By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of  The majority of the eukaryotic genome is transcribed into coding and noncoding RNAs (ncRNAs). ncRNAs fulfill various functions both in the cytoplasm and the cell nucleus. Nuclear ncRNAs are attracted to different genomic regions and mediate the activation or repression of genes located in these regions and are also implicated in the genome three-dimensional organization , 2. In pHelpful in studying nuclear functions of ncRNAs are methods that map the sites of ncRNA associations with the genome. Initially developed for probing genomic interactions of one particular RNA, these methods are now available in an “all-vs.-all” version allowing simultaneous detection of the sites of chromosomal locations for all RNA molecules present in the nucleus  and compared the number of cis contacts between EZH2-precipitated and input fractions. We found that the degree of enrichment in the IP fraction correlated with the percentage of contacts detected in Polycomb-specific and poised promoter-specific chromatin types but not any other chromatin types in the area under study  for each RNA between EZH2-precipitated and input fractions and looked for RNAs showing an elevated number of contacts in the IP fraction. The highest enrichment was observed for antisense RNA KIF5C-AS1, snRNA RNU5B-1, and SNORD3a RNA  experiments H. ImportCollectively, the present study results demonstrate the utility of the RedChIP protocol for identifying RNAs that may target nonhistone proteins to various locations on chromosomes or mediate interactions of these proteins with DNA. The identification of RNAs that are known to target Polycomb complexes to repressed genomic domains strongly supports the validity of the experimental approach, whereas identifying a set of RNAs possessing similar characteristics will stimulate studies of their possible role in Polycomb and CTCF functioning. The RedChIP technique can be used for identifying RNAs associated with genomic regions occupied by any protein of interest.SI Appendix. For sample processing statistics, refer to Dataset S1. For read processing statistics, refer to Dataset S2.Cells are fixed with formaldehyde, DNA is fragmented with NlaIII restriction enzyme, and the ends are blunted and A-tailed. RNA 3′ ends are ligated to a biotinylated bridge adapter followed by ligation of the opposite ends of the bridges with DNA ends in spatial proximity. Ligated complexes are solubilized by sonication, immunoprecipitated, and washed. RNA–DNA chimeras are purified, and DNA is digested with MmeI restriction enzyme. After biotin pull-down, reverse transcription is initiated from the bridge with template switching at the RNA 5′ end, allowing for the incorporation of an Illumina adapter. Another Illumina adapter is ligated to DNA ends, and the chimeras are amplified and paired-end sequenced. The detailed protocol and sequencing data processing are described in Supplementary FileSupplementary FileSupplementary File


Defining the physiological features of these “extreme” corals, as well as their relationships with the, often symbiotic, organisms within their microbiome, could increase our understanding of how corals will persist into the future. To better understand coral-microbe relationships that potentially underpin coral persistence within extreme mangrove environments, we therefore conducted a 9-month reciprocal transplant experiment, whereby specimens of the coral Pocillopora acuta were transplanted between adjacent mangrove and reef sites on the northern Great Barrier Reef. Bacterial communities associated with P. acuta specimens native to the reef environment were dominated by Endozoicomonas, while Symbiodiniaceae communities were dominated by members of the Cladocopium genus. In contrast, P. acuta colonies native to the mangrove site exhibited highly diverse bacterial communities with no dominating members, and Symbiodiniaceae communities dominated by Durusdinium. All corals survived for 9 months after being transplanted from reef-to-mangrove, mangrove-to-reef environments (as well as control within environment transplants), and during this time there were significant changes in the bacterial communities, but not in the Symbiodiniaceae communities or their photo-physiological functioning. In reef-to-mangrove transplanted corals, there were varied, but sometimes rapid shifts in the associated bacterial communities, including a loss of “core” bacterial members after 9 months where coral bacterial communities began to resemble those of the native mangrove corals. Bacterial communities associated with mangrove-to-reef P. acuta colonies also changed from their original composition, but remained different to the native reef corals. Our data demonstrates that P. acuta associated bacterial communities are strongly influenced by changes in environmental conditions, whereas Symbiodiniaceae associated communities remain highly stable.It has been proposed that an effective approach for predicting whether and how reef-forming corals persist under future climate change is to examine populations thriving in present day extreme environments, such as mangrove lagoons, where water temperatures can exceed those of reef environments by more than 3°C, pH levels are more acidic  and O Given previous observations of rapid adjustments in bacterial assemblages , and salinity (15.5) , were selected for our reciprocal transplant experiment: a mangrove lagoon (Woody Isles) and an adjacent reef crest-slope (Low Isles reef) . The Woo5.5) see . In comp2, and pH) were tested using independent samples t tests with Welch’s corrections to test for any differences between reef and mangrove environments (GraphPad Prism v.7).To characterise daily and seasonal temperature shifts, we deployed HOBO Pendant data loggers  at both sites , which recorded data for the 9-month duration of the experiment. In addition, spot measurements were periodically taken for temperature, salinity and oxygen using a multi-meter  and pH using a pH meter . Environmental data series  and <1 m  on the lowest astronomical tide to ensure consistency across the two sites. All colonies were subsampled to yield a partial colony fragment, which was then split in half (ca. 10 cm diameter). The two resulting fragments subsequently served as either a native (remaining within site) or non-native (transplanted between sites) specimen, where colonies were either retained within their native mangrove (mangrove-mangrove), or reef (reef-reef), or were moved from the mangrove to the reef (mangrove-reef), or reef to mangrove (reef-mangrove). All split colonies were attached onto one of five aluminium racks with cable ties within each location, such that each rack contained either a replicate mangrove-mangrove or reef-mangrove coral colony within the mangrove lagoon, or replicate reef-reef or mangrove-reef coral colony on the reef . Two small fragments (<5 cm each) of P. acuta were collected from each of the 20 split colonies at the start of the transplantation  for characterisation of the Symbiodiniaceae and bacterial communities, and Symbiodiniaceae photo-physiology via Pulse Amplitude Modulation (PAM) fluorometry. All fragments were collected using sterile pliers and placed in zip lock bags filled with seawater from the respective location before being returned to the sampling vessel within 30 min for immediate processing. One fragment was used for microbiome characterisation and rinsed with sterile artificial seawater before immediate flash freezing in liquid N2, while the second fragment was analysed using PAM fluorometry. Further samples were collected using this approach three days after transplantation (t3d) to determine any immediate transplant effects on the coral microbiome or photophysiology. The experiment continued with samples collected 2 months (t2M), 3 months (t3M), 6 months (t6M), and 9 months (t9M) after transplantation, which were all immediately processed for microbiome and PAM fluorometry analysis.Five colonies of –2 s–1) for 20 min and then exposed to an increasing light gradient of ca. 0, 180, 210, 360, 450, 670, 1070, 1550, and 1980 μmol photons m–2 s–1. Minimum  and maximum  fluorescence yields were obtained to calculate the excitation energy dissipation terms [1-C] and [1-Q], which are the photochemical and dynamic non-photochemical coefficients, respectively (see t tests (GraphPad Prism v.7).Photophysiology was characterised using a diving PAM set to conduct a fluorescence-light response curve as previously detailed e.g., , where tvely see . To asseg for 10 min, the supernatant was discarded, and pellets were frozen at −80°C prior to subsequent analysis. Pellets were later resuspended in 0.5 mL of extraction buffer  with the addition of 75 μL of Lysozyme (100 mg/ml stock), incubated at 37°C for 1 h and shaken every 15 min, followed by three freeze/thaw cycles in liquid N2 and incubation at 70°C on a heat block. Sodium dodecyl sulphate  was added to samples and then incubated at 70°C for 10 min. Samples were cooled to room temperature before adding proteinase K (20 μL of 20 mg/mL stock) and further incubated for 1 h at 37°C, followed by three additional freeze/thaw cycles. Samples were then added to an equal volume of phenol:chloroform:isoamyl alcohol , mixed by inversion for ten min at room temperature, followed by centrifugation for 15 min at 13,000 g. For each sample, the aqueous phase containing the DNA was transferred into a clean tube and equal volumes of chloroform:isoamyl alcohol (24:1) was added. Samples were mixed by inversion for 10 min and centrifuged for 10 min at 16,000 g. The aqueous layer was transferred to a new tube and mixed with sodium acetate (50 μl of 3M solution), then an equal volume of ice-cold molecular-grade isopropanol was added to precipitate the extracted DNA. Tubes were then centrifuged at 20,000 g for 30 min at 4°C and the supernatant was discarded. The DNA pellets were washed with 500 μL of molecular grade ethanol (70%) and centrifuged for 10 min 20,000 g. Ethanol was removed and samples were air dried for 15 min in the dark, before DNA pellets were resuspended in 30 μL of nuclease free water.Total genomic DNA (gDNA) was extracted using a modified phenol-chloroform protocol. Coral tissue was initially removed from frozen fragments by air-blasting tissue into zip lock bags containing 5 ml of sterile artificial seawater. Coral slurry was then centrifuged at 8,000 Pocillopora from NCBI. All sequences matched with reference sequences for P. acuta  and Pdam-R (5′-CGCCTCCTCTACCAAGACAG-′3) primers, which provide species delineation in pocilloporids . The PCRP. acuta  and confP. acuta, the V3-V4 region of the 16S rRNA gene was amplified using the 341F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CCTAYGGGRBGCASCAG-′3) and 805R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNNGGGTAT CTAAT-′3) primers  were carried out using Bray-Curtis dissimilarity matrices. Differences in alpha diversity and community structure (beta diversity) were analysed using permutational multivariate analysis of variance (PERMANOVA) on square-root transformed data in PRIMER-E + PERMANOVA package v1.0.6. One-factorial PERMANOVA was run with 999 permutations to test for differences between sites (mangrove vs reef). Two-factorial PERMANOVAs were run with 999 permutations to test for differences between sites at different timepoints, and differences within sites between timepoints (fixed factors time and site). To identify differentially abundant bacterial taxa across sites and between timepoints, we used MetagenomeSeq  at the AP. acuta colonies was assessed at t0 (n = 19) and t9M (n = 13) to characterise any changes over time. Specifically, the ITS2 region of the Symbiodiniaceae communities associated with experimental coral colonies was targeted using the ITSintfor2 (5′- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAA TTGCAGAACTCCGTG-′3) and ITS2-reverse (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGATCCATATGC TTAAGTTCAGCGGGT-′3) primer pairs  than for the reef (26°C). Similarly, peak temperature (15:00) was on average 1.5°C higher (p < 0.001) in the mangrove (28.2°C) compared to the reef (26.7°) , pH, salinity and oxygen were lower in the mangrove  compared to the reef  (p < 0.0001)  temperature variation for the mangroves (28°C) was ∼2°C higher ( (26.7°) . Over ea 0.0001) .P. acuta exhibited site-specific ITS2 profiles , while reef corals were dominated by members of the Cladocopium genus , whereby each genus represented >95% of the ITS2 relative abundance at each site. Notably, transplantation to adjacent sites had no effect on coral associated Symbiodiniaceae communities, which remained stable within transplanted corals (9M), ITS2 profiles of the transplants (either within or between sites) were indistinguishable from those at the beginning of the experiment (t0) . In contrast, reef populations exhibited no significant change in quenching dynamics through time, a pattern that was conserved whether colonies were located in native (reef-reef) or non-native (reef-mangrove) habitats  and 3 negative controls were characterised using 16S rRNA amplicon sequencing, which yielded 10,745,579 sequences . Therefore, we further analysed these differences at the family level using metagenomeSeq. Overall, we identified 60 differentially abundant bacterial families between the two sites  and Desulfocapsaceae (2.5% ± 0.3) were relatively more abundant in the mangrove corals at t0  resulted in no significant changes in alpha diversity over time , where these samples as well as those from subsequent time points, became highly dissimilar compared to t0 samples  of transplantation  (0).Coral-associated bacterial communities in corals transplanted from mangrove to reef (mangrove-reef) were significantly altered after only three days  , specifi, 0–t9M) , 7. Each, 0–t9M) . This pa9M , which was characterised partly by the loss of Endozoicomonas ASVs . The bacterial assemblages associated with the corals transplanted from the reef to mangrove (reef-mangrove) became indistinguishable to those of the native mangrove corals (mangrove-mangrove) by t9M nMDS; . Additionas ASVs . In factthe reef .P. acuta strongly respond to changing environmental conditions, but the pressure exerted on the bacterial community is greater in mangrove environments.Coral microbiomes are sensitive to environmental stressors and can dramatically differ across colonies of the same coral species inhabiting dissimilar environments . Within P. acuta between the reef and mangrove environments, which is in line with previous observations at this field site  when the various environmental conditions between the two sites were more similar than towards the end of the experiment in summer   , suggestP. acuta colonies inhabiting mangrove and reef environments. Such difference among sites is consistent with previous studies that have identified highly divergent bacterial communities in P. acuta over small spatial scales  in previous P. acuta studies . This is consistent with previous reports from corals in mangroves and adjacent reefs in New Caledonia , coral associated bacterial communities in the native transplants (reef-reef) underwent very little change in composition, suggesting there had been little to no effect of transplantation on the bacterial communities associated with these corals. In contrast, the bacterial communities associated with the mangrove-mangrove corals differed significantly over all six timepoints sampled, highlighting a dynamic relationship between bacterial communities and the environment.After 9 months of transplantation  remain relatively stable even when subjected to dramatic changes in environmental conditions, including temperature or pollution  underwent strong shifts over the 9-month duration of the experiment. This pattern is in contrast to previous studies, that have shown that the bacterial communities associated with ollution . ChangesP. acuta associated bacterial communities. Coral microbiomes can be altered by shifting environmental conditions such as nutrient availability (Pocillopora) are more influenced by the external environment compared to those with thicker tissue , suggesting that a decrease in pH could even result in the loss of Endozoicomonas (3M), when we observed a significant shift in the bacterial communities of Reef-mangrove colonies from their original state, the pH in the mangrove environment (pH 7.8) was 0.3 units lower than in the reef environment (pH 8.1). The acidic conditions of the mangrove environment are likely responsible for the loss of other coral-associated bacteria among reef corals.Changes in bacterial communities in reef-mangrove corals after transplantation were largely characterised by a rapid decrease in relative abundance, and potential loss, of robiomes , 2017. Ol stress . While aes in pH . One stuicomonas . FollowiAcropora hyacinthus) and polluted sites (in Acropora hemprichii)  , 2019, tprichii) , wherebyprichii) , which mP. acuta in mangrove lagoon and adjacent reef, and characterised the effect of the surrounding environment on the structure of the microbiome by transplanting corals between the sites. We show that corals living in extreme mangrove environments support highly diverse and transient bacterial communities, which could be beneficial and allow them to cope under environmental variability. In comparison, reef corals maintained more consistent bacterial communities with highly dominant members. Transplantation of corals induced major shifts in bacterial composition, but had little to no effect on the Symbiodiniaceae communities. Our results reveal that coral-associated Symbiodiniaceae communities are initially shaped by divergent environmental conditions, but appeared unaffected by longer term (9 month) changes to these conditions. Bacterial communities changed rapidly following transplantation, suggesting strong selective pressure from the environment. The dynamic nature of the bacterial communities in resident mangrove corals reflects that of the microbiomes of corals persisting in other extreme environments – such as A. hemprichii, A. hyacinthus, and P. verrucosa – which have exhibited acclimation and adaptation potential  communities associated with otential , 2019. TPRJNA764039.Raw fastq read files were deposited in the NCBI Sequence Read Archive (SRA), accession number: DS and EC designed the experiments. EC, TH, DS, and JE collected and processed the samples. TH performed all laboratory work and data analysis. TH and J-BR produced the figures. TH, JS, J-BR, DS, and EC wrote the manuscript. All authors edited 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


One common genetic alteration in cancer is gene fusion resulting from chromosomal translocations. The mechanisms that create such oncogenic fusion genes are not well understood. Previously, we provided the direct evidence that expression of a designed chimeric RNA can drive the formation of TMPRSS2-ERG gene fusion. Central to this RNA-mediated gene fusion mechanism is a proposed three-way junction formed by RNA/DNA hybrid and the intergenic DNA stem formed by target genes. In this study, we determined the important parameters for chimeric RNA-mediated gene fusion using TMPRSS2-ERG fusion gene as the model. Our results indicate that both the chimeric RNA lengths and the sizes of unpaired bulges play important roles in inducing TMPRSS2-ERG gene fusion. The optimal length of unpaired bulges was about 35 nt, while the optimal chimeric RNA length was about 50 nt for targeting. These observations were consistent regardless of the target locations within TMPRSS2 and ERG genes. These empirically determined parameters provide important insight for searching cellular RNAs that may initiate oncogenic fusion genes. The knowledge could also facilitate the development of useful genomic technology for manipulating mammalian genomes. Gene fusion is one of the most important chromosomal alterations in cancer . In prosThese results, derived from two independent cases of RNA-driven gene fusion, support a model where the chimeric RNA sequence invades the target genes to stabilize a transient RNA/DNA duplex reminiscent of R-loops ,13,14,15A second fundamental observation derived from our previous studies is that, in addition to the RNA/DNA duplex, an intergenic DNA stem that could be formed by the genomic TMPRSS2 sequence paired with the genomic ERG sequence, may play a role in RNA-mediated TMPRSS2-ERG gene fusion  Figure A. This oA three-way junction is a structural motif commonly found in naturally occurring RNA molecules such as ribosomal RNAs, which contain junction structures ranging from three-way to seven-way junctions, and most of which include stretches of unpaired nucleotides called “bulges” at the branch point ,17. ThesIn this report, we present the empirical data that determines the important parameters—the optimal chimeric RNA length for forming RNA/DNA duplex, and the optimal bulge size for efficient RNA-mediated gene fusion. Studying these parameters is important, as they provide the design principles for developing new technology for manipulating the mammalian genome through large-distance DNA rearrangements. Secondly, these parameters provide valuable bioinformatics guidelines for searching for cellular chimeric-like RNAs that have the potential of inducing oncogenic fusion genes. Lastly, this knowledge could facilitate the development of therapeutic strategies to inhibit the formation of RNA/DNA duplexes and three-way junctions, therefore preventing the formation of cancer fusion genes and future tumors.2 humidified incubator.LNCaP cells are epithelial cells derived from a human prostate carcinoma. For transient transfection experiments, LNCaP cells were routinely cultured in RPMI 1640 medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin  in a 5% COThe chimeric RNAs were expressed using an expression plasmid with a human U6 promoter, a pol-III promoter. The human U6 promoter was constructed by cloning the region (−718 to +40) of the human U6 gene ‘RNU6-1′ from the genomic DNA of HEK-293T cells. This genomic segment contains a sequence upstream of the U6 promoter, the transcription start (+1), and a 40-nt stem loop cap sequence, followed by added multiple cloning sites (Pst-I and Hind-III). The chimeric RNA sequences were designed using target intron sequences of ERG and TMPRSS2 in antisense orientation see . They we5 cells/well and 1 mL/well of culture medium, as described above. Transfection was performed using Turbofect transfection reagent  according to manufacturer’s protocol. Briefly, 1 µg of a particular plasmid was first diluted in 100 µL of the serum-free DMEM followed by immediate mixing by pipetting. Then, 4 µL of the transfection reagent was added to the diluted DNA followed by mixing and incubation for 20 min. The DNA/transfection reagent mixture was then added drop-wise to a well containing LNCaP cells in 1 mL medium. Cells were then incubated in a CO2 humidified incubator at 37 °C for 72 h for the expression of the chimeric RNAs.Twenty hours prior to transfection, LNCaP cells were seeded in 12-well plate  with a density of 5 × 10DHT (dihydrotestosterone) was purchased from Sigma Aldrich . Concentrated stock of 1500 µM was prepared by dissolving 4.3566 mg of DHT powder in 10 mL of 100% ethanol  and then aliquoted in 1 mL tubes and stored at −80 °C. For treating cultured cells, concentrated DHT stock was diluted as 10× working solutions  with the appropriate complete culture medium and used immediately.g for 10 min at 4 °C to separate the mixture into a lower organic phase; an interphase; and an upper, aqueous phase. RNA remained in the aqueous phase while DNA and proteins were in the interphase and organic phase. Then, 400 µL of the upper aqueous phase was extracted in a new tube and 200 µL of 100% ethanol was added followed by immediate vortexing at maximum speed for 5 s to avoid RNA precipitation. The sample was then passed through the filter assembly resulting in the binding of the nucleic acids to the filter. The column was then rinsed twice with wash buffer and total RNA was then eluted in a new tube for further analysis. For detection of residual genomic and plasmid DNA, eluted RNA was subject to PCR reaction with primers specific to intron regions of the house-keeping gene GAPDH, and with primers specific to the transfected plasmid. Total RNA was converted to cDNA only if it is validated as free of DNA contamination.Total RNA from cultured cells was extracted using a Ribopure Kit according to the manufacturer’s instructions . Briefly, cells were homogenized/lysed in 1 mL TRI reagent followed by 5 min incubation at room temperature. This incubation allowed nucleoprotein complexes to dissociate completely. Then, 200 µL of chloroform was added followed by vortexing at maximum speed for 15 s. The mixture was then incubated at room temperature for 10 min. The lysate was then centrifuged at 12,000× Next, 1 µg of total RNA was used for each reverse transcription reaction according to manufacturer’s instruction . RNA was converted to cDNA with oligo dT primer. After the addition of dNTPs, the mixture was denatured at 65 °C for 5 min. This was followed by the addition of a master-mix containing 1× superscript buffer, 10 mM DTT, 5 mM magnesium chloride, RNaseOUT, and SuperScript III reverse transcriptase. Reactions were carried out at 50 °C for 50 min and then terminated by incubation at 85 °C for 5 min. cDNA was then treated with RNase-H for 20 min at 37 °C to degrade RNA in the DNA/RNA hybrid and 1 µL of cDNA was used as template for each subsequent PCR reaction.The induced TMPRSS2-ERG fusion RNA and control GAPDH RNA were detected using one-round RT-PCR. PCR was done with a standard three-step protocol using RED-Taq DNA polymerase  according to the manufacturer’s instruction. The annealing temperature for both TMPRRS2-ERG and GAPDH primers was 57 °C. RT-PCR primers for amplifying induced fusion RNA TMPRSS2-ERG were TMPRSS2 ex-1 F1: 5′-TAGGCGCGAGCTAAGCAGGAG-3′ and ERG ex-4 R1: 5′-CTTGAGCCATTCACCTGGCTAG-3′. RT-PCR primers for amplifying the GAPDH RNA were GAPDH F1: 5′-GCGTCTTCACCACCATGGAGA-3′ and GAPDH R1: 5′-AGCCTTGGCAGCGCCAGTAGA-3′.Tm was calculated using the formula: Tm = 64.9 + 41 * (nG + nC − 16.4)/(nA + nT + nG + nC) where “n” stands for total number of particular nucleotide.Each RT-PCR experiment was repeated three times. GAPDH was used as internal control for the amount of RNA loaded. The band intensities were quantified using ImageJ 1.5.3. In brief, band intensity was calculated for each individual band using ImageJ. Background intensity was subtracted using the averaged background noise obtained from two areas—one from above and one below the band. The corrected band intensity was then normalized to the band intensity of GAPDH. Experiments were repeated thrice and the mean and standard deviation were calculated and presented using Microsoft Office 2016 Excel.Previously we demonstrated that the expression of a designer chimeric RNA can lead to the induction of TMPRSS2-ERG gene fusion in prostate cells . Among aTo determine the optimal bulge size, we designed three series of antisense chimeric RNAs, with each series targeting locations B, C, or D. Each series of chimeric RNAs was designed to test ten different bulge sizes: 2, 16, 20, 25, 31, 35, 40, 45, 50 and 100 nt while maintaining the same intergenic DNA stem. To create different bulge sizes, the chimeric RNAs in the same series employed slightly different target regions so that the appropriate bulge size could be created between the intergenic DNA stem and the RNA/DNA duplex . We keptWe transiently expressed the chimeric RNAs in LNCaP cells by transfection, then treated the cells with DHT for three days. If the expression of the chimeric RNA led to a TMPRSS2-ERG gene fusion, it was expected that the endogenous full-length fusion RNAs would be transcribed from the newly induced TMPRSS2-ERG fusion gene. As we have pointed out in our previous study , the indAs shown in After the effective bulge size was determined, we then set out to determine the lengths of chimeric RNA which contributed directly to the stability of the transient RNA/DNA duplex required for gene fusion induction. To determine the optimal RNA length, we designed three series of antisense chimeric RNAs with each series targeting locations B, C, and D. Each series tested four different RNA lengths: 30/30, 50/50, 75/75, and 100/100 nt. For example, a chimeric RNA with a length of 30/30 nt contains 30 nt complementary to ERG gene sequence followed by 30 nt complementary to TMPRSS2 gene sequence. To create different chimeric RNA lengths targeting each specific location, our designs maintained the same intergenic DNA stem, fixed the bulge size at 35 nt, and then employed progressively longer target sequences until the appropriate RNA lengths are reached see B, the chAs shown in Previously we reported an unappreciated RNA-driven mechanism in which the expression of a designer chimeric RNA induce specified gene fusions in mammalian cells ,7. The pThree-way junctions have long been proposed to play important roles in many biological mechanisms by stabilizing nucleic acid interactions ,21,22. INaturally occurring RNA molecules such as ribosomal RNAs contain numerous three-way junctions, most of which include stretches of unpaired nucleotides called “bulges” at the branch point ,17. Thes5 to 106 [The lengths of chimeric RNA contribute directly to the stability of the transient RNA/DNA duplex. Our results show that chimeric RNA with a length of 50/50 nt is optimal in inducing TMPRSS2-ERG regardless of the target locations within TMPRSS2 and ERG genes. The intensity of induced the TMPRSS2-ERG fusion transcript gradually tapered off when the length was reduced to 30/30 nt or increased to 100/100 nt. This raises the question of why longer RNAs such as 100/100 nt are less efficient for RNA-mediated gene fusion as they should increase the stability of RNA/DNA duplex. Past studies of microarray technologies indicated that hybridization of DNA oligos to rRNA, which also forms an RNA/DNA duplex, is mainly affected by the secondary structures of the RNA molecules. The presence of secondary structures in RNA can reduce the binding of a DNA oligo by a factor of 105 to 106 ,25. In aA second intriguing question is whether a short chimeric RNA of 30/30-nt is sufficient to specify two parental genes for gene fusion. The well-studied guide RNA used in CRISPR technology only has a 20-nt target recognition sequence ,27. Yet,In summary, by focusing on three independent targeting locations, we showed that both the unpaired bulge sizes and the chimeric RNA lengths play important roles in RNA-mediated gene fusion. The optimal length of an unpaired bulge is about 35 nt, while the optimal chimeric RNA length is about 50 nt for targeting. These observations are consistent regardless of the target locations within TMPRSS2 and ERG genes, with each location involving different targeted sequences and different bulge sequences. These empirically determined parameters for RNA-mediated gene fusion are important, as they provide valuable insight for formulating bioinformatics guidelines to search for cellular chimeric-like RNAs that may initiate oncogenic fusion genes. Secondly, these parameters provide the design principles for developing new technology for manipulating the mammalian genome through large-distance DNA rearrangements . Lastly, this knowledge could facilitate the development of therapeutic strategies to inhibit the formation of RNA/DNA duplexes and three-way junctions, therefore preventing the formation of cancer fusion genes and future tumors.


Pentatricopeptide repeat (PPR) proteins are the prime factors that are involved in RNA editing; however, many have not yet been identified. Here, we screened the plastid-targeted PLS-DYW subfamily of PPR proteins belonging to Arabidopsis thaliana and identified ORGANELLE TRANSCRIPT PROCESSING 970 (OTP970) as a key player in RNA editing in plastids. A loss-of-function otp970 mutant was impaired in RNA editing of ndhB transcripts at site 149 (ndhB-C149). RNA-immunoprecipitation analysis indicated that OTP970 was associated with the ndhB-C149 site. The complementation of the otp970 mutant with OTP970 lacking the DYW domain (OTP970∆DYW) failed to restore the RNA editing of ndhB-C149. ndhB gene encodes the B subunit of the NADH dehydrogenase-like (NDH) complex; however, neither NDH activity and stability nor NDH-PSI supercomplex formation were affected in otp970 mutant compared to the wild type, indicating that alteration in amino acid sequence is not necessary for NdhB function. Together, these results suggest that OTP970 is involved in the RNA editing of ndhB-C149 and that the DYW domain is essential for its function. RNA editing is essential for compensating for defects or mutations in haploid organelle genomes and is regulated by numerous  RNA editing is considered an indirect repair strategy that is required for the functional expression of organelle genomes . RNA ediIn the plant kingdom, C-to-U RNA editing was first discovered in mitochondria ,8. Two yArabidopsis, more than 40 and more than 600 C-to-U editing sites have been detected in RNAs transcribed from the chloroplast and mitochondrial genomes, respectively [cis-elements located upstream (5′) of the editing site [trans-factors have been identified in the RNA editing process in plastids, including those involved in the editing of ndhB transcripts; the ndhB gene encodes the B subunit of the NADH dehydrogenase-like (NDH) complex that is involved in cyclic electron transport (CET) around the PSI. For example, CHLORORESPIRATORY REDUCTION 28 (CRR28), CRR22, EDITING LACKING INSERTIONAL MUTANT 1 (ELI1), ORGANELLE TRANSCRIPT PROCESSING 82 (OTP82), QUINTUPLE EDITING FACTOR 1 (QED1), CHLOROPLAST RNA EDITING FACTOR 7 (CREF7), and OTP84 are required for ndhB editing at sites 467, 746, 830, 836, 872, 1255, and 1481, respectively [trans-factors that are specifically required for the RNA editing of ndhB site 149 have not yet been identified. In ectively ,17. In ving site ,19,20. Tectively ,24,25,26Arabidopsis and 477 in rice (Oryza sativa) [Pentatricopeptide repeat (PPR) proteins comprise an extraordinarily large protein family in land plants , with 45 sativa) . The PPR sativa) . The PPR sativa) . Analysi sativa) ,32,33. T sativa) ,35, and  sativa) ,36,37. ndhB; An otp970 T-DNA insertion mutant in Arabidopsis did not accumulate OTP970 and exhibited defective RNA editing at the ndhB-C419 site. Additionally, the OTP970 protein was found to bind to a cis-element surrounding ndhB-C149 in vivo. The body of work described above shows that PLS proteins are crucial for RNA editing. However, the functions of most PPR proteins belonging to the PLS subfamily remain unidentified. In this study, we characterized the role of the OTP970 protein in the RNA editing of Arabidopsis ecotype Columbia (Col-0) was used as the wild type in this study. Seeds were sown on half-strength Murashige and Skoog (MS) medium containing 2% sucrose and were allowed to synchronize their germination at 4 °C for 3 days. Plants were grown at 22 °C under a 12 h light/12 h dark cycle. The otp970 mutant (SALK_150217) was obtained from the Arabidopsis Biological Resource Center, OH, USA. The T-DNA insertion site in the otp970 mutant was identified via the PCR amplification of genomic DNA and T-DNA borders using gene-specific primers (The  primers . otp970 mutant and wild-type plants using the Plant RNA Kit (Magen), and 5 μg total RNA was used for first strand cDNA synthesis, which was conducted using the PrimeScript RT Reagent Kit (TaKaRa). For the RNA editing analyses, 34 published RNA editing sites were amplified and sequenced using specific primers that spanned the editing site [otp970 was amplified using gene-specific primers. RT-qPCR was carried out using the SYBR Premix Ex Taq™ (TaKaRa) on a real-time RT-PCR System . Actin 2 was used as a reference gene. The primers used for RNA editing, RT-PCR, and RT-qPCR analyses are listed in Total RNA was extracted from the ing site . The lev35S:OTP970-FLAG transgenic and wild-type plants, as previously described [RIP assay was performed using 7-day-old escribed . Primersf-AS08 306, PsaB-AS10 695, PsbO-AS06 142-33, and ATPB-AS05 085. Signals were detected using the enhanced chemiluminescence method. Thylakoid membranes were prepared as previously reported  and wereChlorophyll fluorescence was detected with the MAXI version of the Imaging-PAMM–Series chlorophyll fluorescence system. Before measurement, plants were adapted to the dark for 30 min, and light–response curves were then measured as previously reported . Transieotp970 mutation was further confirmed by immunoblot analyses and by sequencing the editing site. The wild-type At1g18485 genomic fragment (2910 bp) was PCR-amplified using gene-specific primers  and was The OTP970 protein was fused with the green fluorescent protein (GFP) as previously described  but withndhB in the chloroplasts, we focused on the phylogenetic analyses of the PPR proteins that belong to the PLS-DYW subfamily, which are predicted to be targeted to the plastids because previous research studies showed that the trans-factors involved in ndhB RNA editing all belong to the PLS-DYW subfamily. Because functionally related genes are believed to undergo co-evolution, we analyzed the co-evolution of the genes encoding PLS-DYW PPR proteins in Arabidopsis by means of hierarchical clustering. The results showed that several of the PPR genes that are involved in ndhB RNA editing were clustered together (ndhB RNA editing: QED1/OTP81 (AT2g29760), CREF7 (At5g66520), OTP82 (At1g08070), ELI1 (At4g37380), and CRR28 (At1g59720). Among these, OTP970 was present, which is an intron-less gene that encodes a putative DYW PPR protein with 970 amino acids. OTP970 contains 23 characteristic PPR motifs, including PPR-related motifs in the E and DYW domains, as predicted by a web portal (https://ppr.plantenergy.uwa.edu.au/ accessed on 12 December 2021). We obtained an otp970 mutant, the T-DNA was inserted in the coding sequence of At1g18485  was confirmed by PCR  fused with two tandem FLAG tags in the otp970 mutant under the control of the 35S promoter. The RNA editing efficiency at the ndhB-C149 site in the transgenic plants was similar to that in the otp970 mutant  6fb complex, PSII, and chloroplast F0F1-ATPase, respectively. The results showed that all of these photosynthetic complexes were not affected in the otp970 mutant (otp970 (otp970 destabilizes the NDH-PSI supercomplex, BN-PAGE was used to analyze the accumulation of this supercomplex, and the crr2-2 mutant was used as a PSI-NDH supercomplex-less control [opt970 mutant was similar to that in the wild type but was reduced compared to the crr2-2 mutant (We also determined the accumulation of PsaB, Cyt0 mutant A. Consis (otp970 . To asse control . The abu2 mutant B, band Iopt970 by detecting the transient increase in chlorophyll fluorescence after turning off AL. The results showed no differences in chlorophyll fluorescence and post-illumination between the wild type and otp970 mutant (otp970. Taken together, these data suggest that the conversion of Ser-50 to Leu-50 in NdhB is not essential for the function of the NDH complex.Although CRR22, CRR28, and CRR21 are involved in the alteration of NDH activity, these proteins are not essential for stabilizing the NDH-PSI supercomplex and NDH complex . To dete0 mutant C, demonsndhB-C149 site varied among different tissues, we conducted RT-PCR on the root, stem, rosettes, flower, and silique of wild-type plants during the reproductive phase. The RT-PCR sequencing products showed that RNA editing at the ndhB-C149 site was dramatically reduced in the root compared to in other tissues (ndhB-C467 and ndhB-C1255 sites were also dramatically reduced in the root, whereas that at the ndhB-C586 site showed a slight reduction in the root compared to other tissues (The extent of RNA editing varies among different tissues during plant development ,52. To d tissues . Moreove tissues . ndhB-C149 site that has not been identified in Arabidopsis plastids. RNA editing is an important post-transcriptional regulation in plastids. Although some regulators have been identified in the RNA editing process in plastids , there aArabidopsis mutants lacking NDH subunits [The chloroplast NDH complex is divided into four subcomplexes, including the membrane, lumen, and stroma-exposed A and B subcomplexes, based on the characterization of the eubacterial NDH complex and subunits . The memsubunits . The actsubunits ,56. ndh transcripts alters the amino acid sequence of the encoded subunit proteins at certain sites; thus, the functional importance of amino acids varies with site changes. For example, defects in RNA editing at the ndhB-C746 and ndhD-C887 sites in crr22, ndhB-C467, and ndhD-C878 sites in crr28 and at the ndhD-C383 site in crr21 can impair NDH activity [otp970 mutant was defective in RNA editing at ndhB-C149; nevertheless, NDH activity and stability of the NDH complex were unaffected compared to in the wild type (otp82 mutant, in which the RNA editing defects at the ndhB-C836 and ndhG-C50 sites did not affect NDH function [The C-to-U editing of the activity ; howeverild type . This isfunction . NDH-dependent CET plays a crucial role in response to various stresses. For example, tobacco mutant NDH complex defects show no differences in photosynthetic activity when compared to wild-type ,57. HowendhB-C149 site was 100% in the stem, rosette, flower, and silique tissues, but only ~10% in the root sample (ndhB-149 in the stem is fully edited, but the expression patterns indicated that OTP970 was expressed at very low levels in stems (trans-acting factors. Previous studies have shown that RNA editing at individual sites occurs in some but not all species . Additiot sample . Notablyin stems A, implyiOTP970, a gene encoding the PLS-type PPR protein, which is required for RNA editing in plastids. The sequencing results of RT-PCR products showed that otp970 mutant was impaired in RNA editing at the ndhB-C149 site. RIP analysis demonstrated that OTP970 was associated with ndhB-C149 site. Furthermore, we discovered that the DYW domain of OTP970 is required for RNA editing at the ndhB-C149 site. These findings increased our understanding of the mechanism in plastid RNA editing.In this paper, we identified


Escherichia/Shigella and Enterococcus, but lower abundances of Dorea longicatena, Blautia wexlerae, and Bacteroides ovatus as compared with non-HTGAP group. Correlation analysis revealed that gut bacterial taxonomic and functional changes were linked with local and systemic complications, ICU admission, and mortality. This study revealed that alterations of gut microbiota were associated with disease severity and poor prognosis in HTGAP patients, indicating a potential pathophysiological link between gut microbiota and hypertriglyceridemia related acute pancreatitis.Hypertriglyceridemia-associated acute pancreatitis (HTGAP) is linked with increased severity and morbidity. Intestinal flora plays an important role in the progression of acute pancreatitis (AP). However, pathogenetic association between gut microbiota and HTGAP remains unknown. In this study, we enrolled 30 HTGAP patients and 30 patients with AP that is evoked by other causes. The V3–V4 regions of 16S rRNA sequences of the gut microbiota were analyzed. Clinical characteristics, microbial diversity, taxonomic profile, microbiome composition, microbiological phenotype, and functional pathways were compared between the two groups. Our results showed that the HTGAP group had a higher proportion of severe AP (46.7% vs. 20.0%), organ failure (56.7% vs. 30.0%), and a longer hospital stay (18.0 days vs. 6.5 days). HTGAP group also had poorer microbial diversity, higher abundances of  Acute pancreatitis (AP) is one of the most common gastrointestinal diseases requiring acute hospitalization, with an annual incidence of 34 per 100,000 . AP is cGallstone 45%) remains the leading cause of AP, followed by alcohol abuse (20%) . Speck f% remainsEnterobacteriaceae and Enterococcus, and a lower relative abundance of beneficial bacteria, such as Bifidobacterium as compared with healthy controls ) for abnormally distributed continuous variables, and as number (percentages) for categorical variables. The P values were calculated by the Chi-square or Fisher test for categorical variables. The t-test or non-parametric Kruskal–Wallis was used for continuous variables. A two-sided P value of less than 0.05 was considered to be statistically significant.p = 0.004), had higher body mass index (BMI) , and had a higher proportion of fatty liver  in the HTGAP group as compared with those in the non-HTGAP group. The HTGAP group also showed a higher CRP level .A total of 60 patients were enrolled in this study and they were divided into two groups based on triglyceride level. p = 0.028), higher incidence of systemic inflammatory response syndrome (SIRS) , acute respiratory distress syndrome (ARDS) , organ failure , ICU admission , and had a longer hospital stay  as compared with patients in non-HTGAP group.Furthermore, patients in the HTGAP group had a higher risk of developing SAP  and 26,085 sequences  . The Venp = 0.440) and Shannon’s diversity index (p = 0.571) between the HTGAP and non-HTGAP groups, but there was a tendency for reduced richness in the HTGAP group. The PCoA of weighted UniFrac distances for the beta-diversity assessment did not show significant differences in the microbial communities between the HTGAP and non-HTGAP groups   and Peptoniphilus gorbachii (ASV1776) , but lower abundances of Dorea longicatena (ASV1564) , Dorea longicatena (ASV2046) , Blautia wexlerae (ASV1659) , Bacteroides ovatus (ASV2006) , and Bacteroides xylanisolvens (ASV2135)  when compared with non-HTGAP group (The volcano plot a and heaAP group c. At theAP group .Blautia wexlerae (ASV1332 and ASV1659), Bacteroides ovatus (ASV2006), and Dorea longicatena  were positively correlated with microbial gene functions related to amino acid metabolism, biosynthesis of secondary metabolites, nucleotide biosynthesis, NAD+ kinase, glutamate synthase (NADPH/NADH) small chain, 4-alpha-glucanotransferase, formate C-acetyltransferase, and glycogen phosphorylase.Bugbase analysis was used to explore the differences in bacterial metabolic phenotypes between HTGAP and non-HTGAP groups, and the predictions of oxidative stress tolerance and mobile element containing are shown in Escherichia/Shigella, which belong to the family Enterobacteriaceae, had moderate to strong positive correlations with shock, ICU admission, ICU stay, acute necrotic accumulation, Balthazar score, and walled-off necrosis. Enterococcus within Enterococcaceae was positively correlated with sepsis, infection, liver damage, ICU admission, ICU stay, SOFA score, organ failure, and shock, but Peptoniphilus gorbachii within Peptoniphilaceae presented a negative correlation. Infected necrosis, a disorder of consciousness, and death were strongly positively correlated with Escherichia/Shigella within Enterobacteriaceae. Hypertriglyceridemia, myocardial injury, and disease severity were negatively correlated with Akkermansia muciniphila within Verrucomicrobiaceae and Dorea longicatena within Lachnospiraceae.We performed a Spearman correlation analysis to explore the relationship between intestinal microflora and clinical indicators such as disease severity and clinical outcomes, and the result is shown in In this study, we investigated the relationship between gut microbiota and the severity and prognosis of patients with HTGAP. We found that patients in the HTGAP group had a worse outcome than that those in the non-HTGAP group, and significant correlations existed between the altered intestinal microflora and prognostic markers, including disease severity, local and systemic complications, ICU admission, and mortality.To our knowledge, this is the first study to investigate the relationship between intestinal flora and prognosis among patients with HTGAP. Consistent with previous studies ,37, patiFirmicutes, Bacteroidetes, and Proteobacteria were the most abundant phyla in the AP patients. In the present study, the microbial diversity in the HTGAP group, especially species richness, was lower than that in the non-HTGAP group, and lower microflora richness was related to adiposity, insulin resistance, dyslipidemia, and a more inflammatory state [We performed bioinformatics analysis to examine microbial diversity, richness, and community structure. Consistent with a previous study , Firmicury state . We postLachnospiraceae and Bacteroidaceae were less abundant in the HTGAP group when compared to the non-HTGAP group. Some Lachnospiraceae species, including Dorea longicatena and Blautia wexlerae, can ferment carbohydrates to produce SCFAs, which are mainly composed of acetate, propionate, and butyrate [Blautia wexlerae could contribute to the maintenance of intestinal immune homeostasis, and its depletion was associated with obesity and metabolic complications [Dorea longicatena was beneficial to maintaining the remission of Crohn’s disease [Bacteroides species within the family Bacteroidaceae, including Bacteroides ovatus, Bacteroides xylanisolvens, and Bacteroides uniformis, were essential species to induce the production of gut IgA, which acted as a protective factor for maintaining the stability of the intestinal environment by limiting the bacteria and endotoxin invasive intestinal epithelial cells, facilitating bacterial clearance, and regulating bacterial colonization [At the family level, butyrate ,44. SCFAbutyrate . It was ications . The stu disease . These rnization ,49,50.Finegoldia and Enterococcus had a higher relative abundance in the HTGAP group when compared with the non-HTGAP group. Escherichia/Shigella and Enterococcus were the most common bacterial pathogens for infected pancreatic necrosis, which were significantly associated with the poor prognosis of AP patients [Enterococcus was related to higher disease severity [Peptoniphilus and Anaerococcus were more abundant in the HTGAP group, while Akkermansia was more abundant in the non-HTGAP group. Anaerococcus, Peptoniphilus gorbachii, and Finegoldia magna, like many opportunistic pathogenic bacteria, were able to cause bacterial translocation and infected necrosis that may aggravate AP. Akkermansia muciniphila was important not only for the regulation of mucus layer thickness and gut barrier integrity, but also for improving the immune system and lipid metabolism [At the genus level, patients . Consistseverity ,14. In atabolism ,52.Blautia wexlerae, Bacteroides ovatus, and Dorea longicatena were positively correlated with microbial gene functions that were related to amino acid metabolism, biosynthesis of secondary metabolites, nucleotide biosynthesis, NAD+ kinase, carbohydrate, and energy metabolism, and formate C-acetyltransferase. Amino acid, carbohydrate, and energy metabolism were the primary life activities of bacteria, which provided a basis for the stress tolerance mechanism. Nucleotide coenzymes, including NAD+, NADP+, NADH, and NADPH, were closely associated with energy metabolism, antioxidation stress, and immunological functions [Blautia wexlerae, Bacteroides ovatus, and Dorea longicatena could be potential probiotics, as they could defend against oxidative stress, regulate the production of SCFAs, and maintain gut homeostasis.Besides, there were significant differences in gut microbial metabolic phenotypes between the HTGAP and non-HTGAP groups. The relative abundance of bacteria containing mobile elements in HTGAP was significantly higher than that in non-HTGAP, which might be a marker for translocation of gut microflora. Besides, stress-tolerant bacteria were less abundant in the HTGAP group as compared to the non-HTGAP group, indicating that intestinal flora in the HTGAP group was more difficult to tolerate oxidative stress and maintain mucosal barrier function. We analyzed the signaling pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to characterize the functional role of the intestinal flora. We found that unctions . FormateThere are several limitations to this study. Firstly, we investigated the association between the prognosis of HTGAP patients and intestinal microflora at a single time point, lacking longitudinal monitoring of the changes in intestinal microflora with disease development. Secondly, the sample size was relatively small, and it was from a single center. Multicenter studies with a larger sample size are required to test our findings. Thirdly, our study was based on 16S rRNA gene sequencing rather than the entire genomes, which provides limited information regarding bacterial genes and their functions.Our study revealed that patients with HTGAP had altered gut microbiome, which, in turn, was associated with disease exacerbation and poor prognosis. The correlation analysis and functional prediction analysis provided insights into disease progression in AP patients complicated with hypertriglyceridemia. We speculate that the HTGAP caused gut microecological disturbance, which may take an important role in the worsening of acute pancreatitis. Further validation and exploration via functional studies are required to prove the causal relationship.In conclusion, there were significant differences in the composition and function of gut microbiota in HTGAP patients compared to non-HTGAP patients, and changes of intestinal microflora were related to poor prognosis of HTGAP. Further studies are needed to determine the mechanisms underlying the effects of the microbiome on mucosal barrier and inflammatory response in HTGAP patients.


RNA editing, a vital supplement to the central dogma, yields genetic information on RNA products that are different from their DNA templates. The conversion of C-to-U in mitochondria and plastids is the main kind of RNA editing in plants. Various factors have been demonstrated to be involved in RNA editing. In this minireview, we summarized the factors and mechanisms involved in RNA editing in plant organelles. Recently, the rapid development of deep sequencing has revealed many RNA editing events in plant organelles, and we further reviewed these events identified through deep sequencing data. Numerous studies have shown that RNA editing plays essential roles in diverse processes, such as the biogenesis of chloroplasts and mitochondria, seed development, and stress and hormone responses. Finally, we discussed the functions of RNA editing in plant organelles. C-to-U rpl2. They demonstrated that RNA editing could also occur within plastids. RNA editing phenomena have been widely reported in many plants. RNA editing events have already been observed in Arabidopsis, rice, wheat, tobacco, maize, and soybean  family proteins are site-specific editing factors that recognize and directly bind to the s of RNA . In 2005s of RNA . Pentatrs of RNA . Based os of RNA . The lasMultiple organellar RNA editing factor (MORF) was the second RNA editing factor discovered in the mitochondria of angiosperms . MORF waOrganelle RNA recognition motif-containing (ORRM) proteins have been revealed to be critical for RNA editing in the organelles of angiosperms. Disruption of ORRM members can severely affect plant growth . There ippo1 mutant showed defects in 18 plastid RNA targets, leading to the decreased accumulation of the NDH complex and decreased synthesis of chlorophyll (Protoporphyrinogen IX oxidase 1 (PPO1) catalyzes the conversion of protoporphyrinogen IX into protoporphyrin IX and has also been identified to function in the RNA editing process in plant plastids. A orophyll . PPO1 caoz1 mutant showed decreased editing efficiency at 16 sites and impairment at 14 sites in plastids and consequently exhibited a yellow phenotype, suggesting its vital role in plant development. Organelle zinc finger 1 can interact with other factors, including PPR, ORRM1, and MORF proteins, indicating, that is, has a function in the assembly of the plastid RNA editing complex (Organelle zinc finger 1 (OZ1) is a RanBP2-type zinc finger protein family member. It was observed to act as an important RNA editing factor by interacting with ORRM1 . The oz1 complex .nuwa mutants showed a decreased level of RNA editing at various sites in both the plastids and mitochondria of mature leaves. In addition to PPR proteins, NUWA proteins were found to be coimmunoprecipitated with MORF proteins in vivo, which revealed their collaboration with other factors in RNA editing  and maize mutants (Zm-orrm1) lost 12 and 9 RNA editing sites, respectively  and nad9 in the mitochondria of Arabidopsis. The slo1 mutants displayed abnormal phenotypes, such as darker and shrunken seeds, late germination, and delayed development (Arabidopsis (Opaque and growth retardation 1 (OGR1) encodes a PPR protein in rice. Kim et al. generated an ogr1 RNA editing mutant in rice and found that OGR1 is required for mitochondrial RNA editing in rice and is essential for seed germination and the growth and development of plants (Pentatricopeptide repeat 2263 (PPR2263) encodes a DYW-subgroup PPR protein responsible for mitochondrial RNA editing in nad5 and cytochrome b (cob). Sosso et al. identified the ppr2263 mutation in maize and found that the mutants exhibited editing defects at the nad5-1,550 and cob-908 sites. In addition, the authors demonstrated that mitochondrial RNA editing is vital for mitochondrial biogenesis and the growth in maize , plays a vital role in RNA editing of elopment . Furtherbidopsis . Opaque f plants . Pentatrin maize . Anotherin maize .Arabidopsis, yellow seedlings 1 (YS1) encodes a PPR protein that is localized to chloroplasts and contains a DYW motif. Zhou et al. found that YS1 is essential for the editing of the housekeeping gene rpoB. During the early stage, YS1 is required for the differentiation of chloroplasts  encodes a chloroplast-localized PPR protein and contains 10 tandem PPR motifs. Lin et al. revealed that interruption of ASL3 disturbed the transcriptional levels of genes involved in chloroplast development and photosynthesis and caused impaired development of chloroplasts and the growth of seedlings , National Natural Science Foundation of China (31900256), Natural Science Foundation of Shandong Province (ZR2020QC026), Modern Agricultural Industry Technical Economic Evaluation System Green Development Position of Henan Province, Soft Science Project of Henan Province (202400410185) and State Key Laboratory of Cotton Biology Open Fund.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study introduces the self-construction methods of consumers and the tendency characteristics of experiential purchase to study the effects of physical purchase and experiential purchase on wellbeing. The dependent self-builders obtain higher happiness from experiential purchase; however, the independent self-builders get higher happiness from physical purchase. Furthermore, consumers with a high purchase experience get higher happiness from experiential purchase. Consumers with high material consumption tendency get significantly higher happiness than physical purchase from experiential purchase. Consumers with high materialism tendency gain higher happiness in experiential purchase, which is in line with the expectations of self-construction and consumption theories. This study provides the first evidence for the impact of self-construction methods on wellbeing with different consumption choices. The disposable income of Chinese residents has been increasing since the mid-2010s. After meeting the basic living needs, they can consume more and more products to meet their material and spiritual needs. At this point, obtaining more happiness through consumption has become a research hot spot of scholars . CurrentSelf-construction as an important personality trait of consumer self-mediation also has important implications for the wellbeing evaluation of consumer behavior. Research of scholars on how self-construction affects consumption patterns is mainly based on group research and individual consumer research. Based on the perspective of group study, At this stage, due to the lack of research on the self-construction mode of consumers on consumption effect, this study explores the influence of self-construction mode and experiential purchase intention on consumer purchase intention and the evaluation of consumption effect through empirical research.The rest of this study is organized as follows. The “Theoretical review and hypotheses” section reviews the theoretical background and explains the hypotheses in this study. The “Empirical results” section discusses the empirical results. The “Conclusion” section concludes.Happiness is a very subjective positive experience after individuals evaluate their own life experiences according to their standards. According to the study by per capita, which is called the “Easterlin Paradox.” Happiness comes from a balance of positive and negative emotions; although the concept of happiness is narrower than SWB, Aristotle equated happiness with subjective happiness. It is believed that subjective happiness is in a happy state, and despite the differences, the researchers have always taken “happiness” as a measure of happiness. Happiness tests characterize the wellbeing of people who are reporting the happiness they feel.Happiness is a narrow concept than subjective happiness. There are many factors affecting happiness, and the most mentioned are material conditions and consumption, as well as family and social relations. Happiness increases with income and also decreases with income, and the relationship between income and happiness is shown in parallel in individuals with high-income levels. It is found that the overall national level of national happiness will not change over time and is not related to the increase of gross domestic product (GDP) The concept of wellbeing adopts the concept of objective wellbeing, drawing on the wellbeing measurement scale developed by Different consumers have different emphases on daily consumption tendencies. Some consumers tend to buy physical products such as computers, cars, and clothing. Some consumers tend to buy experience products such as tourism and outdoor activities. Scholars summarize this behavior phenomenon into two types of consumption, namely, experiential purchase, and physical purchase. Experiential purchase refers to purchases for life experience or experience, such as watching movies, traveling, and listening to concerts. Physical purchase refers to purchasing to own something, such as a bag and a mobile phone .Different consumption tendencies bring different happiness. Physical purchase emphasizes the preservation and possession of goods. Also, in experiential purchase, consumers can only feel and enjoy in a limited time . DiffereResearchers study the impact of purchase types on the happiness of consumers from two large levels. Experiential purchase over time has different impact mechanisms from a physical purchase on happiness from the individual level. The positive experience is better over time, and even negative experiences will pass through time. Through the processing of the consumer, the memory becomes a special experience; experience is part of the self-concept of consumers, and the life of an individual is the synthesis of their life experience. Also, the physical purchase over time will gradually fade until the happiness of the consumer is lost. From the interpersonal perspective, experiential purchase is based on personal feelings, which is more subjective than social comparison. In contrast, the physical purchase is easy to reduce the happiness of consumers in the social comparison; experiential purchase is happier than physical purchase , 2016, wH1: Individuals get more happiness from experiential purchase than physical purchase.Independent self suggests that the characteristics of every person are unique. When they respond to the social environment, the response is not only for the environment itself but also for the needs of the social situation which is also important. Still, they only serve as reference standards to strengthen the inner self. Independent self-builders can feel the immediate pleasure brought by consumption and are more likely to yield to the desire for consumption. The physical object has the characteristics of “here and now,” which is the objective existence of the present and can bring immediate pleasure to consumers.Individuals define the self through their relationship with others, believing that neither self nor others can be separated from the situation but integrated with specific situations. The core concept is the harmony between self and others and creates harmonious relationships by adjusting themselves . The indExperiential consumption is easier for others to share, promoting relationships and gaining respect, conducive to improving individual wellbeing . IndepenH2: There are significant differences in the self-construction of happiness gained in different consumption activities.H2a: Self-builders gain more happiness in their experience and buying.H2b: Independent self-builders gain more happiness in physical purchases.Materialists (non-experience buyers) regard the acquisition of material wealth as the main goal of life struggle and even ignore other goals such as belonging and self-acceptance. Materialists have lower satisfaction with life and higher depression. They do not attach importance to their interpersonal relationship and are not satisfied with the quality of their interpersonal relationship . Lack ofH3: The consumption tendency is different, and consumers feel different happiness in different ways of consumption.H3a: Those who experience purchase tendency can get significantly more happiness in experiential purchase than physical purchase.H3b: Materialists get significantly more happiness in experiential purchase than in physical purchase.p < 0.000, indicating that the reliability and validity of the scale are good, which is suitable for the analysis of this questionnaire.At present, the measurement of self-construal is mainly quantitative measurement. p < 0.000, and Cronbach’s alpha of the Experiential Purchase Tendency Scale is 0.812, KMO is 0.887, and p < 0.000, indicating that the reliability and validity of the two scales are good, which are suitable for the analysis of this questionnaire.According to the Richins’ Materialism Scale, which has nine items, the last is the reverse question, and The purpose of this experiment is to verify hypothesis H1.The purpose of the pretest was to determine whether the description of “physical purchase” and “experiential purchase” in Experiment 1 was appropriate. This study used the method developed by Here are two descriptions. Description 1: the mobile phone you are about to buy is a relatively high-end mobile phone in the market. The metal fuselage, ceramic touch, fuselage fine grinding, corrosion resistance, high stability of the six-layered spraying process, and unique camera design make the vision clearer and wider. Everyone knows that the mobile phone is very advanced. When you hold it in your hand, it is very attractive.Description 2: the mobile phone you are about to buy is a relatively new and high-end model. The screen of the mobile phone is very touchy and warm. The camera shooting function of the mobile phone is more powerful, and it can take a clearer picture of the wonderful moments in your life. It can also edit photos according to your preferences to have more different and better experiences.physical = 2.55, which is significantly lower than the median value 4 , indicating that description 1 is the physical description in manipulation. Mexperience = 5.88, significantly higher than the median 4 , indicates that description 2 is the experience description in manipulation, and the data show that the manipulation is successful, so in the experiment, description 1 is purchased as physical, and description 2 is purchased as experience.Then, let the pretest subjects answer the following two questions: (1) you think description 1 focuses more on physical description or experience description, using a 7-point Likert scale  score and (2) you think description 2 focuses more on physical description or experience description, using Likert scale score. Through the analysis of 40 sample data, MSD = 3.46).The formal participants were 135 undergraduate students from a university in Guangzhou, with 119 valid respondents, including 43 male students (36.1%) and 76 female students (63.9%), with an average age of 21.46 years , whereas description 2 belonged to physical or experience description, Mexperience = 5.45, which was significantly higher than the intermediate value 4 . The manipulation test data show that physical description and experience description are successfully manipulated.Subjects were asked to score whether descriptions 1 and 2 belonged to physical or experience descriptions; description 1 belonged to physical or experience description, MF = 54.25, p < 0.000, Mphysical = 4.03, (SD = 1.38), and Mexperience = 5.72, (SD = 1.04), indicating that there was a significant difference in happiness between physical purchase and experiential purchase. Thus, hypothesis H1 was verified.By calculating the average happiness of the participants who choose physical purchase and those who choose experiential purchase in the questionnaire, the descriptive statistical scale in The experiment verifies that “experiential purchase” can bring consumers more pleasure and happiness than physical purchase . PurchasThis experiment verifies the hypotheses H2, H2a, and H2b that self-construction of different people in the happiness of physical and experiential purchase significantly differs.SD = 3.62).The formal participants were 245 undergraduate students from a university in Guangzhou, with 237 valid respondents, including 87 male students (36.7%) and 150 female students (63.3%), with an average age of 21.22 years , indicating that the reliability and validity of the scale were good, which was suitable for this survey.This experiment adopted 2 (construction mode) × 2 (consumption type) between-group experiments. First, the reliability and validity of the self-construction scale in the questionnaire were analyzed. The Cronbach’s alpha was 0.895, and the KMO was 0.856  = 70.805, p < 0.000, as shown in experience of the dependent construction is 5.625, and the Mphysical is 4.581, whereas the Mexperience of the independent construction is 4.723, and the Mphysical is 5.694, F = 31.09, p < 0.000, which has a significant difference. Self-construal of different people in different consumption patterns of happiness has been significantly different, and hypothesis H2 is verified.Taking the happiness degree of the subjects as the dependent variable and the construction type and the consumption type as the independent variables, the single-factor ANOVA is carried out. The results showed that the interaction between different construction methods and consumption types was significant F = 33.162, p < 0.000, indicating significant differences in the degree of happiness in the same self-construction context experienced by participants in different consumption types. The degree of happiness experienced by the physical purchase subjects Mphysical = 4.58 (SD = 1.222), the degree of happiness experienced by the experimental purchase subjects Mexperience = 5.63 (SD = 0.669), and the degree of happiness between experiential purchase and physical purchase is significant . This evidence shows that in the interdependently constructed group of subjects, the degree of happiness that the subjects feel from experiential purchases is significantly higher than that of physical purchases. It is assumed that the happiness of H2a-interdependent self-construal in the experiential purchase is significantly higher than that in physical purchase.In the context of dependent construction, the happiness level of consumption patterns was significantly different F = 3.191, p < 0.000, indicating that in the context of independent self-construal, there is a significant difference in the degree of happiness that physical purchase and experiential purchase subjects can experience. The degree of happiness that Mphysical object is 5.69 (SD = 0.738), the degree of happiness that Mexperience is 4.72 (SD = 0.615), and the degree of happiness that physical purchase and experiential purchase are significantly different . The results showed that subjects felt more happiness from the physical purchase than physical purchase in the independent self-construal group. It is assumed that H2b-independent self-construal obtains significantly higher happiness in physical purchase than experiential purchase.In the context of independent self-construal, there is a significant difference in the level of happiness among different consumption types Experiment 2 explored the difference in happiness between consumers with different self-construction methods in the physical purchase and experiential purchase. The interdependent self-construal indicated more happiness in the experiential purchase, while the interdependent self-construal obtained more happiness in physical purchase. The interdependent self-construal tended to define themselves through the relationship with the outside world. The experience consumption process is social, increasing interpersonal intimacy to meet the interdependent self-construal needs for social relations and improve their happiness. Independent self-construal pays more attention to themselves, and the physical purchase is often completed alone, which can meet the needs of independent self-construal self-realization. Thus, the hypotheses H2, H2a, and H2b are verified.The purpose of this experiment was to verify H3, H3a, and H3b that people with different materialistic tendencies and experiential purchase tendencies have significant differences in obtaining happiness in the physical purchase and experiential purchase.The participants in the formal experiment were 268 undergraduate students from a university in Guangzhou, 256 valid respondents, 116 male students (45.3%), and 140 female students (54.7%), with an average age of 21.66 years (SD = 4.646).Step 1: Tell the subjects that we are experimenting on consumption choices and get them to make a purchase decision. The preamble to the experiment is as follows: Suppose you are facing a dilemma in consumption decisions because budgets are limited, and you can only choose one from two. Listen to the concert of a favorite singer or buy a large brand of clothing that you have long loved. The concert will make you happy. You can melt yourself into music and embark on a dream journey with melody. And you have a long-loved brand of clothing, and it has a unique design and superior supplies, which will make you feel that you have very grade clothes. Then, let the subjects choose whether they would like to go to the concert or buy their favorite clothes.Step 2: Let subjects score the degree of happiness based on the above decisions.Step 3: Then, let the subjects fill in the Materialism Scale and the Experiential Purchase Tendency Scale.Step 4: Finally, ask the subjects whether they know the purpose of the investigation and fill out the personal information.p < 0.000. The Cronbach’s alpha of the Experiential Purchase Tendency Scale was 0.812, and the KMO was 0.887, p < 0.000, indicating that the reliability and validity of the two scales were good, which were suitable for the analysis of this questionnaire. Variables are calculated and converted for the reverse questions of the Materialism Scale. Then, a simple descriptive analysis is carried out on the happiness obtained by participants with different consumption tendencies and consumption types, as shown in This experiment adopted 2  × 2 (consumption type) between-group experiments. First, the reliability and validity of the Materialism Scale and the Experiential Purchase Tendency Scale were analyzed. The Cronbach’s alpha of the Materialism Scale was 0.766, and the KMO was 0.836, F = -1.763, p = 0.185, as shown in experience of experiential purchase propensity = 6.174, Mphysical = 5.423, Mexperience of materialism consumption propensity = 5.962, Mphysical = 5.413, F = 15.527, p < 0.000, showing significant differences. Assuming that H3 has a different propensity to consume, the significant difference in happiness obtained by participants in the experiential purchase and physical purchase is verified, indicating that participants with different propensity to consume have a significant difference in happiness obtained in the experiential purchase and physical purchase.The results showed that the interaction between different consumption tendencies and consumption types was insignificant F = 10.226, p < 0.002, indicating that, in the context of high experiential purchase tendency, there were significant differences in the degree of happiness that different consumption types of subjects can experience. The degree of happiness experienced by physical purchase subjects was 5.42 (SD = 1.027), the degree of happiness experienced by experiential purchase subjects was 6.17 (SD = 0.677), and the difference between experiential purchase and the physical purchase was significant . This evidence shows that subjects with high experiential purchase tendency feel more happiness from the experiential purchase than physical purchase. Assuming that H3a is verified, experiential purchasers tend to enjoy experiential purchases significantly higher than physical purchases.In the context of experiential purchase tendency, the main effect of the level of happiness of consumption types was significant F = 9.324, p < 0.003, indicating that in the context of high physical consumption tendency, there was a significant difference in the degree of happiness that physical purchase and experiential purchase subjects can experience. The happiness level of physical purchase subjects was 5.41 (SD = 1.402), the happiness level of experiential purchase subjects was 5.96 (SD = 1.123), and the happiness of different consumption types was significantly different . This result shows that subjects with a high propensity to physical consumption still feel significantly higher happiness from the experiential purchase than physical purchase. Assuming that H3b is verified, consumers with a high propensity to physical consumption get a higher happy experience from experiential purchase.In the context of physical consumption tendency, the main effect of the happiness level of consumption types was significant The experimental data show that subjects with different consumption tendencies have significant differences in happiness in physical and experiential purchases. Assuming that H3 is validated, subjects with a higher propensity to experiential purchase gain more happiness from experiential purchase. Assuming that H3a is verified, subjects with higher materialistic propensity to consume also have significant differences in happiness from experiential purchase and physical purchase, and experiential purchase brings happiness to subjects significantly higher than physical purchase. We observed that H3b is verified. Although participants with high materialistic consumption tendency expect to obtain more happiness from physical consumption, they often fall into the “materialistic supremacy” trap. Excessive pursuit of material satisfaction is self-defeating behavior, which brings more negative emotions and depression. Experiential purchase is a kind of social consumption, which can meet the social needs of materialism and correct their extreme emphasis on material pursuit and neglect of the construction of interpersonal relationships. Therefore, subjects with a high propensity to materialistic consumption have significantly higher pleasure in experiential purchase than physical purchase.The main conclusions are as follows: interdependent self-construal gains more happiness from experiential purchase, and independent self-construal gains more happiness from physical purchase. Subjects with a high propensity to experiential purchase gain more happiness from experiential purchase, and subjects with materialism tend to gain more happiness from experiential purchase.First, it enriches the related research of physical purchase and experiential purchase. Previously, the research on the influence of physical purchase and experiential purchase on the happiness of consumers mainly analyzed the influence of experiential purchase and physical purchase on happiness from construal level theory, self-concept, social comparison, and self-determination theory. This study introduces the self-construction of the personality traits of consumers, and consumption tends to study and makes an empirical study on the influence of different construction methods on consumption types and the influence of different consumption tendencies on consumption types, which enriches the related research of physical purchase and experiential purchase.Second, it reveals the influence of different construction methods on consumption types, which affects the explicit behavior and implicit psychology of the consumers. Experiment 2 proves that interdependent self-construal has a significantly higher degree of pleasure in experiential purchase than physical purchase because interdependent self-construal defines themselves by the relationship with others, they attach importance to the relationship with others, the core of their attention is to create a harmonious interpersonal relationship, and experiential purchase due to its social can meet the needs of interdependent self-construal of interpersonal relationships and can increase the intimacy between people. Independent self-construal is more likely to experience the immediate pleasure of physical purchases. Physical purchase is usually an individual behavior, which is relatively isolated. Independent self-construal perceives the difference between themselves and the outside world in the physical purchase and obtains more happiness.Third, it enriches the research on materialism and consumption propensity. Researchers often suggest that materialism is a personality trait but ignore that experiential purchase propensity is also a personality trait that significantly affects the purchase behavior of consumers. This study verifies through experiments that although materialists attach importance to physical purchase, the physical purchase cannot bring the expected happiness, the experiential purchase can help materialists establish the relationships of family and friends, and the support of family and friends can promote the improvement of happiness.It is also important to pay attention to consumer experience, the consumer experience as an important element of enterprise marketing strategy to manage, enterprise product form whether pure physical products, physical products with experience or experience products, consumers will produce use experience, consumer experience is good or bad affect the enterprise product sales. From the enterprise level, we should pay attention to consumer experience and have channels to collect and understand consumer product experience, pay attention to consumer experience feedback, and constantly improve the consumer experience. Pure physical products should increase the function or characteristics to stimulate consumer experience. Experience products should pay attention to the feelings brought to consumers in the experience process and constantly improve or upgrade experience to avoid the negative impact of repeated experience on consumers.In addition, the personality traits of consumers are fully considered to incorporate advocacy and packaging strategies. Self-construal consumers tend to focus on the context of stimulus, and their motivation to consume is often concentrated on sharing and maintaining contact with others. They pay more attention to the social experience of products, so such consumer companies can strengthen the value of products to promote interpersonal relationships, i.e., presenting social scenes and slogans to stimulate audience awareness and recognition of products. The background rarely stimulates consumers with independent self-construal. They pay more attention to self-expression and possession of things. Their uniqueness should be highlighted in publicity and packaging for such consumer products and the significance of self-representation to consumers.The experimental design method adopted in the study is to construct the consumption type through the literal description, which is not a real shopping scene. Participants only use literal reading to virtualize shopping choices. Therefore, different results may appear in the real consumption scene. Future research can be based on real consumption scenes and real consumption. As a personality trait, materialism has been paid much attention to and studied by the academic circle.Finally, The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.AX was responsible for the questionnaire design of the article. LL, SL, LW, and WP were responsible for the data collection of the article and the writing of the article. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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