Escherichia coli Cas6(dEcCas6) allowed ligand (CBS)-activated split-Venus complementation. We name this platform as Cas6 based Fluorescence Complementation (Cas6FC). In living cells, Cas6FC could detect target RNAs with nearly free background noise. Moreover, as minimal as one copy of CBS (29nt) tagged in an RNA of interest was able to turn on Cas6FC fluorescence, which greatly reduced the odds of potential alteration of conformation and localization of target RNAs. Thus, we developed a new RNA tracking platform inherently with high sensitivity and specificity.Given the fact that the localization of RNAs is closely associated with their functions, techniques developed for tracking the distribution of RNAs in live cells have greatly advanced the study of RNA biology. Recently, innovative application of fluorescent protein-labelled Cas9 and Cas13 into live-cell RNA tracking further enriches the toolbox. However, the Cas9/Cas13 platform, as well as the widely-used MS2-MCP technique, failed to solve the problem of high background noise. It was recently reported that CRISPR/Cas6 would exhibit allosteric alteration after interacting with the Cas6 binding site (CBS) on RNAs. Here, we exploited this feature and designed a Cas6-based switch platform for detecting target RNAs in vivo. Conjugating split-Venus fragments to both ends of the endoribonuclease-mutated  The current live-cell RNA tracking techniques fall into two types: fluorescence-enrichment (FE) type and fluorescence-activation (FA) type. An FE-type platform usually comprises two key components: a fluorescent protein-conjugated RNA binding protein (RBP-FP) that acts as a reporter or tracker, and a specific RBP binding sequence (RBS) genetically constructed on target RNAs. A prominent example of the FE-type is the MS2-MCP RNA tracking system in which MCP stands for MS2 phage coat protein . MCP binRecently, Cas9 and Cas13 gene editing tools were proposed to RNA imaging . BenefitAll FE-type RNA tracking platforms inherently suffer from high background noise due to the application of florescent reporters. This concern is partly addressed by the FA-type RNA tracking/imaging platforms. The FA-type platforms are based on bimolecular fluorescence complementation (BiFC). For example, MCP and PCP  were fused with N- and C- fragment of a certain FP, respectively; the resultant two reporters, MCP-FPN and PCP-FPC, would not reconstitute a fully functional florescent protein until they are brought together by adjacent MBS and PBS (PCP binding site) on an RNA target ,8. As suEscherichia coli-originated Cas6 (EcCas6) protein into an FA-type reporter. The catalytic domain-mutated EcCas6 (dEcCas6) was appended at its N- and C-termini with the non-fluorescent moieties of protein Venus, Venus-N (VN) and Venus-C (VC), respectively. The resultant chimera VN-dEcCas6-VC was not fluorescent until binding to CBS on the RNAs of interest. Because Fluorescence Complementation is mediated by the allosteric switch of Cas6, we name this platform as Cas6 based Fluorescence Complementation (Cas6FC). Thus, we designed a new FA-type RNA tracking platform with favorable sensitivity and specificity.Cas6 is a core component of type I-E CRISPR complex. It binds and cleaves pre-crRNA by recognizing a specific stem-loop RNA element designated as CBS (Cas6 binding site) ,10. CBS-The annotations and important sequences of all the plasmids used in this study are presented in ® 2000 Reagent (Invitrogen) was used for COS-7 cell transfection according to the manufacturer's instruction with the modified dose of 1 μl per well on 24-well plate. Entranster™-R4000 (Engreen Biosystem) was used for HEK293T and Hela cell transfection according to the manufacturer's instruction with the modified dose of 0.5 μl per well on 24-well plate. The plasmid usage was listed in Table HEK293T, COS-7 and HeLa cell lines were purchased from China Center for Type Culture Collection (CCTCC). HEK293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). COS-7 cells were cultured in RPMI Medium 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco). Lipofectamined-lysine coverslips, and then these cells were co-transfected with 200 ng VN-dEcCas6-VC-GK and 800 ng Actin-16 × CBS-GK or hTERC-16 × CBS-GK. Single molecule inexpensive fluorescence in situ hybridization (smiFISH) was employed for RNA imaging  and 320 V laser voltage for green fluorescence channel (FITC), respectively. Fluorescence compensation parameters were 4.8 for red fluorescence channel and 12.6 for green fluorescence channel, respectively. A total of 10 000 single living cells were sorted from all the cells according to FSC (Forward Scatter) and SSC (Side Scatter). As the co-transfected cells should express DsRed-Monomer fluorescent protein, the red fluorescence positive (Red+) cells were then sorted and taken for analysis of VN-dEcCas6-VC background and specific fluorescence. The Actin-GK + pDsRed-Monomer-C1 co-transfection group worked as control group; the VN-dEcCas6-VC-GK + pDsRed-Monomer-C1 co-transfection group (named as without CBS group) was used for analyzing initial background fluorescence; the VN-dEcCas6-VC-GK + Rm-20 × CBS-C1 group (named as with CBS group) was used for analyzing specific fluorescence. The maximum fluorescence intensity of Red+ control group cells was used as a threshold for determining initial background fluorescence or specific fluorescence. The Red+ without CBS group cells fluorescence whose intensity was higher than the above threshold was determined as initial background fluorescence; the Red+ with CBS group cells fluorescence whose intensity was higher than the above threshold was determined as specific fluorescence.The HEK293T cells in 24-well glass-bottom plate were transfected with plasmids according to the experiment design Table . Four reFor RNA imaging in living cells, the transfected cells in 24-well plates were observed on ZEISS Vert.A1 microscope platform at 24 h post transfection. For FISH imaging, the coverslips were mounted and observed on ZEISS Vert.A1 microscope platform. Exposure time of each fluorescence channel was set as 5 s, and that of bright field as 100 ms. For the observation of fixed cells by using confocal microscope, the transfected cells were fixed with 4% PFA for 15 min at 37°C, and washed with 1 × PBS (pH7.4) for 5 min three times, followed by permeabilization with 0.5% Triton X-100 for 15 min, and these cells were rewashed with 1 × PBS (pH 7.4) for 5 min three times. After being stained with 0.1 μg/ml DAPI and washed with 1 × PBS (pH7.4) for 5 min three times, the cells were observed with Nikon A1R HD25 Confocal Microscope.Escherichia coli (EcCas6) and investigated its application in tracking RNAs in mammalian cells at physiological temperature (37°C). We constructed a catalytically inactive EcCas6 mutant (an H→A mutation at Position 20 according to a previous study (EcCas6 (dead EcCas6). To confirm that a. EcCas6 could recognize and cleave CBS and b. dEcCas6 lost the endoribonuclease activity but retained its binding activity to CBS in mammalian cells, we test them in de novo designed turn-off and turn-on reporter systems. In the turn-off system, 1 × CBS sequence was inserted into the 5′-UTR of the EGFP mRNA. Since the 5′ cap is pivotal for mRNA translation and EcCas6-mediated cleavage would remove 5′-cap from EGFP mRNA, a successful EcCas6-CBS interaction would lead to reduction of EGFP fluorescence intensity. In HEK293T cells, co-transfection of EcCas6-expressing vector significantly reduced EGFP fluorescence intensity, which was not observed when dEcCas6-expressing construct was used instead  mRNA. LIN28 RNA nuclear retaining signal would retain the host mRNA in nucleus , resulting in juxtaposing the N and C termini of TtCas6 , we, weEcCasEcCas6 protein with diverse peptide linkers, generating a series of dEcCas6-split FP fusion proteins (EcCas6-VC (53 kDa), was isolated.To construct an FA type fluorogenic reporter, we linked split-FP fragments to the ends of dproteins . The FA EcCas6-VC fusion protein consists of sequentially linked Venus-N terminal fragment (1–153), linker1, dEcCas6, linker2 and Venus-C fragment (154–238)  RNA tracking platform. As all its unique characters are empowered by the Cas6 protein, we name this new RNA tracking platform as Cas6 based Fluorescence Complementation (Cas6FC).This VN-d) Figure . To demod Figure . Howevers Figure . Similars Figure , indicatin situ hybridization) technique. To exclude the potential bias introduced by technique discrepancy between them, the FISH probes were designed aiming at the natural non-CBS sequence of the target RNAs and the same batch of cells were tested by both Cas6FC and RNA smiFISH  lncRNA of Hela cells were chosen as the target RNAs for the comparison. The mRNA of ACTB is cytoplasm-localized and encodes a housekeeping cytoskeleton protein , the cells co-transfected with the VN-dEcCas6-VC-GK vector (expressing the reporter of Cas6FC platform) and pDsRed-Monomer-C1 vector had almost no detectable Venus signal (0.78% positive cells versus 0% of background)  displayed strong Venus fluorescence. Thus, the VN-dEcCas6-VC made negligible noise in the absence of CBS sequence in target RNAs.To further evaluate the sensitivity of the Cas6FC platform in tracking target RNAs, we utilized flow cytometry which is relatively more sensitive in differentiating signal from noise than fluorescent microscopy. Compared to the background reference provided by the cells co-transfected with Actin-GK vector (expressing β-actin protein as control) and pDsRed-Monomer-C1 vector (expressing ) Figure . In contSimply increasing the copy number of RBP binding motif could improve the sensitivity of FE type RNA tracking platforms. For instance, 24 × MBS (MCP binding sites) were often used in the MS2-MCP system to achieve optimal signals . HoweverEcCas6, we first analyzed the interactions between VN-dEcCas6-VC and CBS cognates in vivo. Five CBSes derived from different species were selected for this purpose , loop-localized nucleotides (Positions 12–15) and the rest non-stem–loop positions. Accordingly, a series of DsRed-Monomer-4 × mutant EcCBS gene embedded vectors were constructed. To test the specificity of the stem-localized nucleotides, we individually introduced single-nucleotide bulge mutations, transition/transversion mutations and stem-length mutations. As shown in Figure EcCas6-VC, manifested by lack of fluorescence. Similarly, either transition/transversion mutation or stem-length mutation could completely abrogate their VN-dEcCas6-VC interaction .As for the third group of nucleotides, U5, A22, U23 and A24 were predicted to interact with en bonds . Howeverf TtCas6 ; howeverA Figure . Indeed,EcCBS core motif in the transcriptomes of several mammalian species. According to our data above and a previous study  of Homo sapiens, Rattus norvegicus and Mus Musculus. It turned out that no endogenous RNAs derived from these three mammalian species carry this core EcCBS motif. Taken together, the Cas6FC platform has advantages in both sensitivity and specificity over the current mainstream FE and FA RNA tracking platforms.Finally, to interrogate potential off-target effect resulting from CBS-like sequence in mammalian transcriptomes, we analyzed occurrence frequencies of us study , the corBy labeling an RBP with an FP, RBS-tagged RNA targets can be detected. Such a tracking system is simple but unavoidably introduces high background noise which is always a weakness of the FE-based platforms. In contrast, fluorescence complementation (FC) technique could solve this problem with much improved signal-to-noise ratio. As such, FA type platforms, including BiFC and TriFC RNA tracking systems, have achieved prominent progress. However, for achieving an ideal performance in using the current FA platforms, a large tag carrying multiple RBS repeats has to be inserted in the target RNAs, which could potentially alter the structure or location of the target RNAs.EcCas6-based FA-type RNA tracking platform (referred to as Cas6FC) to overcome these problems. The Cas6FC platform reconstructs a split fluorescence by employing the feature of allosteric alteration after Cas6-RBS binding. In this case, one RBP and one RBS can serve as a fully functional unit of FC, avoiding the need of designing two distinctive RBSes on one target RNA. Our data indicate that as minimal as one RBS (CBS in this case) unit is enough to generate a distinguishable signal, superior to the sensitivity of BiFC/TriFC RNA tracking platforms. These facts poise the intermolecular-allosteric-switch-based Cas6FC platform ideal for intracellularly tracking the location and abundance of target RNAs.As a prototype of fluorescence complementation (FC), protein BiFC makes use of protein-protein interaction to reconstruct fluorescence; current BiFC or TriFC RNA tracking platforms adopted this method, relying on juxtaposing two RBPs which carries the moieties of one fluorescent protein by binding to the adjacent corresponding RBSes. It thus imposes a requirement of optimal distance between the RBSes on the target RNAs: if designed too far, the fluorescent protein could not be successfully assembled; if too close, docking of two RBPs will be limited by room. Current solution is to insert multiple repeats of RBSes, aiming at providing more opportunity for the occurrence of RBP interaction. However, increasing the size of inserts is liable to disrupting the genuine structures of RBS as well as target RNAs which may in addition abrogate RBP binding. In this study, we developed an in situ RNA hybridization. The specificity of Cas6FC platform was interrogated and confirmed from two aspects: 1. EcCBS is both structure- and sequence-specific for VN-dEcCas6-VC binding; 2. the EcCBS motif for EcCas6 is unique without overlapping with the RNA sequences in the transcriptomes of multiple mammalian species. These properties preclude potential off-target effects to the most extent.The authenticity of the signal generated from the Cas6FC system was verified by in vivo. Led by guide RNAs, Cas9/Cas13 could track genuine RNA free of RBS tags. However, they suffer from high background problem as other FE-type platforms. In this study, we again show that the CRISPR-Cas family is a precious resource for imaging RNAs. The Cas6FC reached almost free of background noise and needs only as minimal as one CBS tag for signal detection. One CBS is only 29nt long, which alleviates the workload of genetic manipulation. Moreover, a tag as small as one CBS reduces the risk of structural/locational alteration of an RNA target.Recently, Cas9 and Cas13 were shown to be used to track RNAs of interest The Cas6FC RNA tracking platform could be further developed or optimized in a couple of ways: (i) many microbe species own Cas6-like enzymes and they could recognize different CBS from each other, enriching the toolbox of Cas6FC; (ii) various FPs with diverse excitation and emission spectrum could be examined for their amenability for Cas6FC; (iii) it is conceivable that simultaneous application of different Cas6-based reporters with different colors of FP could track multiple target RNAs in the same cell; in such a case, study of RNA–RNA interaction could be possible.gkac014_Supplemental_FilesClick here for additional data file.


MaMPK14 and MaMYB4 in banana fruit delayed fruit ripening, confirming the negative roles in the ripening. The ripening negative regulator MaMYB4 could repress the transcription of genes associated with ethylene biosynthesis and fruit softening, such as MaACS1, MaXTH5, MaPG3, and MaEXPA15. Furthermore, MaMPK14 phosphorylated MaMYB4 at Ser160 via a direct interaction. Mutation at Ser160 of MaMYB4 reduced its interaction with MaMPK14 but did not affect its subcellular localization. Importantly, phosphorylation of MaMYB4 by MaMPK14 enhanced the MaMYB4-mediated transcriptional inhibition, binding strength, protein stability, and the repression of fruit ripening. Taken together, our results delineated the regulation pathway of MAPK module during banana fruit ripening, which involved the phosphorylation modification of MaMYB4 mediated by MaMPK14.Mitogen-activated protein kinase (MAPK/MPK) cascades play crucial parts in plant growth, development processes, immune ability, and stress responses; however, the regulatory mechanism by which MAPK affects fruit ripening remains largely unexplored. Here, we reported that MaMPK14 cooperated with MaMYB4 to mediate postharvest banana fruit ripening. Transient overexpression of individual  Fleshy fruit undergo dramatic changes during ripening, including variations in color, flavor, aroma, and texture . Fruit rCYP71B15 via phosphorylating WRKY33 [FaMAPK5 and FaMAPK10 are related to ABA-mediated fruit ripening in strawberry [Mitogen-activated protein kinase (MAPK/MPK) is one type of serine/threonine-type protein kinase. A typical MAPK cascade consists of three interconnected protein kinase modules (MAPKKK-MAPKK-MAPK) . The MAPg WRKY33 . Poplar g WRKY33 . Similarg WRKY33 , while Mg WRKY33 . Recentlg WRKY33 . FaMAPK5rawberry . These fArabidopsis thaliana are related to the formation of root hairs [MdMYB308L increased or decreased anthocyanin contents [Over the past decade, multiple TFs have been revealed to engage in fruit ripening, including those necessary for ripening , and others responsible for more subtle effects, such as ERF, bHLH, and WRKY –15. Amonot hairs , 19, whiot hairs . Membersot hairs . In addiot hairs , potato ot hairs , and maiot hairs  take parot hairs . The SlMot hairs . In applcontents . Interescontents , and phocontents , suggestMaMPK14 delayed fruit ripening in banana. Furthermore, MaMYB4, one of the negative regulators in banana fruit ripening which could be degraded by MaBRG2/3 ubiquitination [The MAPKs can be classified into four groups, in which the investigated best are MPK3/6 in group A and MPK4 in group B, while relatively little functional information has been reported about groups C and D , 28. Hertination , was ideMusa acuminata v1, 2012) genome possesses 25 MPKs [M. acuminata v2, 2016) through the banana genome database (https://bananagenome-hub.southgreen.fr/) , ethylene-induced ripening and 1-MCP-delayed ripening bananas  fruit  fruit . Meanwhid fruits . Collectin vivo CoIP assay was conducted by utilizing tobacco leaves transiently expressing MaMYB4-GFP and MaMPK14-His, with the combination of GFP and MaMPK14-His serving as a negative control. in vitro GST pull-down assay employing recombinant MBP-MaMPK14 protein and GST-MaMYB4 protein were performed  screening. Since pGBKT7-MaMPK14 displayed self-activation, 3-amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3 protein, was used for preventing its self-activation. By carefully screening, we obtained a positive clone MaMYB4 (Ma01_g19610) that interacted with MaMPK14 in yeast. To verify their interaction, we fused MaMPK14 and MaMYB4 to pGADT7 (AD) or pGBKT7 (BD) vectors, respectively. Further Y2H assays revealed that MaMPK14 actually interacted with MaMYB4 in yeast cells. As the negative control, MaMPK14 did not show interaction with the empty AD vector in Y2H assays . Then weerformed . These dAGE gels . AltogetMaACS1, MaACO13/14, MaEXP2, MaPL2, and MaXTH5) as well as MaMYB4  in MaMPK14-overexpressed banana fruit during ripening, qRT-PCR analyses were conducted. Results showed that expression of these ethylene synthesis-related genes  and cell wall modification-related genes  were significantly repressed in banana fruits overexpressing MaMPK14  to mimic the non-phosphorylation form of MaMYB4 . Interestingly, overexpression of MaMPK14 alone also repressed the activities of MaACS1, MaXTH5, MaPG3, and MaEXPA15, implying that MaMPK14 might affect gene transcription through interaction of other uncharacterized mechanism. Notably, additionally repressive effects on the transcription of MaACS1, MaXTH5, MaPG3, and MaEXPA15 were observed when MaMPK14 and MaMYB4 were co-expressed, indicating that MaMPK14 enhances MaMYB4-mediated transcriptional repression of ripening-associated genes. However, no further repressive effects of MaACS1, MaXTH5, MaPG3, and MaEXPA15 were detected when S160AMaMYB4 was transiently expressed  were hand-harvested from an orchard near Guangzhou, China. Three treatments were used based on our prior study [−1 ethylene treatment for 16 h followed by storage at 22°C), and 1-MCP-delayed ripening group (0.5 μL L−1 1-MCP treatment for 16 h followed by storage at 22°C). During storage, samples were collected according to the ripening progression which was evaluated by ripening parameters. All samples were rapidly frozen via immersion in liquid nitrogen and stored at −80°C pending analyses. Each treatment had three replicas.The 70–80% mature stage of banana fruit  plants were cultivated at 22°C under long daylight conditions (8 h of darkness/16 h of light) in a growth chamber and selected from 4 to 6 weeks old for transient expression experiments.Tobacco . Each pair includes at least six biological replicates.The transcriptional effects of MaMYB4, MaMYB4eviously . Target MaMPK14, MaMYB4, and S160AMaMYB4 were individually cloned into pCXUN-HA vector, which were controlled by the maize (Zea mays) Ubiquitin promoter [S160A-HA or control pCXUN-HA were then injected into green banana fruit by distal injection, respectively, at 22°C, incubated for 24 h, as previously described [−1 ethylene at 16 h and stored at 22°C for 7 days with 90% relative humidity. Samples were collected for measurement of fruit color, ethylene production, pulp firmness, and gene expression, and each assay was referred to our previous report [The coding sequences (CDSs) of promoter . The recescribed . The infs report .MaMPK14 was constructed into pGBKT7 as bait, operated according to the handbook of Y2H screening kit [S160A, they were constructed into pGBKT7 (BD) or pGADT7 (AD) vectors as bait and prey, respectively, as per the instructions. Possible interactions were assessed based on plates could be grown on SD/−Leu-Trp-Ade-His (containing 3-AT) plates as well turn blue in the presence of the chromogenic substrate X-α-Gal.The CDSs of ning kit . A selecMaMYB4, S160AMaMYB4, and MaMPK14 were constructed into pUC-pSPYNE or pUC-pSPYCE vectors, respectively [MaMYB4, S160AMaMYB4, and MaMPK14 alone was used as negative controls, while NLS-mCherry fluorescence signal was used as an indicator for the nucleus, which was then manipulated as mentioned previously [The CDSs of ectively . Expresseviously . FluoresMaMYB4, S160A,MaMYB4 and MaMPK14 were cloned into pEAQ-HT-mCherry and pEAQ-HT-GFP vectors, respectively. The GFP vector, mCherry vector and fusion plasmid were electroporated into Agrobacterium strain EHA105, followed by transient expression in tobacco leaf as described previously [The CDSs of eviously . The empMaMPK14, MaMYB4, and S160AMaMYB4 were introduced into pMAL-c2X and pGEX-4 T-1vector, respectively, then transformed into Escherichia coli strain BM Rosetta (DE3). They were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) to express GST-MaMYB4/GST- MaMYB4S160A or MBP-MaMPK14 as previously reported [The CDSs of reported . The aboGST pull-down assay was conducted by reference to the Cold Spring Harbor Protocols with minor modifications . BrieflyMaMPK14 and MaMYB4 were constructed into pEAQ-HT (with His-tag) and pEAQ-HT-GFP vectors, transfused into Agrobacterium tumefaciens strain EHA105 and injected into tobacco, respectively. CoIP assay were undertaken as described previously [The CDSs of eviously . Samplesin vivo phosphorylation analysis, tobacco leaves injected with MaMYB4-GFP + MaMPK14-His and MaMYB4-GFP + His fusion proteins were subjected to a mixture containing sodium pyrophosphate, 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, β-glycerophosphate, EDTA, Na3VO4, and leupeptin  in IP buffer for lysis. Enrichment of MaMYB4-GFP protein was performed using anti-GFP . After immunoprecipitation, the samples were separated in 10% SDS-PAGE gels, and the protein bands corresponding to MaMYB4-GFP were sectioned and analysed using LC–MS/MS at Jingjie PTM BioLab (Hangzhou) Co. Ltd.For N. benthamiana leaves, followed by transient expression in tobacco leaf as described previously [For protein stability assay, MaMYB4-GFP with or without S/A substitution at S160 was transiently co-expressed with MaMPK14-His and MaBRG2/3-His  , 1 μL of ALP  was mixed into 25 μL of incubation solution. Each sample was gently mixed and incubated at 30°C for 25 min, then separated by 10% (w/v) SDS-PAGE with 0.1 mM MnCl2 and 0.1 mM Phos-tag , followed by detection of the proteins with GST antibody .Phosphorylation assay eviously , 44. BriS160A, and MBP-MaMPK14 proteins as described are detailed in the section on GST pull-down assays. Biotin labeling of the DNA fragments (50 bp) containing YYYACCWAMYW [MaACS1 and EMSA reaction, referring to the kit mentioned in Fan et al. [S160A protein in reaction buffer containing 20 mM Tris–HCl buffer, 20 mM MgCl2, 100 mM NaCl, 10 mM ATP, 2 mM DTT, for 0.5 h at 30°C before performing the EMSA.Expression and purification of GST-MaMYB4, GST-MaMYB4ACCWAMYW   was used to determined statistical differences between two samples, and a one-way ANOVA at the P < 0.05 level was used for statistical comparisons of multiple samples.A Student’s A list of all primers used in the present study is given in This work was funded by the National Natural Science Foundation of China (Grant No. 31830071) and China Agriculture Research System of Ministry of Finance (MOF) and Ministry of Agriculture and Rural affairs (MARA) (Grant No. CARS-31).W.L. conceived and designed the experiments. Y.Y. performed most of the experiments and analysed the data. C.W., W.W., and Y. Z. performed some of the experiments. Y.Y. wrote the manuscript. J.K. revised the manuscript. W.S., J.C., and Y. J. gave advice for the manuscript. All the authors have read and approved the final version of the manuscript.All relevant data can be found within the manuscript and its supporting materials.The authors declare no conflicts of interest.Horticulture Research online.Web_Material_uhac243Click here for additional data file.


Most cases of colorectal cancers (CRCs) are microsatellite stable (MSS), which frequently demonstrate lower response rates to immune checkpoint inhibitors (ICIs). RNA editing produces neoantigens by altering amino acid sequences. In this study, RNA editing was induced artificially by chemoradiation therapy (CRT) to generate neoantigens in MSS CRCs. Altogether, 543 CRC specimens were systematically analyzed, and the expression pattern of ADAR1 was investigated. In vitro and in vivo experiments were also performed. The RNA editing enzyme ADAR1 was upregulated in microsatellite instability–high CRCs, leading to their high affinity for ICIs. Although ADAR1 expression was low in MSS CRC, CRT including oxaliplatin (OX) treatment upregulated RNA editing levels by inducing ADAR1. Immunohistochemistry analyses showed the upregulation of ADAR1 in patients with CRC treated with CAPOX (capecitabine + OX) radiation therapy relative to ADAR1 expression in patients with CRC treated only by surgery (p < 0.001). Compared with other regimens, CRT with OX effectively induced RNA editing in MSS CRC cell lines  via the induction of type 1 interferon-triggered ADAR1 expression. CRT with OX promoted the RNA editing of cyclin I, a neoantigen candidate. Neoantigens can be artificially induced by RNA editing via an OX–CRT regimen. CRT can promote proteomic diversity via RNA editing. However, accumulating evidence from recent decades suggests that molecular targeted therapy can prolong the survival of patients with CRC. Immune checkpoint inhibitors (ICIs) have emerged as “game changers” in cancer therapy; in CRC treatment, ICIs respond mainly to mismatch repair-deficient CRCs, which exhibit microsatellite instability–high (MSI-H) tumors. However, nearly 85% of CRCs are microsatellite stable (MSS)2 and frequently demonstrate lower response rates to ICIs. One of the reasons for this phenomenon is differences in the tumor mutation burden (TMB). MSS CRCs have a TMB lower than that of MSI-H CRCs; thus, cytotoxic T lymphocytes are unable to target neoantigens (a protein that forms on cancer cells when certain mutations occur in tumor DNA), resulting in “cold,” or non-immunogenic tumors 3. Recent research efforts have aimed to identify mechanisms by which cold tumors can be changed to “hot” tumors 3. In this context, RNA editing has emerged as one of the crucial processes that allows increased production of neoantigens in MSS CRCs, thus artificially transforming poorly immunogenic tumors into highly immunogenic and hot tumors.Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the USA5. A comprehensive RNA editing neoantigen profile analysis of 12 cancer types was recently published6. Adenosine-to-inosine RNA editing is mediated by a group of enzymes known as adenosine deaminases that act on RNA (ADARs)7. Although RNA editing is a mechanism whereby neoantigens can be produced in cancer cells8, leading to an improved response to ICIs6, the process by which RNA editing is regulated and produces neoantigens remains unclear.RNA editing is a recently identified epigenetic mechanism that produces neoantigens by altering amino acid sequences via RNA modification8. Overall, our results suggest that neoantigens can be artificially induced by RNA editing activated by an OX–CRT regimen; this epigenetic modification may also compensate for lower TMB in MSS CRC during immunotherapy. Thus, our study will provide a novel molecular basis for CRT and ICI combination therapy in CRC.In this study, how RNA editing can be controlled to transform immunogenically cold tumors into hot tumors was illustrated. Whether RNA editing was induced by existing treatments, such as chemotherapy and radiotherapy, using clinical specimens and an in-vitro experimental model was determined. Chemoradiation therapy (CRT), including oxaliplatin (OX), was shown to upregulate RNA editing levels by inducing the RNA editing enzyme ADAR1, which is triggered by type 1 interferon. Of note, CRT with OX promoted RNA editing of cyclin I (CCNI), which was previously reported to be a candidate of neoantigens induced by RNA editing in patients with melanoma9. MSI CRCs are classified as CMS1. The relationship between the MSI status, CMS classification, and RNA editing as a potential new source of neoantigens for immunotherapy was first analyzed.Whether immune-related markers are associated with any consensus molecular subtype (CMS) subgroups in CRC, a classification based on heterogeneity at the gene expression level, was assessed10.The patients with CRC classified as CMS1 showed strong immune activation, with upregulation of CD8, the surface marker of cytotoxic T cells , PD-1 , and PD-L1  analysis was performed in clinical specimens. Because ADAR1 is expressed in both the nucleus and cytoplasm, ADAR1 expression levels were observed separately in each compartment. IHC analysis revealed strong staining for ADAR1 in CRCs treated with CRT (CAPOX-RT: capecitabine + OX + RT) Fig. A. ADAR1 Next, the best CRT regimen for the activation of ADAR1 expression was identified. First, whether RNA editing could be activated by chemotherapy was assessed. HT29 (ADAR1 high) and Caco2 (ADAR1 low) CRC cells were selected for cell line-based analyses  with radiation at doses of 8 or 16 Gy Fig. E. CCNI R16.Of note, a combination therapy of OX and radiation increased the upregulation of CCNI editing to the greatest extent. OX was previously reported to be a reagent that can introduce immunogenic cell death, which supports the present resultsThe above-mentioned analysis was further validated using a resistant cell model (the Caco2 cells), in which the expression of ADAR1 is normally lower than that in HT29 cells. ADAR1 was upregulated in Caco2 cells treated with 5FU (p < 0.05) but not in cells treated with CPT-11 or OX Fig. A. CCNI RFrom a clinical viewpoint, a stabilized RNA editing induction system is generally preferred. Finally, CRT was tested as a means of stable induction of RNA editing. ADAR1 expression was promoted in Caco2 CRC cells treated with 5FU (p < 0.05), CPT-11 (p < 0.001), or OX (p < 0.001) and at a dose of 8 Gy, or with 5FU (p < 0.01), CPT-11 (p < 0.05), or OX (p < 0.001) and at a dose of 16 Gy Fig. E. CCNI RCompared with monotherapy, CRT combined with OX and radiation showed a significant upregulation of ADAR1, followed by promotion of CCNI RNA editing, in both HT29 and Caco2 cells p < 0.05; Fig. ; Fig. 5AWhether the aforementioned trend was exclusive to CCNI or affected other targets as well was also analyzed. Upregulation of RNA editing by chemoradiation was detected in other typical RNA editing sites in HT29 and Caco2 cells, e.g., AZIN1 , GLI1 , and APOBEC3D (both p < 0.01) , in comparison with ADAR1 expression in CRC lesions treated with a FOFOXIRI (5FU + OX + CPT-11) chemo-regimen  and IFNβ (p < 0.001) were induced by an OX–RT CRT regimen men Fig. A. ADAR1 Overall, this study presented new evidence that showed that CRT can generate neoantigens and promote proteomic diversity via RNA editing Fig. C. This t23. In particular, adenosine-to-inosine RNA editing mediated by ADARs is the most prominent form of RNA editing in humans24. In this study, CRT with OX (CAPOX-RT) was demonstrated to efficiently induce the RNA editing enzyme ADAR1 in CRC clinical specimens. RNA editing is expected to alter MSS CRC with low TMB from a cold to a hot tumor; however, a method that can efficiently induce RNA editing and generate neoantigens has remained elusive. In our experiments, CRT, particularly in regimens including OX, was found to induce ADAR1 and upregulate RNA editing, including CCNI RNA editing levels, in MSS CRC cell lines. A preclinical mouse model was used to functionally validate the findings of this study and the upregulation of RNA editing was confirmed in this experimental system. Edited CCNI has been reported to be a candidate neoantigen induced by RNA editing in melanoma and a target of immunotherapy8. CRT can generate such neoantigens via RNA editing for cancer immunotherapy, as confirmed in this study.RNA editing has emerged as an important epigenetic modification involved in evolution and disease progression in various cancers, including CRC13; however, the mechanism by which these effects are orchestrated has yet to be determined. The upregulation of RNA editing followed by production of neoantigens was speculated to be the basis of this phenomenon in CRC cell lines. CCNI RNA editing induced by CRT may therefore represent a new method by which epigenetic diversity can be controlled for the purpose of cancer immunotherapy. AZIN1, GLI1, and APOBEC3D RNA editing, which are the typical editing targets of ADAR1, were upregulated depending on ADAR1 expression by CRT. IHC analyses also showed that ADAR1 was upregulated in patients with rectal cancer treated with CAPOX-RT. Indeed, CRT tended to upregulate RNA editing, suggesting that neoantigens are generated for cancer immunotherapy via CRT.To the best of our knowledge, this is the first report to demonstrate that CRT can induce RNA editing and upregulate epigenetic diversity. Previous studies have demonstrated that chemotherapy or radiation shows high affinity to ICIs26. This may have been caused, at least partially, by epigenetic induction of neoantigens via RNA editing. Our data suggest that the addition of OX to a Cape-RT regimen, i.e., CAPOX-RT, produces more neoantigens and thereby improves the response to ICI. Moreover, OX has already been reported as a drug that can induce immunogenic cell death16. Simultaneous, rather than sequential, administration of OX, RT, and ICI is required to change cold CRC to hot CRC.Particularly for locally advanced rectal cancer, the combination of CRT and immunotherapy may be an improvement over the “watch and wait” strategy. In clinical trials (VOLTAGE-A), the combination of preoperative CRT (Cape-RT) and ICI for the treatment of locally advanced rectal cancer has improved the pathological complete response rateCRT induces inflammatory interferons, but despite the increase in ADAR1 expression, it is only considerably elevated in the nucleus; therefore, the inflammatory cell death induced by the enhanced protocol is expected to promote the neo-antigen specific immune response by converting tumors from cold to hot. OX–RT did not interfere with ZBP1, RIP1, and RIP3, accelerators of PANoptosis, which is in agreement with our theory  could not be confirmed6. Edited CCNI is presented on HLA-A*028; thus, it is difficult to investigate this phenomenon in a mouse model. Our future studies will focus on the treatment of CRC with CAPOX-RT + ICI using a humanoid mouse.A previous study reported that loss of ADAR1 in tumors enhances the response to ICIs and overcomes resistance to immunotherapy; however, this study did not examine the correlation between actual RNA editing level changes and immunityIn summary, this study provides novel evidence of a method to control epigenetic diversity using CRT-meditated RNA editing. This study highlights the biological and clinical significance of RNA editing for immunotherapy in patients with CRC, especially in locally advanced rectal cancer. Our findings suggest that CRT with OX is an effective treatment for accelerating immunoreactivity for ICIs. In particular, this technology may be useful as a watch and wait therapy in patients with rectal cancer.https://cancergenome.nih.gov/)30 and 31 clinical specimens  obtained from Okayama University Hospital (Supplementary Table 9. The labels for TCGA primary CRC CMS were obtained from Sage Bionetworks Synapse (syn4978511)32 (https://www.synapse.org/Sage). The diagnosis of each CRC was confirmed for all enrolled patients based on their clinicopathological findings. The Tumor Node Metastasis staging system from the American Joint Committee on Cancer was used for pathology staging. The institutional review board  approved the study (1907–001). Written informed consent was obtained from each patient. All methods were performed in accordance with the relevant guidelines and regulations.This study included the analysis of 543 CRC cases, which comprised 512 CRC specimens from the Cancer Genome Atlas (TCGA) dataset . Color development was achieved using an EnVision + Dual Link Kit  and slides were counterstained with hematoxylin. Negative controls were run in parallel. The level of ADAR1 staining was evaluated using intensity scores . The total RNA from tissues and cell lines was isolated using a miRNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. The cDNA was synthesized from 1.0 µg of total RNA using a High Capacity cDNA Reverse Transcription Kit .33. In brief, specific primers for the wild-type and edited AZIN1, GLI1, and APOBEC3D sequences were designed. Based on the difference in the Ct values, the ratios between the edited and wild-type sequences were calculated using the formula 2−(Ct Edited − Ct Wild-type). Primer sequences for the PCRs are shown in Supplementary Table The degree of RNA editing of AZIN1, GLI1, and APOBEC3D was analyzed using RESSq-PCR as previously reported−(Ct Edited − Ct Wild-type). Primer sequences for the PCRs are shown in Supplementary Table The degree of RNA editing of CCNI was analyzed using a PrimeTime 5′ Nuclease Assay , as previously describedThe HT29 and Caco2 CRC cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank  3 months before the experiment began. These cells were authenticated annually by the JCRB Cell Bank using short tandem repeat analysis. All cell lines were cultured according to the manufacturer’s specifications. All experiments were performed using cells that did not exceed 15–20 passages.Cells were cultured for 48 h in a medium to which 5 μM of 5-FU, 5 μM of CPT-11, or 30 μM of OX was added. Radiation therapy was administered at a single dose of 8 or 16 Gy.34. Anti-ADAR1 , and an anti-β-actin antibody  was used as the loading control.Western immunoblotting experiments were performed as described previouslyA radar chart was used to visualize ADAR1 expression and RNA editing levels for multiple comparisons. The levels of ADAR1 expression and CCNI editing, respectively, were compared among the chemotherapy, radiation, and chemoradiation (OX + radiation) groups.6 cells/injection site) with 100 μl of Matrigel (Corning). Mice were monitored for 4 weeks following the injection, and subcutaneous tumors were measured every week. At 4 weeks postinjection, all animals were sacrificed. The protocol for CRT was as follows: OX: 200 μg, days 1, 8, and 15, intraperitoneal administration; radiation: 2 Gy, days 1, 2, 3, 8, 9, 10, 15, 16, and 17.Male Balb/c mice were obtained from CLEA  at 5 weeks of age and maintained under controlled conditions (12/12-h light/dark cycle) with food and water provided ad libitum. The animal protocol was approved by the Institutional Animal Care and Use Committee of Okayama University. To establish a xenograft tumor model, Colon26 cell lines (mouse CRC cell line) were subcutaneously injected into the left and right flanks of 12 mice  was used to perform statistical analyses. Differences between groups were estimated using the Wilcoxon signed rank test, χWritten informed consent was obtained from each patient, and the institutional review board  approved the study (1907–2001).Supplementary Information.


Trypanosoma brucei is a parasite of mammals and Tsetse flies, and control of mRNA stability is critical for parasite survival in the two different hosts. T. brucei RBP10 is a protein with a single RNA Recognition Motif (RRM) which is expressed only in the mammalian (bloodstream) form. Numerous observations suggest that RBP10 binds to procyclic-specific mRNAs and targets them for destruction, and there is also some evidence for selective binding of RBP10 to RNAs containing the motif UA(U)6. We here investigated this binding further.6, and the other two copies of a mutant version, UACUCUCU, which is inactive in regulation. An N-terminal segment of RBP10, including the RRM domain and 90 residues to its C-terminus, could be produced as soluble protein. This could bind both probes in vitro with similar affinities in the low micromolar range, which is not atypical for a single RRM. Soluble RBP10 therefore did not distinguish between UA(U)6 and UACUCUCU. Since no other sequences were tested, the requirements for RBP10 RNA binding remain to be determined.We tested in vitro binding of RBP10 to two different probes in solution. One contained two copies of UA(U) The RNA Recognition Motif (RRM) is a sequence of approximately 90 residues which interacts with single-stranded with RNA or DNA . In someTrypanosoma brucei is a parasite of mammals and Tsetse flies, and control of mRNA stability is critical for parasite survival in the two different hosts. T. brucei RBP10 is a protein with a single RNA Recognition Motif (RRM) which is expressed only in the mammalian (bloodstream) form. Numerous observations, including the effects of RBP10 depletion in bloodstream forms and of inappropriate expression in the procyclic (Tsetse fly midgut) form, and the results of pull-down followed by RNA-Seq, suggest that RBP10 binds to procyclic-specific mRNAs and targets them for destruction [truction , 5.6. The role of this motif in causing mRNA instability had previously been demonstrated in the mRNAs encoding EP procyclin, a major surface protein of procyclic forms [EP1 3′-UTR [6 motif was required for the regulation [The 3′-untranslated regions (3′-UTRs) of the RBP10-bound mRNAs were strongly enriched in the motif UA(U)ic forms , and in ic forms . Reporte1 3′-UTR  were dev1 3′-UTR  containigulation . After igulation .6, but it is difficult to see how this could be possible since there is only a single RRM and no evidence for interaction with other RNA-binding proteins. We here further investigated the binding of RBP10 to RNA.The observations described above suggested that RBP10 binds specifically to the sequence UA(U)E. coli were transformed with the plasmids expressing proteins illustrated in Fig. 2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0) then resuspended in 5 ml binding buffer with 1 mM PMSF and protease inhibitor cocktail without EDTA. Lysozyme was added to a final concentration of 1 mg/ml. All subsequent steps were at 4 °C. The cells were sonicated, then the lysate was centrifuged at 13000g for 20 min. The supernatant was added to 500 µl nickel beads, and protein allowed to bind at 4 °C for 1.5 h with mixing. The beads were then transferred to an empty column, and washed 3 times with 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0. Elution was with 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0. Glycerol was added to a final concentration of 1%, and eluted samples were stored at − 70 °C. Sometimes proteins were dialyzed against 10 mM Tris (8.0), 50 mM NaCl and 10% glycerol, using a 6–7 kDa pore membrane.BL21 We first attempted to express and purify full-length RBP10 . 5% non-denaturing polyacrylamide gels (Tris-borate-EDTA) were pre-run at 85 V for 1 h in 0.5 × TBE before sample loading.RNA gel shifts were done as described in . To labeWe measured binding of the various RBP10 preparations with two probes: the 26mer that is required for stability of the mRNA encoding EP procyclin, and a mutant version that does not give regulation [We next tested various fragments Fig. A. The onCAT mRNAs with various 3′-UTRs. These were the EP1 wild-type 3′-UTR and a version lacking the regulatory 26mer; the PGKB 3′-UTR, which also has two copies of UA(U)6, and a deleted version [CAT that gave satisfactorily specific signals. Problems included the low expression levels of the reporter mRNAs with wild-type EP1 and PGKB 3′-UTRs relative to mutant versions or the ACT 3′-UTR, and RNA degradation.For in vivo pull-downs, we used bloodstream forms in which all RBP10 bore an N-terminal tandem affinity purification (TAP) tag at the N-terminus (TAP-RBP10). Since RBP10 is essential, and the cells grew normally, the tagged version must have been functional: otherwise, cells expressing only the tagged version would not have been viable. We used cells that had chloramphenicol acetyltransferase (CAT) reporters integrated into the alpha–beta tubulin locus, so were transcribed by RNA polymerase II. The reporters expressed  version ; and as  version . RNAs we version . AlthougWe here showed that an N-terminal segment of RBP10 that includes the 48 N-terminal residues, the RRM domain, and 85 further residues towards the C-terminus, could be produced as soluble protein, and could bind two pyrimidine-rich RNAs in vitro. The binding affinity was well within the range expected for a single RRM, but the purified proteins did not distinguish between a sequence that is active in developmental regulation in vivo, and one that is inactive. A major limitation was that we tested in vitro binding with only two RNA sequences, so we do not know whether RBP10 might also bind purine-rich RNA. Proteins with only 10 residues, or no residues, C-terminal to the RRM had at least tenfold lower affinities for the probes, suggesting that at least part of the region from residues 133–218 either directly contributes to RNA binding, or is important for RRM-domain folding.The results of TAP-RBP10 pull-downs from cells expressing CAT reporters were irreproducible. More conclusive results—to either confirm, or disprove, the sequence specificity of RBP10 mRNA binding—might be obtained by using an RNA polymerase I promoter to drive expression of the reporter mRNAs, and an open reading frame with reliable real-time PCR primers for quantitation.


Drosophila embryogenesis, the recruitment of RNA polymerase II precedes active gene transcription. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. In addition, the composition of the RNA polymerase II “paused” complexes associated with promoters at different developmental stages are described in detail. For this purpose, we performed ChIP-Seq analysis using antibodies for various modifications of RNA polymerase II  as well as for subunits of the NELF, DSIF, and PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that during metamorphosis, similar to mid-embryogenesis, the promoters were bound by RNA polymerase II in the “paused” state, preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in a “pause” state was phosphorylated at Ser5 and Ser2 of Pol II CTD and bound the NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the “paused” RNA polymerase II complex included Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex was phosphorylated at Ser5 of Pol II CTD, but not at Ser2. These results indicate that, during mid-embryogenesis, RNA polymerase II stalls in the “post-pause” state, being phosphorylated at Ser2 of Pol II CTD (after the stage of p-TEFb action). During metamorphosis, the “pause” mechanism is closer to classical promoter-proximal pausing and is characterized by a low level of Pol II CTD Ser2P.According to previous studies, during  The development of a multicellular organism occurs due to the timely activation of genes in cells of a certain type, which develop in the form of “transcriptional waves” ,2. The tDrosophila in particular) RNA polymerase II contains an additional stoichiometric, tightly interacting Gdown1/POLR2M subunit [Drosophila, are represented by 45 repeats, of which only two correspond exactly to the evolutionarily conservative consensus) [Drosophila has highly divergent CTD repeats, they are also actively phosphorylated at Ser2 and Ser5, which makes all of the above described information relevant [The RNA polymerase II enzymatic complex contains 12 subunits that are highly conserved from yeast to humans . Metazoa subunit . The pro subunit . Evolutinsensus) . An unphnsensus) . Phosphonsensus) ,9. Phospnsensus) . At all nsensus) . Recentlrelevant .The multistage nature of the transcription cycle allows its various stages to be used for regulatory purposes. A large portion of inactive Metazoan genes are in a state of transcriptional “pause” when RNA polymerase II is recruited to gene promoters but no productive transcription is observed . It has The most studied regulators of eukaryotic transcription “pause” are the NELF (Negative Elongation Factor) and DSIF (DRB Sensitivity Inducing Factor) complexes ,17,18,19Drosophila Brd4/Fs(1) is recruited to chromatin not only through interaction with acetylated histones, treatment of Drosophila cells with selective BET inhibitor JQ1 (specific to Brd4/Fs(1)h) leads to a decrease in Brd4/Fs(1)h binding, but not to the background level [During transcription activation, the NELF and DSIF subunits, as well as Ser2 CTD Rpb1, are phosphorylated by the p-TEFb complex (positive Transcription Elongation Factor), releasing the PrPP state . DSIF bend level . Drosophnd level ,35,36.Promoter proximal pausing is not the only means of post-initiation transcription regulation in Metazoans. Transcription can also be controlled by the poised RNA polymerase II when the enzyme is recruited to the promoter in an inactive state (RNA synthesis is not initiated) ,38. Thertup, sog, and eve patterning genes of early embryogenesis, carry “paused” RNA polymerase II at the promoters [Elongation control of RNA polymerase II is meant to be a way of coordinating various regulatory stimuli . It was romoters ,44. The romoters ,46. In aDrosophila embryogenesis, the presence of RNA polymerase II was found on gene promoters at the developmental stages preceding the stages of their active transcription. Interestingly, the authors of the study found pre-recruited RNA polymerase II not only in the tissue where transcription of these genes would be activated, but also in the other tissue where these genes would remain silent. This suggests a model in which these genes are induced in a tissue-specific manner by factors that release the “pause” of RNA polymerase II. This observation suggests that temporal stimuli (such as hormones) induce RNA polymerase II recruitment in both target and non-target tissues, while spatial stimuli (such as master regulators and morphogens) affect the transcriptional elongation. However, this assumption is preliminary and certainly needs further confirmation.The role of the RNA polymerase II “pause” in the coordination of developmental temporal signals was described in most detail by Gaertner and colleagues . In thisdhr3, hr4, eip78c, etc.), preparing them for the upcoming transcription during Drosophila metamorphosis [Drosophila development by analyzing nonspecific pools of genes that dynamically change their transcription. We obtained a new set of ChIP-Seq data at different stages of Drosophila development. These data include ChIP-Seqs of various forms of RNA polymerase II and regulators of transcription elongation NELF-E, Spt5, PAF1 , and Brd4/Fs(1)h. This allowed us to assess in detail the composition of the “paused” RNA polymerase II complexes formed on promoters at the stages of development preceding the stages of their active work.Recently, we demonstrated that RNA polymerase II and NELF are present on the promoters of ecdysone-induced genes . giant, snail, thisbe, and twist. For the analysis of the larvae and prepupae, we selected genes activated by ecdysone: early sgs3 and br and early–late dhr3 and hr4. The pattern of changes in the gene transcription in the analyzed tissues corresponded to the expected pattern (previously published) [For the ChIP-Seq analysis, we obtained the material at two time points in embryogenesis (at 2–4 h and at 6–8 h after egg laying (AEL)). We specifically decided to study early and middle embryogenesis in order not to move far from the point of synchronization, i.e., egg laying. In addition, important processes of embryogenesis occur at the following stages: at the 2–4 h stage, active transcription of the genome begins for the first time, and at the 6–8 h stage, specification and determination of cell fate occurs. This is why these embryonic stages are often chosen by researchers ,50. We ablished) . We concDrosophila developmental stage [To double-check the previously obtained information regarding the role of the RNA polymerase II “pause” in preparing the promoters for the upcoming transcription in embryogenesis, we started from the analysis of various pools of genes activated at this al stage .First, for the analysis, we selected a pool of genes whose transcription is induced at the 6–8 h AEL (after eggs laying) stage by at least five times relative to the 2–4 h AEL stage (“6–8 h genes”). The total number of selected genes was 420 (which corresponded to 1253 transcripts). The selection of this set of genes was performed using data previously published by Graveley and colleagues  . We testWe obtained the averaged binding profiles of RNA polymerase II isoforms and factors that control the elongation on the resulting set of “6–8 h genes” . Our ChIWe observed the presence of some RNA polymerase II and elongation regulators at the 2–4 h AEL stage preceding the transcriptional activation stage of the 6–8 h AEL gene pool. This was especially evident from the substantial enrichment of the promoters by NELF-E at the 2–4 h AEL stage. However, it did not appear that the genes induced at 6–8 h AEL actively used the “paused” RNA polymerase II to prepare the promoters for the upcoming transcription. It is also possible that the level of this “paused” RNA polymerase II was rather low and it could not be easily detected on averaged profiles. The level of RNA polymerase II on these promoters increased significantly during the transition from 2–4 h to 6–8 h AEL.To estimate the exact proportion of “6–8 h gene” promoters bound by RNA polymerase II at the 2–4 h stage, we performed a cluster analysis according to the amount of Rpb3 . This analysis demonstrated that 20.8% of the “6–8 h gene” promoters (corresponding to Cluster 1) possess RNA polymerase II before their activation at the 6–8 h AEL stage . It impoNext, we decided to test whether genes from the later stages of embryogenesis used the RNA polymerase II “pause” to prepare for the upcoming transcription. We selected genes whose transcription was induced at least five times at the 10–12 h AEL stage compared with the 6–8 h stage level (“10–12 h genes”) and another set of genes that were induced at the 14–16 h AEL stage at least five times compared with the 10–12 h AEL genes (“14–16 h genes”). The total number of selected “10–12 h genes” was 462 (which corresponded to 1188 transcripts) and 548 for the “14–16 h genes” set (which corresponded to 1379 transcripts). The quality of the selection was tested in the same way as was carried out for the pool of “6–8 h genes” by obtaining the averaged profiles of the RNA-Seq signal . The lisWe found the presence of a substantial amount of RNA polymerase II on the promoters of “10–12 h genes” at the 6–8 h AEL developmental stage . RNA polTo estimate the exact proportion of the “10–12 h genes” promoters bound by RNA polymerase II at the 6–8 h stage, we performed a cluster analysis according to the amount of Rpb3  . This anThe generation of averaged profiles in the region close to TSS revealed the presence of an additional peak of RNA polymerase II, phosphorylated for both Pol II CTD Ser2 and Pol II Ser5, within the distal part of the promoters, which cannot be correlated with the accumulation of any analyzed elongation control factors .To determine how much in advance, before transcription, developmental genes recruit RNA polymerase II to their promoters, we analyzed the pool of “14–16 h genes” in our datasets (lower panels of Drosophila gene in ChIP-Seqs at 2–4 h and 6–8 h provided a similar result: the level of Brd4/Fs(1)h binding decreased when moving from 2–4 h to 6–8 h AEL (Drosophila development when the Brd4/Fs(1)h regulator of elongation is not involved in gene transcription.At this point in the study, we already analyzed three different pools of genes . In all the analyzed pools, we observed a low level of Brd4/Fs(1)h binding to promoters in embryogenesis (with the exception of the presence of some Brd4/Fs(1)h on the promoters of the “6–8 h genes” at the 2–4 h AEL stage). The generation of the Brd4/Fs(1)h binding profile for the average –8 h AEL . We were–8 h AEL . We founDrosophila metamorphosis and performed ChIP-Seqs on homogeneously staged L3 PS1-3 full gut, white prepupa, and prepupa 10 h after puparium formation (APF). For analysis, we selected two pools of genes: genes induced in wandering larvae during the puparium formation, i.e., “WL genes” (wandering larvae (WL)), and genes activated 24 h after the formation of the white prepupa, i.e., “WPP +24 h genes” (white prepupa (WPP)). The total number of “WL genes” that increased their transcriptional level by at least five times at the L3 PS7-9 empty gut stage compared with the L3 PS 1-3 full gut stage was 303 (which corresponds to 852 transcripts). The total number of “WPP +24 h genes” that increased their transcriptional level by at least five times at the WPP +24 h stage compared with WPP + 12 h stage was 62 (which corresponds to 108 transcripts). The lists of selected regions corresponding to “WL genes” and “WPP +24 h genes” are provided as To explore whether the RNA polymerase II “pause” is used to prepare promoters for upcoming transcription at other developmental stages, we took advantage of First, we analyzed the state of RNA polymerase II at the promoters of the “WL genes” at different stages of metamorphosis . The proWith active transcription of the “WL genes”, we observed a significant increase in binding of the DSIF and PAF complexes and Brd4/Fs(1)h in the gene bodies. Certainly, these regulators are closely associated with RNA polymerase II not only in the promoter but also during its movement in the gene bodies. This result is in complete agreement with the previously described behavior of these complexes during active transcription ,52,53,54To estimate the exact proportion of the “WL gene” promoters bound by RNA polymerase II at the L3 PS1-3 full gut stage, we performed a cluster analysis according to the amount of Rpb3  . This anDrosophila uses the RNA polymerase II “pause” to prepare promoters for transcription at previous stages of development, but not too much in advance.As in the study of embryogenesis, we decided to look at whether RNA polymerase II is recruited to genes that are activated later in development than “WL genes”. We analyzed the obtained ChIP-Seqs on the pool of genes activated 24 h after puparium formation, i.e., “WPP +24 h” . To control our selection scheme, we decided to analyze a pool of genes selected according to a completely different principle. We chose a pool of genes that were repeatedly activated in development, being induced by the 20-hydroxyecdysone hormone (ecdysone). Since we obtained the ChIP-Seqs for the material of whole embryos, larvae, and prepupae (but not for tissues), we chose a pool of ecdysone-inducible genes from the study characterizing the ecdysone response in many cell lines at once  . The lisFirst, we checked at what developmental stages a selected pool of ecdysone-inducible genes is activated . As expeDuring metamorphosis, the pool of ecdysone-inducible genes was found to be regulated in the same way as the general pool of genes activated at the WL stage. We observed the presence of RNA polymerase II on the promoters of ecdysone-inducible genes up to the stage of their active transcription, and its level did not increase upon activation. We detected an increase only in the degree of RNA polymerase II phosphorylation at Ser2 Rpb1 CTD . That isDrosophila uses the RNA polymerase II “pause” to prepare promoters for active transcription at the next stage of development. The main purpose was to determine whether the “pause” is involved in the preparation of genes for transcription at various stages of development. Drosophila development provides a very convenient opportunity for this by allowing us to obtain material that is highly synchronized in terms of developmental stages, not only during embryogenesis but also during the metamorphosis phase.This study aimed to fill a gap in the knowledge regarding how Analyzing the pools of “6–8 h genes”, “10–12 h genes”, and “WL genes” activating during mid-embryogenesis and metamorphosis, we observed RNA polymerase II binding to promoters at the stages preceding the stages of their active transcription . The comEverything described above regarding the RNA polymerase II “pause” both in embryogenesis and metamorphosis is related to the RNA polymerase II peak formed in the proximal part of the promoters. However, a close inspection of the distribution of RNA polymerase II across TSS regions revealed that the “paused” promoters, both at 6–8 h of embryogenesis and at the L3 PS 1-3 full gut stage of metamorphosis, had an additional downstream peak of RNA polymerase II and its isoforms Pol II CTD Ser5P and Pol II CTD Ser2P . We founThe performed cluster analysis showed that most of the promoters were not associated with RNA polymerase II before their activation, and our conclusions are valid only for some of the genes preparing for transcription. We attribute this to the limitations of ChIP-Seq. Because we analyzed the entire embryo and larva, we were not able to detect tissue-specific binding events. Single-cell techniques may help to overcome this problem, and the implementation of such techniques appears to be a good development for the current work.Drosophila development that we analyzed, the recruitment of elongation regulators to the promoters occurred together with the recruitment of RNA polymerase II.It would seem that this is a natural conclusion, since elongation regulators directly interact with RNA polymerase II, but this is not very obvious. The process of elongation regulators recruitment to RNA polymerase II is very unclear and it may well be a multistage process. Additionally, the step of this multistage process may well be the recruitment of elongation regulators onto chromatin through interaction with DNA-binding proteins, and not directly with RNA polymerase II. With this recruitment mechanism, even in the absence of RNA polymerase II, we would detect binding of some elongation regulators to promoters due to their recruitment by DNA-binding proteins. However, in all the analyzed pools of genes that did not contain RNA polymerase II on the promoters (the pools of 14–16 h of embryogenesis and WPP +24 h), we did not observe the binding of elongation regulators with the promoters. Moreover, the cluster analysis showed that all the analyzed elongation regulators fell into the cluster of genes containing RNA polymerase on the promoters. That is, at least at the stages of It is worth noting that, in previous studies, we detected the binding of elongation regulators with DNA in the absence of RNA polymerase II. In our recent works, we described that the NELF-A subunit of the NELF complex is able to bind not only promoters but also enhancers and PRE elements containing a relatively low level of RNA polymerase II ,48. The Drosophila Brd4/Fs(1)h protein was previously found to be present not only in promoters and enhancers but also in sites enriched in architectural proteins, mostly not associated with RNA polymerase II [The erase II . That isIt seems that some elongation regulators can indeed be recruited by DNA-binding proteins as a preliminary step in their binding to RNA polymerase II; however, judging by the data of this article, this does not occur on promoters.Drosophila development, genes prepare in advance for the upcoming transcription by pausing the RNA polymerase II at their promoters. It is assumed that productive transcription of these genes at the appropriate stage is achieved by resolving this “pause”. In the case of promoter-proximal pausing, this is the recruitment of the p-TEFb complex to promoters or its activation if it is pre-recruited in an inactive HEXIM-suppressed state [Our data suggest that, during ed state . In the ed state .A not entirely clear but interesting question concerns how the increase in the concentration of “pause-releasing” complexes on the targeted promoters is achieved. Is it gene-specific, as in the case with heat shock genes activated by recruitment of HSF1, which stimulates elongation ? Or can The most advanced works in this area, namely, the control of gene transcription through the intracellular level of coregulators, refers to genes controlled by poised Pol II and released by TFIIH complex. Some time ago, it was demonstrated that a change in the concentration of TFIIH  is controlled by the level of glucose . More reDrosophila’s rapid development is a convenient experimental model for this goal.The transcription of developmental genes is under the control of a variety of regulatory systems that control the timing and specificity of transcription in a particular tissue as well as under the influence of master regulator proteins that control transcription in a particular part of the body. It takes time to implement and coordinate all these stimuli. Not surprisingly, developmental genes control their transcription by controlling productive elongation. This approach helps to form a transcriptional hub in the promoter region and ensures the specificity of all the necessary interactions with RNA polymerase II and GTFs. The study of the dynamics of such hubs in development can help us to better understand the mechanisms of transcription regulation in general. Drosophila), only flies aged 3–5 days after eclosion were used to collect embryos.The flies of Oregon-R-modENCODE stock were used (corresponds to Bloomington stock #25211). Embryos were collected in the fly cages for 2 h using apple juice agar plates. Then plates were incubated at the 25 °C for the required period of time. The time after egg laying (AEL) was calculated starting from the moment the agar plates were placed in the fly cages. To reduce an effect of the retention of embryos inside flies  were collected according to description of this developmental stage: white motionless prepupae starting to evert their anterior spiracles. Prepupae corresponding to later developmental stages were collected at the stage of white prepupa and then incubated at the 25 °C for the required period of time.2, 15 mM HEPES pH7.6, 0,5% Triton X-100) containing 0.7% of formaldehyde for 10 min and incubated for 5 min with 125 mM Glycine. Then cells were washed for three times with homogenization buffer. The remaining ChIP protocol was performed as described previously [For the ChIP-Seq experiments embryos were dechorionized, washed and homogenized in a buffer . Only the library fragments of 350–500 bp were subjected to NGS sequencing. Next generation sequencing was performed by Evrogen (evrogen.ru) with the Illumina NovaSeq6000 sequencer. For each of the ChIP-Seq libraries approximately 3–8 millions of unique paired-end reads were obtained. The paired-end reads in FastQ format were mapped to the Drosophila genome assembly dm6 using HISAT2 [The chromatin immunoprecipitation (ChIP) was performed and analyzed exactly as previously described ,61. ChIPg HISAT2  and filtg HISAT2 . BigWig g HISAT2 . The fing HISAT2 . The ChIDrosophila development were selected using information from Supplementary Table 9 of previously published study  [Sets of genes induced at the specific stages of  stages) . For exaThe set of genes induced in wandering larvae “WL genes” was selected by comparing transcriptional levels of genes at L3 PS7-9 empty gut and L3 PS1-3 full gut stages. We had to use L3 PS7-9 empty gut instead of white prepupa 0 h stage as the latter was absent in analysis performed by Graveley et al. We consider this substitution to be insignificant since the L3 PS7-9 empty gut and the white prepupa 0 h stages are separated by only a few hours and represent a common transcriptional induction wave that is primarily a response to ecdysone. Our data on the distribution of various forms of RNA polymerase II on this gene set confirms this conclusion, we saw a substantial increase in the phosphorylation level of Ser2 Rpb1 CTD at these genes during the transition between the L3 PS1-3 full gut and white prepupa stages, indicating the active work of this gene set at the stage of white prepupa.Drosophila developmental transcriptome [To verify our selection strategy, we provided averaged profiles of RNA-Seqs at the selected pools of genes in each figure. We used raw RNA-Seq data obtained from ModENCODE web-server, which were previously used by Graveley and colleagues for the analysis of criptome ,64. We dThe Galaxy-P platform  was used for analysis of ChIP-Seq data . All obthsp70 gene induced by heat shock [Rabbit polyclonal antibodies against Rpb3 (1–275 aa), Spt5 (917–1117 aa), NELF E (2–279 aa), PAF1 (1–234 aa), Fs(1)h/Brd4 (120–386 aa) were obtained and described previously in our lab ,66. All at shock . AntibodAntibodies against RNA polymerase II CTD repeat (phospho S2) (Ab5095), and RNA polymerase II CTD repeat (phospho S5) (Ab5131) were purchased in Abcam .g for 20 min. Then supplemented with SDS loading buffer containing DTT and loaded on a polyacrylamide gel.To obtain protein extracts for Western blotting of various embryogenesis stages, embryos were collected using apple juice agar plates in the same way as was performed for the collection of ChIP-Seq material (the only difference is 1h-increment between different time points). For Western blotting embryos were dechorionized, washed, and homogenized by triple sonication in a RIPA buffer . Protein extracts were treated with DNAse I at 37 °C for 15 min and centrifuged at 16,000×