Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted — and in some cases even shown — to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1. ADAR1 is an interferon-induced enzyme that catalyzes editing of adenine to inosine across the transcriptome as part of the immune response. Here the authors establish how ADAR1 recognizes non-CpG RNA sequences to facilitate the formation of A-Z junctions. Because inosines tend to disrupt double-stranded regions, such RNAs are not recognized by PKR, MDA5, and RIG-I, which are enzymes that trigger an immune response through binding to foreign and therefore unedited double-stranded regions5.Distinguishing between self and non-self RNA is a hallmark of the innate immune system. In humans, self RNAs are edited by an adenosine deaminase that acts on RNA (ADAR1), which converts some adenosines to inosines8. Upon invasion by a pathogen, the cell launches an interferon (IFN) response, resulting in the expression of a longer p150 isoform, which contributes to resisting the infection by editing self RNAs in the cytoplasm9  of ~9 nM23. Furthermore, the addition of CpG repeat sequences to ADAR1p150 substrates significantly increases the deamination of neighboring adenosines24. Whether Zα also binds to less idealized sequences, with what Kd, and following what binding process are fundamental questions that remain to be answered.ADAR1p150 catalyzes A-to-I editing within Alu “foldback” structures, where an Alu element pairs with an inverted Alu element located a few hundred nucleotides away on the same RNAion Fig. 20,21. Ba3 RNA duplex showed that binding specificity is achieved through a critical tyrosine residue (Tyr177), which makes a C–H…π interaction with the syn purine in the CpG Z-step . This reduction in editing leads to the Aicardi-Goutières syndrome28, a neurodevelopmental disorder characterized by hyperactive immune responses. Zα is clearly important for the integrity of the editing process, but how does that relate to its ability to bind Z-DNA and/or Z-RNA?The crystal structure of Zα in complex with a (CpG)tep Fig. 25,26. Ty29. This observation indicates that many dinucleotide sequences can adopt a Z-conformation, as observed in RNA for an even broader range of sequences  have been predicted to shift to Z-RNA within the heavily edited Alu foldback structures36. In further support of a broad recognition of RNA sequences by Zα, pull-down assays revealed that Zα binds to ribosomal stem-loops sometimes devoid of CpG repeats37. This broader RNA sequence specificity is in support of the Zα-mediated increase in ADAR1’s specificity and activity proposed by Rich24, and could also explain the recently described surge in mouse anti-viral response upon sensing endogenous retroviral elements by the Zα domain of Z-DNA-binding protein 138.Although most studies of Zα were done with CpG repeats, Zα can also recognize TpA, CpA, GpC, and TpG in DNA12 are associated with widespread Z-RNA formation at CpG as well as non-CpG sequences within Alu foldbacks. To begin to address this possibility, we applied NMR, circular dichroism (CD), isothermal titration calorimetry (ITC), and analytical ultracentrifugation (AUC) to characterize the binding of Zα to RNA fragments with various frequencies of CpG repeats. We demonstrate binding of Zα at specific sites on a fragment from an Alu foldback rich in UpG steps  and on hairpins from bacterial and human ribosomal RNAs that contain various types of YpR repeats 37. Our NMR results show that binding results in the formation of A-Z junctions in these RNAs and that RNAs with regions of low helical stability adjacent to a Z-forming sequence are better Z-RNA adopters. For the Alu fragment and for the E. coli h43 hairpin in particular, we further determine that two Zα molecules bind every four to five base pairs, and that binding is cooperative, with KD values of 1–9 µM and 40–110 nM. In sum, when binding to AluSx1Jo, Zα adopts a conformation that has characteristics of both a B-Z DNA junction and the (CpG)3 RNA repeat. Overall, our work offers a rationale for earlier observations that ADAR1 binds in multiple steps to double-stranded RNA regions often flanked by regions rich in non-Watson–Crick pairs40. This study also provides a framework for better understanding widespread and pervasive editing in cells.We hypothesize that the >10,000 editing sites in the human transcriptomeCathepsin S (CTSS) gene on human chromosome 1, as well as hairpin 43 from the small subunit of the E. coli ribosome comprising hairpin h43  CpG step together with several UpG and CpA steps. Based on current knowledge of Zα recognition, it is not immediately apparent how these two RNAs could adopt the Z-RNA conformation required for Zα binding.The core model systems of our study are an element from the AluSx1Jo foldback within the h43 Fig. . This re3 and two A-Z junctions, we first carried out CD experiments, which have traditionally been used to monitor transitions to the Z-conformation in DNA and RNA41. Upon titrating increasing amounts of Zα to the control (CpG)3 fragment (from 12:1 to 1:6 RNA:Zα), we observed the characteristic decrease in the A-form peak at 266 nm, the appearance of a peak at 285 nm, and the flip from negative to positive at 295 nm , we observed instead a reduction of the peak at 266 nm and smaller-in-magnitude increases to the molar ellipticities values at 285 and 295 nm  nm Fig. . TherefoTyr177Ala)—resulting in impaired Z-RNA binding capabilities42—which led to smaller absolute magnitudes of the shifts at these three wavelengths for the (CpG)3 RNA and resulted in a growth of the molar ellipticity at 266 nm for the A-Z junction  as the growth in molar ellipticity at 285 and 295 nm and decrease in molar ellipticity at 266 nm of the fully saturated RNA, normalized to the molar ellipticity at 266 nm of the free form 6, EZ ~ 1 6, G10C10 duplex, as well as for cUUCGg and cGAAAg hairpins; Fig. EZ ~ 0.3 and ~0.2, respectively, confirming further that these RNAs are very similar to the control A-Z junctions 3 control  from 3. EZ scores ranged from ~0.1 to 0.47  and for h41 from E. coli (0.47), which we monitored by AUC (see next paragraph). These duplexes were nonetheless interesting, as their predicted secondary structure  and to RNA length (R = 0.75) 3, cUUCGg hairpin, h43 (E. coli), H25 (E. coli), H66 (H. sapiens), H66 (E. coli), h41 (H. sapiens and E. coli), and the AluSx1Jo RNA. Specifically, we compared the fitted molecular weights from the AUC measurements with the theoretical molecular weights from increasing stoichiometric ratios of Zα:RNA. For each fragment except the negative cUUCGg control (major peak (78%) at 7.8 kDa, corresponding to unbound Zα), all potential Zα binding sites according to RNA length were occupied We also measured h43 at 1:2 and 1:4 stoichiometric ratios of RNA:Zα, which gave molecular weights of 21.3 kDa (at 2:1 [RNA]:[Zα]) and 27.9 kDa (at 4:1 [RNA]:[Zα]), indicating 1:2, and 1:3 h43:Zα complexes, respectively n RNA/DNA repeats43 and B-Z junctions32 can be used as a reference for A-Z junction formation. Specifically, it was shown that Z-RNA formation within the (CpG)3 repeat mostly follows a two-step process. First, one Zα binds to one side of the repeat, which begins to convert the RNA to the Z-form. This event promotes binding of a second Zα molecule, which stabilizes the Z-conformation45. The conversion from the A-form to the Z-form in (CpG)3 DNA/RNA causes the imino proton of the guanine base within CpG repeats contacted by Zα to experience slow exchange between the chemical shift positions of the A- and Z-forms43. For B-Z DNA junctions, on the other hand, the adoption of the Z-DNA in the Z-forming sequence is inhibited by neighboring B-DNA32. Therefore, these regions must be destabilized by initial Zα binding before the DNA can adopt the Z-conformation, which causes the imino protons in such regions to disappear into the noise32. Following this initial destabilization, the Z-forming sequence is shifted to the Z-conformation and can either resemble the (CpG)3 case with slow exchange of the syn purines from the B-Z junction, or chemical shift perturbations (CSPs) can be observed instead, depending on the sequence32.The observations that Zα binds to a variety of Alu and ribosomal sequences and that binding is correlated to the number of non-canonical base pairs raise the question of its sequence specificity. To investigate the context for Z-RNA formation at the nucleotide level upon Zα binding, we used 1D 1H-1H NOESY assignments and full 1D imino titrations in Supplementary Figs. 14, U22, U8, U18, G17, and G9 of AluSx1Jo disappeared into the noise in a manner similar to what had been observed for the neighboring B-DNA in B-Z junctions31 and the A-Z 1 control, indicating those base pairs were destabilized , and h43, usually having a high concentration of G–C base pairs and one or more (YpR) steps. Binding also coincides with destabilization of the adjacent base pairs , we also observed CSPs and line broadening (indicating the increase in complex size) in the base pairs that were not destabilized below 1:1 RNA:Zα. These results indicated loading of additional Zα proteins onto the different RNAs, in agreement with our CD and AUC results Fig. –S5. Plot3 RNA23, with the exception of U11 and G21 of H25 . This could be for a number of reasons, the most likely being that the formation of Z-RNA in RNAs with some sequence contexts is more dynamic and thus the conformations are exchanging on a faster timescale.For H25, Zα appeared to target the lower portion of the stem which contained only two (YpR) steps and two G.A base pairs Fig. . However31 suggests that this is not the case. In addition, we did not observe the re-emergence of these peaks at higher concentrations of Zα which we would expect as the complex becomes more stable in the case of intermediate exchange.While the observed disappearance of imino peaks could be caused by line broadening due to intermediate exchange, the striking similarity to titrations done on the B-Z DNA junctionEZ score  with generally the highest stability (low free energy) and tends to destabilize those with the lowest stabillity .Comparing the regions on AluSx1Jo and H66 which are bound or destabilized by Zα to predicted base-pair stabilitiesity Fig. . The regity Fig. , which lity Fig. . However32. Thus, the AluSx1Jo RNA is the best Z-adopting sequence from our selection, followed by H66, h43, and finally H25.Tracking the disappearance of the imino peaks versus Zα concentration revealed that AluSx1Jo, h43, H66, and H25 are destabilized at different concentrations. Specifically, the concentration of Zα required to reduce the imino peak signals to 90% of their initial value is about 2.5-fold less for AluSx1Jo than for h43, twofold less than for H66, and eightfold less than for H25 Fig. . The concorr, calculated from Eq. Having determined that Zα induces novel A-Z junctions in our RNA constructs, we sought to characterize how Zα interacts with such elements. To characterize the binding of Zα to AluSx1Jo and h43, we used ITC and NMR measurements. For AluSx1Jo titration into Zα, these experiments showed an initial endothermic binding event up to a stoichiometric ratio of 2:1 of Zα:AluSx1Jo, followed by a second exothermic event which continued until saturation Fig. . The firKd of 1.14 ± 8.85 µM when the ratio of Zα:AluSx1Jo is ≤2 and 37.6 ± 103.8 nM when ≥2 n repeats (Kd of 241.5 ± 1300 nM for r(CpG)3, Fig. Kd of 9 nM for (CpG)6, according to bio-layer interferometry measurements in ref. 23), although with more complexity.ITC measurements of Zα titrated into h43 showed exothermic binding heat with injections up to a Zα:RNA molar ratio of 2:1, followed by decreasing binding heat up to a ratio of 4:1 Fig. . This su3 RNA and DNA repeats differently than B-Z DNA junctions32. For (CpG)n repeats, significant CSPs (>0.2 ppm) to the backbone residues of Zα were observed for β1–α2 and β2–loop–β3 regions of Zα43, which reflect the binding of two Zαs and conversion to the Z-conformation. For B-Z DNA junctions, Zα experiences less extreme CSPs (<0.15 ppm) and the disappearance of residues 173–177 due to intermediate exchange, which was proposed to reflect an “initial contact conformation” where Zα binds to the Z-adopting sequence and destabilizes the neighboring B-form regions32.Previous NMR studies showed that Zα interacts with (CpG)15N-labelled Zα to investigate how similar the binding site of AluSx1Jo on Zα is to that of (CpG)n repeats and to that of a B-Z DNA junction. The CSPs 3 DNA but not RNA, possibly due to differences in the hydrogen bonding networks23. Our observation of a significant CSP for His159, combined with CSPs at residues 190–194 that do not occur when bound to d(CpG)3, suggests that Zα may invoke a hybrid binding mechanism between Z-DNA and Z-RNA.Our analysis also revealed significant CSPs for Glu141 and His159. Perturbations in Glu141 were unexpected as the amino acid lies near the back side of Zα, adjacent to the binding helix. Glu141 may make specific interactions with A-Z junctions as this residue did not shift when bound to a B-Z DNA junction3 RNA. To answer this question, we acquired Carr-Purcell-Meiboom-Gill (CPMG) measurements at 2:1 Zα:AluSx1Jo  and compared them to previous CPMG measurements on the (CpG)3 DNA and RNA also measured at a 2:1 ratio of Zα:RNA23. CPMG profiles were globally fit (using Eq. kex) between the bound and unbound states of 634 s−1 and major-state population of 96.2% 3 DNA , and were therefore roughly 200-fold higher than for the (CpG)3 RNA23. Thus, Z-RNA formation in our RNA constructs is more dynamic than for the canonical (CpG)3 repeat, and this may explain why we did not observe slow exchange between the A- and Z-form peaks in our imino titrations , we wondered whether the dynamics of Z-RNA formation might be faster in our constructs relative to the (CpG)32. NMR characterization of the binding of Zα to AluSx1Jo suggests that the mechanism for A-Z junction formation upon Zα binding lies somewhere between that of binding to the canonical (CpG)3 RNA and that of B-Z junction formation. In addition, our NMR results suggest that the initial interaction of Zα with such RNAs involves the proposed “initial contact formation”32 where Zα begins to destabilize the neighboring A-form regions, followed by the binding of an additional Zα molecule and adoption of the Z-conformation  will require structural determination of these complexes.Our findings suggest that a wider variety of sequence contexts than previously assumed are able to adopt a Z-conformation, as had been shown for Z-DNA32. It follows from our analysis that the stability of A-form regions adjacent to a Z-forming sequence in RNA would pose a similar barrier, and that Zα may favor binding to regions which can be converted to the Z-conformation more easily (dsRNA regions adjacent to regions with low stability). The sequence of the Z-adopting region is likely also important, as for Zα converting B- to Z-DNA49. The number of hydrogen bonds at the protein-DNA interface is maximal for CpG but minimal for TpA29. We similarly notice in our study that Alu adopts the Z-form at a lower protein concentration than H66 (extended duplex) > h43 > H25. From this analysis we conclude that (CpG)n sequences are better Z-adopting sequences compared to other combinations, but that Gs in any configuration at the binding site  tend to be preferred. Therefore, sequence specificity is likely driven by both the Z-forming propensity of the binding site and the stability of the adjacent regions  plasmid  was a gift from Drs. Peter Dröge and Alekos Athanasiadis. Zα was expressed and purified similarly to56. Briefly, the plasmid was transformed and expresed in BL21(DE3) E. coli. The cultures were grown in Luria Broth and induced with 1 M IPTG at an OD600 of 0.6 and allowed to express Zα for 4 h at 37 °C, then centrifuged to collect the cell pellets. Pellets were resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM Imidazole, 5 mM β-Mercaptoethanol (BME)) and sonicated. Lysate was centrifuged and the supernatant was applied to a His-trap column, washed with 40 mL of lysis buffer, 80 mL of wash buffer (50 mM Tris-HCl (pH 8.0), 1 M NaCl, 10 mM Imidazole, 5 mM BME), and eluted in 20 mL of elution buffer (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 500 mM Imidazole, 1 mM BME). The eluted fraction was then dialyzed into thrombin buffer (5 mM Tris-HCl (pH 8.0), 37.5 mM NaCl, 62.5 mM CaCl2, 0.5 mM DTT), and incubated with bovine thrombin  at room temperature overnight. The resulting samples were then reapplied to a His-trap column and the flow-through was collected. The flow-throughs were then concentrated to ~2 mL and applied to a Superdex 75 Gel Filtration Column  and peak corresponding to pure Zα was collected and concentrated using an Amicon 3 kDa cutoff centrifugal filter . ZαTyr177Ala mutant was ordered from GenScript and prepared the same way as Zα. Proteins were dialyzed into 20 mM potassium phosphate (pH 6.4), 25 mM NaCl, 0.5 mM EDTA for NMR titrations , and for ITC, proteins were dialyzed in the same beaker with the RNA (to match buffer conditions) in 20 mM potassium phosphate (pH 7.0), 25 mM NaCl, 0.5 mM EDTA, and 1 mM DTT. Proteins were concentrated using Amicon 3 kDa cutoff centrifugal filters. For CD and AUC, proteins were diluted into respective buffers from concentrated stocks. The buffers were as follows: 20 mM potassium phosphate (pH 6.4), 25 mM NaCl, 0.5 mM EDTA, 1 mM DTT for CD, and 20 mM potassium phosphate (pH 7.0), 25 mM NaCl, 0.5 mM EDTA, and 1 mM DTT for AUC.The N-terminal Zα domain of 2O was added to 5% prior to NMR measurements. The RNAs for CD and AUC were resuspended at 1 mM in RNAse free ddH2O and diluted to the concentrations indicated for the experiments. For ITC, the RNAs were dialyzed in the same beaker as the proteins in order to match the buffer conditions and concentrated afterwards using 3 kDa cutoff centrifugal filters.RNA constructs were purchased from Horizon Discovery  with HPLC purification. RNAs for NMR titrations were dialyzed into 20 mM potassium phosphate (pH 6.4), 25 mM NaCl, 0.5 mM EDTA, and concentrated to 300 μL using Amicon 3 kDa cutoff centrifugal filters followed by heat denaturing at 95 °C for 1 min and rapid cooling on ice. DTyr177Ala at 42 °C for 30 min and then bringing the samples down to 25 °C over a period of 20 min. Control experiments were run to ensure that the absorbance of Zα and ZaTyr177Ala alone at the same concentrations was minimal in the 250–320 nm range (the range which reports on RNA secondary structure) ) in a 0.1 cm quartz cuvette in 20 mM potassium phosphate (pH 6.4), 25 mM NaCl, 0.5 mM EDTA, and 1 mM DTT at 25 °C. Two scans were acquired and averaged. RNAs were heat denatured at 95 °C for 1 min followed by rapid cooling on ice and titration samples were prepared by incubating 50 µM of the RNA constructs with the specified amount of Zα or ZαWe refer to either the RNA:Protein or the Protein:RNA ratio, with the first molecule mentioned being the one that is being titrated into, for CD, ITC, and NMR measurements.EZ). EZ is based on the CD intensities at wavelengths 285 and 295 nm (Int285 and Int295), both of which have been shown to be characteristic of Z-conformation41, and the decrease in intensity at 266 nm (Int266), which reports on the reduction of the A-form. We assumed that the growth of the intensities at 285 and 295 nm and decrease at 266 nm are proportional to the presence of Z-conformation with an offset accounting for intensities measured when no Z-conformation is present. We calibrated the following equation from CD intensities measured from (CpG)3, where the intensities reach plateaus at 100% Z-conformation:EZ score of the (CpG)3 RNA would be equal to one.To experimentally quantify the extent of Z-conformation present in an RNA construct, we introduced a Z-conformation score derived from CD spectra  and 1 (each nucleotide 100% in Z-conformation). Values between 0 and 1 indicate that the molecule is partially in Z-conformation. This may arise from different contributions from the various nucleotides such that a specific Ez score can be achieved by different Z patterns. For example, if only a subset of the nucleotides adopts Z-conformation at 100%, EZ will be lower than 1.EZ scores by taking into account the difference between the (CpG)3 and (CpG)6 RNAs  and the difference in the EZ score between repeat measurements on h43 E.coli.An error of 0.1 was determined to be appropriate for the calculated EZ score , respectively.The fractional decrease in the ore Fig.  was calcn repeat 3 control (which has the same concentration of Zα as the other constructs).Our method is based on the assumption that the RNA in question starts off predominantly in the A-form. For other conformations, our approximation would be less rigorous. We consider not only the changes in the molar ellipticities at 285 and 295 nm, but also at 266 nm because the loss of the A-form peak is directly related to the transition to the Z-conformation. For Z-RNA junctions where only a portion of the RNA adopts the Z-conformation, the growth at 285 and 295 is not as extensive compared to the (CpG)−1. The titrations of (CpG)3 into Zα were measured with twenty 2 µL injections of 200 µM RNA into 50 µM of protein. The titration of Zα into h43 E. coli was measured with eighty consecutive 0.5 µL injections of 1 mM Zα into 50 µM of RNA. The titration of h43 E. coli into Zα was measured with twenty 2 µL injections of 400 µM RNA into 20 µM of protein. The titration of AluSx1Jo into Zα was measured with twenty 2 µL injections of 200 µM RNA into 20 µM of protein. All ITC thermograms were analyzed and fit using Microcal Analysis version 7 SR4 (Origin); the details of fitting are detailed in ref. 58.RNA constructs for ITC and the Zα protein were dialyzed overnight into 20 mM potassium phosphate (pH 7.0), 25 mM NaCl, 0.5 mM EDTA, and 1 mM DTT (in the same beaker to match buffers) and concentrated to ~500 µM using Amicon 3 kDa cutoff centrifugal filters. Binding heat was measured on a Malvern ITC200 instrument ) at 25 °C and 750 RPM, with 180 s injection delays and a reference power of 10 µcals59 using a specific volume which was normalized to the weight-average of RNA and protein60. The complex stoichiometry was chosen according to which theoretical weight was the closest to the measured weight. Error in the measured molecular weight by AUC can be caused by deviation of the predicted specific volume or viscosity of the sample from the actual values. In addition, if one of the binding sites is weaker than the others resulting in decreased site occupancy, this may cause a deviation in the observed molecular weight from the predicted complex size.For the interactions between Zα and the different RNAs )_equipped with a 5/3 mm triple resonance 1H/13C/15N/19F cryoprobe (CP2.1 TCI). 1D 1H NMR titrations for h43 E. coli, H66 H. sapiens, and H25 E. coli with Zα were carried out on the Varian 600 MHz spectrometer, while the titration for the AluSx1Jo RNA was done on the Bruker 600 MHz spectrometer using a W5 scheme for water suppression . The number of scans for all titration points was 128 with a relaxation delay of 1.6 s, and the spectral width was 24 ppm. 2D 1H-1H NOESY spectra were recorded on the Varian 900 MHz spectrometer for h43 E. coli, the Varian 600 MHz spectrometer for H66 H. sapiens and H25 E. coli, and the Bruker 600 MHz spectrometer for the AluSx1Jo RNA . The 2D 1H-1H NOESY recorded for h43 E. coli was acquired with a mixing time of 200 ms, 1470 × 800 complex points , a 1.3 s recycle delay, and 32 scans. The spectral widths were 22 × 22 ppm for both 1H dimensions. The 2D 1H-1H NOESY recorded for H66 H. sapiens was acquired with a mixing time of 320 ms, 1386 × 400 complex points (162 of the points were collected following a 40% NUS sampling schedule), a 1.3 s recycle delay, and 32 scans. The spectral widths were 20 × 20 ppm for both 1H dimensions. The 2D 1H-1H NOESY recorded for H25 E. coli was acquired with a mixing time of 200 ms, 1396 × 400 complex points (162 of the points were collected following a 40% NUS sampling schedule), a 1.3 recycle delay, and 32 scans. The spectral widths were 20 × 20 ppm for both 1H dimensions. The 2D 1H-1H NOESY recorded for AluSx1Jo was acquired with a mixing time of 300 ms, 1024 × 400 complex points, a 2 s recycle delay, and 32 scans. The spectral widths were 24.5 × 24.5 ppm for both 1H dimensions. 1D spectra from the Bruker spectrometer were processed using TopSpin; all other data were processed using the NmrPipe/NmrDraw/NlinLS package version 10.961. All NUS data were reconstructed using the hmsIST software62 (a part of NMRPipe). All assignments were done in ccpNmr analysis version 2.4.263.All NMR experiments were carried out on Varian 600 and 900 MHz spectrometers (run using VNMRJ version 4.2 Revision A (Agilent)) equipped with 5 mm triple resonance 15N-HSQC spectra for the titration were measured with 1048 (1H) × 60 (15N) complex points with a 1.6 s recycle delay and 32 scans. The spectral widths were 16 × 35 ppm for the 1H and 15N dimensions, respectively. The concentration of Zα was 200 μM. The R1 relaxation experiments were measured with 1048 (1H) × 64 (15N) complex points, a recycle delay of 2 s, 16 scans, and relaxation delays of 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, and 1200 ms. The spectral widths were 16 × 35 ppm for the 1H and 15N dimensions, respectively. The R1ρ relaxation experiments were measured with 1048 (1H) × 64 (15N) complex points, a recycle delay of 2 s, 32 scans, and relaxation delays of 0, 10, 20, 30, 40, 60, 80, 100, 120, and 160 ms. The spectral widths were 16 × 35 ppm for the 1H and 15N dimensions, respectively. During the R1ρ relaxation time, a 15N spin-lock field of 1500-Hz strength was applied. The transverse relaxation rate R2 was calculated from R1 and R1ρ using the following equation:−1(2πΔv/γNB1), Δv is the resonance offset, |γNB1/2π| is the strength of the spin-lock field B1, and γN is the gyromagnetic ratio of the 15N spin.For the titration of AluSx1Jo into Zα, all NMR measurements were carried out on the Varian 900 MHz spectrometer. The τcorr was calculated from the ratio of R2/R164. The τcorr for free Zα was calculated from R1/R1ρ measurements done with a 500 μM Zα sample instead of the 200 μM sample used in the titration.1H) × 64 (15N) complex points, a recycle delay of 1.2 s, 32 scans, and 11 νCPMG values (T = 40 ms) ranging from 10 to 1000 Hz. The spectral widths were 16 × 35 ppm for the 1H and 15N dimensions, respectively. Dispersion profiles were fit to a two-state fast CPMG exchange model with the following equation:R2a and pa are the R2 relaxation rate and the population of state A, Δω is the difference in the chemical shift between states A and B, kex is the rate of exchange between states A and B, and νCPMG is the effective field strength of the refocusing pulse train.The CPMG experiment at 2:1 Zα:AluSx1Jo  was measured on the 600 MHz Bruker spectrometer with 1024  was measured on the 600 MHz Bruker spectrometer with 1024 (1H) × 64 (15N) complex points, a 1 s recycle delay, and 16 scans. The spectral widths were 16 × 35 ppm for the 1H and 15N dimensions, respectively. A weak 15N B1 field of 5 Hz strength was applied during a 400 ms relaxation time. A total of 66 datasets were acquired corresponding to a chemical shift range of 102–132 ppm with steps of 0.5 ppm. From 115 to 116 ppm, the steps were decreased to 0.25 ppm to acquire additional points around Tyr177. In all experiments, 1H decoupling was achieved using a 90x240y90x composite pulse.The 15N-HSQC titration of AluSx1Jo into 15N-labelled Zα were calculated using the following equation66:δH is the chemical shift of a peak in the 1H dimension, and δN is the chemical shift of a peak in the 15N dimension. The KD was determined by fitting CSPs to the following equation66:P] is the concentration of protein in solution (divided by two because the ligand has two binding sites), [L] is the ligand concentration, and CSPmax is the maximum CSP measured over the series of titration points.CSPs from the 15N-HSQC titrations of Zα with the AluSx1Jo RNA and those measured for the (CpG)3 DNA and RNA23 and the B-Z DNA junction32, CSPs were estimated from the published bar graphs showing CSPs vs residue number.For comparison of our measured CSPs from 46  and plotted vs. base-pair identity. The regions identified as being bound and destabilized by Zα binding were overlayed over the plots.The base-pair-specific stability energies for AluSx1Jo, h43, H66, and H25 (Fig. Further information on research design is available in the Supplementary informationPeer Review FileReporting Summary


Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo. Some transcription factors have been proposed to functionally interact with RNA to facilitate proper regulation of gene expression. Here the authors demonstrate that human Sox2 interact directly and with high affinity to RNAs through its HMG DNA-binding domain. For example, the PANDA lncRNA acts as a molecular decoy through its interaction with nuclear transcription factor Y (NF-YA), thereby titrating the transcription factor from its target genes to decrease their expression3. The transcription factor Yin Yang 1 (YY1) binds nascent transcripts, increasing its retention at actively transcribed promoters and the subsequent activation of its target genes4. p53 can bind to the 5ʹ-untranslated region of mdmx mRNA to regulate translation5 and its interaction with RNA may regulate oligomerization and DNA-binding activity6. RNA can also serve as a scaffold for transcription factor assembly, as proposed for the HSR1 RNA-dependent trimerization of the heat shock factor 1 (HSF1) transcription factor to activate heat shock-responsive target genes7. While an expanding set of transcription factors are being found to associate with the transcriptome15, the majority of these studies do not distinguish between direct and indirect association of the transcription factor with RNA and little is known about the biochemical or structural basis for the interactions16.A significant number of transcription factor-RNA associations have been reported that are proposed to regulate gene expression in multiple ways, suggesting that these interactions are quite prevalent18 in embryonic stem cells through expression of pluripotency-associated transcription factors and repression of lineage-specific genes20. Sox2 is essential for embryonic development and its knockout or dysregulation leads to terminal differentiation and embryonic lethality23. In combination with several other transcription factors, including Oct425, Sox2 reprograms somatic cells back to a pluripotent state. Further, induction of pluripotency can be achieved with just Sox2 and Oct4 alone under specific conditions26, highlighting the core role Sox2 plays in the maintenance of pluripotency. Sox2’s role further extends throughout neuronal development28, as Sox2 maintains neural progenitor cells (NPCs) in an undifferentiated, slowly proliferating state by repressing the function of pro-neuronal differentiation proteins29. To fully understand how Sox2 governs these processes, the networks of interactions with partners governing its regulation and function are important to elucidate.A transcription factor of high interest that is also proposed to be associated with RNA is the transcription factor Sox2. Sox2 regulates pluripotency31 and promoting neuronal differentiation32. The lncRNAs ES1 (AK056826) and ES2 (EF565083) are proposed to regulate pluripotency in a Sox2-dependent manner based on stem-cell specific expression, downregulation upon differentiation, and changes to the global transcriptome upon knockdown that were similar to Sox2 RNAi31. Association between Sox2 and these lncRNAs is based upon a pull-down-type experiment in which Sox2 was immunoprecipitated and the ES1 and ES2 transcripts quantified in the bound fraction, although whether this is direct or mediated via other proteins was not determined31. This type of analysis also identified a Sox2 association with the rhabdomyosarcoma 2-associated transcript (RMST), a lncRNA proposed to regulate the Sox2-dependent expression of neuronal differentiation genes32. More recently, analyses of the protein interactome of Sox2 in mouse embryonic stem cells identified a number of RNA-binding protein partners, indicating the potential for broader association of Sox2 with the transcriptome33. A separate approach that identifies proteins involved in ribonucleoprotein (RNP) complexes also implicated Sox2 as associating with RNA, but again, whether this association is direct or indirect remains unknown34.In addition to its well-defined roles in direct transcriptional regulation at the DNA level, Sox2 was found associated with lncRNAs involved in maintaining pluripotencyES2 lncRNA using fluorescence anisotropy (FA) supplemented with an electrophoretic mobility shift assay (EMSA). We observed that Sox2 binds ES2 directly with high affinity through its DNA-binding HMG domain. Using a minimization approach and a library of RNA hairpins designed to test structural contributions to affinity, we found that Sox2-HMG does not bind a single specific site within ES2 but rather generally binds RNAs with a high degree of double-stranded character. To further define the RNA-binding characteristics of the Sox2-HMG domain, we compared the binding modes of DNA and RNA using biochemical and biophysical approaches including NMR spectroscopy and alanine scanning mutagenesis. These data revealed that the HMG domain uses the same surface to bind DNA and RNA, but only a partially overlapping set of amino acids are used to bind each with high affinity. To complement the in vitro biochemical data, we used formaldehyde RNA immunoprecipitation (fRIP) and UV-RNA immunoprecipitation (UV-RIP) in mouse embryonic stem cells (mESC) to establish that Sox2 directly interacts with RNA in vivo. Collectively our data determine that Sox2 directly binds RNA in vitro and in vivo—with high-affinity and low-specificity substrate properties.To determine the nature of the association of Sox2 with RNA, we investigated its ability to directly interact with RNA both in vitro and in vivo. In this study, we measured the binding affinity of Sox2 for 35, a direct interaction has yet to be established. To unambiguously determine whether Sox2 directly binds RNA, we used fluorescence anisotropy (FA) to monitor binding in vitro with highly purified components. The interaction of Sox2 with a fluorescein-labeled DNA containing the FGF4 enhancer element  of 2.1 ± 0.6 nM ((n = 4) standard error of the mean (s.e.m.)) for the high-affinity transition . As evidence for site-specific DNA binding, Sox2 binds poorly to single-stranded DNA containing the consensus site and DNA lacking the consensus site. Each of these ligands binds with >200-fold lower affinity than the FGF4 enhancer sequence 31) was performed. This RNA was selected for initial study due to the experimental tractability of its short length compared with other proposed interacting transcripts35 . The FA assay reveals that Sox2 binds ES2 with a KD,app of 18 ± 1 nM   Fig. . This isDNA Fig. . These d41, we hypothesized that the HMG domain was responsible for interaction with RNA. The binding affinity of the Sox2-HMG domain (amino acids 40–123) was evaluated for the same nucleic acids described above. We observe that Sox2-HMG fully recapitulates the binding profile exhibited by the full-length protein  was further divided into three RNAs that preserve both the sequence and computationally predicted secondary structure of the parental RNA element ) as the RNA duplex  hairpin RNA. DNA and RNA competitively bind Sox2-HMG with apparent inhibition constants  consistent with directly measured dissociation constants ; RNAFAM with DNAunlabeled = 1.0 ± 0.2 nM ) ) Fig. . As a coKD . Thus, the stoichiometric binding properties of Sox2 to DNA and RNA are the same.While the Sox2-HMG domain engages site-specific DNA as a monomer, it is possible that RNA binding is achieved as dimer or other higher-order multimer. The binding stoichiometry of Sox2-HMG to DNA and RNA was assessed using an electrophoretic mobility shift assay where the concentration of labeled nucleic acid greatly exceed the KD Fig. . In thisKD Fig.  for eachKD Fig.  correspoKD Fig. . The B1 46; if the Sox2-HMG domain recognizes RNA in a similar fashion as site-specific DNA, then it is expected to be similarly electrostatically driven. To assess the electrostatic contributions to nucleic acid binding, the salt dependence of Sox2-HMG/nucleic acid interactions was determined by measuring KD,app as a function of monovalent salt concentration , corresponding to 8.2 counterions released. This observation is consistent with the 9 basic amino-acid side chains within 5 Å of the phosphate backbone in a crystal structure45 and 10 of these type of contacts present in an NMR structure of the Sox2-HMG/DNA complex46. Using a similar approach for RNA binding, we observed a slope of −6.0 ± 0.4 , corresponding to 8.7 released counterions. This result indicates that Sox2-HMG uses a similar number of electrostatic interactions to contact DNA and RNA. By comparison, the slopes associated with binding ssDNA and non-specific DNA are substantially less  and −3.8 ± 0.2 , respectively), suggesting that the mode of RNA recognition is more similar to that of site-specific DNA binding than to that of non-specific binding. Separating electrostatic (ΔGel) and non-electrostatic (ΔGnel) components of binding48 reinforces this conclusion, as DNA and RNA have a similar ΔGel that is significantly larger than ssDNA and non-specific DNA , and a fully base-paired RNA hairpin  hairpin RNA was measured using the FA assay; a range of KD,app values from 0.7 ± 0.2 ) to 590 ± 30  nM were observed. To use this data to visualize the DNA and RNA-binding surface of the HMG domain /KD,app(wild type)) of each mutant  have a large impact on binding of DNA and RNA, while several residues involved in the hydrophobic core of the minor wing (His101 and His105) have essentially no effect. These data indicate that the folding and stability of the major wing of the HMG domain is critically important for high-affinity recognition of both DNA and RNA.54. First, we validated the specificity of the Sox2 antibody and ability to specifically and quantitatively immunoprecipate Sox2 with magnetic beads using western blot  crosslinking immunoprecipitation followed by deep sequencing (UV-RIP-seq), a technique that retrieves directly cross-linked RNA–protein interactions Fig. 54. Firstp-value of <0.05. Using this approach, we identified 54 RNA species that were enriched with UV crosslinking, suggestive of Sox2-direct RNA interactions in mESC  with gene annotation derived from GENCODE (vM16). A regression-based statistical test was used to identify enriched RNA species in the IP relative to the input. An RNA species was considered to be enriched if it had a >1.5-fold change (FC) and false discovery rate (FDR)-adjusted ESC Fig. .57. fRIP has the advantage of not only detecting direct Sox2-RNA interactions, but also interactions that arise due to proximity to Sox2. Four independent biological replicates of fRIP were performed in which mESCs were cross-linked with 0.1% CH2O and subsequently treated using the same methods as in UV-RIP. In contrast to UV-RIP, in fRIP we found many more enriched RNA interactions with Sox2—over a thousand compared with 54 in UV-RIP. Importantly, ~75% of UV-RIP interactions were also detected in fRIP , demonstrating the reproducibility of direct interactions in fRIP as well as a more expansive interactome  Fig. 57. fRIP ome Fig. . ConsistES2 lncRNA and a broad set of smaller RNA hairpins with affinities comparable to that of experimentally validated Sox2 DNA-binding sites. Strikingly, we observed that the Sox2-HMG domain has a tighter affinity for both RNA and RNA–DNA duplexes than non-specific DNA, suggesting a strong non-sequence selective binding mode for nucleic acids with non-B-form helical geometry. Analysis of the NMR structure of the closely related SRY–DNA complex58 revealed that protein binding distorts the B-form MIS DNA to a helical geometry much more A-form in character than B-form 59. This preference for bent DNA is consistent with its role as a pioneer transcription factor, as Sox2 binds compact, inaccessible DNA wrapped in nucleosome structures by recognizing partial DNA-binding sites on the nucleosome surface37. The pre-bent structure of nucleosomal DNA is believed to facilitate binding of Sox2 to relaxed consensus sequences through shape complementarity of the widened minor groove37. A similar phenomenon is observed for TATA-binding protein (TBP), another minor-groove binding protein that binds pre-bent DNA targets with an affinity 100-fold tighter than the unbent state60. The preference of the Sox2-HMG domain for DNA deformed from B-form geometry likely also enables it to bind RNAs with A-form double-stranded elements in a non-sequence-specific fashion with high affinity.In this study we demonstrate that Sox2 directly interacts with RNA using its HMG domain. In vitro, the Sox2-HMG domain binds the 37. We propose that it is this feature that enables Sox2 to interact with high affinity to a spectrum of double-stranded non-B-form nucleic acids. This hypothesis is consistent with the observation that Sox2-HMG, like other HMG domains that bind DNA in a sequence-specific fashion, exists in a weakly structured unbound state61. The flexible character of the Sox2-HMG domain would allow it to accommodate a range of nucleic acid geometries. This feature is essential for its duality as a pioneer transcription factor and an RNA-binding protein. Given the proclivity of many HMG domains to also bind non-B-form DNA, it would be anticipated that other HMG domains also have significant RNA-binding activities.The ability of Sox2 to bind pre-bent nucleosomal DNA has been ascribed to its structural flexibility62. On the other hand, fRIP is capable of not only identifying direct interactions, but also those that occur through ribonucleoprotein complexes57. Our fRIP assay captured ~20-fold more potential Sox2-RNA targets than UV-RIP, suggestive that Sox2 may be also be a component of RNPs. While our in vivo data demonstrate that Sox2-RNA interactions occur broadly within the mESC transcriptome, our findings do not provide information about the mechanism(s) by which RNA binding impacts Sox2 function or vice versa. However, our observation of the substantial association of Sox2 with RNA is consistent with previous studies, all of which point to functional Sox2-RNA interactions in cells63.Comparison of the results of the UV-RIP and fRIP experiements reveals a number of associations with the transcriptome via a combination of direct protein–RNA and potentially indirect interactions. Our in vitro and UV-RIP experiments support that Sox2 interacts directly with RNA in vivo. The relatively small set of RNAs captured by this experiment may reflect a limited set of direct Sox2 interaction partners in vivo or the relative inefficiency of crosslinking to double-stranded nucleic acids66. For example, Sox2 promotes alternative splicing in transitional carcinoma cells67. Together, these data suggest that Sox2 may play multiple roles in RNA metabolism either through direct interaction with RNA or via protein partners.Our in vitro studies demonstrate that the Sox2-HMG domain interacts non-sequence specifically with small RNA hairpins. These elements are expected to be a pervasive structural element in RNA, thus it is surprising that Sox2 interacts with a limited set of transcripts in vivo. This selective binding to RNA by Sox2 in vivo may be conferred through interactions with other proteins. The Sox2 proteo-interactome, which involves at least 100 proteins, contains many RNA-binding proteins involved in RNA splicing and processing15, although relatively few of these have yet been characterized extensively in vitro. Of those that have, a greater theme of RNA structural recognition emerges in light of the sequence recognition observed for DNA. This departure from classical models of transcription factors requires a reassessment of how these proteins establish proper patterns of gene expression across the genome.The high-affinity RNA-binding activity of Sox2 revealed by this study underscores a newly appreciated complex interplay between transcription factors and the transcriptome. The large number of transcription factors identified in unbiased transcriptome-wide protein association surveys points toward the pervasive nature of these interactions68 and the resultant vector sequence verified  at 37 °C for 4 h. The cells were pelleted at 2400 × g, resuspended in Sox2 lysis buffer , and lysed by flash freezing in liquid nitrogen and thawing at 37 °C three times prior to incubation for 1 h with 20 U/mL DNase I. The resulting lysate was pelleted at 22,500 × g and the supernatant discarded. The pelleted inclusion bodies containing Sox2 were washed three times with inclusion body wash buffer  prior to resuspension in Sox2 denaturing buffer . The denatured Sox2 solution was then incubated with Ni-NTA agarose (Qiagen) at room temperature (RT) for 30 min. The Sox2-bound Ni-NTA agarose was washed in denaturing high salt buffer  and eluted with Sox2 denaturing elution buffer . Purified protein was flash frozen in liquid nitrogen and stored at −80 °C until ready for use, at which point the protein aliquot was refolded by rapid dilution into a physiological salt buffer .A gene encoding Sox2 was cloned into the pET28a vector as an N-terminal hexahistidine fusion using standard molecular biological approachesE. coli cells expressing Sox2-HMG were grown in LB medium supplemented with 100 µg/mL carbenicillin inoculated with a saturated overnight culture, grown at 37 °C until the OD600 reached 0.6. Protein expression was induced overnight with 1 mM IPTG at 25 °C. The cells were pelleted at 2400 × g, resuspended in Sox2-HMG lysis buffer , and lysed via C3 cell homogenizer. The lysate was clarified at 22,500 × g and incubated with Ni-NTA agarose at 4 °C for 1 h. The bound resin was washed in high salt wash buffer  and eluted in lysis buffer supplemented with 300 mM imidazole. Sox2-HMG was further purified by cleavage of the hexahistidine affinity tag by overnight incubation with bovine alpha-thrombin  and separated using a HiLoad 16/600 Superdex G-75 prep grade column  (GE Life Sciences). Fractions containing Sox2-HMG were pooled and dialyzed in physiological salt buffer supplemented with 50% glycerol and stored at −20 °C until use. For the truncated hairpin assays, Sox2-HMG domain was cloned into pET30b as a N-terminal His8-MBP fusion. Using a similar purification protocol as above, the MBP tag was removed using HRV-3C protease. This Sox2-HMG domain was verified to have comparable binding affinities as the Sox2-HMG expressed using the prior approach.The Sox2-HMG domain (amino acids 40–123) was cloned into pET15b as an N-terminal hexahistidine fusion. BL21(DE3) 69. Protein purification and expression was performed using the same protocol as wild type Sox2-HMG but with the Sox2-HMG lysis buffer supplemented with of 0.1 mg/mL lysozyme and cell lysis achieved by three freeze/thaw cycles. Proteins were purified using HisPur Ni-NTA spin columns (ThermoFisher) using the same buffer conditions listed above. The hexahistidine affinity tag was not removed; Sox2-HMG showed no difference in binding with or without the hexahistidine tag.Alanine mutations of Sox2-HMG were made using PCR-mediated site-directed mutagenesis15NH4)2SO4 and purified as described above. Protein concentrations were calculated using the molar extinction coefficients determined using a molar extinction coefficient of 13,980 M−1 cm−1 for the Sox2-HMG domain, 37,360 M−1 cm−1 for full-length Sox2, 8480 M−1 cm−1 for the W51A and W79A mutants of the Sox2-HMG domain and 12,490 M−1 cm−1 for the Y110A mutant of Sox2-HMG at 280 nm.Isotopically labeled Sox2-HMG for NMR experiments was grown in standard minimal media supplemented with (ES2 lncRNA (EF565083) was cloned into pUC19 using standard restriction cloning techniques68 .71. Briefly, the 3ʹ terminal ribose vicinal diols were oxidized in freshly prepared periodate oxidation buffer  and conjugated to fluorescein-5-thiosemicarbazide (FTSC) by incubating the RNA in freshly prepared fluorescent labeling solution  at 37 °C for 1 h. The labeled RNA was ethanol precipitated and washed several times in cold ethanol prior to resuspension in 0.5x T.E. buffer and passaged over a G-25 column  to remove residual fluorophore. Purified FTSC-labeled RNA was stored at −20 °C until use. FAM-labeled DNA oligonucleotides were chemically synthesized (Integrated DNA Technologies). Prior to use, RNA was heated to 65 °C for one min and snap-cooled and DNA was annealed as described above. 3ʹ-FAM-labeled truncated RNA hairpins were purchased (Integrated DNA Technologies) and used without further purification.RNAs used for FA were labeled at the 3ʹ terminus as describedTo determine the apparent binding affinities, appropriate concentrations of protein were incubated with either 2 nM DNA or 3 nM RNA in binding buffer  for at least 1 h prior to FA measurements. Polarization was measured by a BMG Labtech CLARIOstar microplate reader using a 482 nm excitation filter with a 16 nm bandpass and a 530 nm emission filter with a 4 nm bandpass. Each titration was performed in triplicate and the polarization of each triplicate was measured three times to account for instrument error.KI was calculated from the IC50 by the Nikolovska–Coleska equation72. For salt dependence of select binding interactions, the monovalent salt concentration was varied but maintained a physiological 9:1 KCl:NaCl ratio. Binding curves were fit in Kaleidagraph using the following two-transition binding Eq.  buffer and ran at 200 V for 30 min at room temperature. Gels were dried, imaged using a Typhoon PhosphoImager (Molecular Dynamics), and quantified by measuring the integrated signal density of the free and bound bands by the ImageQuant software suite. Data were fit in Kaleidagraph as a Langmuir binding isotherm. For stoichiometric EMSAs, the above reactions were assembled with either trace radiolabeled RNA or DNA supplemented with 2 µM RNA or DNA, respectively.To validate select apparent binding affinities, binding reactions were assembled as in the fluorescent polarization experiments described above using trace DNA or RNA radiolabeled with 15N-HSQC spectra were collected on an Oxford 600 MHz magnet equipped with an Agilent console and HCN PFG cold probe. Spectra were collected with ~500 µM 15N-labeled Sox2-HMG in NMR Buffer  as free protein or in fully-saturated 1:1.5 Sox2-HMG:nucleic acid complex with DNA or RNA at pH 6.5 and 25 °C. Spectra were analyzed via NMRPipe73 and visualized by CcpNmr Analysis74.Male mouse embryonic stem cells were maintained on 0.1% gelatin (EmbryoMax) coated plates in mESC media , 12.6% fetal bovine serum (EmbryoMax), 1x MEM non-essential amino acids (Gibco), 1x GlutaMAX supplement (Gibco), Pen-Strep , 100 µM 2-mercaptoethanol (Gibco), 1000 U/mL LIF (ESGRO), 1 µM PD0325901 (Sigma-Aldrich), and 3 µM CHIR99021 (Sigma-Aldrich) to maintain pluripotency. Media was changed daily and cells were passaged every 2–3 days.57. Using 15-cm plates, mESC media was aspirated and the cells were washed once with 25 mL of RT 1x phosphate buffered saline (PBS) followed by submersion in 15 mL of room temperature PBS. Formaldehyde was added to 0.1%, allowed to crosslink for 10 min at room temperature, and quenched with the addition of glycine to 125 mM for 5 min. Cells were lifted, collected, and at 4 °C, spun down at 230 × g for 5 min. The pellet was washed twice with 10 mL of ice-cold PBS with centrifuge speeds of 230 × g 5 min for the first wash and 500 × g 5 min for the second wash, and followed by resuspension into 1.6 mL of ice-cold PBS. The suspension was centrifuged at 4000 × g for 75 s at 4 °C. The supernatant was removed, and the pellet was immediately flash frozen in liquid N2 and stored at −70 °C.The fRIP protocol was adapted from a previous study2 of 254 nm UV radiation . Cells were collected mechanically and at 4 °C, centrifuged at 500 × g for 5 min. The cell pellet was resuspended in 1 mL of ice-cold PBS and centrifuged at 4000 × g for 75 s at 4 °C. The supernatant was removed, and the pellet was immediately flash frozen in liquid N2 and stored at −70 °C.Using 15-cm plates, mESC media was aspirated, replaced with 13 mL of ice-cold PBS, and subjected to 400 mJ/cmg for 10 min and the supernatant was collected. The number of cells collected after crosslinking for fRIP and UV-RIP were normalized to ~6.5 million cells. To preclear the supernatant for both fRIP and UV-RIP, 33 µL of Dynabeads Protein G (Invitrogen) and 550 µL of binding/washing buffer  was added and allowed to rotate for 45 min at 4 °C. From the pre-cleared supernatant, 50 µL was used as the input and 25 µL for a pre-Sox2 IP western blot with both being frozen at −20 °C. To the remaining supernatant, 5 µg of Sox2 antibody , and 100 U/mL of RNAseOut (Invitrogen)), allowed to sit for 10 min, and sonicated (Bioruptor UCD-200) in an ice bath with high power setting and 30 s on/off for 10 min. At 4 °C the lysed cells were centrifuged at 16.1k × g for 10 min at 4 °C. The aqueous layer was transferred, mixed with 500 µL of isopropanol and 1 µL of GlycoBlue Coprecipitant (Invitrogen), and allowed to sit 10 min at 4 °C. Samples were centrifuged for 10 min at 12,000 × g at 4 °C, the supernatant was decanted, and the pellet allowed to air dry for 5 min. To input and bead samples, 85 µL of nuclease-free H2O, 10 µL of DNAse I reaction buffer, 1 µL of DNAse I (New England Biolabs) was added, and allowed to sit at room temperature for 15 min. RNA was cleaned up using RNeasy MinElute Cleanup Kit (Qiagen) following quick start protocol and eluted in RNAse free H2O and stored at −70 °C. The cDNA library was generated using KAPA RNA Hyperprep kit with RiboErase , 8 bp xGen dual index UMI adaptors (Integrated DNA Technologies), and sequenced on an Illumina NextSeq 500 using paired end sequencing of 34 bp.At RT, to the input and beads, 83 µL of nuclease-free reverse-crosslinking buffer  was added and allowed to mix well. From the resuspended bead sample, 3 µL was removed, frozen at −20 °C, and stored for a post-Sox2 IP western blot 76. The reads that are not uniquely mapped were discarded. The unique molecular identifiers (UMIs) associated with sequencing reads were counted for gene bodies in the annotation by using featureCounts77. The enriched genes were identified by comparing pull-down with inputs. The fold changes of pull-down relative to input and their statistical significance were calculated by DESeq278 for the genes that have three or more UMIs in at least one sample. Specifically, the total number of UMIs of uniquely mapped reads were used as a normalization factor, and biological replicate information were considered as a covariate.Sequencing reads were mapped by STARFurther information on research design is available in the Supplementary InformationPeer ReviewReporting Summary


Large predators play important ecological roles, yet many are disproportionately imperiled. In marine systems, artificial reefs are often deployed to restore degraded reefs or supplement existing reefs, but it remains unknown whether these interventions benefit large predators. Comparative field surveys of thirty artificial and natural reefs across ~200 km of the North Carolina, USA coast revealed large reef-associated predators were more dense on artificial than natural reefs. This pattern was associated with higher densities of transient predators  on artificial reefs, but not of resident predators . Further analyses revealed that this pattern of higher transient predator densities on artificial reefs related to reef morphology, as artificial reefs composed of ships hosted higher transient predator densities than concrete reefs. The strength of the positive association between artificial reefs and transient predators increased with a fundamental habitat trait–vertical extent. Taller artificial reefs had higher densities of transient predators, even when accounting for habitat area. A global literature review of high trophic level fishes on artificial and natural habitats suggests that the overall pattern of more predators on artificial habitats is generalizable. Together, these findings provide evidence that artificial habitats, especially those like sunken ships that provide high vertical structure, may support large predators. Across terrestrial and aquatic ecosystems, large predators provide valuable services, such as enhancing ecosystem resilience and production , 2, faciEnsuring that large predators have access to suitable habitats is challenging because of widespread habitat degradation in terrestrial and aquatic systems . In coasContinued introduction of artificial habitats to marine ecosystems globally can have both positive and negative ecological effects , 18. EcoDirect comparisons of fish communities occupying artificial versus natural habitats have found conflicting results. Some studies suggest that artificial habitats sometimes underperform natural habitats by hosting lower abundances and species richness , 27, as Because large predators play critical roles in marine ecosystems , it is iWe used observational data from thirty artificial and natural warm-temperate reefs to test how density of large reef-associated predators differed on artificial versus natural reefs . The dat2) belt transects seasonally established along prominent reef features, following Paxton et al. [On each of fourteen artificial reefs and sixteen rocky reefs within Onslow Bay and Long Bay, North Carolina , scuba-dn et al.   and guachanche (Sphyraena guanchancho), at the genus level as Sphyraena sp. because similarities in appearance made it difficult for divers to distinguish them at the species level. The species that we classified as predators are likely mesopredators or top predators depending on the ecosystem specific context, as well as food web dynamics, such as whether these species exert diffuse or concentrated predation pressure [We calculated the density of large reef-associated predators on each 120 mFishbase  and regiFishbase  to classFishbase , 39. In pressure .We further classified the large reef-associated predators by their residency to reefs . We desiStatistical analyses were conducted in R version 3.5.3 . We usedWe used GLMMs and generalized linear models (GLMs) to investigate potential mechanisms, such as reef morphology and vertical relief, behind observed patterns in predator densities by reef type. First, we examined the relationship between specific reef morphologies and predator density. We did this by refitting the GLMM model above , and conducting accompanying LRTs, with reef morphology replacing reef type as a fixed effect. Reef morphology is a factor containing four levels, two for each reef type . Morphology levels for artificial reefs relate to characteristics, such as reef material and spatial extent, as ships are metal, isolated structures, whereas concrete structures are often dozens to hundreds of individual structures dispersed across a broader area. Morphology levels for natural reefs also relate to reef characteristics, as pavement-and-rubble reefs are often low-lying, ephemeral habitats that can be covered and uncovered by sand, whereas ledges are more pronounced, less ephemeral habitats.Second, we examined the relationship between reef vertical relief and predator density. We calculated vertical relief as the difference between the deepest and shallowest point on each reef transect, as recorded by a pressure transducer  moved continuously along the transect  AND  AND (fish community OR fish assemblage). This search yielded 479 potentially relevant studies. We imported titles and abstracts from all 479 studies into Colandr [To test whether patterns observed in diver data were generalizable, we conducted a literature search and accompanying analysis of reef-associated predators on artificial versus natural habitats in marine and coastal ocean environments . We set  Colandr  and screHaemuleon aurolineatum) which have a high trophic level on Fishbase but are not reef-associated predators in many locations. Our refined list came from six unique studies, as eight of the original fourteen studies did not include large predatory fish, as defined on the basis of trophic level  differed among studies, we could not carry out a formal meta-analysis to estimate a global effect. Since a formal, precision-based weighting was not possible, we treated all studies with equal weight and because the abundance metrics varied by study, we summarized the data using a binary variable computing whether the species was more dense on artificial than natural habitats (= 1) versus not (= 0).For each reef-associated predator species from each study, we extracted metadata for the study, including the title, date, authors, geographic location, survey method , artificial reef type , and reef depth. We then extracted the abundance, density, or biomass of each species from each study on artificial versus natural habitats. We also extracted any available measures of precision, such as standard error, standard deviation, or We estimated the log odds of reef-associated predators exhibiting higher values  on artificial reefs than natural reefs by fitting a logit link binomial GLMM  with theComparative field surveys of fourteen artificial reefs and sixteen natural reefs across ~200 km of the North Carolina, USA coast revealed that large reef-associated predators were 4.3 times more dense on artificial than natural reefs. Mean observed predator density across species per sampling transect conducted on artificial reefs was 1.00 ± 0.24 SE, whereas mean density across species on natural reefs was 0.23 ± 0.04 SE. This pattern is based on relative densities of large fish predators observed during diver-conducted visual surveys (236 transects) conducted seasonally on the reefs in 2013–2015 , this pattern was driven by higher densities of transient predators on artificial reefs . These more transient predators, which often associate with reef structures and the water column above, include jacks , mackerel , and sharks . Our model estimated that transient predators were five times as dense on artificial than natural reefs, as the estimated mean transient predator density per transect was 0.31 ± 0.15 on artificial reefs and 0.08 ± 0.04 on natural reefs at the average reef depth (20 m) during both the summer and fall seasons . Season did not significantly relate to predator densities, although reefs sampled during summer and fall seasons tended to have higher predator densities than reefs sampled in spring and winter . The amount of variation in random effects accounted for by reefs (SD = 0.06) and sampling events (SD = 0.15) is smaller than that of species . The model estimated that mean transient predator density per transect was over twice as high on ships (0.37 ± 0.18) than on concrete (0.16 ± 0.09) at the average reef depth (20 m) during the summer . This positive effect of artificial reef relief held for transient predators on artificial reefs . This pattern was also preserved across a subset of artificial reefs for which we could normalize transient predator density by habitat area . The subset of data that we normalized was for artificial reefs that were ships of known lengths. For these ships, we used their length (length prior to sinking) as an estimate of habitat area.Further analyses investigating potential mechanisms that may explain the pattern of elevated predator densities on artificial reefs revealed that reef morphology and reef height were associated with predator densities. Including reef morphology in the model instead of reef type improved the model fit (ΔAIC = 15.7) and suggested that higher densities of transient predators on artificial reefs were largely driven by ships (p = 0.03). Specifically, on the basis of this analysis, we estimate that the odds of large predator species being more abundant on an artificial than natural reef is 2.25 . Study-to-study variation in this quantity was estimated to be small (SD = 0.04).We documented twenty-six cases (reports or instances of a species within a study) in which values of large reef-associated predatory fishes were reported on both artificial and natural habitats  as nearby reefs in our study system . First, In addition to reef morphology and vertical relief, several other reasons may explain why artificial reefs host higher densities of predators in general. First, artificial habitats may augment the capabilities of existing habitats to support large predators. For example, artificial habitats installed as replacements for degraded habitats may replace functions formerly associated with once healthy habitats. Similarly, artificial habitats deployed in locations with healthy, natural habitats may supplement, rather than replace, their function for large predators. Second, in areas that are habitat limited, artificial habitats may form otherwise unavailable sources of habitat capable of supporting large consumers. Specifically, in North Carolina, where our comparative field studies were conducted, differences in habitat availability may relate to why transient predators, but not resident predators, were more abundant on artificial than natural habitats. In this system, natural habitats are often extensive networks of ledges and flat pavements that are not degraded, so artificial habitats of both ship and concrete morphologies supplement existing functions of healthy rocky reefs. The artificial habitats of ships or fields of concrete structures are often isolated ‘islands’ of habitats, whereas the natural rocky reefs are more continuous .The possibility remains that large predators are less likely to be detected on more extensive rocky reefs than on the more island-like artificial habitats simply based upon encounter rates in surveys relative to the overall area of natural reefs. Acoustic telemetry of large predators could help address this by investigating spatial movements and habitat use questions. Comparisons of predator density across the entire area of habitats, rather than standardized sampling transects as conducted here, would also help disentangle whether habitat area may drive the patterns in predator densities that we observed, as would examining reef spatial distributions and proximity to one another. Future research should determine whether large reef-associated predators receive net energetic or fitness benefits from artificial reefs through foraging or whether these large predators are merely resting on the reefs; answering this question will help determine whether artificial habitats provide an aggregation or production function for these fishes. Moreover, while the studied reefs are all open to fishing, we do not know whether differential fishing pressure among sites may relate to our findings. In particular, spearfishing is allowed on these reefs and may result in fish being more wary around our divers conducting fish transects and less likely to be detected on the transects .While there are multiple approaches for the management of large marine predators, our findings initially indicate that strategic installation of artificial habitats may also be a potential tactic for managing these predators; however, this would not be a sound approach if artificial reef aggregate rather than produce predators. Based on the findings of our observational study, we propose that this method would be best suited for transient or mobile species, like jacks and barracuda, which move locally among habitats , 42. ThiWith these uncertainties in mind, we cannot advocate for the widespread and rapid introduction of artificial habitats globally in marine ecosystems. Instead, we suggest that in areas that may be habitat limited and also historically hosted or currently host populations of large predators that marine artificial habitats might assist predators and in such a way that these artificial habitats may facilitate conservation and management objectives.S1 Data(DOCX)Click here for additional data file.S1 File(ZIP)Click here for additional data file.


MaMYB4, an R2R3-MYB repressor TF, characterized by the presence of EAR (ethylene-responsive element binding factor–associated amphiphilic repression) and TLLLFR motifs. MaMYB4 expression was induced by the accumulation of anthocyanins. Transgenic banana plants overexpressing MaMYB4 displayed a significant reduction in anthocyanin compared to wild type. Consistent with the above results, metabolome results showed that there was a decrease in all three identified cyanidins and one delphinidin, the main anthocyanins that determine the color of banana leaves, whereas both transcriptome and reverse transcription–quantitative polymerase chain reaction analysis showed that many key anthocyanin synthesis structural genes and TF regulators were downregulated in MaMYB4 overexpressors. Furthermore, dual-luciferase assays showed that MaMYB4 was able to bind to the CHS, ANS, DFR, and bHLH promoters, leading to inhibition of their expression. Yeast two-hybrid analysis verified that MaMYB4 did not interact with bHLH, which ruled out the possibility that MaMYB4 could be incorporated into the MYB-bHLH-WD40 complex. Our results indicated that MaMYB4 acts as a repressor of anthocyanin biosynthesis in banana, likely due to a two-level repression mechanism that consists of reduced expression of anthocyanin synthesis structural genes and the parallel downregulation of bHLH to interfere with the proper assembly of the MYB-bHLH-WD40 activation complex. To the best of our knowledge, this is the first MYB TF that regulates anthocyanin synthesis that was identified by genetic methods in bananas, which will be helpful for manipulating anthocyanin coloration in banana programs in the future.Anthocyanins spatiotemporally accumulate in certain tissues of particular species in the banana plant, and MYB transcription factors (TFs) serve as their primary regulators. However, the precise regulatory mechanism in banana remains to be determined. Here, we report the identification and characterization of  Musa spp.) are a nutritional fruit that is consumed worldwide, and they are also one of the most important commercial crops in tropical and subtropical developing countries. During the life cycle of banana plants, certain tissues accumulate a large amount of anthocyanin at a particular time  (BrMYB4 (for Brassica rapa)  (VvMYB4 (for Vitis vinifera) (NtMYB2 (for Narcissus tazetta) (TrMYB133 (for Trifolium repens) (PtrMYB194 and PtrMYB165 (for Populus trichocarpa) (FaMYB1 (for Fragaria × ananassa) (Musa acuminata var. DH-Pahang) genome, 298 R2R3-MYB genes have been identified  , BrMYB4 ca rapa) , MdMYB16mestica) , VvMYB4 inifera) , NtMYB2 tazetta) , TrMYB13 repens) , PtrMYB1hocarpa) , and FaMnanassa) . R2R3-MYnanassa) . Two connanassa) . The EARnanassa) . Anothernanassa) . In the entified . Compareynthesis .Vitis vinifera cultivar Carmenere, the expression pattern of VvMYB4-like was the opposite to that of anthocyanin biosynthesis to prevent ectopic accumulation of anthocyanins , a photon flux density of 300 μmol m–2 s–1 throughout a 12-h photoperiod, and a relative humidity of 60–80%. Eight-leaf-stage plantlets were used in the experiment. The leaves from three plants were cut into pieces (1.5 cm × 1.5 cm) and mixed well. Aliquots of the mixed tissues were frozen in liquid N2 and stored at −80°C until use. All samples were collected in triplicate.The embryogenic cell suspensions (ECSs) that were used for transformation were induced from the male flower of MaMYB4 homolog open reading frame was amplified with the following primer set: forward (5′-ATGAG GAGCCCTTGTTGTGATAAG-3′) and reverse (5′-TTAGC GAAAGAGAAGGAGCGTCG-3′) and then introduced into the pMD18-T vector for sequencing. The right MaMYB4 open reading frame fragment was collected and subcloned into the pBI121 vector after a SacI and XbaI double digest. The plasmid pBI121: MaMYB4 was digested with EcoRI and HindIII and then cloned into the pCAMBIA1301 vector (CAMBIA). The obtained pCMABIA1301:MaMYB4 construct was transformed into Agrobacterium tumefaciens strain EHA105 using a previously described method  in the dark. After this, the ECSs were cultivated in regenerated medium and subcultured every 3 weeks for three times until resistant mature somatic embryos appeared.The full-length d method . Brieflygus coding region (forward 5′-GACGATTGCGTCGCATCGAC-3′ and reverse 5′-CGAAAAGTTCGACAGCGTCTCC-3′). Only those yielding the expected PCR fragments by the above primers were chosen for the Southern blot analyses. Four independent transgenic plants were obtained, named MaMYB4OE, MaMYB4-2OE, MaMYB4-3OE, and MaMYB4-4OE. Southern blot analyses were done as we previously described with minor modified (EcoRV and fractionated on 1.0% agarose gel. DNA fragments containing the 303-bp fragment of the third exon of MaMYB4 gene were amplified from the pCMABIA1301:MaMYB4 construct with the forward primer (5′- AGAAGCTCACAAGCATG-3′) and reverse primer (5′-AGCGAAAGAGAAGGAGC-3′), and used as a hybridization probe and labeled with digoxigenin. DNA of wild-type plantlets used as positive control and empty plasmid pCAMBIA1301 was digested with BamHI used as negative control. Labeling, hybridization, and washing were carried out using the DIG labeling and Luminescent Detection Kits . Chemiluminescence was detected on the nylon filters using a chemiluminescence enabled gel-documentation system. Because of MaMYB4OE and MaMYB4-2OE accumulated the least anthocyanin content, they were chosen for subsequent quantitative analysis, and MaMYB4OE was used for subsequent qualitative analysis.The genomic DNA, respectively, from samples including non-transformed controls and hygromycin-resistant plantlets was isolated by the improved CTAB method of modified . Total gThe full-length amino acid sequences of MaMYB4 from banana and R2R3-MYB repressors from other plants were aligned using ClustalW software. A phylogenetic tree was constructed using MEGA 5.05 software . Node suMaMYB4 gene was amplified by PCR and cloned into the pYL322-d1 vector containing a red fluorescent protein (mCherry) reporter gene (digested with EcoRI in advance) to produce the fusion construct pYL322-MaMYB4-mCherry under control of the cauliflower mosaic virus 35S promoter by using a One Step Cloning Kit . The fusion construct and the control vector (pYL322-ICE1-GFP) were transferred into rice protoplasts  and 2 mL of buffer B , respectively, for 30 min at 4°C with ultrasonic vibration. The extracts were then centrifuged for 10 min at 12,000 rpm. The resulting supernatant (2 mL) was used to measure the A510 values, and (A510 at pH 1.0 - A510 at pH 4.5) × 484/24825/fresh weight × 100 was considered as the anthocyanin content. All experiments were independently repeated six times.The anthocyanin content was determined based on a previously reported protocol  with modTM reverse transcription–PCR (RT-PCR) kit protocol . Primer pairs for RT–real-time quantitative PCR (RT-qPCR)  by using the LightCycler® Premix EX Taq  kit protocol . The efficiency of primers for the target gene and internal control gene  was assessed. No-template controls were also set for each primer pair as a blank control. All experiments were independently repeated three times.Total RNA was extracted from different tissues using a plant RNA isolation kit  according to the manual’s instructions. For each sample, the first-strand cDNA was synthesized according to the Prime ScriptPCR) see  were desbHLH were cloned in-frame with the GAL4 DNA-binding domain sequence in the bait vector pGBKT7. Next, we fused the coding sequence of MaMYB4 in-frame with the sequence of the GAL4 activation domain in the prey vector pGADT7. The bait plasmid pGBKT7-bHLH and the prey plasmid pGADT7-MaMYB4 were cotransformed into yeast strain AH109 by the lithium acetate method and grown on DDO medium  for 3 days according to the manufacturer’s protocol. Transformed colonies were plated onto QDO medium , and the possible interaction between MaMYB4 and MabHLH was evaluated according to their growth status. The bait plasmid pGBKT7-ICE1 and the prey plasmid pGADT7-MAPK3 were used as a positive control. Additionally, the bait plasmid pGBKT7-ICE1 and the prey plasmid pGADT7-MAPK9 were used as a negative control.The yeast two-hybrid assay was performed using the Matchmaker Gold Yeast Two-Hybrid Systems (Clontech). The coding sequences of MaCHS, MaDFR, MaANS, and MabHLH promoter activity by MaMYB4, transient transcription dual-LUC assays using ECSs of banana were performed. The protoplast isolation and polyethylene glycol–mediated transformation were carried out as previously described  was transfected into protoplasts and incubated overnight. The luciferase activity was measured using a luminometer  with the Dual-Luciferase Reporter Assay System , according to the manufacturer’s instructions. All experiments were independently repeated six times.For the analysis of transrepression of escribed . We isolMaMYB4-overexpressed plants and wild-type plants in three biological replicates as previously described  of 1 were set as the threshold for significantly differential expression. Gene ontology enrichment analysis of differentially expressed genes was implemented by the GOseq R package, in which gene length bias was corrected. Gene ontology terms with a corrected P value less than 0.05 were considered significantly enriched by differentially expressed genes. All sequences generated in this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive1 with project number PRJNA662185.Total RNA was isolated from the leaves of eight-leaf-stage plantlets of escribed . RNA samg for 10 min, the extracts were absorbed  and filtered  before liquid chromatography–mass spectrometry (LC-MS) analysis.For sample preparation and extraction, each freeze-dried leaf was crushed using a mixer mill  with a zirconia bead for 1.5 min at 30 Hz. Next, 100 mg of powdered leaf was extracted overnight at 4°C with 1.0 mL 70% aqueous methanol. Following centrifugation at 10,000 × The sample extracts were analyzed using an LC–electrospray ionization–tandem MS (ESI-MS/MS) system, high-performance LC, Shim-pack ultrafast LC Shimadzu CBM30A system, quadrupole-linear ion trap hybrid mass spectrometer (Applied Biosystems 4500 QTRAP). The analytical conditions were as follows: high-performance LC: column, Waters Acquity ultrahigh performance LC HSS T3 C18 ; solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program, 100:0 vol/vol at 0 min, 5:95 vol/vol at 11.0 min, 5:95 vol/vol at 12.0 min, 95:5 vol/vol at 12.1 min, 95:5 vol/vol at 15.0 min; flow rate, 0.40 mL/min; temperature, 40°C; injection volume: 5 μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap mass spectrometer.Linear ion trap and triple quadrupole scans were acquired on a triple quadrupole–linear ion trap mass spectrometer, API 4500 QTRAP LC/MS/MS system, equipped with an ESI turbo ion-spray interface, operating in a positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550°C; ion spray voltage 5,500 V; ion source gas I, gas II; and curtain gas were set at 55, 60, and 25.0 psi, respectively; the collision gas was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in triple quadrupole and linear ion trap modes, respectively. Triple quadrupole scans were acquired as multiple reaction monitoring (MRM) experiments with collision gas (nitrogen) set to 5 psi. Declustering potential and collision energy for individual MRM transitions were performed with further declustering potential and collision energy optimization. A specific set of MRM transitions was monitored for each period according to the metabolites eluted within this period. All samples were independently collected and tested in triplicate.t test (P < 0.05 or 0.01).Each experiment contained a minimum of six plants per treatment and was repeated three or six times. The data were recorded as the mean ± standard errors of three or six independent biological replicates. Statistical differences between samples were analyzed by Student 2 and the MYB conserved domain (PF00249) from HMMER3, we identified 298 R2R3-MYBs. Among them, 21 R2R3-MYBs were found that contained the EAR motif, and four of them also contained the TLLLRF motif, namely, Ma06_g11140.1, Ma03_g21920.1, Ma08_g16760.1, and Ma10_g19970.1, and they all belong to subgroup 4 according to the classification of MYB in A. thaliana (MaMYB3 (Ma06_g11140.1) has been proven to be related to decreased degradation of starch and delayed fruit ripening in banana, but it is unrelated to the regulation of anthocyanin  were identified, with 39 DEFs exclusively in wild type, 10 DEFs in MaMYB4 overexpressors only, and 5 DEFs in both. Compared to the wild type, the number of flavonoids that were decreased in abundance (43) was three times more than that which was increased in abundance (11) in the leaves of MaMYB4-overexpressed plants , two chalcone isomerase (CHI), one flavonoid 3′-monooxygenase (F3’H), two dihydroflavonol 4-reductase (DFR), one leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS), and two anthocyanidin 3-O-glucosyltransferase (UFGT) genes  genes . MoreoveMYB  and two bHLH genes (Ma03_g18660.1 and Ma06_g24180.1), which were homologous to the member of the MYB-bHLH-WD40 complex in Arabidopsis. Unexpectedly, the two R2R3-MYBs (Ma08_g16760.1 and Ma10_g19970.1) with EAR and TLLLFR motifs were downregulated in MaMYB4 overexpressors , and they contained at least one conserved MYB-recognition element, suggesting that MaMYB4 can bind to their promoter. We then analyzed whether MaMYB4 affected the transcriptional activity of four representative genes on its target promoters using a transient transcription assay in banana protoplasts. The LUC activity level from the pGreen-CHSpro:LUC, pGreen-DFRpro:LUC, pGreen-ANSpro:LUC, and pGreen-bHLHpro:LUC reporter was 2. 4-, 2. 8-, 4. 8-, and 6.7-fold lower, respectively, when we coexpressed MaMYB4 in the protoplasts relative to coexpressed GFP  was three times more than increased (11) in the leaves of MaMYB4 overexpressors  and TFs (including the MYB-bHLH-WD40 complex) have been found to promote anthocyanin biosynthesis, whereas R2R3-MYB repressors can prevent anthocyanin accumulation by acting upon MYB-bHLH-WD40 complex, restraining the abundance of bHLH and MYB activators, and directly binding to the promoter of enzyme-coding structural genes  and two bHLH genes (Ma03_g18660.1 and Ma06_g24180.1)  . Subsequ24180.1) . Ten antFaMYB1-like–type repressors inhibited anthocyanin biosynthesis mainly at three levels. First, they are able to interfere with the proper assembly of the MYB-bHLH-WD40 activation complex by bridging through their bHLH factors, resulting in the repression of transcription of late anthocyanin structural genes, such ANS, DFR, and UFGT. Second, FaMYB1-like–type repressors restrain the abundance of bHLH and MYB activators. Third, they inhibit the expression of anthocyanin structural genes , DFR (Ma04_g10620.1), ANS (Ma05_g03850.1), and bHLH (Ma06_g24180.1) promoters  reduced expression of anthocyanin synthesis structural genes and (ii) parallel downregulation of the bHLH to interfere with the proper assembly of the MYB-bHLH-WD40 activation complex.In our study, three MYB genes and two bHLH genes, which were highly homologous to y MaMYB4 , and it ynthesis . Consistromoters . Our resbHLH to interfere with the proper assembly of the MYB-bHLH-WD40 activation complex. To the best of our knowledge, this is the first MYB TF that regulates anthocyanin synthesis that has been identified by the genetic method in bananas, which will be helpful for manipulating anthocyanin coloration in banana plants in the future.Our data indicate that MaMYB4 acts as a repressor of anthocyanin biosynthesis in banana, which is likely due to a two-level repression mechanism consisting of a reduction of the expression of anthocyanin synthesis structural genes and the parallel downregulation of https://www.ncbi.nlm.nih.gov/sra/) with project number PRJNA662185.All sequences generated in this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive (G-MD and SZ conceived the project. G-MD, SZ, and Q-SY performed most of the experiments. C-YL, G-JY, and F-CB performed the genomic expression analysis. H-JG, TD, and OS performed the RNA-Seq analysis. C-HH and W-DH analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.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.