assay has been widely used in both vertebrates and invertebrates. The sucrose concentrations and puffing durations, however, vary in different studies . It is unlikely that the different results in SV priming in the was used . As showpe worms , consistpe worms . These rWe have described more details how to perform the sucrose puff experiments at the worm NMJ in the “Materials and methods”.“A pulsed application of sucrose  was applied onto the ventral nerve cord using a Picospritzer III (Parker). The glass pipette containing sucrose was placed at the end of the patched muscle .”unc10(K77/79E) mutant by puffing hypertonic sucrose . […] The unchanged SV priming in the unc-10(K77/79E) mutant was also confirmed by puffing a lower concentration of sucrose .”“Nevertheless, we assessed the RRP size in the UNC-13L(K49E) rescue animals and the The reviewers have discussed the reviews with one another and with the Reviewing Editor. They found most revisions satisfactory, but there are still some concerns that are summarized below to help you prepare a revised submission.1) The authors produced new extrachromasomal lines for the UNC-13 constructs tagged with C-terminal Apple, which allowed them to measure comparable expression levels, but these were not the arrays used to do the functional electrophysiological analysis in the paper. It is the case that arrays can vary in expression levels from one injection to the next. The authors only provide assurances that the behavior was rescued but did not show the data or the extent of the rescue. More concerning, they did not repeat the electrophysiological analyses on these strains. Without this, how can we be certain that these are similar to the original array-expressing strains and that the same results would be found.unc-13 mutants rescued by mApple-tagged UNC-13 proteins . Basically, we found very similar results in these rescue strains, compared to those observed untagged UNC-13 rescue strains, indicating that the UNC-13 with C-terminal tag is fully functional. These results have been incorporated into the revised manuscript as a supplementary figure .Both mEPSCs and evoked EPSCs were analyzed in the unc-13 mutants, and the phenotypes were similar to those rescued by corresponding untagged UNC13L constructs ”“All mApple-tagged UNC-13L constructs rescued the SV release in the 2) As for the hyperosmotic traces, only those for the unc-10(k77/79E) were reexamined in 0.5M sucrose and the requested leak currents for the two data sets (i.e. 0.5 and 1M) were not provided. The concern is that at 1M sucrose, if there were substantial leak, the large inward currents observed would arise not from sucrose-induced release but poor seals. This problem has arisen in the past in analyses of fly embryos in which large inward currents were observed in mutants that were subsequently shown to have no primed vesicles; the reason was leaky recordings. If the authors could have produced these data for the unc-13 null and the UNC-13 rescuing arrays tagged with Apple, this would have greatly improved confidence in the conclusions of the paper. The leak currents should be provided and the data should be interpreted considering these issues.unc-13(s69) and UNC-13L rescue, under the lower sucrose condition . The results are similar with what we obtained when puffing 1M sucrose (see eLife), consistent with our findings in this study. We hope that the reviews can agree that our current protocol for sucrose puffing  is reasonable.We now added the quantification for two more mutants, rose see . MoreoveWe remade the following figures with all sucrose traces. The leak current can be seen on each individual traces. We also quantified the leak current for all the sucrose recordings . The results have been incorporated into the revised manuscript as a supplementary figure . As you can see, the leak current is controlled very well in our recordings. The averaged leak current for each genotype is between -20pA and -30pA.“It should be noted that recording of the sucrose-evoked current requires a tight gigaohm seal between the recording pipette and the cell, as a loose seal often leads to an unreal current. We therefore analyzed the leak currents for each sucrose recordings. Our data showed that all recordings were controlled well with acceptable leak currents .”3) In the decision letter a critical discussion of the use of extrachromosomal-array-based expression was provided. Clearly, overexpression via this approach could mask effects that would become detectable with WT-like expression levels. This issue should be discussed briefly as a possible caveat.We agree with this point and have discussed the issue of rescue using extrachromosomal arrays.unc-13 mutants rescued by single copy of UNC-13.”“It should be noted that all the UNC-13 transgenes in this study are extrachromosomal arrays, and that the UNC-13 proteins are overexpressed in these transgenic animals. The overexpression of UNC-13 may cause differential effects with wild-type expression of this protein. Despite this, our findings  are similar with Zhou et al. results which were obtained from


Technological advances over the past decade have unraveled the remarkable complexity of RNA. The identification of small peptides encoded by long non-coding RNAs (lncRNAs) as well as regulatory functions mediated by non-coding regions of mRNAs have further complicated our understanding of the multifaceted functions of RNA. In this review, we summarize current evidence pointing to dual roles of RNA molecules defined by their coding and non-coding potentials. We also discuss how the emerging roles of RNA transform our understanding of gene expression and evolution. Benefiting from the advances in science and technology, our understanding of the complexity of organisms is constantly increasing. The “central dogma” of molecular biology states that genetic information is typically processed from DNA to RNA to protein, and this decides cellular and organismal phenotype . In the In higher organisms, only a small fraction of genetic transcripts (less than 3%) have the capability to encode proteins, despite pervasive transcription across genomes. This raises the question of whether the remaining non-protein-coding transcripts are transcriptional “noise” or contain more genetic information. Large-scale projects for the systematic annotation and functional characterization of genes (such as ENCODE and FANTOM) have reported that at least 80% of mammalian genomic DNA is actively transcribed and elaborately regulated, with the vast majority of this considered to be noncoding RNA (ncRNA) genes . The numLong non-coding RNAs can regulate gene expression in various ways, including epigenetic, transcriptional, post-transcriptional, translational and protein location effects. Corresponding to functional diversity, the modes of action of lncRNAs are also quite varied. lncRNAs can recruit epigenetic factors to modify chromatin state , assemblBased on the “noncoding” definition, the modes of action of lncRNAs mentioned above are exerted primarily through ncRNAs. Intriguingly, recent bioinformatics analyses of large-scale data from ribosome-protected RNA fragments (ribosome profiling or ribo-profiling) have revealed that a considerably large part of these transcripts tends to contain sORFs and binds with ribosomes , suggestIn the present article, we will review current studies of the bilateral functionality of lncRNAs and mRNAs in terms of their coding potential, as well as the advancement of high-throughput techniques that would facilitate a deeper recognition of functional diversity of RNAs. This review will highlight the cases that illuminate the contrapositive roles between lncRNAs and mRNAs, and briefly discuss the biological significance of these discoveries for gene expression and evolution.The original definition of lncRNAs concerns their low/non-coding potential. However, with accumulating evidence from bioinformatics and ribosome transcriptome profiling, lncRNAs have been shown to display strong ribosomal associations in many species, varying from plant to animal, indicating a potential coding capacity in lncRNA sORFs . In receSRA was initially identified as a noncoding gene with multiple RNA isoforms, which is critical in many biological processes, such as acting as a co-activator of nuclear receptors and a regulator of steroid receptor-dependent gene expression  of introns/extrons ; it can directly interact with SERCA to decrease the affinity of this ATPase for Ca2+ and inhibit Ca2+ entry into the SR   via the spliceosome, thereby forming lncRNAs in a circular shape . Throughnslation . Moreovec-SHPRH) .circ-ZNF609 was initially screened out in a functional genetic screen, and is differentially expressed during myogenesis  in flies and humans, with a function of competing with pre-mRNA splicing  and short (CD47-SU), and only the CD47-LU, which is AU-rich, can interact with the RNA-binding protein TIS11B to form a membraneless organelle with a specific biochemical and biophysical environment which is separate from the cytosol  in codons led to a misshapen structure, resulting in loss of the IRES activity of p47 (one isoform of p53) and an abrogated affinity of hnRNPC, but with better binding to Mdm2, which is an E3 ubiquitin-protein ligase in mediating p53/TP53 ubiquitination, and an augmented ability of p53 to activate apoptosis. These facts indicate that TP53 has intricate regulatory roles at both the RNA and protein levels, suggesting that the functions of the RNA and protein molecules are closely intertwined  is attributed to HIST1H1C-mRNA-mediated biological activity, through complementation with the terminal stem-loop sequence of the P6b region of hTR, in a base-pairing pattern. These results extend the functional potency of mRNA ORF regions in a non-traditional and noncoding direction  of the c-myc gene, which has an extra ˜639-nucleotide extension of the 5′ UTR when compared with two major isoforms (P1 and P2) of c-myc mRNA. Ectopic expression of the 5′ UTR of the c-myc P0 transcript alone in HeLa cells results in significantly increased expression of the c-Myc1 (p67) and c-Myc2 (p64) proteins as well as incremental apoptosis sensitivity, but decreased tumorigenicity, all of which are likely attributable to competitive regulation of gene expression in the c-myc locus. These results demonstrate that the 5′ UTR potentially functions in trans to perform gene regulation (The gulation .In recent years, researchers have begun to pay close attention to the development of bifunctional RNAs, and have discussed the evolved roles of RNAs with multiple functions . In the With recent estimates of ribosome profiling, small peptides encoded by lncRNAs have significantly expanded the extent and diversity of the proteome, and predictions suggested that a large fraction of the annotated lncRNAs in various eukaryotic organisms would be translated with sORFs . ProteogMany lncRNAs share similar features with classical mRNAs, such as transcription by polymerase II with a 5′-cap and 3′-polyadenylated tail, and frequent accumulation in the cytoplasm . TherefoHowever, in view of the huge quantity, diversified mechanisms of action, and intricate functions of lncRNAs, it is inappropriate to regard lncRNAs just as a pool for evolved peptides. In terms of RNA alone, its roles are diverse, including the potential to be retro-transcribed into DNA, or to act as an enzyme to participate in complex biochemical processes . MoreoveIn recent decades, because of the addition of the huge family of noncoding genes, RNAs have provoked great interest for their mysterious roles in organisms. lncRNA-encoded peptides expand the horizon of functional mechanisms for these bio-macromolecules. To date, thousands of peptide products have been identified in human cells, with limited understanding of their function. The current review has summarized the recently discovered micropeptides implicated in various biological processes. We also discussed the potential noncoding roles of mRNAs as a regulator. The continued discovery and functional characterization of bifunctional RNAs will provide new insights into important cellular processes and organismal evolution.Both authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Arabidopsis by trimming their 3′ ends. Here, we report the crystal structure of Arabidopsis SDN1 (residues 2-300) in complex with a 9 nucleotide single-stranded RNA substrate, revealing that the DEDDh domain forms rigid interactions with the N-terminal domain and binds 4 nucleotides from the 3′ end of the RNA via its catalytic pocket. Structural and biochemical results suggest that the SDN1 C-terminal domain adopts an RNA Recognition Motif (RRM) fold and is critical for substrate binding and enzymatic processivity of SDN1. In addition, SDN1 interacts with the AGO1 PAZ domain in an RNA-independent manner in vitro, enabling it to act on AGO1-bound microRNAs. These extensive structural and biochemical studies may shed light on a common 3′ end trimming mechanism for 3′→5′ exonucleases in the metabolism of small non-coding RNAs.A family of DEDDh 3′→5′ exonucleases known as Small RNA Degrading Nucleases (SDNs) initiates the turnover of ARGONAUTE1 (AGO1)-bound microRNAs in  Small RNA degrading nucleases (SDNs) can degrade short RNAs. Here the authors report the crystal structure of Arabidopsis SDN1 in complex with a single-stranded RNA, and provide new insight into 3′ end trimming mechanism of 3′ to 5′ riboexonucleases in the metabolism of various species of small RNAs. Within RISC, the 5′ and 3′ ends of the miRNA are accommodated by binding pockets in the AGO protein3.MicroRNAs (miRNAs) are a class of endogenous small RNAs that impact nearly all biological processes by controlling gene expression at the posttranscriptional level. The critical biological functions of miRNAs necessitate tight control of their own abundance in vivo. Both biogenesis and degradation contribute to the steady-state levels of miRNAs. The biogenesis of miRNAs is a multi-step process that results in a mature miRNA loaded into its effector ARGONAUTE (AGO) protein to form the RNA-induced silencing complex (RISC)4. In hen1 mutants, full-length miRNAs are greatly reduced in abundance while miRNA species with 3′ truncation and 3′ uridylation are produced5. These species are associated with the miRNA effector AGO17, suggesting that trimming and tailing occur while the miRNAs are AGO1-bound. In Drosophila, endogenous siRNAs associated with Ago2 are methylated by dmHen19. In dmhen1 mutants, Ago2-bound siRNAs are 3′ truncated and uridylated10. In Arabidopsis, the nucleotidyl transferases HESO1 and URT1 are responsible for miRNA uridylation while the exonucleases SDN1 and SDN2 are responsible for miRNA 3′ trimming15. In vitro, SDN1 can almost completely degrade free miRNAs, generating products of a very small size16, but can only trim AGO1-bound miRNAs by a few nucleotides15. This trimming activity is critical as it removes the last nucleotide that is 2′-O-methylated to allow HESO1 and URT1 to act on the unmethylated, trimmed species15.The degradation of AGO-bound small RNAs in vivo entails their 3′ trimming and tailing. In plants, miRNAs and siRNAs are methylated by the small RNA methyltransferase HEN1 to protect them from degradationDrosophila and Arabidopsis, highly complementary target or target mimic RNAs lead to the specific degradation of cognate miRNAs in vivo20. In fly extracts, fully complementary target RNAs cause the 3′ trimming and tailing of cognate, Ago1-bound miRNAs10. Structural studies reveal that a fully complementary target dislodges the 3′ end of the guide strand from the PAZ domain of an AGO protein21. Highly complementary target RNAs are also known to completely dislodge miRNAs from human Ago2 in vitro22. Despite evidence implicating the role of target RNAs in miRNA degradation, it is not known how miRNA trimming or tailing enzymes engage target RNAs or AGO proteins to exert their effects on AGO-bound miRNAs.Target or target mimic RNAs induce the degradation of RISC-associated small RNAs. In Arabidopsis SDN1 and SDN2 belong to the DEDD 3′→5′ exonuclease superfamily, characterized by an active core consisting of three separate sequence motifs with four invariant acidic amino acids (DEDD). The DEDD superfamily can be further divided into two subfamilies, DEDDh and DEDDy, distinguished by a histidine or a tyrosine present in motif III. The Arabidopsis SDN family belongs to the DEDDh subfamily. Many DEDD superfamily members act in the 3′ end trimming or maturation of small non-coding RNAs. For example, Eri-1 was identified in C. elegans for its negative effects on RNA interference24. It trims 2–4 nucleotides from the 3′ overhang of an siRNA/siRNA* duplex in vitro, thus making the duplex ineffective in RNA silencing;24 it is also involved in 26 G endo-siRNA maturation25. Triman is another DEDD exonuclease that functions in the 3′ end maturation of some Dicer-independent small RNAs connected to heterochromatin formation in S. pombe26. Neurospora crassa QIP was found to interact with the Argonaute protein QDE-2 and trim the 3′ end of QDE-2-loaded precursors of miRNA-like RNAs (milRNAs) into mature milRNAs together with the exosome28. Drosophila Nibbler is a DEDD exonuclease that functions in the 3′ end maturation of AGO1-bound miRNAs30 and PIWI-interacting RNAs (piRNAs)32. PARN-1 in C. elegans and its homolog PNLDC1 in Bombyx mori are DEDD exonucleases that trim piRNA precursors to generate mature piRNAs34. AGO/PIWI-mediated protection and DEDD exonuclease-mediated truncation may be universal mechanisms of small RNA 3′ end formation or small RNA degradation.Arabidopsis SDN1 (residues 2–300) in complex with a 9 nt single-stranded (ss) RNA at 2.8 Å resolution and the crystal structure of SDN1 C-terminal domain (CTD) at 2.05 Å resolution. The SDN1 DEDDh catalytic domain interacts extensively with the N-terminal domain (NTD) and binds four nucleotides from the 3′ end of the RNA via its catalytic pocket. The structure of the SDN1 CTD revealed a non-canonical RRM fold with two extended β strands. Through biochemical studies, we showed that the C-terminal RRM domain acts cooperatively with the DEDDh domain in substrate recognition and is critical for enzymatic processivity. We revealed that the RRM domain binds the 5′ regions of ssRNA substrates or target strands of miRNA/target RNA duplexes, in coordination with the DEDDh domain that attacks the 3′ ends of miRNAs. In addition, we showed that SDN1 interacts with the PAZ domain of AGO1 in an RNA-independent manner and the RRM domain is critical for the trimming of AGO1-bound miRNAs in vitro. Our work reveals how SDN1 acts on free miRNAs and provides a model on how it might trim AGO1-bound miRNAs.To understand the mechanistic basis of SDN1’s activities in miRNA metabolism, we conducted structural and biochemical studies. Here, we report the crystal structure of Arabidopsis SDN1 (residues 2–409) and a 10 nt ssRNA (5′-AGCCCAUUAG-3′) were used in crystallization. The crystals diffracted up to 2.8 Å and the structure was refined to an Rwork of 25.2% and an Rfree of 26.1% with good stereochemistry , and the DEDDh domain interacts with the RNA substrate , the catalytic subunit of Pan2-Pan3 deadenylation complex36. Structural superimposition and sequence alignment of the SDN1 DEDDh domain and hISG20 reveals that the two proteins are highly similar not only in overall structures but also in amino acid sequences , which connects β4 and α3, formed H-bonds with the phosphate group of A9 and the 2′-OH group on the ribose of U8  ions were observed at occupancies of 0.57 for MgA and 0.97 for MgB Fig. . The sidRwork of 17.5% and an Rfree of 23.3% with good stereochemistry  abolished enzymatic activity under enzyme-limiting conditions Fig. , indicatIn the CTD structure, residues 315–409 adopts a βαββαβββ RNA Recognition Motif (RRM) fold Fig. , but the15N-labeled SDN1 RRM (residues 305–409) and a 10 nt ssRNA (5′-AGCCCAUUAG-3′). During the NMR titration, various residues showed large perturbations in chemical shifts or disappearances in the cross peaks  Fig. , which cRNA Fig. , consistRNA Fig. . These rRNA Fig. . The croRNA Fig.  and bindRNA Fig. , support2FA). Although Phe356 may not interact with RNA according to NMR titration and EMSA assays  degraded to a small size in the absence of cold RNA  in the RRM domain replaced by Ala  that mimic miRNA/target mRNA duplexes to test whether SDN1 could act on such RNAs. DsRNAs with a differing number of single-stranded nucleotides at the 3′ end of the miRNA, namely miR166:T0, miR166:T2, miR166:T4, and miR166:T6, were used as substrates , confirming that the RRM domain is involved in RNA duplex processing. To investigate how SDN1 acts on such RNA duplexes, UV-crosslinking assays were carried out with full-length SDN1 or SDN1 ΔC proteins and miR166:T6 RNA duplexes. The RNA duplexes were 5-iodoU modified at different positions in the complementary strand, as illustrated in Fig. The strong activity that SDN1 exhibits towards the miR166:T6 RNA duplex raises a question: as the 3′ single-stranded overhang is not supposed to be long enough for the simultaneous binding by both the DEDDh and the RRM domains, is RRM required for substrate binding or enzymatic activity and if so, where in the RNA duplex does RRM bind? The RRM mutant SDN138. The recombinant SDN1 protein was redesigned with a TEV cleavage site inserted between the DEDDh and the RRM domains  was performed to pulldown AGO1 and associated endogenous miRNAs, and the AGO1 immunoprecipitate was used as the substrate for in vitro SDN1 enzymatic assays. Small RNA sequencing of the enzymatic products showed that SDN1 acted on many AGO1-bound miRNAs15. Using a similar approach, we sought to determine whether the RRM domain is required for the trimming of AGO1-bound miRNAs. First, we purified recombinant wild-type SDN1, the catalytic mutant SDN1D283A and the RRM mutant SDN12FA  and sequence specificities, SDN1 RRM binds RNA weakly in a sequence independent mode. Moreover, canonical RRMs usually fold into a βαββαβ topology, while our structural and NMR titration studies show that SDN1 RRM is composed of a six-stranded antiparallel β sheet with the unconventional extension of β4 and β5, and the two extended β strands are involved in RNA binding. Despite its low RNA binding affinity, the RRM domain is critical to the overall RNA binding affinity and processive enzyme activity of SDN1 by working cooperatively with the DEDDh domain—while the DEDDh domain attacks the 3′ end of an ssRNA, the RRM binds the 5′ region of the RNA.42 may result in the dissociation of miRNA/target mRNA duplexes from AGO1, in analogy to miRNA release from hAgo2 induced by highly complementary target RNAs observed both in vitro and in cells39. Our in vitro data suggests that SDN1 may trim the miRNA 3′ ends in released miRNA/target mRNA duplexes with the RRM domain binding to the target mRNA strand to facilitate trimming.In vitro, the SDN1 RRM domain is important for the activity of SDN1 on other types of substrates, such as RNA duplexes with single-stranded termini and AGO1-bound miRNAs. The activity of SDN1 on RNA duplexes with unpaired 3′ termini may be relevant in vivo. For example, stem-loop RNAs with 3′ overhangs such as pri-miRNAs could be in vivo targets of SDN1. The high complementary between miRNAs and target RNAs in plantsDrosophila10, along with structural support for target RNA displacing the 3′ end of the guide strand from AGO PAZ21, implies that target RNAs promote the access to or activities on AGO1-bound miRNAs by the trimming or tailing enzymes. In our in vitro SDN1 assays on AGO1-bound miRNAs, we found that the RNA binding activity of the SDN1 RRM domain is critical. We propose a model on how target RNAs and SDN1 act in concert to enable the trimming of AGO1-bound miRNAs. In this model, the target RNA displaces the 3′ end of the miRNA from the AGO1 PAZ domain, enabling SDN1 to gain access to the miRNA 3′ end for trimming. Meanwhile, the RRM domain binds the target strand to enhance processivity of the enzyme, as illustrated in Fig. The 3′ end trimming of an AGO1-bound miRNA requires the 3′ end to be released from the AGO1 PAZ domain. The observation of target RNA-triggered trimming and tailing of AGO1-bound miRNAs in Drosophila Nibbler was co-immunoprecipitated with AGO1 and interacts with PIWI to process pre-piRNA 3′ ends in ovaries;32Neurospora QIP interacts with QDE-2 (an AGO protein) to unwind and trim pre-milRNAs;27 Dis3l2 binds hAgo2 to truncate some hAgo2-bound miRNA species in an RNA-dependent manner;43 silkworm Trimmer indirectly interacts with PIWI through the Tudor protein Papi/Tdrkh34. Most of the exoribonucleases that act on AGO-associated small RNAs could interact with RISC directly or indirectly. The interaction between SDN1 and AGO1 PAZ may help to stabilize the reaction complex and enhance the trimming process.In this study, we also revealed a direct interaction between SDN1 and the AGO1/10 PAZ domain. The phenomena of exonucleases interacting with Argonaute proteins to facilitate the truncation of AGO/PIWI-associated small RNAs have been observed elsewhere. Arabidopsis SDN1 cDNA was cloned into pET28-SMT3 (with a His-SUMO tag) for protein expression in E. coli BL21 (DE3) cells. The mutation sites of the SDN1 CTD mutant (E329A/E330A/E332A) were predicted by the UCLA MBI SERp Server to enhance protein crystallizability44. The point mutations were introduced into SDN1 by PCR with a pair of overlapping primers containing the mutations with the QuikChange Site-Directed Mutagenesis Kit (Stratagene). All other SDN1 mutants were constructed using this method. DNA fragments corresponding to AGO1 or AGO10 PAZ and MID-PIWI domains were PCR amplified from AtAGO1 or AtAGO10 constructs45 and cloned into a pET28-MBP vector in which the MBP gene was inserted between NdeI and BamHI sites of pET28. All constructs were validated through DNA sequencing.Full-length or truncated E. coli BL21 (DE3). In general, cells were cultured at 37 °C to an OD600 of 0.6, then isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, after which cells were grown for about 16 h at 18 °C. Cells were collected via centrifugation, resuspended in Lysis Buffer  and lysed via an ultrahigh-pressure homogenizer (JNBIO). Cellular debris were removed by centrifugation and the supernatant was applied to a HisTrap FF prepacked column . The target protein was eluted via AKTA purifier  using elution buffers with a gradient concentration of imidazole . Then ULP protease was used to remove the 6xHis-SUMO-tag, and dialysis was applied to remove the high salt . Then the sample was loaded onto the HisTrap column again and this time the flow-through was collected. The protein sample was diluted to a final NaCl concentration of 100 mM and further purified through ion exchange chromatography with a Q FF column . The protein was eluted with a linear gradient of 0.1–1 M NaCl mixed with a buffer containing 20 mM Tris, pH 8.0, and 0.1% β-Mercaptoethanol via AKTA purifier. The eluted protein was concentrated and further subjected to size exclusion chromatography with a Superdex 75 HiLoad 16/60 column  to achieve high homogeneity in the protein sample . The final purified SDN1 protein was analyzed on a 15% SDS-PAGE gel, and concentrated to approximately 40 mg/mL. Then the sample was aliquoted, flash frozen by liquid nitrogen and stored at −80 °C.All SDN1 proteins and AGO proteins were expressed in 13C/15N-labeled SDN1 proteins were expressed in BL21 (DE3) in M9 medium supplied with L-selenomethionine or with 15NH4Cl and [13C] glucose, respectively. Derivative SDN1 proteins were purified in the same way as native SDN1 proteins.Seleno-methionine (Se-Met) or isotope 2, 50 mM MES, pH 5.6, 1.8 M Li2SO4, and optimized to larger crystals in 10 mM MgCl2, 50 mM MES, pH 5.2, 1.45 M Li2SO4. Crystals of Se-Met-substituted SDN1-ssRNA complex were grown under the same condition. SDN1 CTD  crystals were obtained by hanging drop vapor diffusion at 20 °C in 21% PEG 4 K, 0.2 M NH4Cl, and crystals of Se-Met-substituted SDN1 CTD were grown in 23% PEG 3350, 0.2 M NH4-citrate. All crystals were cryo-protected by the reservoir solution supplemented with 20% ethylene glycerol (SDN1-ssRNA) or 20% glycerol (SDN1 CTD) before flash cooling in liquid nitrogen.SDN1 protein was diluted in GF Buffer to 15 mg/mL and a 10 nt single-stranded RNA was added in a protein/RNA molar ratio of 1:1.2. The sample was incubated on ice for 30 min and screened for crystals using hanging drop vapor diffusion at 20 °C. The initial crystals were obtained in 10 mM MgClAll diffraction data were collected at beamline BL-17U at the Shanghai Synchrotron Radiation Facility (SSRF). Data were processed and scaled using the HKL2000 package. Initial phases were determined by Single-wavelength Anomalous Diffraction (SAD) using the Se-Met-substituted SDN1 proteins or by Molecular Replacement (MR) using Se-Met SDN1 ΔC-ssRNA structure as search model.46. Some of the residues were manually built based on the electron density map using Coot47. The models were then refined against the diffraction data using the Refmac5 program of CCP448 and phenix.refine in PHENIX46. The diffraction data from a native SDN1-ssRNA crystal was collected and processed via MR using Se-Met SDN1 ΔC-ssRNA structure as the search model. The final model of the SDN1 ΔC-ssRNA was built based on the improved density map of the native dataset after refinement. During refinement, at least 5% of randomly selected data was set aside for free R-factor cross validation calculations. Water molecules were added either automatically or manually using Coot47. Sulfate, citrate, and metal ions were modeled in the refinement until the last few cycles. Structural figures were prepared with Pymol49.The Se-Met SDN1 ΔC-ssRNA structure and the RRM structure were solved using SAD with the AutoSol and AutoBuild programs embedded in the PHENIX suite13C/15N-labeled SDN1 CTD (residues 305–405) was used in the standard NMR spectroscopy experiments to assign its 1H, 13C, and 15N backbone and sidechain chemical shifts, and to obtain the NOE-based restraints51. The spectra included the two-dimensional (2D) 13C-edited Heteronuclear Single Quantum Coherence (HSQC) and 15N-edited HSQC; the three-dimensional (3D) HNCO, HNCACO, HNCA, HNCACB, CBCA(CO)NH, HBHA(CO)NH, 15N-resolved HSQC-TOCSY, HCCH-TOCSY for both aliphatic and aromatic regions, 15N-resolved HSQC-NOESY and 13C-resolved HSQC-NOESY in both aliphatic and aromatic resonances52. All NMR experiments were performed at 297 K using a Varian Unity Inova 600 MHz NMR spectrometer, with all spectra processed with NMRPipe53 and analyzed using Sparky54.2HPO4, pH7.4, 100 mM NaCl, 10% D2O; and the pH was adjusted to pH 7.4 by addition of NaOH. Then the RNA was gradually added to the 15N-labeled SDN1 C-terminus (309–405) to a protein/RNA ratio of 1:0, 1:0.5, 1:0.7, 1:1, 1:1.2, 1:1.5, 1:1.7, and 1:2. The 1H-15N HSQC spectrum was recorded every time when a portion of the RNA was added and the weighted average chemical shift perturbations (Δδavg) of each backbone amide resonance were calculated using the equation: Δδavg = [ΔδH2 + (0.2 × ΔδN)2]1/2, where ΔδH and ΔδN are the differences in the 1H and 15N chemical shifts, respectively. Amino acids with chemical shift changes greater than 0.05 p.p.m. are considered as ones showing large changes.The RNA (5′-AGCCCAUUAG-3′) was dissolved in 50 mM Na32P at their 5′ ends. Labeled miR166 was annealed to its cold target RNA in a 1:1.5 molar ratio, and the RNA duplexes were analyzed on a native gel to ensure that all miR166 was in duplexed form. 50 ng/µL SDN1 wild-type or mutant proteins were incubated with 2 nM labeled RNA substrates at room temperature for 30 min in 50 mM Tris, pH 8.0, 135 mM KCl, 2.5 mM MgCl2 (or 2.5 mM MnCl2 as indicated), 1 mM DTT, 40 units RNasein (Promega). Reactions were stopped by adding deionized formamide followed by boiling at 95 °C for 10 min. The RNAs were then resolved on 15% denaturing polyacryladmide gels.All RNA oligonucleotides were synthesized at Dharmacon or IDT, and labeled by 32P-labeled RNAs on ice for 30 min in 50 mM Tris, pH 8.0, 100 mM NaCl, 1 mM DTT in a total volume of 10 µL. The samples were analyzed on 8% native PAGE gels with cold 0.5× TBE buffer.Wild-type SDN1 or mutants (0.5 µM–0.5 mM) were incubated with 2 nM 32P-labeled, and gel purified before annealing to miR166 in 1:1.5 molar ratio. Ten nanomolar RNA was incubated with 0.5 mM wild-type or mutant SDN1 proteins in 50 mM Tris, pH 8.0, 100 mM NaCl, 1 mM DTT in a total volume of 10 µL at room temperature for 30 min. The mixture was subjected to UV irradiation for 30 min on ice. The UV light was filtered through a polystyrene Petri dish to retain UV light at the wavelength of approximately 312 nm. For the SDN1TEV mutant, crosslinking products were digested with 5 units of TEV protease  on ice for 1 h. All samples were incubated with SDS loading dye at 95 °C for 10 min before being resolved on 12% or 15% SDS-PAGE gels.5′-iodoU modified miR166 target RNAs were ordered from Dharmacon, 5′ end 32P-labeled miR166 (approximately 25 fmoles for each reaction) was first incubated with 5 pmoles of wild-type or mutant SDN1 proteins for 20 min in buffer containing 20 mM Tris-HCl, pH 8.0, 135 mM KCl, 1 mM DTT, 0.5 mM EDTA, pH 8.0, and 5% Glycerol. This buffer allows substrate binding but does not support catalytic reactions due to the lack of Mg2+. Then MgCl2, or a mixture of MgCl2 and 0.1 nmoles of cold miR166, was added to initiate exonucleolytic reactions. The final concentration of Mg2+ was 5 mM, and the final molar ratio of labeled miRNA: enyzme: cold RNA competitor was 1:200:4000.The 5′ end E. coli as described below. First, 1 mL medium was prepared by swelling and washing 0.2857 g freeze-dried, CNBr-activated Sepharose 4B powder with 60 ml HCl (1 mM) for 15 min. 5–10 mg SDN1 recombinant protein was dialyzed against coupling buffer , concentrated to 1.5 ml (3.3–6.7 mg/ml) and mixed with the medium. The mixture was incubated on an end-over-end mixer for 1 h at room temperature or overnight at 4 °C. The medium was packed into a column and excess SDN1 protein was washed away with at least 5 column volumes (cv) of coupling buffer. The medium was transferred to 0.1 M Tris-HCl buffer, pH 8.0 or 1 M ethanolamine, pH 8.0 for 2 h to quench any remaining active groups. The medium was then washed with two buffers for at least three rounds. In each round, the medium was first washed with 0.1 M NaAc, pH 4.0, 0.5 M NaCl, followed by another wash with 0.1 M Tris-HCl, pH 8.0, 0.5 M NaCl. Then the medium was washed with TBS . Rabbit serum was diluted 1:2 with TBS, passed through a 0.2 µm filter and incubated with the medium on an end-over-end mixer overnight at 4 °C. The medium was then washed with 5 cv of TBS, 10 cv of 0.5 M NaCl, 20 mM Tris-HCl, pH 7.4, 0.2% Triton- X-100, and 5 cv of TBS. The antibody was eluted in fractions with 0.15 M NaCl, 0.2 M Glycine-HCl, pH 2.0, and 10% (v/v) of 2 M Tris-HCl, pH 8.5, was added to each fraction. The fractions containing anti-SDN1 antibodies were determined by dot blotting followed by protein staining. The fractions containing SDN1 antibodies were pooled, aliquoted and stored at −80 °C.SDN1 polyclonal antibodies were generated by immunizing a rabbit with the full-length SDN1 recombinant protein. The antibodies were affinity-purified using SDN1 protein expressed from rdr6 sdn1 sdn2 12-day-seedlings were extracted, and western blotting was performed using 1 µg/ml SDN1 antibody (1: 2000 dilution).To demonstrate that the SDN1 antibodies recognize SDN1, total proteins from wild-type and 2, 1 mM DTT, 5% Glycerol and 0.2 mg/ml BSA. Then 20 μL pre-equilibrated dextrin beads  were added to each mixture and the samples were incubated at 4 °C overnight on a shaker. All samples were then centrifuged at 86×g for 30 s to collect beads. The beads were washed ten times using wash buffer , and SDS loading dye was added to the beads. After being heated at 95 °C for 10 min, the samples were resolved on 12% SDS-PAGE gels followed by western blotting experiments with 1:1000 diluted anti-SDN1 antibody and 1:1000 diluted HRP-labeled goat anti-rabbit IgG (Beyotime).Approximately 40 μg of purified MBP-tagged, truncated AGOs and MBP alone were mixed with 1 μg of purified wild-type or mutant SDN1 proteins in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM MgCl13. In brief, 1 g of 12-day-old wild-type seedlings was ground to a fine powder in liquid nitrogen and resuspended in 1.5 ml IP buffer. The extract was incubated with 4 μl anti-AGO1 antibody (Agrisera). Then the protein-antibody complexes were captured by 30 µL pre-cleared Dynabeads-Protein-A (Life Technologies). A portion of the IPs was subjected to western blotting to detect AGO1. The rest of the AGO1 IPs was resuspended in reaction buffer , and aliquoted to three equal portions, to which wild-type SDN1, SDN1D283A, and SDN12FA were each added. Each reaction contained 275 pmoles of SDN1 proteins and less than 5 pmoles of small RNAs present in AGO1 IPs. After incubation at room temperature for 2 h, the beads were collected for RNA extraction and small RNA library construction. The RNA extraction, small RNA library construction and bioinformatics analysis were performed as described15.Immunoprecipitation (IP) of AGO1 was performed as describedSupplementary InformationPeer Review File


HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the α-helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2-RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of full-length HuR  HuR/ELAVL1  is a ubiquitously expressed RNA-binding protein implicated in several vital processes such as cell proliferation, differentiation or responses to stress and immune stimuli. Not surprisingly, its knockdown in mice is embryonic lethal . The funDespite this extensive cellular control of HuR abundance, localization and function, the protein is upregulated in many cancer types and its expression levels and cytoplasmic localization are correlated with malignancy. The tumorigenic effect of HuR is proposed to result from the stabilization of mRNAs contributing to cancer development through enhanced cell proliferation and survival, proangiogenic properties, evasion from recognition by the immune system and increase of invasive and metastatic potential of cancer cells . Thus, tHuR specifically recognizes adenine and uridine-rich elements (ARE) and uridine-rich sequences in 3′ untranslated regions (3′UTRs) of mRNAs ,13. PhotDrosophila melanogaster  and in Caenorhabditis elegans (EXC-7). The group of Hu/ELAV proteins show remarkable sequence conservation and all bind to U- and AU-rich sequences. However, they have different cellular localizations  and molecular functions   .In vivo, two groups reported a distinct effect of overexpression of HuR with a deletion of RRM3 on the stability of a reporter mRNA with c-fos ARE  by a less conserved, flexible hinge region of variable length ,26. The -fos ARE ,35, and -fos ARE ,36. In vreported ,38. RRM3reported ,40. Finareported ,37,41. NHere, we report crystal structures of HuR RRM3 free and bound to U- and AU-rich RNAs. Combined with NMR, SAXS and additional biophysical experiments, we characterize RNA binding and dimerization of RRM3 and full-length HuR. We show that the three HuR RRM domains are dynamic in the absence of RNA but adopt a more compact arrangement upon binding to target RNAs. Full-length HuR with mutations that affect RRM3 RNA binding or dimerization has an impaired function, when overexpressed in colon cancer cells (RKO). Our results reveal an important role of RRM3 in RNA binding and functional activity of HuR.http://www.helmholtz-muenchen.de/en/pepf/materials/vector-database/bacterial-expression-vectors/index.html). Highest expression and solubility was obtained with pETM44 . The following constructs were created based on full-length HuR: sHuR (18–323) in pETM44, RRM1,2 (1–186) in pETM11  and RRM3 (243–326) in pET GST-1a . The tags are cleavable with tobacco etch virus protease (TEV) (pETM11 and pET GST-1a) or human rhinovirus 3C protease (3C) (pETM44). Mutations in HuR and HuR RRM3 were introduced using the Quick Change® Site-Directed Mutagenesis Kit (Stratagene) according to manufacturer's protocol. HuR GGS in pETM11 was created by replacing the linker region between RRM1,2 and RRM3 (186–239) with 18 GGS repeats. Additionally, for crystallization, RRM3 was linked to the C-terminus of Escherichia coli thioredoxin 1 (Trx) via a short uncleavable GSAM linker (The codon optimized sequence of human HuR (NP_001410.2) was obtained from Eurofins and cloned into a series of modified pET-24d vectors with various tags (M linker .The HuR-V5 construct (residues 1-326) was cloned into the pEGFPN1 vector along with a C-terminal V5 tag and a stop codon following the tag. HuR-V5 F247/Y249A and W261E were derived from HuR WT by mutagenesis. HuR-V5 ΔRRM3 was subcloned using the In-Fusion technique (Clontech) following the manufacturer's instructions.PAGE-purified synthetic RNAs were obtained from IBA GmbH. Before use RNAs were resuspended in RNase-free water at 10 mM concentration.E. coli BL21(DE3) in LB medium. The proteins were purified with Ni-NTA agarose (Qiagen) under standard conditions. Tags were cleaved with TEV/3C proteases and removed with a second Ni-NTA purification step. Subsequently, the proteins were purified by size-exclusion chromatography (SEC) on a Superdex™ 75 16/60 prep grade column. HuR WT, its point mutants and sHuR were additionally applied on an ion exchange Resource S column before SEC. 15N- and 15N-,13C-labeled proteins were expressed in minimal (M9) medium supplemented with 15NH4Cl and 15NH4Cl, 13C-glucose, respectively. For deuterated protein production, the expression was carried out in M9 D2O medium supplemented with 15NH4Cl and 2H-glucose.Unlabeled proteins were expressed in T1ρ experiment, the HuR GGS concentration was 150 μM (5.4 mg/ml). The higher concentration was required to obtain sufficient signal-to-noise ratio for the NMR T1ρ measurements. For backbone assignment of free RRM3, free RRM1,2 and RRM1,2 bound to AU12 (AUUUUUAUUUUA), the following spectra were collected: HNCA, HNCACB, CBCACONH and 15N-edited NOESY. For NMR titrations, RRM3 and RRM1,2 were titrated with increasing amounts of selected RNAs. Chemical shift perturbations were calculated using the following equation: (Δδ(1H)2 + (0.2 × Δδ(15N))2)1/2. NMR spectra were recorded at 298 K on Bruker 800 and 600 MHz spectrometers equipped with cryoprobes, processed with NMRPipe , 200 mM NaCl, 5 mM DTT and 1 mM EDTA. Concentrations of NMR samples of HuR WT, W261E and GGS were in the range of 20 μM (0.7 mg/ml) to 40 μM (1.4 mg ml). For  NMRPipe  and anal15N backbone T1ρ relaxation experiments were performed at 800 MHz and 298 K. TROSY detection and temperature compensation were employed, according to , setting the experimental error to the standard deviation of the spectral noise. Uncertainties in T1ρ were estimated by 1000 Monte Carlo runs.rding to . The 15NIsothermal titration calorimetry (ITC) measurements were carried out at 298 K using MicroCal ITC200 and PEAQ-ITC calorimeters . Before calorimetry, proteins were dialyzed against 50 mM sodium phosphate buffer (pH 7.0), 200 mM NaCl and 2 mM β-mercaptoethanol. RNAs were injected into the cell containing WT or mutated HuR and RRM3 constructs. After correcting for heat of dilution, the data were fitted to a one-site binding model using the Microcal Origin 7.0 software.http://pymol.sourceforge.net/). For structure refinement statistics see Table For crystallization, HuR RRM3 was expressed with a Trx tag in order to increase its solubility. The protein was concentrated to 6 mg/ml in 20 mM Tris (pH 7.0), 200 mM NaCl, 2 mM β-mercaptoethanol and 6% glycerol. Crystals of the complex were grown at room temperature by vapor diffusion in sitting drops composed of equal volumes (2.25 μl each) of protein solution and crystallization buffer (23% (w/v) PEG 2000 MME, 0.1 M potassium thiocyanate) with addition of 10 mM spermidine tetrahydrochloride . For crystallization of RNA-bound HuR RRM3, untagged RRM3 was co-concentrated with U6 (UUUUUU), AU15 (AUUUUUAUUUUAUUU) and AU6tnf (UAUUUA) to a concentration of 6, 3 and 4 mg/ml, respectively. Crystals of RRM3–U6 complex were grown at room temperature by vapor diffusion in sitting drops composed of equal volumes (1 μl each) of protein solution and crystallization buffer (0.1 M Tris pH 8.5 and 2.25 M ammonium sulfate). Crystals of the RRM3–AU15 complex were grown at room temperature by vapor diffusion in sitting drops composed of equal volumes (200 nl each) of protein solution and crystallization buffer  PEG 8000). Crystals of RRM3–AU6tnf complex were grown at room temperature by vapor diffusion in sitting drops composed of equal volumes (200 nL each) of protein solution and crystallization buffer  PEG 4000). They were cryoprotected by serial transfer into reservoir solution containing 20% (v/v) glycerol. Cryogenic data were recorded at beamline ID23 of the European Synchrotron Radiation Facility (ESRF)  and beamline X06DA at Swiss Light Source (RRM3–U6 complex) . At minimum, three concentrations were measured from each sample and normalized to concentration to exclude concentration-dependent effects. Pair distance distributions, low resolution models, rigid body models, and ensembles were calculated with the ATSAS package v2.7.0.1  and at the beamline BM29 at ESRF Grenoble with a SEC SAXS setup. The BioSAXS1000 measurements were v2.7.0.1 . Molecul2–95% air.RKO colon carcinoma cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine at 37°C in a humidified atmosphere of 5% CORKO cells were transfected with HuR-V5 WT, HuR-V5 ΔRRM3, HuR-V5 F247/Y249A and HuR-V5 W261E plasmids by using Lipofectamine 2000 (Invitrogen), and stably transfected pools of cells were selected with Geneticin (G418) (Gibco).Cells were lysed in buffer  and centrifuged . Protein concentration was determined by using the BCA Protein Assay Kit (Thermo Scientific). After quantification, 10 μg of protein were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and transferred onto membranes. Membranes were blocked with 5% nonfat dry milk in TBS pH 8.0 containing 0.1% Tween-20 (TBST-0.1%), for 1 h at room temperature (RT), washed three times with TBST-0.1% and incubated overnight at 4°C with V5 antibody  and α-tubulin antibody (1:5000 Sigma-Aldrich). Membranes were then washed three times with TBST-0.1% and incubated for 1 h at RT in blocking solution containing secondary anti-mouse antibody conjugated to horseradish peroxidase . Immunoreactive proteins were detected by Western Lightning Enhanced Chemiluminescence reagent  and exposed to X-ray films (Amersham) in a Curix 60 Developer (AGFA).Cell cycle distribution was determined by measuring the cellular DNA content using flow cytometry. In brief, the cells were synchronized in G0 phase by serum deprivation for 16 h and then were released from growth arrest by re-exposure to 10% fetal bovine serum for 6 h, collected by trypsinization and washed with PBS. The collected cells were fixed in 70% ethanol. After the incubation with 10 mg/ml RNase A for 15 min at RT, the cells were resuspended in 0.5 ml 10 μg/ml propidium iodide solution (PI) for staining. The stained cells were monitored by a FACSCanto cytometer (Becton Dickinson). The percentage of cells in the G0/G1, and G2/M phases of the cell cycle was determined using the software FlowJo.et al., 2011). The protein lysates for the RNP-IPs were obtained from stably expressing RKO cell lines after overnight serum starvation and 6 h serum re-addition. Cells were washed twice with 1× PBS and lysed in buffer containing 100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP-40, 1 mM DTT, RNaseOUT (100 U/ml) and Complete protease inhibitor cocktail (Roche). Homogenates were centrifuged 30 min at 14000 rpm, 4°C, and the supernatant was used for IP of RNA–protein complexes. Fresh whole-cell lysate (150 μg) was first precleaned with 15 μg of IgG2 control (BD Pharmingen) and 25 μl of Protein G-Sepharose beads (Sigma-Aldrich) for 30 min, 4°C with agitation. After spin centrifugation, the supernatant was incubated  with 1 ml of a 50% (v/v) suspension of Protein G-Sepharose beads previously precoated with 30 μg of either IgG2 (BD Pharmingen) or V5 (Invitrogen) antibodies, and washed twice using NT2 buffer . After incubation, the beads were washed four times  with 1 ml of ice-cold NT-2 buffer. After the last wash, beads were incubated with 100 μl NT2 buffer containing 20 U DNase I (RNase-free) (Ambion) for 15 min at 37°C, washed with NT2 buffer, and further incubated in 100 μl of NT2 buffer containing 0.1% SDS and 0.5 mg/ml Proteinase K (Roche) for 15 min at 55°C for the isolation of RNA from the immunoprecipitated material. Following centrifugation, the supernatant was collected. RNA from this supernatant was extracted with acid–phenol–CHCl3 and precipitated overnight in the presence of 5 μl glycoblue (Ambion), 25 μl sodium acetate pH 5.2 and 625 μl 100% ethanol. Next day, the precipitated RNA was collected by centrifugation, the pellet washed with 70% ethanol, air dried and resuspended in 20 μl of RNase free water (Sigma-Aldrich). Finally, the RNA was analyzed by real time PCR as described below.RNA-protein complexes were immunoprecipitated as described before  and its concentration and integrity were determined. PCRs were performed using Applied Biosystems ViiA7 thermocycler by the ΔΔCt method, using GAPDH as reference gene.In order to investigate the role of RRM3 in HuR function, we set out to optimize its expression and purification conditions to obtain the domain in amounts and purity suitable for biochemical and structural studies. High expression of soluble RRM3 was obtained as fusion with an N-terminal His-GST tag, but tag cleavage lowered the solubility of the protein. To overcome this limitation, we produced RRM3 fused to an N-terminal thioredoxin 1 (Trx) tag via a short uncleavable Gly-Ser-Ala-Met linker Figure  to improKd of 156 μM and 19 μM, respectively  using untagged RRM3. We initially selected two 6-mer RNA substrates: a sequence comprising the AUUUA pentamer derived from an ARE in TNF-α 3′UTR, (AU6tnf – UAUUUA), and a poly-U RNA that can be found in class III ARE elements without AUUUA repeats (U6 – UUUUUU). Both RNAs were bound by RRM3, with a ly Table . The higly Table  and othely Table . Based oly Table . It thusWe next wished to determine the structural basis for RNA recognition by RRM3. We observed a substantial increase in RRM solubility after RNA binding and were thus able to use untagged RRM3 incubated with selected RNA ligands for structural studies of the complex. This allowed us to perform a comprehensive and comparative analysis of RRM3 interaction with three distinct RNA ligands: AUUUA motif-containing (AU6tnf), a U-rich motif with sparsely distributed adenosine residues AU15) and a poly-U motif (U6). The complexes were crystallized and diffracted to 1.35, 1.90 and 2.01Å, respectively  were added to 15N-labeled untagged RRM3 domain  upon addition of three RNAs that present adenosine bases at specific positions of the RNA  Figure . RNP2 TyKd increased by more than 8-fold (from 40 nM to 351 nM). The RNP1 mutation (F287/F289A) in full-length HuR increased the Kd by >4-fold (194 nM), while the F279A mutation had only a moderate effect on RNA binding by RRM3 (Kd from 19 to 45 μM) and no effect on full-length HuR–RNA interaction (29 nM). Thus, the ITC data confirm a primary role of RNP2 and, to a lesser extent, of RNP1 for RNA binding by RRM3, as well as a marginal contribution of the Phe279 in the β2β3 loop. Moreover, our data clearly demonstrate that RRM3 contributes to RNA binding by full-length HuR in vitro.In order to validate the structural data further, we used ITC to measure the affinities of wildtype (WT) and mutant RRM3 and full-length HuR titrated with various RNA ligands (U6 for RRM3 and AU17 for full-length HuR) Table . We mutaThe dimerization of HuR RRM3 was proposed to be mediated by Trp261, a surface exposed residue conserved in the RRM3 of HuR paralogues and orthologues, but absent in other RRMs that are structurally homologous to HuR RRM3 Figure  40). Ne. Ne40). E. coli Trx 1 is monomeric in solution and is thus not expected to contribute to dimerization of the Trx-RRM3 fusion protein  and oligomerization. Therefore, in addition to WT HuR, we also used its two mutants: HuR W261E (as monomeric protein) and HuR GGS (to increase solubility and stability) Figure . We conf15N-labeled RRM3 bound to AU17 RNA in the presence and absence of unlabeled RRM12, and of 15N -labeled RRM1,2 bound to AU17 RNA in the presence and absence of unlabeled RRM3. In these experiments, each spectrum shows only residues from the 15N-labeled part of HuR  and the signals undergoing spectral changes can be unambiguously identified  upon RNA binding indicating a formation of a more compact entity engaging all three RRMs in RNA binding  of the selected ensemble for HuR W261E shows a high frequency at small Rg compared to the random pool, indicating a prevalence of more compact structures  and Kratky plots derived from the SAXS data , in order to obtain low-resolution structural information in solution for the monomeric full-length HuR (W261E). Data analysis was conducted using the ensemble optimization method (EOM) that is compatible with the existence of variable conformations of the protein. This is advised as the HuR RRMs are connected by unstructured flexible linker regions, consistent with our NMR data, and dynamic RRM arrangements in RRM1,2 that has been reported previously . The disructures . The ensructures , most saa Figure . As thesa Figure  based ona Figure  and on ta Figure . In conca Figure  and F.in vitro and contributes to the RNA-binding affinity of the full-length HuR protein. To confirm this in a cellular context, stable RKO cell lines expressing comparable amounts of V5-tagged HuR WT, ΔRRM3, F2447/Y249A and W261E were created .,61.60,61in vivo    in positions 1 to 4 are comparable (U/A–U–U–U/A versus A–U–U–U)  over AU-rich (UAUUUA) targets. This binding preference matches very well with the major binding motifs, (i) identified by PAR-CLIP for full-length HuR leotide) ,15,62, (UUGUUUU) , (iii) aUUGUUUU) . The incA–U–U–U) . StackinA–U–U–U) .D. melanogaster ELAV RRM3, three short sequences responsible for RRM3 dimerization in vitro were identified  data  data . It is pKd of HuR interaction with c-fos 3′UTR RNA (AUUUUUAUUUUAUUUUU) (in vitro (Albeit RNA recognition by RRM3 is of moderate strength (19 to 156 μM depending on RNA target), it contributes to RNA binding of full-length HuR, as the mutation in RRM3 RNP2 (F247/Y249A) significantly increased the in vitro .Importantly, we detected significantly impaired binding of HuR Y247/F249A to several well-established mRNA targets in human RKO cells Figure –E. This in vitro data indicate that the binding affinity of HuR W261E to an RNA target with a single HuR binding site is not decreased. Nonetheless, targets that exhibit two or more HuR binding sites can be more efficiently recognized by dimeric/oligomeric HuR complexes  also resulted in decreased binding of full-length HuR to some cellular targets. Our s Figure , thus exin vitro and in vivo will be an interesting topic for future investigation.Recently, it has been reported that RRM3 may exhibit posttranslational modifications. For example, Lys283, Lys313 and Lys326 are neddylation sites, while Ser304 and Ser318 are phosphorylation sites ,36. Thesin vitro binding studies we demonstrate that RRM3 is a bona fide RNA binding domain with a canonical mode of RNA recognition expected for RRM domains. Residues centered around RNP2 and RNP1 motifs specifically recognize a UUU/A RNA sequence. (ii) Mutation of key residues involved in RNA recognition reduces the RNA binding capacity of full-length HuR both in vitro and in human cells. (iii) We identified structural details of the HuR dimerization interface localized on the α-helical surface of RRM3 and thus spatially separated from the RNA-binding region. The interaction is mediated by the conserved Trp261 residue. (iv) Our analysis of the full-length HuR conformation in solution indicates that the three RRMs are flexibly arranged in the absence of RNA, while RNA binding induces a more rigid domain arrangement where all three RRMs contribute to RNA binding. (v) Finally, we demonstrate that RNA binding by RRM1,2 together with RRM3 and the dimerization involving Trp261 in RRM3 are required for functional activity of HuR in vivo.We have studied the overall structure and domain arrangements of HuR with a particular focus on the role of RRM3 and its contribution to full-length HuR function. (i) Using high-resolution structural analysis and Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data bank under accession numbers 6GD1 (RRM3-Trx), 6GD2 (RRM3-AU15), 6GD3 (RRM3-AU6tnf) and 6G2K (RRM3-U6). Amide NMR chemcial shifts have been deposited with the BMRB with accession codes 27669 (HuR RRM1-3), 27670 (HuR GGS), and 27671 .Supplementary DataClick here for additional data file.