Homo sapiens and Chaetomium thermophilum, for which crystal structures are known. Preferential binding of G-quadruplex RNA is conserved, surprisingly using different protein elements. Key RNA-binding residues are spread out along the surface of EZH2, with other subunits including EED also contributing, and missense mutations of some of these residues have been found in cancer patients. The unusual nature of this protein-RNA interaction provides a paradigm for other epigenetic modifiers that bind RNA without canonical RNA-binding motifs.Polycomb repressive complex 2 (PRC2) is a key chromatin modifier responsible for methylation of lysine 27 in histone H3. PRC2 has been shown to interact with thousands of RNA species in vivo, but understanding the physiological function of RNA binding has been hampered by the lack of separation-of-function mutants. Here, we use comprehensive mutagenesis and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify critical residues for RNA interaction in PRC2 core complexes from  PRC2 spotein 4) . The comotein 4) .How human PRC2 is recruited to specific sites and regulated on chromatin in mammals is an important unanswered question. Multiple factors including chromatin marks, DNA sequence elements and other DNA-binding proteins (e.g. JARID2) have been suggested to recruit PRC2 to its genomic targets . AlthougPRC2 binds RNA promiscuously in vitro and in vivo , 2015, mWhat elements within PRC2 bind RNA has been unclear, especially since none of the core PRC2 subunits has an RNA recognition motif (RRM). An unstructured region (residues 342–368) of mouse EZH2 that has been proposed to bind RNA  is not pChaetomium thermophilum (ctPRC2) (ac/hsPRC2 containing EZH2 from the American chameleon (Anolis carolinensis) and the remaining subunits from H. sapiens  , a chime sapiens  and an a sapiens . SurprishsPRC2  from a thermophilic fungus (Kdapp) of purified ctPRC2 with a variety of synthetic homopolymeric 40-mer RNAs. ctPRC2 had specificity for poly (G) RNA, while having the lowest affinity for poly (A) RNA (ctPRC2 preferred binding to RNA sequences capable of folding into G-quadruplexes ((GGAA)10, (G4A4)4 and G40) (10 and (GA)20 RNA have the same nucleotide composition, but ctPRC2 has strong preference for (GGAA)10, which is capable of forming G-quadruplexes. Third, the affinity for G-quadruplex-forming RNAs was cation-specific, as illustrated by the fold change of Kdapp in K+ buffer versus Li+ buffer 10, (G4A4)4 and G40  RNA . Second,and G40) . For exa+ buffer . K+ stim and G40 . The catd hsPRC2 .ctPRC2 shares the binding specificity for G-quadruplex RNAs with hsPRC2, we first used ctPRC2 as a model to identify its RNA-binding residues using mutagenesis. Our initial approach involved site-directed mutagenesis of conserved positive-charged residues on the surface of ctPRC2, but 26 such mutations were found to have little or no effect on RNA-binding affinity  and a catalytic moiety   . We expr (10 RNA . Purifieing site .10 RNA (Kdapp > 5 µM), and the CXC-SET domains bound to (GGAA)10 RNA with a 60-fold higher Kdapp compared with wild-type ctPRC2. These results support the idea that the catalytic moiety may not contain the major RNA-binding sites. In contrast, in the regulatory moiety we identified a minimal RNA-binding complex consisting of EBD and BAM domains of EZH2 and the entire EED subunit, and it had RNA-binding affinity comparable to that of intact core ctPRC2. Strikingly, this minimal RNA-binding complex and the regulatory moiety maintained the binding specificity for G-quadruplex RNA (10 and (GA)20 RNAs. This result indicated that this minimal RNA-binding complex contains the major RNA-binding site(s) that provides the specificity for G-quadruplex RNA. The minimal complex consists of EBD and BAM domains of EZH2 and the entire EED subunit, while EED alone binds RNA weakly (Kdapp = 2.7 µM). Addition of the EBD and BAM domains to EED increases the RNA binding by about 50-fold, suggesting that key determinants for RNA binding are located on these two domains of EZH2, with EED providing a platform for their proper 3D architecture.To further identify important domains for RNA binding, we expressed and purified a few well-defined domains from both the regulatory moiety and the catalytic moiety. For the components of the catalytic moiety, the VEFS and SANT2L domains showed little binding to (GGAA)plex RNA , tested The WD-repeat (WD: Trp-Asp dipeptide) protein EED provides a rigid scaffold for mutagenesis experiments on the minimal RNA-binding complex . Thus, tctPRC2 has a high electrostatic potential and contains a few positively charged residues that might be responsible for interacting with RNA. We mutated two patches of residues (mutant 1 and 2) in the context of the ternary ctPRC2 complex (EZH2-EED-VEFS of SUZ12)  in its BAM domain.It is interesting that the BAM domain of EZH2 in f SUZ12) ; mutant f SUZ12) . We furtf SUZ12) . Thus thctPRC2 and hsPRC2, and of the four key RNA-binding residues in the BAM domain of ctPRC2, only D266 is found in hsPRC2. Thus, the RNA-binding residues and sites on hsPRC2 could be different from ctPRC2, and we sought to identify the differences.The amino acid sequence of the BAM domain is not well conserved between 40, (GA)20 and (GGAA)10, the last of which forms G-quadruplex structures. The 3 m hsPRC2 shared very similar RNA-binding specificity with the 5 m hsPRC2 complex previously characterized  fusion co-expressed with full length EED purified from yeast was comparable to that purified from insect cells 10 RNA was dramatically disrupted (Kdapp > 2 µM), suggesting a potentially important role of this basic helix in RNA binding. However, the loss of RNA binding by this truncation could also be indirect and may be caused by charge repulsion after deletion of basic N-terminal helix and release of the adjacent acidic SANT1 domain 10 quadruplex was annealed as described previously, pre-incubated with hsPRC2 3 m complex at 8:1 molar ratio (RNA:3 m hsPRC2) prior to HDX studies. Deuterium uptake was measured at five time points over a period of 480 min for comparison of hsPRC2 against the RNA-hsPRC2 complex. A deuterium uptake profile of 258 (EZH2), 149 (EED) and 114 (SUZ12) peptides, generated from inline digest, provided 91.3%, 96.4%, 99.0% sequence coverage for each subunit, respectively.Deletion analysis and mutagenesis have the inherent risk of perturbing protein folding, so effects on RNA binding might be indirect as well as direct. As an alternative approach, we performed hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis of the hsPRC2 3 m complex. Comparative deuterium uptake for the RNA bound and apo-EZH2 subunit highlighted four distinctly differentiated regions, 32–42 (Z1), 210–224 (Z2), 512–531 (Z3) and 645–665 (Z4) of human EZH2. Reduction in deuterium uptake for EZH2 in complex with (GGAA)10 RNA suggests that these regions could be on the binding interface with G-quadruplex or impacted in an allosteric fashion (ac/hsPRC2 complex results in the following assignments: (1) Z1 is located in N-terminal alpha helix of EZH2 preceding EED binding site; (2) Z2 is located in the disordered region of SANT1; (3) Z3 is located in the disordered linker between SANT2 and cysteine-rich domain of hsEZH2 catalytic and also overlaps the beginning of the first Zn binding motif of EZH2 CXC; and (4) Z4 maps to i-SET region of EZH2 catalytic domain  and 338–350 (E2) with minor but statistically significant  changes in deuterium uptake  and SUZ12-VEFS S583D subunits of 3 m m uptake . Mapping regions . Evidencm hsPRC2 , bringinm hsPRC2 . SUZ12-Vm hsPRC2 . PRC2 re10 RNA. Alanine scanning mutagenesis was used to explore the role of specific residues in RNA binding in short structured Z1 and Z4 regions while NAAIRS - serine ) sequenchsEZH2 helped us to identify the most important residues in this region for RNA binding, including F32, R34 , D36 and K39. The K39A point mutation caused a 5-fold reduction in RNA binding, and the triple mutant [F32A D36A K39A] and the quadruple mutant [F32A R34A D36A K39A] led to additional reduction in RNA binding (Alanine scanning mutagenesis of 32–42 (Z1) in  binding .hsEZH2, we conducted NAAIRS sequence substitution mutagenesis to 208–213, and a moderate reduction in RNA binding was observed (For Z2 (210–224) of observed . It is pac/hsPRC2-3 protein complexes previously used for structural work (acEZH2 corresponding to residues 488–510 in hsEZH2 to a glycine-serine linker caused a significant reduction in binding to (GGAA)10 RNA, while deletion of the previously reported RNA-binding region (acent to Z3) as being critical for interaction, because mutation of this region in ac/hsPRC2 caused loss of crosslinking of the RNA to PRC2 (hsPRC2 complex. The 489–494 NAAIRS substitution mutant (PRKKKR to NAAIRS) retained its methyltransferase activity, with a 10-fold reduction in RNA binding. NAAIRS substitutions for other residues in the 489–510 region had only a modest effect on RNA binding and also had negative effect on methyltransferase activity suggesting that even if those binding effects are real they could be the consequence of a large conformation change.Z3 (512–531) is adjacent to a positive-charged and unstructured region (489-510) that has no HDX-MS coverage . RNA-binral work  showed tg region  did not g region . Additio to PRC2 . This cr10 RNA 4–5 fold weaker compared to the wild-type hsPRC2 concurrent with 50% increase of methyltransferase activity, while R653A had a modest change in RNA binding and also showed reduced methyltransferase activity, suggesting an important role of K656 in G-quadruplex RNA binding.Z4 (645–665) is located at the I-SET alpha-helix that is critical for maintaining catalytically competent PRC2 in basal state via interaction with SAL and this region interacts with SRM in the activated PRC2 structure resulting in further increase in catalytic activity. We picked two positive-charged amino acids (R653 and K656) in this region and substituted each of them with alanine.  The K656A mutant bound (GGAA)Kdapp to (GGAA)10 RNA increasing by around 12-fold compared to the wild-type 3 m hsPRC2 complex (hsPRC2 (EZH2-SUZ12-EED-RBBP4-AEBP2), the mutant [F32A R34A D36A K39A and PRKKKR489-494NAAIRS] complex had no defects in assembly compared to the wild-type complex  in the CXC domain and K656 in the I-SET helix of EZH2. Importantly, H3 methylation activities were tested on all mutants and most had no change in catalytic activity , except  complex . In addi complex , but exhxclusive . ImportahsEZH2 and a (GGAA)10-MS2 RNA. The MS2 RNA stem loop is used to stabilize the (GGAA)10 RNA, and the RNA expression has been verified previously using a Northern blot experiment  experiment was performed in HEK293T cells using exogenously expressed FLAG-tagged periment . Here, wproteins . The K39Numerous chromatin-binding proteins have been reported to interact with RNA in the last decade , includihsEED also contains two regions - 290–308 (E1) and 338–350 (E2) - that are identified in HDX-MS and potentially important for interacting with RNA. The RNA-binding residues are dispersed across the protein surface, which may explain why identifying them by mutagenesis has been so challenging: if any single binding site is affected by mutation, the RNA can still bind to another site(s) and the resultant change in Kdapp is minimal. It will be interesting to see whether other epigenetic modifiers employ this non-canonical mode of RNA binding.Here, we undertook a comprehensive study of the non-canonical RNA-binding regions of PRC2. First, we discovered that the RNA-binding specificity of PRC2 is conserved over vast evolutionary distance, from a fungus to human, which provides new evidence for its functional importance. We also identified several key RNA-binding residues of PRC2 from both of these species through a comprehensive analysis using mutagenesis and HDX-MS . The majC. thermophilum and H. sapiens) share overall structural similarity and domain organization of the minimal catalytic unit (ctPRC2 (this study), 3 m hsPRC2 (this study) and 5 m hsPRC2  due to steric hindrance. Thus ctPRC2 could not use the N-terminal helix for RNA interaction as hsPRC2 does, and instead needs an alternative site to achieve the RNA-binding specificity.This study also reveals that the 3 m PRC2 complex contains elements sufficient for the RNA-binding specificity. Surprisingly, almost none of the identified key RNA-binding residues are conserved between  of EZH2 , and thihsEZH2 residues critical for RNA binding, including R34, K39 and R494, have been identified in cancer patients . However, it remains unclear what physiological consequences these mutations have in cancer, because each of these heterogeneous mutations was identified in one out of hundreds of patients investigated and in different type of cancers (R34L and K39E were both identified in lung adenocarcinoma patients). Given that RNA binding inhibits PRC2’s ability to act as a histone methyltransferase, these mutant PRC2 proteins might be expected to have increased activity in vivo, a possibility that remains to be investigated. Thus, in addition to EZH2 overexpression, mutation of RNA-binding residues may also drive PRC2 gain-of-function in cancer.Notably, missense mutations of some of the hsEZH2. Our study was unable to support this finding using three independent experiments: [1] deletion of residues 342–368 in a purified mouse 4 m PRC2 (EZH2-EED-SUZ12-RBBP4) results in no change in binding to a RepA noncoding RNA (hsPRC2 complex (EZH2-EED-VEFS(SUZ12)) doesn’t affect binding to a (GGAA)10 G-quadruplex RNA 10 RNA binding or the crosslinking to a 24 nt G-quadruplex RNA. Although it is possible this region is important for interaction with some other RNA that was not tested in this study, this may be unlikely given that different RNAs compete for binding and show similar RNA-protein crosslinking patterns  in mouse EZH2 were reported to be important for RNA interaction , and thiding RNA ; [2] delpatterns . It alsopatterns .N-hydroxy-succinimide (NHS)-biotin labeling of surface lysine residues , minimal RNA-binding complex , EED  and CXC-SET  were expressed in Escherichia coli strain Rosetta 2 (Novagen). Cell pellets were resuspended in lysis buffer containing 100 mM Tris-HCl (pH 8.5), 300 mM NaCl, 1 mM dithiothreitol (DTT), 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and protease inhibitors, and lysed using sonication. Lysate was then cleared using centrifugation and incubated with Ni-NTA Agarose resin (Qiagen) for 2 hr. Resin was washed stepwise by 50-fold resin volume of buffer A , 50-fold resin volume of buffer B , 20-fold resin volume of buffer C containing 10 mM imidazole, and finally eluted with buffer C containing 250 mM imidazole. Elutions were concentrated and loaded to Superdex 75 size-exclusion chromatography column with running buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 2 mM DTT. Peak fractions were pooled, analyzed with denaturing gel and concentrated for downstream analysis. For point mutations in the context of the intact 3 m ctPRC2, mutants and wild type proteins were expressed in S. cerevisiae strain CB010 and purified using Ni-NTA agarose resin as described above followed by resolving using Superdex 200 size-exclusion chromatography column.Wild-type 3 m escribed . Two polctSANT2L and pGex4T1-ctVEFS plasmids in Rosetta 2 (DE3) competent cells, and VEFS protein alone was expressed by only transforming the pGex4T1-ctVEFS plasmid. When OD600 reached 0.6, protein expression was induced by 0.5 mM IPTG at 20°C for 20 hr. Cells were lysed by sonication in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 2.5 mM DTT, and 1 mM PMSF. Cell lysate was clarified by centrifugation and the supernatant was mixed with Glutathione Agarose (Pierce) for batch binding at 4°C for 2 hr. The glutathione agarose was washed by wash buffer 1 containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 2.5 mM DTT, 0.1% NP-40, and then by wash buffer 2 containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% Glycerol, 2.5 mM DTT. Sumo protease was added to cleave the His6-Sumo-ctSANT2L (EZH2) fusion protein overnight, after which the glutathione agarose was washed by wash buffer 2 . Thrombin was then added to cleave the GST-ctVEFS (SUZ12) protein overnight, and the binary complex was eluted by elution buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2.5 mM DTT. The SANT2L-VEFS complex or VEFS alone was further purified on a Superdex 75 gel filtration column in gel filtration buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl and 2 mM DTT.SANT2L-VEFS complex was expressed by co-transforming pSumo-32P-ATP. RNA was diluted in TE pH 7.5, heated for 10 min at 95°C, and snap-cooled on ice for 3–5 min. RNA was then allowed to fold for 30 min at 37°C in binding buffer , 5% v/v glycerol). Protein samples were diluted with binding buffer, and then mixed with refolded radiolabeled RNA  at 30°C for 30 min. Sample was loaded to non-denaturing 0.7% agarose gel  buffered with 1XTBE at 4°C and resolved at 66 V for 90 min in a 4°C cold room. Gels were vacuum dried for 60 min at 80°C on a Hybond N + membrane  and a sheet of Whatman 3 mm chromatography paper. Dried gels were exposed to phosphorimaging plates and signal acquisition was performed using a Typhoon Trio phosphorimager . For PRC2-RNA binding in LiCl buffer, 100 mM KCl was substituted with 100 mM LiCl in binding buffer.Binding assays were conducted similarly as previously described . All 40-hsPRC2.Two expression systems were used for purification of wild type and mutant S. cerevisiae expression system  and transformants were selected on synthetic complete media lacking uracil and leucine. All the constructs harboring mutations were generated with the site-directed mutagenesis strategy. Cell starters were grown in synthetic complete drop-out medium without uracil and leucine (Sc-Ura-Leu) supplemented with 2% raffinose at 30°C for 24 hr. Protein expression was induced by inoculating the cell starters into the Sc-Ura-Leu medium supplemented with 2% galactose at 30°C for an additional 24 hr. Protein purification for human PRC2 followed a similar procedure as detailed previously  and SUZ12-VEFS   and amplified to high-titer passage two (P2) stocks. Protein overexpression was conducted in exponentially growing Sf9 or Sf21 insect cells (depending on the optimum cell line for specific constructs) infected at 2 × 106 with P2 viral stock at MOI = 1. All wild-type and mutant hsPRC2 were purified identically. They were purified from cell lysate using Flag-affinity chromatography. Cells were lyzed in 50 mM Tris 8.0, 200 mM NaCl, 5% glycerol, 0.25 mM TCEP supplemented with EDTA-free protease inhibitor cocktail (Roche). 1.5 ml of lysis buffer was added per 1 g of frozen biomass. The clarified lysate was obtained by centrifugation of cell lysate at 10,000 g for 1 hr at 4°C. 5 ml of Anti-FLAG M2 Agarose (Sigma) was added per 5 L of biomass and incubated for 3 hr at 4°C (batch binding). Flag resin bound to PRC2 was washed with 20 column volumes (CV) of 50 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 0.25 mM TCEP followed by elution of PRC2 using 3 CV of 50 mM Tris 8.0, 200 mM NaCl, 5% glycerol, 0.25 mM TCEP supplemented with 200 g ml−1 of FLAG Peptide (DYKDDDDK). As expected, single step FLAG purification allowed stoichiometric capture of all three desired subunits of PRC2. PRC2 was further purified using S200 26/600 column  pre-equilibrated with 2 CV of 25 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 0.5 mM TCEP buffer. Peak fractions containing hsPRC2 monomer were concentrated to 2–20 mg ml−1, flash frozen in small aliquots using liquid nitrogen and stored at −80°C.For insect cell expression system, expression of the 3 m ; S583D) . MutantshsPRC2 complexes were expressed and purified as previously described (4 m and 5 m escribed .hsPRC2 samples (5 µM) were prepared with and without the addition of RNA (40 µM) in stock buffer  for differential HDX-MS analysis. Deuterium exchange was conducted on a temperature-controlled HDX2 autosampler (Leap Technologies). Aliquots of sample (4 µL) were mixed (1:4) with deuterium exchange buffer  at 4°C. Exchange was conducted across five time points  and run in replicates (triplicate/quadruplicate). After a defined exchange time, the sample was transferred by a chilled syringe, and quenched/denatured at 1°C with addition of 20 µL of chilled quench buffer . Quenched samples were injected into the chiller box, which housed the sample loop, protease column and trap/analytical columns at 3°C. Blank injections were inserted between every sample to minimize potential carryover.Insect cell-expressed 3 m A three pump scheme was employed for peptide detection using Vanquish UPLC pumps (Dionex). A loading pump running 0.1% formic acid at 200 µl/min carried sample sequentially across the pepsin/protease XIII immobilized column (NovaBio Assay) and 2.1 × 5 mm CSH C18 trap column(Waters) for 2 min. Peptides were desalted for an additional 30 s, while the protease column was back-flushed with 0.05% TFA. Peptides were subsequently eluted/separated across a Kinetex C8 2.1 × 50 mm analytical column via a 5.7-min gradient of mobile phase B  employing a binary pump gradient with mobile phase A (0.1% formic acid) and mobile phase B (acetonitrile 0.1% formic acid) at 150 µL/min.Data acquisition and processing involved multiple software platforms. Mass spectra were acquired on a Thermo Fusion-Lumos mass spectrometer running XCalibur 2.1 across a scan range of 375–1300 m/z. Orbitrap resolution was set at 60,000 for charge state selection of +2 to + 5 peptides. Peptide fragmentation for peptide identification employed both HCD and EThcd tandem MS2 acquisition. Electrospray source settings were set at high-gas flow  and temperature (150°C) to handle the 150 µl/min LC flow rate, while minimizing potential deuterium back-exchange. Peptide pools were generated in Thermo Proteome Discoverer 2.1 with a directed search against the protein sequence, using Sequest HT search and a fixed value PSM validator (0.05 Delta Cn). Tolerances were set at five ppm mass accuracy with 0.6 Da fragment. Deuterium exchange was determined with HDExaminer software. For peptide deuterium uptake, the first two residues and prolines were excluded from calculations.ctPRC2, 600 nM of purified wild type or mutant protein was incubated with 12 µM 14C-labelled SAM (PerkinElmer) and 2.4 µM histone H3.1 proteins  for 30 min in a 1X reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5 mM ZnCl2, 0.1 mM CaCl2, 2 mM 2-mercaptoethanol. Reactions were then loaded on a Nupage 4–12% Bis-Tris gel (Life Technologies). Gel electrophoresis was carried out for 60 min at 150 Volts, and the gel was vacuum dried at 80°C for 30 min on Whatman 3 mm chromatography paper. Dried gels were exposed to phosphorimaging plates, and signal acquisition was performed with a Typhoon Trio phosphorimager .For hsPRC2 and mutants, methyltransferase activity was measured using a radioactive filter-binding assay as previously described  and 4 µL of 3H SAM  in a final volume of 80 µL , and incubated at 30°C in a buffer containing 25 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 2.5 mM MgCl2, and 5 mM DTT. After 30 min, 10 µL of the reaction mixture was transferred to a tube to stop the reaction with 2 mM of unlabeled SAM, spotted onto phosphocellulose filters (Reaction Biology Corporation), and allowed to dry. Filters were washed five times with 50 mL of 50 mM Na2CO3/NaHCO3, pH 9.0 and once with 30 mL of 100% acetone and dried. Filters were immersed in 4 mL of scintillation fluid and counted with a scintillation counter set to output readings as disintegrations per minute (DPM). All experiments were performed in triplicate, and activities of mutants were normalized to that of the wild-type protein.For insect cell-expressed escribed . For S. 32P-ATP. Trace amounts of hot RNA were refolded  and then incubated with 200 nM ac/hsPRC2 protein for 30 min at 30°C. Each sample was moved to siliconized glass coverslips on ice, placed in a Stratalinker with 365 nm bulbs, and crosslinked for 30 min. SDS loading dye was added and samples were then resolved on a Nupage 4–12% Bis-Tris gel (Life Technologies) for 60 min at 180 V. Gels were vacuum dried at 80°C for 30 min on Whatman 3 mm chromatography paper. Dried gels were exposed to phosphorimaging plates and signal acquisition was performed using a Typhoon Trio phosphorimager .4-thio-U incorporated RNA (sequence: UUUGGGU(4-thio-U)UGGGUUGGGUUGGGUU) was synthesized by Dharmacon and then end- labeled with gamma-10 RNA) or 5’(dsDNA) Alexa-488 fluorescent dye. (GGAA)10 RNA was refolded and bound to various concentrations of PRC2 complexes the same way as described in the EMSA binding assay, except yeast tRNA was omitted in the binding buffer. (GC)30 dsDNA was incubated with PRC2 complexes in a low-salt binding buffer (10-fold less KCl and MgCl2) to facilitate binding (because DNA binding is much weaker compared to RNA binding). After 30 min of incubation, fluorescent polarizations were measured in a 384-well plate using a Synergy two multi-mode plate reader (BioTek).Fluorophore-attached nucleic acids were commercially synthesized with a 3’((GGAA)2O and 33 µL 3X reverse-crosslinking buffer , and the 25 µL input sample was mixed with 33 µL 3X reverse-crosslinking buffer and 42 µL ddH2O. Protein degradation and reverse-crosslinking were performed for 1 hr at 42°C and 1 hr at 55°C. 1 mL Trizol  was then added and RNA extraction was performed following manufacturer's instructions. Extracted RNA was treated with RQ1 RNase-free DNase (Promega M6101) for 1 hr at 37°C, and then extracted using phenol: chloroform: isoamyl alcohol followed by ethanol precipitation. All precipitation steps were facilitated by addition of 2 µg glycogen per sample. RNA pellet was re-suspended in 20 µL ddH2O, and 2 µL was used for making cDNA using SuperScript III (Life Technologies), 1 µM random hexamer and 500 µM dNTPs. qPCR was performed using SYBR Select Master Mix (ThermoFisher) and primers to the MS2 region as previous described , P_MU6-(GGAA)10-MS2 and pMS2-eGFP (Addgene:#27121) using Lipofectamine 2000 reagent (Life Technologies). At 48 hr post transfection, media were aspirated and cells were fixed and crosslinked by adding 1XPBS containing 0.1% formaldehyde. After 10 min of incubation at room temperature, crosslinking was quenched by addition of 125 mM glycine. Cells were harvested by scrapping using cell lifters (Corning), washed once in cold 1XPBS, and stored at −80°C. Cell lysis and immunoprecipitation protocols were adapted from . Cells hescribed . FractiohsEZH2. Transfections of the three plasmids were performed similar as described above, except at a smaller scale in 6-well plates. 24 hr after transfection, media were aspirated and cells were harvested by adding 200 µL 1X NuPAGE LDS Sample Buffer (Invitrogen NP0007) and 2 µL Benzonase Nuclease (Sigma E1014) per well. Lysate was incubated at 37°C for 30 min to completely digest nucleic acids. Samples (2.5 µL and 0.625 µL) of lysate were resolved in a NuPAGE 4–12% bis-tris protein gel (ThermoFisher) and then transferred to a Hybond ECL membrane (GE# RPN78D) in 1X transfer buffer  at 0.5 Amps constant for 1 hr. The membrane was blocked with 10 mL StartingBlock T20 (PBS) Blocking buffer (Thermo # 37539) at room temperature for 30 min, incubated with 2 µL anti-FLAG M2-Peroxidase (HRP) antibody (Sigma A8592) in 10 mL blocking buffer for 1 hr, washed 3 times with 10 mL 1XPBS with 0.05% tween20 and once with 10 mL 1XPBS, and developed using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Scientific 34080).Western blot experiments were used to compare expression levels of wild type and mutant  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 two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and John Kuriyan as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Conserved RNA Binding Specificity of Polycomb Repressive Complex 2 Is Achieved by Dispersed Amino Acid Patches in EZH2" for consideration by All of the reviewers and Editors are positive about the study and find it to be a major contribution to the polycomb field. Therefore, we are ready to move forward with the acceptance of the manuscript for publication. Prior to formal acceptance, we wanted to give you the opportunity to textually address the minor comments/suggestions made by expert reviewers. We have included the reviewers comments at the bottom of this letter for you and look forward to receiving the revised version of your study.Reviewer #1:PRC2 binds RNA molecules promiscuously, and the authors of this manuscript have recently reported that this occurs with a preference for G-rich sequences and G-quadruplex structures .Here, they show that RNA binding preference is the same for evolutionarily distant ctPRC2 as for human PRC2, and they characterize the binding interface on PRC2 for both species. Experiments and interpretations of data are thorough and of high quality, and identification of binding interfaces are convincing.The physiological relevance of promiscuous RNA binding by PRC2 is not established in the literature. The authors have made great efforts to identify separation-of-function mutants that can aid in addressing the physiological function of RNA binding, but they have not applied their mutants towards such investigations. The authors report a mutant with reduced RNA binding (to approximately 25%), and it is not clear, if this reduction is sufficient to facilitate investigation into physiological relevance.eLife cannot be recommended.In summary, the technical quality of this study is very high and the authors address a very interesting question. Unfortunately, the authors have not used their RNA-binding PRC2 mutants to address the physiological role of RNA-binding for PRC2 function. Thus, it appears that the authors have stopped half-way, and publication in Reviewer #2:Long et al. investigated the molecular basis of RNA-binding by PRC2. Using the previously crystallized conserved catalytic core comprising EED, EZH2 and a short fragment of SUZ12 from both Chaetomium and human PRC2, they perform rigorous biochemical binding assays combined with HDX-MS mapping studies to identify residues that are important for RNA interaction. Binding studies with PRC2 versions containing mutations in these residues confirm their importance for RNA interaction. Surprisingly, the location of Chaetomium and human PRC2 amino acid residues required for RNA binding (i.e. G-quadruplex RNA) are not conserved, and they are also not located on the same surfaces in the two complexes.eLife. There is a vast amount of literature accompanied by considerable hype about RNA-binding by PRC2. Yet, as the authors state, to date it still is completely unclear whether RNA binding by PRC2 is of any physiological significance at all. The regions identified in this study will allow performing the relevant mutational analyses (i.e. EZH2 knock-in mutations in mice) to clarify this issue. Such experiments are beyond the scope of this study but will hopefully be done in the near future.This is a very nice study of excellent technical quality. The paper definitely merits to be published in I only have a few minor comments of editorial nature that the authors should address.1) 2) 3) Figure 8 is a nice summary. So it would be helpful to clearly indicate in the figure that panel B is C.t. PRC2 and C is H.s. PRC2. It may also be helpful to include a C.t and H.s. EZH2 domain architecture diagram (i.e. like in 4) Discussion, first paragraph: I don't think that the term "RNA binding domain" is helpful in this case. The term "domain" should be reserved for a portion of a protein with a defined fold. Here, the authors show that the interaction of PRC2 with RNA occurs via positively charged amino acid residues that are dispersed over a large protein surface and are not even in a conserved location on the complex when comparing C.t. and H.s. PRC2.5) The authors state that "missense mutations of some the H.s. EZH2 residues critical for RNA binding have been identified in cancer patients." The https://portal.gdc.cancer.gov website lists about 150 different amino acid changes that have been identified in EZH2 (which is a 746 amino acid protein) and almost all of them – including the residues mentioned in the text – were identified a single time and in completely different tumours. So it is not clear how meaningful these specific particular mutations are. Moreover, I assume that these are all heterozygous mutations in tumours where the homologous chromosome carries the EZH2 wild-type allele. Please clarify this in the text. Reviewer #1:[…] In summary, the technical quality of this study is very high and the authors address a very interesting question. Unfortunately, the authors have not used their RNA-binding PRC2 mutants to address the physiological role of RNA-binding for PRC2 function. Thus, it appears that the authors have stopped half-way, and publication in eLife cannot be recommended.While we agree that in vivo studies are important, they are beyond the scope of the current study. The epigenetic silencing field has been waiting for the identification of RNA-binding sites of PRC2 for a long time, and this is a very competitive field. By publishing this analysis now, we will facilitate the in vivo studies of many groups around the world.Reviewer #2:[…] I only have a few minor comments of editorial nature that the authors should address.1) As suggested, the original label “H3K27me3” has been changed to “H3K27me”.2) The original 3) Figure 8 is a nice summary. So it would be helpful to clearly indicate in the figure that panel B is C.t. PRC2 and C is H.s. PRC2. It may also be helpful to include a C.t and H.s. EZH2 domain architecture diagram  Discussion, first paragraph: I don't think that the term "RNA binding domain" is helpful in this case. The term "domain" should be reserved for a portion of a protein with a defined fold. Here, the authors show that the interaction of PRC2 with RNA occurs via positively charged amino acid residues that are dispersed over a large protein surface and are not even in a conserved location on the complex when comparing C.t. and H.s. PRC2.“Domains” has been changed to “sites” to be consistent with the terms used two sentences earlier (“RNA binding to these proteins seems to occur at multiple sites of the protein.”)5) The authors state that "missense mutations of some the H.s. EZH2 residues critical for RNA binding have been identified in cancer patients." The https://portal.gdc.cancer.gov website lists about 150 different amino acid changes that have been identified in EZH2 (which is a 746 amino acid protein) and almost all of them – including the residues mentioned in the text – were identified a single time and in completely different tumours. So it is not clear how meaningful these specific particular mutations are. Moreover, I assume that these are all heterozygous mutations in tumours where the homologous chromosome carries the EZH2 wild-type allele. Please clarify this in the text.We totally agree with the comments that it is not clear how meaningful these specific cancer mutations are, and a few sentences have been added in the paragraph to explain the limited information for the biological consequences of these mutations.In addition to addressing the above comments, we made several other revisions to make the story clearer and more convincing:1) We added new results in 2) To test these mutations in the context of the holoenzyme (5m hsPRC2), we added new results in 3) We also fixed the color labeling of E1 and E2 region in 4) Materials and methods sections have been updated.5) Supplemental figures in the original manuscript file have been incorporated with each main figure to become “Figure X—figure supplement X”.


The RNAzol RT reagent was used to provide pure RNA from human cells. We develop a protocol using RNAzol RT reagent to extract pure RNA from plants tissues and demonstrate that this RNA extraction method works not only at room temperature but also at elevated temperatures and provides the simplest and most effective single-step method to extract pure and undegraded RNA directly from tropical plants in the field. RNA extraction directly in a complex field environment opens up the way for studying gene-environment interactions at transcriptome level to decipher the complex regulatory network involved in multiple-stress responses. In 2010, Chomczynski and co-workers published a new approach to single-step RNA extraction using RNAzol RT reagent, which does not employ chloroform-induced phase separation to obtain pure RNAThe acclimation of plants to field conditions is a coordinated response involving hundreds of genes and a complex regulatory network. These responses are governed by interactions between different environmental factors and the developmental stage of the plantThe recent development of novel high-throughput DNA sequencing methods has provided a new powerful method for transcriptome analysis. Because RNA-Seq provides a far more precise measurement of both the transcriptional structure and level of expression for each gene than other methods, this technique is perfectly suited to deciphering complex regulatory networks involved in multiple-stress responsesTo evaluate the effectiveness of RNAzol RT to isolate RNA samples in field, we compared these RNA with RNA extracted from the same samples in the lab using TRIzol reagent or commercial spin-columns kit. A spectrophotometric analysis, an electrophoretic separation using agarose gel  and the Coffea arabica leaf samples to isolate pure and undegraded RNA (A260/280 ratio of 1.84 ± 0.1 and a RNA integrity number (RIN) value of 8–9. The Agilent 2100 bioanalyser system confirmed the absence of genomic DNA in the RNA samples. The average quantity of total RNA extracted from 100 mg of fresh leaf tissues reached 33.4 ± 18.8 μg. Processing of RNA from more challenging plant-derived samples, such as fruits and roots, yielded RNA with an A260/280 ratio of 1.8–1.9 and a RIN value of 7–8 .The simplified protocol for single-step RNA isolation with RNAzol RT reagent was first adapted to coffee trees using mature aded RNA . A spectCoffea arabica cv. Marsellesa cultivated in Mexico  under full sun or shade (50% of the sun filtered). Here we present the results obtained with Chalcone synthase primers (CHS). CHS enzyme is the first committed enzyme in flavonoid biosynthesis. PCR performed with these RNA samples without reverse transcription showed no amplification of DNA in a 40-cycle PCR. In parallel, the same leaf samples were freeze-dried to be extracted with methanol/water (80/20) and analysed by HPLC for their flavonoid content. http://www.mgx.cnrs.fr). The samples had a quality corresponding to sequencing platform quality standards (BiSA Platform. ISO9001 certification).Quantitative RT-PCR was performed using total RNA extracted in the field from samples of To confirm the value of this RNA extraction method for tropical plants, other agronomically important crops were considered in this study. Mature leaf samples from cassava, rice and maize were collected in the field and total RNA was extracted following the protocol established for coffee tissues. Extracted RNAs were analysed by agarose gel electrophoresis, a spectrophotometer and a bioanalyser . SimilarThe RNAzol RT single-step method has been adapted to provide pure RNA from plant tissues. This reagent isolates pure and undegraded RNA that is ready for use without DNase treatment. We have demonstrated that this RNA extraction method works not only at room temperature but also at elevated temperatures, for both monocots and dicots, and for different plant tissues. This simple and effective method can be used directly in the field for transcriptomic analysis of plants under multiple stress conditions.Basing ourselves on the work by Chomczynski and co-workers (2010), we first adapted their single-step RNA protocol for extracting RNA from coffee plant tissues, and tested its versatility and effectiveness for temperatures above 30 °C. A biological sample (80 (leaf)–250 (root) mg) is lysed in 1 ml of RNAzol RT, and the homogenate is supplemented with 400 μl of water to precipitate DNA, proteins and polysaccharides, while RNA remains soluble in the homogenate. After vortexing 10 sec and shaking for 15 min (500 rpm), the precipitated compounds are removed from the homogenate by centrifugation . The supernatant is transferred to a fresh tube with 10 μl of 4-Bromoanisol and vortexed for 10 sec . After incubation at ambient temperature for 5 min and centrifugation for 10 min at 13 000 g, pure RNA is precipitated from the resulting supernatant by the addition of 1 volume of 2-propanol. After mixing by inversion 5 times, the solution is allowed to stand for 20 min at ambient temperature then centrifuged for 15 min at 12 500 g to pellet RNA. The pellet is washed three times with 1 ml of 75% ethanol  before re-suspension in 50 μl of water.The whole procedure could be achieved under the high temperatures (25–38 °C) encountered in tropical fields. Extracting total RNA from tissue samples directly in the field requires little equipment and few devices  . RNA pelwww.qiagen.com).The total RNA extraction with TRIzol reagent is used as supplied by Invitrogen Life Technologies. QIAGEN RNeasy mini kit is used as fast commercial method to extract total RNA using spin columns .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N = 16). We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens. Results. Both rRNA depletion methods produced consistent data between the technical replicates. We found that the RNase H method produced higher quality RNAseq data as compared to the Ribo-Zero method. In addition, we evaluated the RNAseq data generated from the FFPE tissue samples for noncoding RNA, including lncRNA, enhancer/super enhancer RNA, and single nucleotide variation (SNV). We found that the RNase H is more suitable for detecting high-quality, noncoding RNAs as compared to the Ribo-Zero and provided more consistent molecular subtype identification between replicates. Unfortunately, neither method produced reliable SNV data. Conclusions. In conclusion, for FFPE specimens, the RNase H rRNA depletion method performed better than the Ribo-Zero. Neither method generates data sufficient for SNV detection. Formalin-fixed paraffin-embedded (FFPE) tissue is the most common method of tissue preparation used in clinics. FFPE preservation was developed to maintain morphology without any special considerations of preserving nucleic acids. Therefore, the difficulty of evaluating gene expression levels in FFPE samples remains one of the biggest disadvantages of FFPE preservation because the process of fixing the tissue samples and embedding them in paraffin often leads to RNA degradation and chemical modification. Furthermore, a nucleic acid can be cross-linked with a protein during the formalin fixation process, and most of the RNA isolated from FFPE tissues is highly degraded and reduced to a much lower yield than that of RNA isolated from the same amount of fresh tissues. To that end, RNA isolated from recently embedded tissues will be of better quality than RNA isolated from older embedded tissues. As a result, when amplifying RNA with oligo-dT primers, there is an overrepresentation of 3′ data due to the fragmented nature of RNA isolated from FFPE tissues.Given the aforementioned reasons, gene expression analysis based on FFPE samples has been historically challenging. The most critical step in a FFPE sample based study is tissue preparation, as it ensures the integrity of the yield and data quality. It has been greatly emphasized that improper FFPE tissue preparation can diminish the quality of the nucleic acids from the tissue, limiting their use for gene expression profiling . Yet, FFThe vast majority of cellular RNA (>80%) is composed of noninformative ribosomal RNAs  that require removal prior to cDNA synthesis for a RNA-seq library. For high-quality RNA samples, polyadenylated RNA is enriched from intact RNA using oligo-dT primers. Since the rRNA does not have a poly-A tail, it is removed prior to cDNA synthesis along with other informative, non-polyA RNA species. RNA samples isolated from FFPE tissues have two features that are not compatible with oligo-dT primer selection: fragmented RNA that produces 3′ bias from oligo-dT selection, and, the degradation of the poly-A tail, thereby impacting the yield of recovered mRNA. Currently, there are two major rRNA depletion methods used for RNA isolated from FFPE samples: the Ribo-Zero rRNA removal kit (Epicentre/Illumina) and the RNase H method  . The RibIn the 2000s, microarray technology dominated high-throughput gene expression profiling but has since been replaced by RNAseq technology –9. SucceFurthermore, based on the premise that sequencing data offers exciting opportunities for additional data mining , 14, 16,To evaluate the practicability and effectiveness of gene expression profiling using FFPE samples, we designed a study using four triple negative breast cancer (TNBC) FFPE tumor tissue samples. The H&E slides were reviewed by a study pathologist and tumor tissues were dissected from an unstained FFPE tissue section for total RNA extraction. The tumor tissue sections were stored in a vacuum chamber at 4°C for eight to nine years before RNA isolation was performed. Total RNA was extracted and purified using a Qiagen's miRNeasy FFPE Kit, a kit specifically designed for purifying the total RNA and microRNA from FFPE tissue sections. The input RNA amount for both Ribo-Zero and RNase H rRNA depletion methods was 200 ng each. The quantity and quality of the RNA samples extracted from tumor tissue FFPE sections were checked by Nanodrop  and by separation on an Agilent BioAnalyzer. Total RNA extracted from each of the four tumors was split into two samples . Two rRNA depletion methods were used: Ribo-Zero and RNase H. Each of the eight samples was treated with the two rRNA depletion methods, prepared for library using TruSeq RNA sample Prep Kit v2 (Illumina), and sequenced by BGI Americas. In total, 16 RNAseq libraries were generated following manufacture protocols and sequenced on two lanes . The qualified libraries were amplified on cBots to generate the cluster on the flow cell. The amplified flow cell was sequenced paired-end on the HiSeq 2000 at read length of the 90 base pairs.RNAseq data was thoroughly quality-controlled at multiple stages  following the recommendation by Guo et al. . Raw datTriple negative breast cancer (TNBC) is known to be molecularly and transcriptionally heterogeneous and can be classified into one of six subtypes  based on centroid correlations using gene expression . In ordeNanoString nCounter data was obtained on 302 genes using the same samples. The detailed processing and normalization method is described in . We compWe conducted advanced data mining on our FFPE RNAseq data to extract SNV. We inferred SNVs using Varscan 2 . SNV quaOn average, the Ribo-Zero rRNA removal method produced 16.1 (range: 14.0–17.8) million reads per sample, and the RNase H produced 21.4 (range: 20.2–24.0) million reads per sample. The RNase H method consistently produced more reads than Ribo-Zero. Given that the same amount of RNA was used and the same number of samples was pooled per lane, a higher RNA capture efficiency is probable for RNase H than that of Ribo-Zero. On average, the guanine-cytosine (GC) content of Ribo-Zero was 72.3% (range: 70.0–76.8%), which was above the expected value (50%), whereas the GC content of the RNase H method was 50.6% (range: 39.8–55.1%). The GC content of the reference genome is roughly the expected GC content for the sequenced data. The GC content is 39.3% for the entire human genome, 48.9% for protein coding RNA, 39.7% for lncRNA, and 50.2% for rRNA. The sequenced reads of total RNAseq data are a mixture of protein coding RNA, lncRNA, and other species of RNA. With the expected GC content around 50%, RNase H produced data with GC content closest to the expected value. The raw data quality control only provided partial quality assessment of the samples.t-test p = 0.03).Next, we examined the percentage of the reads that aligned to the coding region . For theCluster analysis showed that, regardless of which RNA isolation kit was used, the repeated sample clustered together based on gene expression. Within repeated samples, the rRNA depletion kits were clustered separately. The cluster analysis results provided additional evidence of quality concern for the RNase H sample two repeat one, as it was the only sample that did not perfectly cluster with its pair within the same RNA isolation kit . The corOverall, correlations to the TNBC subtypes were similar in replicates . RNase HWe computed Spearman's correlation coefficients using the gene expression levels between RNAseq. The correlation dot plot  shows thWe examined four kinds of RNAs: mRNA , lncRNA We inferred SNVs from the FFPE RNA data using VarScan 2. After filtering for high quality SNVs (depth > 20), on average, the Ribo-Zero samples identified 525 SNVs per sample (range: 73–1862), and the RNase H samples identified 57747 SNVs per sample (range: 21932–87146). The RNase H samples clearly identified more SNVs than the Ribo-Zero prepared samples. This is caused by the difference of number of callable sites between the two kits. We defined a callable site to be a genomic position with coverage depth ≥ 20. RNase H produced substantially more callable sites than Ribo-Zero . The calAdditional evidence for problematic SNV inferences was observed in the results of the pairwise heterozygous genotype consistency between samples. In DNA sequencing, we expect the heterozygous genotype consistency rate for technical replicates to be above 0.99. For RNAseq, the consistency rate is expected to be lower but still yield above 80%. However, on average, the consistency rates for both kits were less than 40% which were substantially below expectation. Restricting SNV pairwise heterozygous consistency computation to SNVs with depth greater than 50x for both samples in the pair increased the consistency slightly but still remained <50%. The low heterozygous consistency rates indicate that SNVs inferred from FFPE RNAseq samples contain high false positive rates and are therefore not ideal sources for detecting SNV.Utilization of FFPE specimens for gene expression studies could open a new avenue for molecular epidemiological and clinical research. Yet to date, the low quality of RNA from FFPE specimens for gene expression analysis has been a challenge. Several technologies have been developed for quantifying gene expression from FFPE specimens, such as NanoString  and quanSince gene expression data can yield both molecular subtype classification and predicative markers of risk, efforts have been made to use RNA extracted from FFPE tissue on NanoString and microarray platforms , 52. TriThrough thorough quality control and analyses, we found that expression profiling of coding and noncoding RNA is possible for aged FFPE samples with RNAseq technology. The Ribo-Zero and RNase H method each had strengths and weaknesses in different areas. Our analyses suggested that RNase H is more suitable for studies that target protein coding RNA. On the other hand, Ribo-Zero offered more consistency between repeated samples, which is of pivotal importance, especially for low quality RNA extracted from FFPE tissues. Under the same amount of library input and same multiplexing scenario, RNase H consistently produced more reads than Ribo-Zero. Many reasons could have caused this read counts difference, including batch effect of the cluster on the flow cell, and library efficiencies. The evidences of more total reads sequenced under the same input amount and better rRNA depletion efficiency for RNase H support that RNase H has better library efficiency than Ribo-Zero. RNase H hybridizes directly to the sequences of rRNAs without the requirement of perfect match. The Ribo-Zero uses bait strategy which is similar to enrichment like exome capture with baits and beads. Thus it does not remove degraded, fragmented rRNAs as efficient as RNase H. Our study confirms previous finding that RNase H performed better than Ribo-Zero for low quality RNAs .Furthermore, genes quantified from Ribo-Zero processed RNAseq data also had a slightly higher correlation with genes quantified by NanoString technology. This suggests that Ribo-Zero might offer better repeatability, although the correlation  with NanoString data (FFPE) did not reach the high correlation  between microarray and RNAseq . We suspThe subtyping of gene expression profiles obtained by both methods demonstrated that RNA isolated from stored FFPE samples can be used to determine distinct TNBC subtypes. While TNBC subtypes were similar among replicates, RNase H samples had more consistent TNBC subtype calls between replicates than that of the Ribo-Zero samples, which is potentially due to the more efficient capture of protein coding RNA.By performing SNV detection analysis, we found that SNV detected by FFPE RNAseq data is subjected to quality concerns. It has been suggested that the SNV data inferred from RNAseq data has a high false positive rate . SeveralRecent studies have shown remarkably high consistency between RNAseq data generated from paired freshly frozen and FFPE tissue samples –60. Our


O-methyl modification at the 3′ end of the RNA substrate is inhibitory. Ectopically expressed NEF-sp localizes to the nucleolar/nuclear compartment in mammalian cell cultures and this is dependent on an amino-terminal nuclear localization signal. Finally, mice lacking NEF-sp are viable and display normal fertility, likely indicating overlapping functions with other nucleases. Taken together, our study provides the first biochemical and genetic exploration of the role of the NEF-sp exoribonuclease in the mammalian genome.Ribonucleases catalyze maturation of functional RNAs or mediate degradation of cellular transcripts, activities that are critical for gene expression control. Here we identify a previously uncharacterized mammalian nuclease family member NEF-sp (RNA exonuclease 5 [REXO5] or LOC81691) as a testis-specific factor. Recombinant human NEF-sp demonstrates a divalent metal ion-dependent 3′ → 5′ exoribonuclease activity. This activity is specific to single-stranded RNA substrates and is independent of their length. The presence of a 2′- Spermatogenesis is the process by which sperm cells are produced in the male germline. Early events in this process include differentiation of embryonic primordial germ cells (PGCs) to form prospermatogonia, and later spermatogonia and spermatogonial stem cells (SSCs) . These uRibonucleases play an essential role in control of gene expression. All messenger RNAs (mRNAs) require ribonuclease-mediated processing to create their final 3′ ends: poly(A) tails of most mRNAs or the hairpin structure of replication-dependent histone mRNAs . FunctioNef-sp is exclusively expressed in mouse testes. Recombinant human NEF-sp (hNEF-sp) protein is a 3′ → 5′ exoribonuclease that acts on single-stranded RNA in a distributive manner. Modification of the RNA 3′ end is inhibitory for this activity. Ectopically expressed hNEF-sp localizes to the nucleolus in human cell cultures and this is dependent on an amino-terminal nuclear localization signal. In contrast, mouse NEF-sp is uniformly nuclear in both mouse and human cell cultures. The Nef-sp knockout mice are viable and fertile, indicating potential complementation by other nuclease(s). This study sheds light on the biochemical and physiological relevance of the NEF-sp exoribonuclease.In our search for testis-specific nucleases, we identified the previously uncharacterized mammalian nuclease RNA exonuclease 5 (REXO5 or NEF-sp) be specifically enriched in testicular transcriptomes. We demonstrate that PIWIL2/Mili  encodes for a 774 amino acid (aa) human NEF-sp (hNEF-sp) protein that is composed of an amino-terminal nuclease domain followed by two carboxy-terminal tandem RNA-recognition motifs (RRMs) (Nef-sp: a full-length long isoform encoding for a protein of 784 aa (accession no. KY853396), and a shorter carboxy-terminal splice variant encoding for a protein of 696 aa (accession no. KY853397). Both the mouse proteins have the same domain architecture, but the shorter isoform carries changes downstream from the RRM domains .A search for potential nucleases in testis transcriptomes led to the identification of human NEF-sp (accession no. NP_001185982) as an uncharacterized protein. It is variously known in the databases as LOC81691 or Q96IC2_REXON or 2610020H08Rik or RNA exonuclease 5 (REXO5), but in this report we will refer to it as NEF-sp. We first performed reverse transcription PCR (RT-PCR) analysis using total testes RNA A. SimilaIL2/Mili , the Nefe testes B. We faie testes B. To stus (RRMs) C. We ide2+ or Mn2+) resulted in appearance of an RNA ladder, indicative of nuclease activity (2+ ion (2.5 mM) showed a stronger stimulation of the nuclease activity compared with the Mg2+ ion (25 mM). This is likely due to the relaxed coordination properties of Mn2+, compared with the stringent coordination geometry requirement of Mg2+  version of the protein carrying a single amino acid substitution (D313A) within the motif III. The purified mutant protein behaves identical to the wild-type protein during size-exclusion chromatography, indicating lack of any gross conformational changes  distributed in three sequence motifs . The pre changes A. Import changes C. Taken O-methyl modification on the 3′ terminal nucleotide of RNAs is proposed to act as a stabilizing mark by protecting them from exonucleases  D. The 10ucleases . When hNucleases E. This s2+-dependent 3′ → 5′ exonuclease acting on ssRNAs and is inhibited by the presence of a 2′-O-methyl modification at the 3′ end of the substrate.Next, we examined whether double-stranded RNA (dsRNA) could be used as a substrate. When a 20-nt ssRNA was provided as a substrate, it was cleaved by the NEF-sp enzyme F. HoweveΔNLS version to the cytoplasm . Homozygous Nef-sp−/− males displayed normal testes sizes when compared to wild-type animals , we conclude that perhaps abundance of the endogenous protein is below the detection limit of our antibodies. We demonstrate that hNEF-sp is a 3′ → 5′ exoribonuclease that is active on ssRNA substrates and likely functions in the nucleolar compartment. The importance of the two RNA-recognition motifs (RRMs) in the protein was not examined in this study, but these may perhaps help identify certain RNA substrates. Alternatively, potential protein complexes formed by the enzyme might help select the RNA targets. However, in vitro nuclease assays used RNAs with a variety of sequences and these were all efficiently processed.Here we examined the biochemical properties of the uncharacterized mammalian nuclease family member NEF-sp. We show that Saccharomyces cerevisiae, where RNA exonuclease 1 (Rex1p)/Rnh70p/yGR276  from adult human testes total RNA  or mouse testes RNA, respectively. GenBank accession numbers are human NEF-sp (NM_001199053), mouse Nef-sp long isoform (KY853396), and mouse short isoform (KY853397). For mammalian cell culture expression, the following coding sequences were cloned into the SalI and NotI sites of the vector pCI-neo-N-HA (ΔNLS (58–774 aa), mNEF-sp (1–784 aa), mNEF-spΔNLS (53–784 aa), and mNEF-sp short isoform (1–696 aa).The coding sequences for human (LOC8169) and mouse  RNA exonuclease 5 (Rexo5) or neo-N-HA : hNEF-spHuman epithelioid cervix carcinoma (HeLa) and mouse NIH/3T3 cell lines growing on coverslips in 12-well plates were transfected with 2.5 µg of expression plasmids using X-tremeGENE HP . Subcellular localization of HA-tagged proteins was examined using the anti-HA as primary antibody, followed with staining by secondary antibody anti-mouse-IgG coupled Alexa Fluor 488 (Invitrogen). Cells were examined under an inverted confocal microscope Leica TCS SP2 AOBS.Spodoptera frugiperda or High Five (Hi5) from the cabbage looper, Trichoplusia ni. The full-length human NEF-sp (1–774 aa) or a mutant version (hNEF-spMut) carrying a point mutation (D313A) were cloned into the NheI and KpnI sites of the vector pACEBac2 (6xHis-Strep-SUMO-TEV-fusion)  supplemented with protease inhibitor . The protein was further purified on a Heparin column  and monodisperse fractions collected by gel filtration chromatography  . Proteins were used in nuclease assays without removal of the tags.For production of recombinant proteins using the eukaryotic expression systems we used the following ovary-derived insect cells: Sf21 from the Fall Army worm -fusion) . Protein32P]ATP  and T4 polynucleotide kinase . The labeled probes were resolved in 15% denaturing urea–polyacrylamide gels, and full-length labeled RNAs were gel-eluted with 0.3 M NaCl followed by phenol–chloroform extraction, precipitation and resuspension in 20 µL of RNase-free water.The ssRNA (Microsynth) and ssDNA (Invitrogen) substrates (see list below) were labeled at the 5′ end with [γ-32P]ATP and resolved in 20% in denaturing urea–polyacrylamide gels. The full-length labeled oligonucleotides were eluted in 600 µL of elution buffer  overnight at 4°C followed by precipitation using 1.8 mL of 100% ethanol and 2 µL of glycogen. The pellets were resuspended in 16 µL of water and annealing reactions were performed using 2 µL of 100 µM unlabeled complementary strand and 2 µL of 10× duplex-annealing buffer  followed by heating the samples to 95°C and gradually cooled down to room temperature. The oligonucleotide duplexes were resolved on 15% nondenaturing PAGE followed by gel elution and precipitation as described above.RNA and DNA duplexes were made as described: 100 µM of the top strand was labeled at the 5′ end with ) and incubated at 37°C for 1 h in buffer . Divalent metal ions needed for the reaction were provided by supplementing the reaction with 25 mM MgCl-O-Met_1 E. dsRNA -O-Met_1 F, and ds-O-Met_1 G. HairpiNef-sp locus of the mouse genome was disrupted using the RNA-guided Cas9 endonuclease (Cas9 nickase [Cas9n]) and a pair of guide RNAs (gRNAs) . The Cas9n mRNA and the in vitro transcribed gRNAs (gRNA L1 and R4) were used for injection into C57B1/6J host embryos for mouse generation using one-cell stage injection  at 55°C overnight. DNA was precipitated with isopropanol, washed in 70% (v/v) ethanol and resuspended in 10 mM Tris–HCl, pH 8.0. To identify the F1 animals we used a pair of primers, the forward primer (represented in blue) complementary to the region spanning the deleted region and the reverse primer around 500-bp downstream from the deletion D. To dis2, 2 µL of DNA template, and 0.5 µL of Taq DNA polymerase in a final volume of 25 µL. PCR conditions were 95°C for 5 min, then 40 cycles of 30 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C, and a final step at 72°C for 5 min.Genotyping primers: SS323 (5′-GGTTTCATAACTTGACTGTG-3′), SS330 (5′-CAGATAGCAGTCCCCTCTTT-3′), and SS336 (5′-GGATTACGGAAACTCAAAGTG-3′). The PCR reactions were performed in 1× Taq Buffer (Fermentas), 200 µM dNTPs, 0.25 µM primers, 2.5 µM of MgClTotal testicular RNA was isolated by TRIzol Reagent (Invitrogen cat. no. 15596026). Strand-specific RNA-seq libraries were prepared using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina) after removal of abundant ribosomal RNAs with Ribo-Zero (mouse). Libraries were sequenced with the Illumina HiSeq 2000 platform (EMBL Heidelberg Gene Core facility) for 50 cycles.Nef-sp−/− and Nef-sp+/−. DESeq2 bioconductor package . The software used for processing the data  from the raw data files are in-house tools developed by the Sachidanandam laboratory . Only re package  was usedRNA secondary structures were predicted using the Mfold web server .Deep sequencing data sets generated in this study are deposited with the Gene Expression Omnibus (GEO) under accession number GSE97235.


While intron retention (IR) is considered a widely conserved and distinct mechanism of gene expression control, its regulation is poorly understood. Here we show that DNA methylation directly regulates IR. We also find reduced occupancy of MeCP2 near the splice junctions of retained introns, mirroring the reduced DNA methylation at these sites. Accordingly, MeCP2 depletion in tissues and cells enhances IR. By analysing the MeCP2 interactome using mass spectrometry and RNA co-precipitation, we demonstrate that decreased MeCP2 binding near splice junctions facilitates IR via reduced recruitment of splicing factors, including Tra2b, and increased RNA polymerase II stalling. These results suggest an association between IR and a slower rate of transcription elongation, which reflects inefficient splicing factor recruitment. In summary, our results reinforce the interdependency between alternative splicing involving IR and epigenetic controls of gene expression. Intron retention is a conserved mechanism that controls gene expression but its regulation is poorly understood. Here, the authors provide evidence that DNA methylation regulates intron retention and find reduced MeCP2 occupancy and splicing factor recruitment near affected splice junctions. Alternative splicing (AS) is essential for diverse physiological functions including cell division, differentiation, stress response and lineage specification1234One mode of AS termed intron retention (IR) occurs when an intron is not excised by the spliceosome, and is preserved in the final mature messenger RNA (mRNA). Intron-retaining transcripts typically contain premature termination codons, resulting in their removal by surveillance mechanisms including nonsense-mediated decay (NMD). Whilst AS generates proteomic complexity from most genes, IR is a distinctive mechanism that reduces gene expression, which in turn regulates cellular differentiation127Much is known about the mechanisms that regulate other modes of AS including exon skipping and inclusion, but their relevance to the control of IR remains uncertain. RNA Poll II stalling, which improves splice signal recognition is integral to the ‘kinetic' model of AS that involves epigenetic changes12Alternatively, RNA Pol II stalling near splice junctions of retained introns could occur consequent to inefficient splicing factor recruitment1517In this study, we sought to determine whether IR is regulated by spliceosomal and DNA methylation changes and how they are linked. We show that lower levels of DNA methylation near 5′ and 3′ splice junctions are significantly associated with increased IR in a myriad of primary cells and cell lines. Importantly, both chemical and genetic inhibition of DNA methylation promoted IR in mouse embryonic stem cells, mouse B cells, and in both mouse and human cell lines. We show reduced MeCP2 binding near 5′ and 3′ splice junctions of retained introns in mouse granulocytes and promyelocytes, which corresponds with the physiological decrease in DNA methylation levels. Further, knockdown or knockout of MeCP2 in primary cells and cell lines led to increased IR. To confirm that MeCP2 occupancy influences the efficiency of splicing, we show that reduced MeCP2 occupancy is associated with RNA Pol II stalling. Using immunoprecipitation followed by mass spectrometry (MS), we further show that many splicing factors are associated with MeCP2 including the Srsf family members and Tra2b. Consistent with inefficient splicing factor recruitment as reflected by RNA Pol II stalling, we demonstrate reduced binding of the splicing factor Tra2b to RNA transcripts containing retained introns along with reduced binding of MeCP2 to these transcripts. Our data link the two models of splicing by demonstrating a direct causative role of decreased DNA methylation in promoting IR via reduced MeCP2 occupancy, which in turn perturbs the recruitment of splicing factors such as Tra2b, leading to RNA Pol II stalling.P<0.05, Audic and Claverie test) between the two cell types out of a total of 3,200 and 3,888 introns retained (IR level ≥10%) in promyelocytes and granulocytes, respectively. Of these 737 differentially retained introns, substantially more introns  showed higher levels of retention in granulocytes (22021We performed fluorescence-activated cell sorting (FACS) of primary mouse promyelocytes and granulocytes isolated from mouse bone marrow according to our established protocol 2. Our prulocytes ; consistulocytes  again reP<1.00e-16, t-test, P<0.05, t-test, P<0.05, t-test, Retained introns were significantly shorter in nucleotide length than non-retained introns  showed reduced DNA methylation levels by ≥10% near splice junctions or within introns when IR levels increased in either cell type. This result indicates that DNA methylation-associated IR events may occur at a similar frequency as BS69-associated IR eventsnb1 refs , Ngp , a human lung fibroblast cell line (IMR90), a human colon cancer cell line (HCT116) and human neuron progenitor cells  and 5-Aza-treated leukaemic OCI-AML3 cells compared to respective wild type and untreated cells. The number of introns exhibiting increased IR levels  was significantly more than introns with decreased IR levels in all three cell types  compared to control   treatment or al test) . This diLmnb1, the IR levels were significantly increased by >four-fold with  and without NMD inhibition  and >4-fold , respectively, were achieved following NMD inhibition , these results may be explained by the lack of changes in DNA methylation levels near 5′ or 3′ splice sites despite 5-Aza exposure , and further demethylation by 5-Aza failed to alter IR levels  were observed in 8/12 introns , we measured the specific levels of MeCP2 binding near splice junctions flanking twelve introns that are subject to DNA methylation-associated increased IR in granulocytes compared to promyelocytes . Signifi introns  in assoc introns . No diff introns . In 5-Azobserved ; concomiobserved . MeCP2 ot change ; consistt change . These dMeCP2-knockdown and -knockout cells and tissues by analysing publicly available RNA-seq data. Of the differential IR events, we detected predominantly increased IR in all cell and tissue types examined including: IMR90 , mouse cerebellum , mouse peritoneal macrophage (59/91 64.84%) and mouse visual cortex cells  have previously been reported as MeCP2-binding partners . Histone3539Malat1 and Tra2a, was not significantly different in promyelocytes and granulocytes . Our RNA-IP-reIP assays consistently detected de-enrichment of concomitant Tra2b and MeCP2 binding to ifferent . Thus, wOur data support a model where MeCP2 regulates splicing via recruitment of splicing factors, such as Tra2b . IR may Until recently, IR has been the least understood mode of splicing in terms of its role in normal physiology and disease. As a result of recent work by our group and others, it is now established that IR is a conserved mechanism that regulates cell differentiation and plasticity in normal tissues and is implicated in diverse human cancers12894142278Since DNA methylation is known to regulate other forms of splicing, we explored its role in controlling IR. Two models of splicing regulation via DNA methylation have been proposed: ‘kinetic' and ‘recruitment'. The former involves ‘go slow' signal proteins reflecting DNA methylation changes, to reduce the rate of RNA Pol II elongation and promote recognition of splice sites12Our data indicate that DNA methylation changes are associated with approximately one in every six differential IR events in myeloid cells. This observation reveals the complexity of splicing and IR regulation, which may involve many different epigenetic factors and associated proteins. Indeed, H3.3K36me3 mediated recruitment of BS69 is known to promote IR by antagonizing the activity of the spliceosome via its interaction with snRNPs, including EFTUD2 ref. . While BIt is important to recognize that AS can also be modulated by the ratio of DNA methylation to hydroxymethylationAlthough intragenic DNA demethylation has been associated with reduced gene expression in cancers50In conclusion, we have discovered a causative role of DNA methylation changes in the regulation of IR via MeCP2 binding and splicing factor recruitment. Our findings reveal another mechanism that controls splicing and IR in diverse tissue types. Further understanding of this mechanism in the development of cancers, and its impact in patients receiving DNA methylation inhibitors may contribute to improved therapeutic strategies.Note added in proof: At the proof stage of this manuscript, our software article detailing the IRFinder algorithm, Middleton et al.L-lysine slides . All mouse experiments were performed in a specific pathogen-free facility with approval from the Royal Prince Alfred Hospital Animal Ethics Committee (Protocol no. 2013/045). The FACS strategies for promyelocytes and granulocytes are shown in 2e slides . MPRO ceGenomic DNA extracted from cells was bisulfite-converted using the EZ DNA Methylation-Gold Kit , according to the manufacturers' instructions. For WGBS analyses, libraries were prepared from bisulfite-converted samples and sequenced on an Illumina HiSeq2000 platform at Macrogen Inc. (Korea). For gene-specific clonal bisulfite sequencing, bisulfite-converted DNA was amplified using primers that equally amplify methylated and unmethylated template. Primer sequences are provided in Inhibition of DNA methylation in MPRO cells was performed using 3 μM or 7 μM 5-Aza-2′ deoxycytidine for 120 h with drugs replenished every 24 h. Dead cells were excluded by FACS following 4′,6-diamidino-2-phenylindole staining.Total RNA extraction was performed using Trizol (Invitrogen). RNA 6000 Nano Chips (Agilent Technologies) were used to confirm RNA quality. For qRT-PCR, total RNA was treated with DNAse I, amplification grade (Invitrogen). cDNA was generated using SuperScript III First-Strand Synthesis System (Invitrogen), according to the manufacturers' instructions. qRT-PCR was performed using the CFX96 Real Time PCR Machine (Bio-Rad). Reactions were performed in 20 μl volumes containing 1 × IQ SyberGreen supermix and 0.3 μM of the respective forward and reverse primers. Cycling conditions were: 95 °C for 6 min followed by 95 °C for 30 s, primer-specific annealing temperature for 30 s, and extension at 72 °C for 30 s. Primer sequences are shown in −1) were then added, and an actinomycin D-only control was used to block transcription of genes. Following an additional 4 h of incubation, total RNA was collected for qRT-PCR.NMD inhibition was performed on MPRO cells after 5-Aza-2′ deoxycytidine treatment as previously publishedTotal protein lysates were loaded onto precast SDS-PAGE gels (Invitrogen) and subjected to electrophoresis before being transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% (weight per volume) skim milk for 1 h at room temperature and incubated overnight with a rabbit anti-MeCP2  with 15 cycles of 30 second ON and 30 s OFF at high power setting. Sonicated samples were centrifuged for 10 min at 10,000g. Supernatant was incubated overnight at 4 °C with the antibody of interest mixed with protein A/G magnetic beads. Following washes, crosslinking was reversed at 62 °C for 2 h and DNA was purified using the Agencourt AMPure XP beads . Antibodies used were rabbit anti-MeCP2 , rat anti-Pol II Ser2p , rabbit IgG control (5 μg Abcam ab46540) and normal rat IgG . ChIP DNA was analysed by qPCR with primers listed in ChIP was performed with either the Magna ChIP A/G kit (Millipore 17-10085) or Magna ChIP HiSens kit (Millipore 17-10460) following the manufacturers' instructions, with minor modifications. Briefly, 5 × 10eFor ChIP-seq, sample concentration and size distribution of immunoprecipitated DNA were measured with Qubit dsDNA HS Assay Kits (Invitrogen) and High Sensitivity DNA Analysis Kit (Agilent Technologies), respectively. Approximately 20 ng of the input and ChIP DNA samples were sent to Macrogen Inc. (Korea) for ChIP-seq library preparation and sequencing on an Illumina Hiseq2000 platform.Rapid immunoprecipitation and MS was performed as previously reportedRNA-IP was performed using the Magna Nuclear RNA-IP (cross-linked) kit (17-10520) from Millipore according to the manufacturer's protocol. Antibodies used were rabbit anti-MeCP2  and rabbit anti-Tra2b . Approximately 1 million 0.5% formaldehyde-fixed cells were used for RNA-IP. Immunoprecipitated RNA was purified using Trizol LS Reagent  and subjected to DNase I treatment. iTaq universal SYBR Green one-step kit (Bio-Rad) was used to quantify the amount of immunoprecipitated RNA. Primers used were listed in RNA-IP-reIP was performed using the same kit used for RNA-IP: Magna Nuclear RNA-IP (Cross-linked) kit (17-10520) from Millipore. Approximately 2 million 0.5% formaldehyde-fixed cells were used for each RNA-IP-reIP. Sequential RNA-IP was performed with an additional purification step between the two immunoprecipitation steps. Briefly, immunoprecipitation was performed on fixed and sonicated chromatin lysate using the rabbit anti-Tra2b antibody  together with A/G magnetic beads at 4 °C for 1 h. Following washes according to the manufacturer's instruction, the bead bound RNA-Protein complex were released with the elution buffer supplemented with DTT, RNase inhibitor and protease inhibitor for 15 min at 37 °C. SDS was removed from the eluent with HiPPR Detergent Removal spin column  before the subsequent RNA-IP.The eluent from the first RNA-IP was then used for a second RNA-IP at 4 °C for 1 h with either the Rabbit anti-MeCP2  or the normal rabbit IgG  as control. Following washes as recommended by the manufacturer, the immunoprecipitated RNA was purified using the Trizol LS Reagent  and DNase I treatment was performed. iTaq universal SYBR Green one-step kit (Bio-Rad) was used to quantify the amount of immunoprecipitated RNA relative to the sequential Tra2b-IgG RNA-IP control. The workflow for RNA-IP-reIP is shown in Besides the WGBS and RNA-seq reads generated from promyelocytes and granulocytes, we also analysed WGBS, RNA-seq and MeCP2 ChIP-seq data from the GEO (Gene expression Omnibus) database . All RNA2125556q-value (FDR) cut-off of 0.05. To assess the MeCP2 binding density, ChIP-seq reads were mapped to human (hg19) or mouse (mm10) genomes using Bowtie2 v2.2.3 with default settings. MACS2  cut-off of 0.05. MeCP2 density at each peak was reported as fold enrichment, which is the number of position-adjusted reads in MeCP2-ChIP sample/input. A sliding window of 10 bp was then applied to scan the ±100 bp region of a splice junction or from the middle of an intron. Within each window, average MeCP2 signal was calculated as the sum of fold enrichment divided by the number of peaks.To assess the MeCP2 binding density, ChIP-seq reads were mapped to human (hg19) or mouse (mm10) genomes using Bowtie2 v2.2.3 default settings. MAC2 ref.  was applCS2 ref.  was applThe steps of detecting IR in an individual sample and alternate IR between samples are described below:A STARRNA-seq reads were aligned to the reference genome by STAR using default scoring parameters but excluding multi-mapping reads from the output. The default scoring parameters suit the detection of IR as they favour neither mapping from exon to exon across splice junctions nor mapping from exons into introns. IR is then determined by measurement of both the splicing level and intronic abundance. The key calculated metric is the IR ratio. Abundance of normal splicing is measured by a count of read fragments spliced across the intron. Reads that start in the 5′ exon but end in another exon and reads that start in another exon but end in the 3′ exon are also counted, the larger value being used. Intronic abundance is measured as a trimmed mean of fragment coverage depth. The trimmed mean excludes previously excluded regions  and the highest and lowest 30% of values. This compensates for noise in intronic coverage. The IR ratio is calculated simply as follows:P-value is <0.05; 2) exonic splice abundance for both samples or groups are above 10; 3) IR ratio in one sample or group is above 0.1.To compare the change of IR of a certain intron between a pair of samples or groups, the intronic abundance and exonic splice abundance of this intron is evaluated using a Bayesian statistic adapted for digital countsP-value <0.01 after Benjamini Hochberg correction were considered significant.Gene ontology enrichment analysis was performed using DAVIDt-test was then applied to assess the significance of overall difference across all the sub-windows.Statistical analyses were performed as indicated in the text and/or figure legends. To compare the overall DNA methylation level or MeCP2 fold enrichment across the ±100 bp flanking splice junctions, the whole region (200 bp) was first divided into 20 sub-windows (10 bp each). Within each sub-window, the mean of DNA methylation level or MeCP2 fold enrichment for all IR and non-IR events was calculated respectively and paired together. Paired t-tests were used to compare differential gene expression and levels of GC content and CpG density between retained and non-retained introns. Differential enrichment of MeCP2, Pol II Ser2p and Tra2b assessed by qPCR following ChIP and/or RNA-IP was also determined using t-tests. F-test was used to determine the significance of regression analysis. Analyses were performed using Graph-Pad Prism v.6 . P<0.05 was considered significant for all tests.The significance of the difference between the number of increased and decreased IR events in cells following DNA methylation inhibition or MeCP2 knockdown was determined using the binomial test. Student WGBS and ChIP-seq data that support the findings of this study have been deposited in Gene Expression Omnibus with the accession number GSE85517. All other data that support the findings of this study are available from the corresponding authors upon reasonable request.How to cite this article: Wong, J. J.-L. et al. Intron retention is regulated by altered MeCP2-mediated splicing factor recruitment. Nat. Commun.8, 15134 doi: 10.1038/ncomms15134 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Tables


The identification and tracking of antigen-specific immunoglobulin (Ig) sequences within total Ig repertoires is central to high-throughput sequencing (HTS) studies of infections or vaccinations. In this context, public Ig sequences shared by different individuals exposed to the same antigen could be valuable markers for tracing back infections, measuring vaccine immunogenicity, and perhaps ultimately allow the reconstruction of the immunological history of an individual. Here, we immunized groups of transgenic rats expressing human Ig against tetanus toxoid (TT), Modified Vaccinia virus Ankara (MVA), measles virus hemagglutinin and fusion proteins expressed on MVA, and the environmental carcinogen benzo[a]pyrene, coupled to TT. We showed that these antigens impose a selective pressure causing the Ig heavy chain (IgH) repertoires of the rats to converge toward the expression of antibodies with highly similar IgH CDR3 amino acid sequences. We present a computational approach, similar to differential gene expression analysis, that selects for clusters of CDR3s with 80% similarity, significantly overrepresented within the different groups of immunized rats. These IgH clusters represent antigen-induced IgH signatures exhibiting stereotypic amino acid patterns including previously described TT- and measles-specific IgH sequences. Our data suggest that with the presented methodology, transgenic Ig rats can be utilized as a model to identify antigen-induced, human IgH signatures to a variety of different antigens. Immunoglobulin (Ig) molecules are the primary effectors of the humoral immune response. In theory, Ig can bind to every possible antigen through the large variety of Ig V (variable), D (diversity), and J (joining) gene rearrangements in the bone marrow (BM) and target-oriented affinity maturation in germinal centers . All B cHigh-throughput sequencing (HTS) has been widely applied to study the IgH repertoire in response to vaccination and infection . With thHere, we applied HTS on class switched, BM B cells, rich in serum antibody producing plasma cells, from rats carrying human germline IgH and light chain (IgL) loci, the OmniRat™ –21. Thesn = 4; Serum Institute of India, Pune, India) or of a BaP-TT conjugate construct  (7 PFU of a recombinant MVA expressing the hemagglutinin (H) and fusion (F) glycoproteins of the MV  or the MVA viral vector only  without adjuvant. The control animals received either 330 µg of ALUM alone  or were left untouched . Antigen-specific IgG responses were monitored by ELISA 10 days after immunizations and at sacrifice. All animal procedures were in compliance with the rules described in the Guide for the Care and Use of Laboratory Animals  were developed and bred as previously described –21. Omni, n = 5) , both fo Animals  and acce5 PFU of sonicated MVA (~314 ng), 187.5 ng of TT, or 0.25 µM of BaP-OVA in carbonate buffer . Free binding sites were saturated with 1% bovine serum albumin in Tris-buffered saline at room temperature for 2 h. Serial dilutions of the sera were added for 90 min at 37°C and developed with alkaline phosphatase-conjugated goat anti-rat IgG  and the appropriate substrate. Absorbance was measured at 405 nm. End point titers were determined as the serum dilutions corresponding to five times the background.BaP was coupled to ovalbumin  for ELISA and to purified TT as previously described . The rec® Paque Plus; Sigma-Aldrich). Total RNA was extracted from 108 cells with an RNeasy midi kit following the manufacturer’s protocol (Qiagen) and enriched for mRNA using paramagnetic separation . cDNA was prepared from 300 ng of mRNA using dT18 primers and Superscript III reverse transcriptase (Thermo Fisher Scientific) at 50°C for 80 min. Recombined IgH fragments were subsequently amplified by multiplex PCR using primers for human IgHV region and rat Cγ region with Q5 Hot Start High Fidelity polymerase  as described previously  and demultiplexed by MIDs. Only reads with an unambiguously assigned MID (0 mismatch), identified primers at both ends  and more than 85% of the bases with a quality score above 25 were considered for further analysis. After clipping MIDs and primers, sequences were collapsed and submitted to the ImMunoGeneTics database (IMGT) HighV-QUEST web serverBAM files were extracted from the Torrent SuiteHuman Ig sequences (IgG and IgM) were obtained from the Sequence Read Archive databaseThe number of matches for the 200 most frequent CDR3s (top 200) of a rat A in a rat B was obtained for a series of similarity thresholds and returned as ratio from 0 to 1 . Ratios were determined from 50–100% sequence similarity in 1% increments. The averages for the top 200 matching ratios at each increment were then calculated for all rats within a vaccination group and all rats vaccinated with unrelated antigens. Rats with related antigens were excluded in the pairwise comparison . The average top 200 matching ratios were plotted against sequence similarity along with the first derivatives in GraphPadP-values were adjusted for multiple testing and to determine the false discovery rate (FDR) using Benjamini–Hochberg correction  were immunized with different antigens Table  and somaP-value < 2 × 10−16, Kruskal–Wallis with Nemenyi post hoc test) or immunized with related antigens , indicating that mutual CDR3s are essentially induced by the immunizations  shared less CDR3s with each other than pairs of rats within the same group , of any rat A had a related CDR3 in a rat B either within the same group (intragroup comparison) or between groups (intergroup comparison) allowing for a single amino acid substitution. The same analysis was repeated for two, three, and up to eight amino acid substitutions. The number of top 200 CDR3s found to be present in inter- and intragroup was plotted against the amino acid substitutions expressed as percentage of CDR3 length  and TT and BaP-TT , which reflected the high immunogenicity of the antigens TT and MVA common within these groups. Less overrepresented CDR3s were found in the MVA , the MVA-HF , and TT group . The lowest number of overrepresented CDR3s was found in the BaP-TT group . These overrepresented CDR3s could be considered group-specific and thus immunization induced.Similar to RNA-seq expression experiments, we aimed to identify CDR3s that are differentially represented between groups of rats. Based on an FDR of 1%, 16,727 of the 249,657 (6.9%) unique CDR3s across all groups were found to be overrepresented. One hundred-fold differences in numbers of overrepresented CDR3s were identified in each of the six antigen groups  of the CDR3s, and the IgHV gene families IgVH1 (16.3 ± 13.4%), IgVH3 (14.2 ± 18.5%), and IgVH6 (11.3 ± 11.6%) were predominating the cluster 2 repertoire of one, two, and one rat, respectively. All together we showed that OmniRat™ exhibited large fractions of highly similar, stereotypic CDR3s in response to the applied vaccinations, even across groups with shared antigens.Sequences encoding the overrepresented CDR3s were surprisingly diverse in IgHV gene usage . IgHV genes belonging to one family were largely predominant for each cluster (average 93.3 ± 9.8% of genes belonging to one family per cluster), and the level of associated IgHV genes was comparable between rats of each cluster  (We showed that between 6 and 46% of the BM IgH repertoire correspond to convergent CDR3 sequences. Similar proportions 15–50%) of antigen-specific CDR3 sequences were reported in peripheral blood B cells of patients with acute dengue infections –50% of a, 49. Blo per day , 51. In gM− PI−) . In the gM− PI−) . BecausegM− PI−) ] and onlgM− PI−) , 48. ThiThe IgH repertoire of OmniRat™ displayed a CDR3 length distribution comparable to that of human IgG B cells isolated from peripheral blood and lower than that of the corresponding IgM B cells . This incluster 2 of the animals in the combined vaccination group MVA-HF + MVA exhibited significant differences in IgHV gene family usage across the rats. Similarly, different IgHV gene families were found in convergent CDR3 responses to Dengue infections and antipolysaccharide vaccination in humans implying a convergent evolution  share a high level of similarity, only differing by few nucleotides. As the amplicons of the rat IgH transcripts did not span the whole IgHV sequence, information encoded in the FR1 and CDR1 are lost. This could easily lead to varying gene assignments, explaining the IgHV gene variability of the described CDR3 clusters.Potential influence on the repertoire composition could result from PCR amplification biases introduced during library preparation as well as sequencing errors . We did We found that certain CDR3s have high counts of 80% relatives within a group but very few to none in the unrelated groups. This is in principle comparable to differential gene expression in RNA-seq data. The CDR3 counts followed a negative binomial distribution but, unlike in RNA-seq experiments, our data contained large amounts of CDR3s with zero counts over different samples. These correspond to private CDR3s that are absent in other rats of the same or other groups. On the other hand, some CDR3s exhibited very high counts of 80% relatives within an animal. While such a data distribution is uncommon in RNA-seq, they were nevertheless compatible with our computational approach [DESeq2 ], as demIdentified convergent CDR3 matched to sequences of previously described human monoclonal antibodies against TT protein , 23–25. In conclusion, we demonstrated a strong public IgH response with converging and overlapping CDR3 repertoires in animals exposed to the same antigens. These converging repertoires consisted of similar CDR3 sequences that can be best described using an 80% amino acid similarity threshold. Additionally, we presented an approach to identify such CDR3s by adopting a group-wise expression analysis, similar to RNA-seq approaches. This provides also a valuable tool for large-scale HTS data mining to identify potential candidates for high-affinity targeted antibody design.This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals as approved by the Comité National d’Ethique de Recherche .J-PB and AD contributed equally to the work. J-PB designed and developed the bioinformatics approach, interpreted data, performed data processing, and wrote the manuscript. AD designed and carried out research, prepared samples, interpreted data, and wrote the manuscript. WF supported bioinformatics approaches and data processing and corrected the manuscript. AW-B set up the raw data processing script and performed data processing, data interpretation, and corrected the manuscript. SF and EC provided technical assistance with immunizations, ELISA, and virus culture. RS performed IonTorrent PGM sequencing. CM designed research, interpreted data, corrected the manuscript, and supervised work. All authors have read and approved the final version of the 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.