MEG3, we explore the mechanisms by whichlncRNAs target chromatin. Here we show that MEG3 and EZH2 share commontarget genes, including the TGF-β pathway genes. Genome-wide mapping ofMEG3 binding sites reveals that MEG3 modulates the activity ofTGF-β genes by binding to distal regulatory elements. MEG3 bindingsites have GA-rich sequences, which guide MEG3 to the chromatin throughRNA–DNA triplex formation. We have found that RNA–DNA triplexstructures are widespread and are present over the MEG3 binding sitesassociated with the TGF-β pathway genes. Our findings suggest thatRNA–DNA triplex formation could be a general characteristic of target generecognition by the chromatin-interacting lncRNAs.Long noncoding RNAs (lncRNAs) regulate gene expression by association with chromatin,but how they target chromatin remains poorly understood. We have used chromatin RNAimmunoprecipitation-coupled high-throughput sequencing to identify 276 lncRNAsenriched in repressive chromatin from breast cancer cells. Using one of thechromatin-interacting lncRNAs,  Long noncoding RNAs (lncRNAs) regulate gene expression by associationwith chromatin. Here, the authors show that lncRNA MEG3 regulates theTGF-β pathway by bridging the interactions between polycomb repressive complex2 and the distal regulatory elements of the TGF-β pathway genes via formationof RNA–DNA triplexes. On the other hand,chromatin-binding maps of HOTAIR and Drosophila roX2 lncRNAs revealed thatGA-rich sequences are the preferred binding motif, indicating that GA-rich sequences mayhelp these RNAs to target the chromatinLong noncoding RNAs (lncRNAs) have emerged as key regulators of important biologicalprocesses implicated in development and differentiation12345Drosophila, the recruitmentof PRC2 to the chromatin is mediated by specific sequences known as polycomb responseelements. However, in mammals, it is not clear how sequence-specific recruitment of thePRC2 occurs across the genome. Recent evidence of a strong association between lncRNAand the PRC2 complex raise the possibility that lncRNAs may act as guiding molecules forPRC2 to target the chromatinActive and inactive epigenetic modifications of the chromatin can regulate geneexpression at the transcriptional level. When chromatin is enriched with repressivehistone marks such as H3K27me3 and H3K9me3, it negatively regulates transcriptionSeveral previous studies have focused on the identification of the polycomb-interactinglncRNAs either by using the RNA immunoprecipitation (RIP) technique or thephotoactivatable ribonucleside-enhanced crosslinking and immunoprecipitationtechnique242526MEG3) as a model system, we explored themechanisms by which it recognizes target genes. Consistent with ChRIP-seq data,MEG3 interacts with the PRC2 complex. Through loss-of-function experiments ofMEG3 and EZH2, we found that MEG3 in cooperation with PRC2regulates a common set of genes, including those of the transforming growthfactor-β (TGF-β) pathway. Using a modified chromatin oligo affinityprecipitation (ChOP) method, we fine-mapped genome-wide chromatin-binding sites forMEG3 RNA, revealing some of the TGF-β pathway genes as directtargets. MEG3 binding sites showed enrichment in GA-rich sequences and we foundthat these GA-rich sequences guide MEG3 RNA to its target genes through formationof RNA–DNA triplex structures. Our data demonstrate that RNA–DNAtriplex structures are widespread in vivo, and are also present in the vicinityof the TGF-β pathway genes. Taken together, these results suggest thatRNA–DNA triplex formation may be a general mechanism for target generecognition by lncRNAs.Hence we sought to identify the repressive chromatin-associated lncRNAs on a global scaleand also characterize the mechanisms by which these lncRNAs are targeted to thechromatin. Here we have characterized the repressive chromatin-associated lncRNAs on agenome-wide scale by performing chromatin RIP followed by high-throughput sequencing(ChRIP-seq) using antibodies to H3K27me3 and EZH2 in BT-549 cells. We identified 276lncRNAs that are enriched in repressive chromatin. By using one of thechromatin-interacting lncRNAs  between H3K27me3 and EZH2 ChRIP pull-downs  andnon-annotated lncRNAs (intergenic CUFF.16286 and intronicCUFF.9557) using qPCR assay on ActD-treated and -untreated ChRIPmaterials  in the EZH2 ChRIP-seq data,which indicates possible RNA–protein contact points. We detected twoconverted nucleotides at the 5′-end of the MEG3 RNA in the EZH2immunopurified RNA (MEG3 clones using nuclear RNA from BT-549 cells (hereonreferred to as wild-type (WT) MEG3). It contains all the constitutivelyexpressed exons  and analternatively spliced exon 7 , but not exon 4. Moreover, we detected fewer reads in the nuclearRNA over the alternatively spliced exon with the second T-to-C conversion  and9 bp (340–348) (MEG3 clone. We carried outan in vitro binding assay with WT and mutant (Δ340-348MEG3 and Δ345-348 MEG3) MEG3 RNAs usingpurified PRC2 complex, comprising EZH2, EED and SUZ12 subunits (MEG3(antisense WT MEG3) was used as a negative control. We found that thePRC2 binding was partially compromised in the mutant MEG3 RNAs(Δ340-348 MEG3 and Δ345-348 MEG3) comparedwith the WT MEG3  , overlapsubunits . An anti WT MEG3 . We alsomunoblot . To furtll-downs . Thus, bMEG3 lncRNA, both EZH2 and MEG3 transcripts weredownregulated in BT-549 and HF cells using small interfering RNA (siRNA), andgene expression profiles were measured using microarray. We observed asignificant overlap among the deregulated genes between the EZH2 andMEG3 data sets from BT-549 and HF cells, indicating a functionalassociation between the MEG3 lncRNA and EZH2 –qPCR to validate four target genes uponMEG3 downregulation using an alternative siRNA against MEG3. Compared with thecontrol shRNA, the MEG3 shRNA-transduced BT-549 cells showed activationof the TGF-β pathway genes  RNA (with no known target in the human genome), usedas a negative control, detected neither MEG3 nor MALAT1,highlighting the specificity of the ChOP pull-down assay , indicating a functional role of the MEG3 peaks in theregulation of associated genes  in BT-549 cells  associated with theMEG3-deregulated genes  raises the possibility that the lncRNAs may berecruited to their target genes via RNA–DNA triplex formation. Byusing Triplexator softwareMEG3peak summit (±200 bp from the centre of the peak) than theflanking sequences  located at the 5′-end of the MEG3 RNA andits sequence overlap with the TFOs  with high score thatwere identified by Triplexator  with the GA-rich  MEG3 peak sequencesassociated with the selected TGF-β pathway target genes  in vitro  or control RNA incubatedwith control DNA sequences  . TheGA-ed genes , suggestequences , indicatplexator . Using ein vitro . Consist RNase H . Theserequences . To furtMEG3 TFO . These oequences .MEG3 TFO and acontrol RNA oligo were either used to transfect BT-549 cells  of the peak∼270–280 nm and a strong negative peak at∼210 nm, which are not seen in the sample containing thecontrol ssRNA. The effect is emphasized in the inset in TGFB2 dsDNA oligo with the MEG3 TFO and the control ssRNA.These two features, and especially the strong negative peak at∼210 nm, are often seen for TFOs4243TGFB2 dsDNA and MEG3 TFO (ssRNA) orTGFB2 dsDNA and the control ssRNA. For these artificial spectra, thedifference between the MEG3 TFO and the control ssRNA is much smaller spectroscopy to investigate thetriplex-forming ability of some of the n SMAD2-  and TGFB  backbone and did not bind to nucleic acids whennon-complementary sequences in solutions impeded formation of triple-strandedcomplexes. Also, the antibodies clearly bound to the triplex DNA made withpoly-purine/poly-pyrimidine sequences  containingGA-rich sequences and named this Δ46-56 MEG3 (46–56indicates the position of the nucleotides with respect to the 5′-endof MEG3). We found that the TFO deletion had no effect on the interactionof PRC2 with MEG3 (MEG3 affects the PRC2 interaction of WT MEG3(MEG3 RNAs withdeletions (Δ46-56 MEG3 and Δ345-348 MEG3) bytransfecting BT-549 cells with in vitro-synthesized biotin-labelled WT ormutant MEG3 RNAs (Δ46-56 MEG3 or Δ345-348MEG3). We found that the WT and Δ345-348 MEG3 RNAscould pull-down MEG3 peak sequences associated with theTGF-β genes ,but not when the pull-down was performed with the Δ46-56 MEG3TFO deletion that have been implicated in cancer or other cellular functions, and among thisBDNF-AS1 lncRNA has been shown to regulate gene expression by recruitingPRC2  or pREP4 episomal vector (Life Technologies)using the primers described in MEG3 clone are provided in the Results section. MutantMEG3 RNAs  described in the manuscript were generated usingthe Quik-change site-directed mutagenesis kit (Agilent Technologies). Primersused in the site-directed mutagenesis and for cloning of MEG3 areprovided in in vitro transcription of biotin-labelled and unlabelled MEG3RNA, full-length MEG3 (referred to as WT MEG3) or the mutantMEG3 RNAs, carrying various deletions, were cloned into pGEM-T Easyvector (Promega).Full-length MEG3 and EZH2 or the control siRNAwere used for transfection at a final concentration of 65 nM (detailsof siRNAs are provided in MEG3) were used totransfect BT-549 cells using Lipofectamine 2000 reagent (Life Technologies) andoverexpression of MEG3 RNA was verified by RT–qPCR48 h after transfection, using primer pairs overlapping MEG3exon 3 . For each siRNA transfection, 50,000 BT-549 or HF cells wereseeded per well in 24-well plates 12–16 h beforetransfection. siRNAs against MEG3 were obtained from Sigma (the sequences are providedin 4) were plated in the wells of 24-wellplates and transduced with either control or MEG3 shRNA lentiviralparticles. Lentivirus-transduced BT-549 cells were selected with puromycin toobtain stably integrated shRNA vectors. The stable clones were maintainedthereafter in RPMI containing puromycin(1 μg ml−1). Weverified the downregulation of the MEG3 lncRNA in lentivirus-transducedstable clones by RT-qPCR. Stable clones of MDA-MB-231 cells containingpREP4MEG3 were generated after antibiotic selection with hygromycin(850 μg ml−1), andmaintained in DMEM containing hygromycin(850 μg ml−1). Weverified the overexpression of the MEG3 RNA in MDA-MB-231 stable clonesby RT–qPCR following the same procedure as described above.BT-549 cells were maintained in RPMI (Sigma) supplemented with 10%fetal bovine serum (FBS) (Sigma). MDA-MB-231 and HF cells were maintained inDMEM (Invitrogen) with 10% FBS. BT-549 cells were procured from CLScell line service. MDA-MB-231 cells were kindly gifted by Dr Briegel Karoline and HF cells werekindly gifted by Dr Bengt Westermark .MISSION lentiviral transduction particles expressing non-target shRNA control orshRNA against etal.et al.c-Myc RNA, with a short half-life, by RT–qPCR.After ActD incubation, cells were washed two times with PBS and crosslinked with1% formaldehyde for 10 min with gentle shaking.Crosslinking was stopped by adding glycine to a final concentration of125 mM and incubated for 5 min with gentle shacking. Cellswere subsequently washed twice with PBS followed by crosslinking on ice withultraviolet. Cells were removed by scraping from the plate and resuspended incold PBS. Nuclei were isolated using 1 × nuclei isolation buffer(40 mM Tris-HCl (pH 7.5),20 mM MgCl2, 4% Triton X-100 and1.28 M sucrose) and washed again with PBS. The isolated crosslinkednuclei were resuspended in lysis buffer , 150 mM NaCl and1 ml lysis buffer per 10 million of cells) supplemented with RNasein(Promega) and subjected to sonication  to obtainchromatin fragments of ∼1 kb. An amount of50–60 μg of soluble chromatin was used in eachchromatin immunoprecipitation and incubated with 5 μg ofanti-EZH2 (active motif) and anti-H3K27me3 (Merck-Millipore) antibody.Antibody-bound chromatin was washed according to our earlier published protocolwith buffers and was supplemented with RNasein. Protein A magnetic beads boundto immunoprecipitated chromatin were resuspended in 10 × volume ofelution buffer ,1 mM EDTA and 0.5% SDS) containing proteinase K.Proteinase K treatment was carried out at 55 °C for45 min followed by heating at 95 °C for10 min to reverse crosslinking. Chromatin-bound RNA was extractedwith Trizol (Life Technologies) and subjected to DNase I (Promega) treatment toremove traces of DNA. Since the chromatin-bound RNA yield from one singleexperiment is suboptimal for high-throughput sequencing, we pooled RNA from 6 to8 ChRIP pull-downs, and the sequencing library was made using SOLiD TotalRNA-Seq Kit and sequenced the library using SOLiD platform (Applied Biosystem).SOLiD Total RNA-Seq Kit allows obtaining strand-specific RNA sequencinginformation. Since library preparation in SOLID protocol involves ligation ofRNA adaptors to the RNA fragments by RNA ligase, no contamination from DNAfragments is expected. For input, nuclear RNA was isolated from 4sU- andActD-treated BT-549 cells, and depleted ribosomal RNA using RiboMinus EukaryoteSystem v2 (Life Technologies). For ChRIP validation, we followed the sameprotocol as described above with or without ActD treatment using antibodiesagainst H3K27me3, EZH2, H3K4me2 (Merck-Millipore) and nonspecific Rabbit IgG.ChRIP was performed using BT-549 cells adapting the protocols from Mondal Accession codes: The data associated with this publication have been depositedin European Nucleotide Archive and are accessible through accession numberPRJEB7307.How to cite this article: Mondal, T. et al.MEG3 long noncoding RNA regulates TGF-β pathway genes throughformation of RNA–DNA triplex structures. Nat. Commun. 6:7743 doi:10.1038/ncomms8743 (2015).Supplementary Figures 1-13, Supplementary Methods and SupplementaryReferencesList of the annotated and non-annotated lncRNAs enriched in H3K27me3List of the annotated and non-annotated lncRNAs enriched in EZH2List of the annotated and non-annotated lncRNAs enriched in both H3K27me3 and EZH2List of repressive chromatin-enriched annotated and non-annotated lncRNAs with T to C conversionThe deregulated genes as detected by microarray after MEG3 and EZH2 downregulation in BT-549 cells and the overlap between the MEG3 and EZH2-dependent genesThe deregulated genes as detected by microarray after MEG3 and EZH2 downregulation in HF cells and the overlap between the MEG3 and EZH2-dependent genesList of pathways commonly affected after MEG3 and EZH2 downregulation in BT-549 cells, as detected by both microarray and RNA-seqSummary of MEG3 peaks associated with the deregulated genes after MEG3 downregulation in BT-549 cellsLocation and length of the MEG3 peaks associated with the deregulated genes after MEG3 downregulation in BT-549 cellsList of the oligos used in the study


TA) systems are the means by which bacterial cells become persistent; that is, those cells that are tolerant to multiple environmental stresses such as antibiotics by becoming metabolically dormant. These persister cells are responsible for recalcitrant infections. Once toxins are activated by the inactivation of antitoxins , many toxins reduce metabolism by inhibiting translation . The MqsR/MqsA TA system of Escherichia coli cleaves mRNA to help the cell withstand oxidative and bile acid stress. Here, we investigated the role of secondary structure and 5′ mRNA processing on MqsR degradation of mRNA and found that MqsR cleaves only single‐stranded RNA at 5′‐GCU sites and that MqsR is equally active against RNA with 5′‐triphosphate, 5′‐monophosphate, and 5′‐hydroxyl groups.Toxin/antitoxin ( Persister cells, those cells that are tolerant to antibiotics by becoming metabolically dormant, are one of the main causes of recurring infections  systems  RNA as its substrate   and structures were visualized using PseudoViewer (http://pseudoviewer.inha.ac.kr/). RNA was synthesized directly from the duplex DNA templates via in vitro transcription using the AmpliScribe T7‐Flash Transcription Kit . In brief, in a standard 20 μL reaction mix, 3 μg of the duplex DNA templates along with transcription kit components were incubated at 37°C for 4 h. The unused DNA template (if any) was removed by adding 1 μL containing 1 unit (U) of DNase I for 15 min at 37°C. The RNAs were then gel purified by fractionation through 10% denaturing polyacrylamide gels. Purified RNAs were quantified using a nanodrop spectrophotometer  and stored at −80°C.Duplex DNA oligonucleotides containing the T7 promoter sequence were purchased . Sequences of DNA templates for the ss, ds, sl, and pk RNA are shown in Table μL) for 1 h at 37°C to remove the phosphate groups (5′‐ppp) from the RNA and to create 5′‐OH substrates for the kinase reaction. The CIP was removed by a phosphatase removal reaction of the KinaseMax Kit. The 5′‐OH RNA was phosphorylated for 1 h at 37°C using 10 U of T4 polynucleotide kinase  and approximately 50 pmol of [γ‐32P] ATP  to generate 5′‐p RNA. The 32P‐labeled RNA was purified by the mini quick spin column for RNA as per manufacturer's protocol . The RNA concentration was determined from the CPM from a liquid scintillation counter LS 6500 . RNA was stored at −80°C.The ss, ds, sl, and pk RNA samples were radioactively labeled at their 5′ terminus using the KinaseMax 5′ End‐Labeling Kit . In brief, 25 pmol of RNA was first treated with 0.1 U of calf intestine alkaline phosphatase , CTP (9 mmol/L), and GTP (9 mmol/L) along with other kit reagents for 4 h a 37°C. At this point, the primary transcripts have triphosphate at the 5′‐end (5′‐ppp). An aliquot (15 μL of 445 nmol/L) was treated with CIP to convert it to 5′‐OH ssRNA. Then, an aliquot (15 μL of 324 nmol/L) of this 5′‐OH ssRNA was treated with PNK and 1 mmol/L unlabeled ATP for 30 min at 37°C to make 5′‐p ssRNA internally labeled with [α‐32P] UTP.The ssRNA was radioactively labeled internally in a 20 γ‐32P] ATP at the 5′‐end (5′‐p), approximately 5 × 10−3 pmol of RNA was used as a substrate for cleavage with 107 pmol (1×), 27 pmol (1/4×), 5.4 pmol (1/20×), and 1.1 pmol (1/100×) of purified MqsR , 5.4 pmol (1/20×), and 1.1 pmol (1/100×) of purified MqsR and incubated for 5 min at 37°C to determine whether MqsR could cleave all three types of 5′ ends of ssRNA, and if there was any preference for these 5′ ends of ssRNA. The MqsR cleavage of RNA reaction was stopped by adding an equal volume of 2 × formamide dye . T1 digests with each of the labeled RNAs were prepared using 0.03 pmol of 5′‐end‐labeled RNA or 0.3 pmol of 32P‐UTP‐labeled RNA along with 1 or 10 U of RNase T1 (Life Technologies) and incubated at 55°C for 15 min. The reaction was stopped by adding an equal volume of 2 × formamide dye. RNA samples (MqsR or RNase T1 digested) were heated at 85°C for 3 min and fractionated through 10% denaturing polyacrylamide gels with 1 × TBE gel running buffer. RNA species were visualized with a Typhoon 9410 phosphorimager .For RNA labeled with  ATP to form RNA with 5′‐p ends. Note that MqsR cuts mRNA before and after the G nucleotide in the 5′‐GCU site (Yamaguchi et al. To explore how toxin MqsR degrades mRNA, we designed four ons Fig. . Each ofWe found that MqsR preferentially cleaves single‐stranded 5′‐GCU sites since cleavage was approximately 20‐fold higher than cleavage seen with the 5′‐GCU site in the stem‐loop and pseudoknot configuration Fig.  due to tα‐32P] UTP, used these ssRNAs for MqsR cleavage, and found that MqsR cleaved all three types of ssRNA (Fig. We also investigated whether MqsR has a preference for a 5′‐ppp, 5′‐p, or 5′‐OH ssRNA since some RNases, like RNase E, prefer 5′‐p (Callaghan et al. E. coli MazF/MazE TA system, which also cuts mRNA only at single‐stranded sites as shown by occluding the 5′‐ACA cleavage site with either antisense RNA or DNA (Zhang et al. Mycobacterium tuberculosis cuts only single‐stranded tRNA at 5′‐ACGC sequences (Cruz et al. Our results demonstrate clearly that toxin MqsR cuts primarily single‐stranded RNA. Therefore, secondary structure has a profound impact on the ability of MqsR to degrade mRNA. Similar results have been found with toxin MazF of the E. coli, RNase E, has a preference for RNA containing 5′‐p ends; the 5′‐p interacts with a 5′ binding pocket on the enzyme (Callaghan et al. in vivo.We also found that MqsR can degrade ssRNA independently of whether there is a 5′‐ppp, 5′‐p, or 5′‐OH end. In contrast, the primary endonuclease for degrading mRNA in in vivo, MqsR targets a wide range of mRNAs related to central metabolism (González Barrios et al. E. coli mRNAs since only 14 E. coli mRNAs lack the 5′‐GCU site (Yamaguchi et al. Previous results have found that The authors declare no conflicts of interest.


Diplonema papillatum (Diplonemea), a member of the kinetoplastids’ sister group.RNA ligases 2 are scarce and scattered across the tree of life. Two members of this family are well studied: the mitochondrial RNA editing ligase from the parasitic trypanosomes (Kinetoplastea), a promising drug target, and bacteriophage T4 RNA ligase 2, a workhorse in molecular biology. Here we report the identification of a divergent RNA ligase 2 (DpRNL) from We identified DpRNL with methods based on sensitive hidden Markov Model. Then, using homology modeling and molecular dynamics simulations, we established a three dimensional structure model of DpRNL complexed with ATP and Mg2+.Diplonema was compared with available crystal structures from Trypanosoma brucei, bacteriophage T4, and two archaeans. Interaction of DpRNL with ATP is predicted to involve double π-stacking, which has not been reported before in RNA ligases. This particular contact would shift the orientation of ATP and have considerable consequences on the interaction network of amino acids in the catalytic pocket. We postulate that certain canonical amino acids assume different functional roles in DpRNL compared to structurally homologous residues in other RNA ligases 2, a reassignment indicative of constructive neutral evolution. Finally, both structure comparison and phylogenetic analysis show that DpRNL is not specifically related to RNA ligases from trypanosomes, suggesting a unique adaptation of the latter for RNA editing, after the split of diplonemids and kinetoplastids.The 3D model of Homology modeling and molecular dynamics simulations strongly suggest that DpRNL is an RNA ligase 2. The predicted innovative reshaping of DpRNL’s catalytic pocket is worthwhile to be tested experimentally.The online version of this article (doi:10.1186/s12900-015-0046-0) contains supplementary material, which is available to authorized users. RNA ligase from phage T4, the work horse of molecular biology research, is the best known member of a large protein family encompassing RNA and DNA ligation enzymes . RNA ligRNA ligase 1 enzymes are mainly present in viruses, mammals and fungi . This enTrypanosoma brucei [Kinetoplastids (Euglenozoa) are a group of protozoans, some members of which are causing life-threatening human diseases  . These sa brucei .Diplonema papillatum, a member of diplonemids (Euglenozoa), which are the sister group of kinetoplastids. The corresponding gene was discovered in our search of a candidate enzyme involved in the eccentric post-transcriptional processing in Diplonema mitochondria [Diplonema mitochondrial DNA (mtDNA) is also multi-partite, as it is composed of hundreds of circular chromosomes of two size classes. The difference and uniqueness of the diplonemid mtDNA is that each chromosome contains one short coding region specifying a fragment of a gene. Each gene module is transcribed separately and then trans-spliced to form full-length mRNAs or structural RNAs. The second resemblance with kinetoplastid mitochondria is RNA editing [Diplonema, RNA editing proceeds by uridine appendage at certain module ends, prior to trans-splicing. We hypothesize that an ancestral molecular machinery containing RNA ligase 2 has led to the editosome in kinetoplastids, while it has evolved to perform trans-splicing in the diplonemid branch.Here we report the identification of a putative new member of the RNA ligase 2 family in chondria , 19. Thi editing , 20. Uri2+ dependent [Trypanosoma brucei, notably in apo form as well as complexed with a magnesium ion and ATP [RNA ligases 2 consist of two discrete portions: the N-terminal nucleotidyltransferase domain (amino acids 1–234 in T4) and a C-terminal domain (amino acids 244–329 in T4) responsible for substrate specificity. The ligation reaction of RNA ligase 2 is ATP and Mgependent , 21, 22  and ATP .Diplonema. The model predicts intriguing innovations in the interaction network between ATP and the residues of the catalytic pocket, which are worthwhile to be tested experimentally by resolving the crystal structure. We discuss possible evolutionary scenarios that led to these innovations.In this study, we devise a strategy based on hidden Markov models (HMMs) and structural comparisons to identify proteins of large evolutionary distance to well-studied counterparts in model organisms. Comparative analysis of highly diverged homologs is particularly informative for identifying functionally and structurally important residues that are under elevated selective pressure. Employing this analytic strategy, we identify the gene and model the structure and ligand interactions of a putative RNA ligase 2 from D. papillatum display a low level of sequence similarity with homologs of other taxa, and are difficult to identify with tools based on sequence similarity such as BLAST [Diplonema draft genome assembly (version no. 2). Expression of this open reading frame was confirmed by RNAseq experiments. The corresponding transcript is poly-adenylated and its steady-state level is about 1/10 compared to the expression of Aspartyl tRNA synthase.In general, proteins of as BLAST . Therefoas BLAST , a modelTrypanosoma  and the heterolobosean Naegleria gruberi. Naegleria was chosen because heteroloboseans are the sistergroup of Euglenozoa, and because sequences of this taxon have not been used in building the PFAM HMM. Table E-values. Dp28902_3 has the lowest E-value with the PF09414 model, a value that is 107 times smaller than the second-best match, which was obtained with the HMM of ATP-dependent DNA ligases. Models for proteins that have a different fold  did not yield significant E-values (>0.05) for either Dp28902_3 or the RNA ligases 2 of Trypanosoma. Therefore, Dp28902_3 most likely belongs to the RNA ligase 2 family and will be referred to as DpRNL.For comparison, we also used HMMs for other RNA and DNA ligase super-families in searches against Dp28902_3 and RNA ligases 2 of Naegleria   located ria Fig. ; and strsii Fig. .Fig. 1DeDiplonema protein shows only low sequence similarity to established RNA ligases 2 . DpRNL lacks portions of two loops between domains III and IIIa (TbREL1 amino acid (aa) 163–166 and aa 176–205) that are distinctive for kinetoplastid RNA ligases 2, and that have been shown to interact with RNA [While the five subdomain motifs are well conserved across all RNA ligases 2 and readily recognizable in DpRNL, the rest of the N-terminal portion of the with RNA . Also miwith RNA . Finallyhttp://services.mbi.ucla.edu/SAVES/). Nearly all (96.1 %) amino acids have a stereochemical conformation in the “favored” or “allowed” regions of the Ramachandran plot. Only the seven most C-terminal residues are in an unfavorable environment according to the assessment by the tool Verify-3D [−3) is highly confident. The estimated TM-Score obtained from the standard output of I-Tasser was 0.70 ± 0.12. A TM-score >0.5 usually indicates a model of correct topology, and a TM-score <0.17 means a similarity no better than random. As a whole, the topology of the I-Tasser model of DpRNL is of good quality.The global three-dimensional 3D) model of DpRNL was predicted by I-Tasser   simulation. The Root Mean Square Deviation (RMSD) of the backbone α-carbon atoms remained stable after 10 ns of simulation with a mean of 4.2 Å  structure conservation during the simulation  and the lateral chain of two other residues (DpRNL_R41 with 86 % occupancy and DpRNL_S24 with 46 % occupancy), as well as between the lateral chains of DpRNL_Y203 and DpRNL_Q52. Additional stabilization of this domain comes from a hydrogen bond involving the carbonyl group of DpRNL_K202 in the main chain and the hydroxyl of DpRNL_S49. In conclusion, the 3D model of DpRNL is stable both at the 2D and 3D level. The observed flexibility parallels that of other RNA ligases 2 , providiTrypanosoma or phage T4. Pairwise structural comparison with experimentally confirmed structures , and the putative DNA ligase from Aquifex aeolicus . The subgroup clustering strongly suggests a duplication of RNA ligases 2 in the kinetoplastid branch prior to the speciation of Leishmania and Trypanosoma. In contrast, the phylogenetic position of DpRNL in the tree has virtually no support, and the observed affiliation with a homolog from Naegleria (heterolobosean) might be an artifact known as long-branch attraction [The moderate structural similarity of DpRNL with RNA ligases 2 from the diplonemid sister group raised questions about the phylogenetic relationship of these proteins. We focused our analyses on Excavate taxa, because a broader taxonomic sampling would have resulted in sequences too diverse for meaningful phylogenetic reconstruction. The inferred tree  and loop 2 (TbREL1 aa 190–200) had been predicted based on the crystal structure  and the Diplonema protein wrap themselves around the nucleic acid like a hook, corroborating the predicted position of the RNA substrate in the DpRNL model.In the Nucleotidyltransferase domain of the phage T4RNA ligase 2, RNA interaction is achieved by a patch of positively charged residues located in the exposed region of central beta sheets, as revealed by the crystal structure of T4RNL2 bound to a nicked nucleic acid duplex (PDB id 2HVR). To identify such regions in DpRNL, we computed the electrostatic potential at the solvent-accessible surface of the protein see . We foun2+ assumed stable positions in the catalytic pocket that correspond to those in the crystal structure [RNA ligases 2 typically bind ATP in a covalent fashion during the first step of the catalysis resulting in a ligase-AMP complex  followed by four replicates of unrestrained MD simulation during 35 ns. Second, we conducted three independent ATP-restrained productions of 15 ns, each followed by 30 ns unrestrained MD simulation in order to test whether ATP adopts each time the same position , and detailed molecular dynamics simulations are available . ATP intDiplonema protein the valine is replaced by a tyrosine (DpRNL_Y161), a residue determined as highly exceptional by comparative analysis (see above). This stabilizing interaction reduces greatly the degrees of freedom of the ATP molecule, and gives a significant turn to the interactions in the catalytic pocket by shifting the position of the ligand in DpRNL compared to well characterized RNA ligases. Additional ATP stabilization in DpRNL comes from two hydrogen bonds implicating the amine group of ATP. One hydrogen contacts the carbonyl group of DpRNL_E19  and the other the lateral chain of DpRNL_E18 .The phenylalanine (DpRNL_F101) and tyrosine (DpRNL_Y161), which together sequester the adenine base of ATP in the DpRNL model, establish a π-π stacking interaction with the substrate. This contrasts with the TbREL1 structure, where the base is enclosed by a sandwich composed of the aromatic ring of a phenylalanine , and a valine (TbREL1_V286). In the Diplonema protein  allowing the sugar moiety only little mobility. Four out of these five residues (except TbREL1_I59) are conserved in the ein Fig. , but onlIn the predicted predominant conformation of DpRNL, the triphosphate tail of ATP is stabilized by a network of interactions with three basic residues . In TbREL1, the triphosphate tail is held in place by five residues, TbREL1_I61, TbREL1_K87, TbREL1_R111, TbREL1_K307 and TbREL1_R309  plays a radically different role, rather forming hydrogen bridges with residues in the C-terminal region of the protein (maintained for 75.3 % of the frames). It should be stressed that all simulations with TbREL1 have been performed with a sequence lacking the C-terminal domain , so that interactions with the C-terminal domain are not known. In T4, the crystal structure of the adenylated full-length enzyme revealed a salt bridge between two residues of the C domain, R266 and D292, probably reinforcing its structural integrity .Finally, TbREL1_R309 as well interacts with the triphosphate tail of ATP, and in the homologous position of this residue, we find in the DpRNL model a valine (DpRNL_V177). However, this valine seems not to interact with ATP or any amino acid of the catalytic pocket. The functional homolog of TbREL1_R309 is rather DpRNL_R173. Note that both DpRNL_V177 and DpRNL_R173, are “exceptional” residues, and that a non-basic residue at the position corresponding to V177 in T4RNL2 was demonstrated to prevent ligation of ATP ). The imD. papillatum genome sequence. Detection of this candidate required the most sensitive HMM search method, because molecular sequences of diplonemids are in general highly divergent [In the search of an enzyme responsible for the unique trans-splicing in mitochondria of diplonemids, we identified a candidate RNA ligase 2 in the ivergent .Diplonema protein. To pinpoint deviant amino acids in the 3D model of DpRNL, we computed a score of exceptionality for each residue. The preliminary structural model was refined by first, eliminating structural inconsistencies and second, performing molecular dynamics simulation. The final model was compared with well-characterized RNA ligases 2.To confirm the sequence-based gene assignment, we constructed a preliminary 3D model of DpRNL that we aligned with RNA ligase 2 family members. Based on the structural sequence alignment, we delineated the boundaries of the predicted functional domains of the in-silico modeled 3D structure of the protein. Homology models built from a template that is very distant in sequence space are usually less reliable and tend to be biased toward the template. Even if the main chains of residues interacting with ATP are correctly placed in the DpRNL model, misplacement of their side chains may influence the simulation of ligand binding. To alleviate these difficulties, we have refined the homology model using extensive MD simulation, and have tested the resulting structure using several metrics . The predicted unusual ATP-binding mode in the Diplonema protein must be considered with this precautionary note in mind.Available information on how RNA ligases 2 interact with their substrate and ATP comes from crystal structure analysis and enzymatic assays of trypanosome TbREL1 and bacteriophage T4RNL2. In contrast, the presented ligand-binding mode of DpRNL was inferred from molecular dynamics simulations that were based on an The present model of DpRNL indicates a reorganization of residue-residue and residue-ATP interactions in the catalytic pocket compared to other ligases, entailing that (i) the ribose is less firmly stabilized than in TbTEL1 and T4RNL2, (ii) the conserved lysine DpRNL_K20 in motif I is pulled away from ATP, and (iii) ATP is now contacted by the conserved lysine DpRNL_K175 in motif V. Such a reshaping would most likely impact steps 1 and 2 of the catalysis  was made possible by the subsidiary presence of the lysine DpRNL_K175, which incidentally replaced the original catalytic lysine (DpRNL_K20). In this intermediary step, the system could have reverted back to its previous organization. Yet, the accumulation of mutations in a second step  led to a state with no way back, in the manner of a ratchet . Such a Diplonema but not their predicted function raises the question about the underlying selection pressure. Interestingly, the catalytic lysine of proven RNA ligases 2 , has been suggested to also interact with the RNA substrate, notably in the reaction step 3 [As mentioned before, two residues highly conserved at the structural level are predicted to have a different function in DpRNL compared to orthodox RNA ligases 2. These are the ubiquitous lysine (TbREL_K87|T4RNL2_K35) and arginine (TbREL_R111|T4RNL2_R55), which correspond in the structure alignment to DpRNL_K20 and DpRNL_R41, respectively Fig. . Conservn step 3 .Diplonema proteins. Therefore, we cannot extrapolate from TbREL the biological process in which DpRNL may be involved.We found that sequence- and structure-wise, mitochondrial RNA ligases 2 of kinetoplastids are not the closest homologs of DpRNL. Specifically, the 3D-structure model of DpRNL does not fit better the structure of TbREL compared to that of RNA ligases 2 from a bacteriophage or an archaean. Further, phylogenetic analysis of RNA ligases 2 did not group together the kinetoplastid and diplonemid proteins, but placed DpRNL without support next to a member of the heteroloboseans, a group that emerged prior to Euglenozoa. The large distance between kinetoplastid RNA ligases and DpRNL is probably due to a divergent, accelerated evolution and hyper-specialization of both the kinetoplastid and At present it is unknown whether or not DpRNL acts inside mitochondria. There is no recognizable signal in the inferred protein sequence indicative for import into mitochondria or any other subcellular localisation. After translation, DpRNL may either remain in the cytoplasm or be imported into mitochondria by one of the cryptic signals reported for proteins of several other eukaryotes . If DpRNin-silico analysis suggests that DpRNL activity relies on structure-function innovations not present in the commonly used RNA ligases, which might reveal suitable for future applications in biotechnology.RNA ligase 2 from bacteriophage T4 is widely used as a tool in molecular biology, in particular for massively parallelized RNA sequencing technologies. Enzyme versions have been engineered with higher efficiency and fidelity than the natural protein. Specifically, the truncated version of the RNA ligase from phage T4 produces less concatemer side products and is 10 times more active than the natural enzyme . FurtherD. papillatum nuclear genome obtained from a Mira V3.4.1.1 [Diplonema sequence matching a hypothetical protein (DpRNL). The identification of the domains characteristic for the RNA ligase 2 family was first performed by analysing the alignment of DpRNL with the PF09414 HMM domain in the HMMer result file, then by a multiple alignment of the two ligase paralogs from four Leishmania species  and six Trypanosoma species .We identified RNA ligase 2 in the draft version of the V3.4.1.1  assemblyV3.4.1.1  domain PV3.4.1.1 , 46 usinDiplonema candidate RNA ligase, (ii) the two well characterized RNA ligases from Trypanosoma brucei TREU927  downloaded from TriTrypDB [Naegleria gruberi (XP_002674912.1), a protist diverging basally to Euglenozoa.The specificity of PF09414 in detecting RNA ligases 2 was evaluated by comparing the score of all PFAM domains of DNA and RNA ligases against (i) the riTrypDB , and (iihttp://zhanglab.ccmb.med.umich.edu/I-TASSER/) [http://services.mbi.ucla.edu/SAVES/) and ModFold [The three-dimensional model of DpRNL has been determined by I-Tasser (the Iterative Threading Assembly Refinement program) web server (TASSER/)  using deTASSER/) , and eva ModFold . From th2+ (DpRNL_ATP+Mg2+). In this experiment, we superimposed DpRNL onto TbREL1, the Trypanosoma homolog of DpRNL crystallised with ATP (PDB ID = 1XDN), and manually copied the ATP and Mg2+ residues from 1XDN to the corresponding position in DpRNL. We added hydrogens when needed with WHATIF [Two different molecular dynamics simulation protocol were used for DpRNL. To investigate the stability of our model, we used the apo form of the protein (apo-DpRNL). To examine the interactions between the ligand and the protein, we used DpRNL with bound ATP and Mgh WHATIF  and rendh WHATIF .http://www.gromacs.org/Documentation/How-tos/Adding_a_Residue_to_a_Force_Field). Proteins and ligands were solvated in a cubic box of TIP3P water molecules at a distance of 3 nm (30 Å) from the solute. The net charge of the system was neutralized by addition of six chloride ions for the DpRNL apo system, four chloride ions for DpRNL+ATP+Mg2+ and five sodium ions for TbKREL1+ATP+Mg2+. The cut-off for short-range van der Waals and electrostatic interactions was 1.0 nm , and PME  was used for long-range interactions in all simulations. First, we performed an energy minimisation by steepest descent to remove possible spurious contacts until convergence to a maximum force of 1000 kJ/mol/nm on any atom of the system (850 steps). For all MD simulations, the leap-frog formula was used to integrate the equations of motion. Then two MD equilibrations of 100 ps each  were performed with restrained positions of protein and ligand. For the first NVT  equilibration, the temperature was set to 300K using the V-rescale thermostat [2+.All molecular dynamics (MD) simulations were performed with the Gromacs 4.0.5, 4.6.5, 5.0.1 and 5.0.2 software  and CHARermostat  with sepermostat , 57 was 2+, we performed MD simulations of 50 and 45 ns in total. During a preliminary simulation, ATP escaped from the catalytic pocket. Therefore, as a precaution, we restrained its position during the initial 15 ns of the production simulation (referred to as ATP-restrained phase), to let the protein equilibrate around the ligand, and after lifting the restriction, the simulation was continued. First, we used the same 15 ns ATP-restrained simulation (15R0) that we extended by four independent 35-ns MD simulations (replicates 15R0 + 35_1 to 15R0 + 35_4). Second, we ran three independent 15-ns restrained simulations  followed by 30 ns MD simulations. When measuring the distance between the two molecules during the initial time interval, we noted that the restraint was used in less than 1 % of the frames for all the predominant replicates  and in 18 % of the frames for the deviant replicate (15RIII)  = − Σi=16Pi(l) logPi(l) where i is the category of amino acid , and Pi(l) is the proportion of amino acids belonging to category i at position l. At a given position, amino-acid categories for which Pi(l) is null are ignored. If the entropy is low, then the position is conserved among family members. The entropy is set arbitrarily to 0 when the position in the multiple alignment contains more than 50 % gaps. We designed an exceptionality score S at position l for amino acids of DpRNL as Sl = (Pmax(l) - Pi(l)) / s(l) where Pi(l) is the proportion in the previously computed multiple alignment of the amino acid observed at position l for DpRNL, and Pmax(l) is the proportion of the most abundant amino-acid category (the category that we expect).In order to identify exceptional residues in the candidate RNA ligase of server/, ): we selserver/,  which reTrajectory analyses were performed with R , VMD 6262 and Pyhttp://www.poissonboltzmann.org/) [kBT/e (blue) and +5kBT/e (red). For DpRNL, this procedure was sufficient to reveal a large region with positive potential, having the propensity to bind RNA. In contrast, for TbREL1, the classical potential calculation  –69 and vg Delphi ) identifg Delphi .D. papillatum onto the contig carrying the gene. Library construction and read processing have been described earlier [The expression of the gene coding for DpRNL was assessed by mapping RNA-seq reads from a total-RNA library of  earlier . Cutadap earlier  was used earlier  onto the earlier . Alignme earlier .Diplonema. Sequences were aligned using MAFFT with option "--localpair" . The multiple alignment was refined by successive re-alignment of the sequences on a guiding hmm model built from the alignment with HMMer 3 [We identified RNA ligase 2 proteins in Excavata species by searching with the same PFAM HMM PF09414 as used for  HMMer 3 , 46. TheThe sequence of DpRNL is available under Genbank accession number KT828338. The 3D model is included as additional files in PDB format. Alignment is available on request.


Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export.The NXF1:NXT1 complex  is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains . It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a  Saccharomyces cerevisiae (S. cerevisiae (The export of mRNA from the nucleus through nuclear pores (NPCs) is the culmination of the nuclear phase of the eukaryotic gene expression pathway and releases mature mRNA transcripts into the cytoplasm for translation (reviewed by –3). The revisiae ,5. NXF1:revisiae . NXF1 isrevisiae ). NXT1/previsiae –10. Strurevisiae –13 have revisiae . Efficierevisiae .NXF1 transcript and thus the CTE-mediated enhancement of mRNA export is a mechanism also utilized by metazoans natively  need to have been completed before mature transcripts are exported to the cytoplasm for translation and these processes are coordinated by factors such as the TREX complex ,16. The natively .Xenopus oocytes has identified the RRM and LRR domains of NXF1 to be the minimal requirement for binding viral CTE-RNA  shows an ‘L’-shaped CTE-B was bound by the two NXF1 domains. Phosphate-backbone interactions dominated for the RRM domain, whereas some based flipped interactions were employed in binding to the LRR domain. The two NXF1 domains also formed an ‘L’-shaped configuration, forming a ‘mutual molecular embrace’  and NXT1 and show that it has a novel homodimeric domain-swapped configuration generated by a interaction between the linker between the LRR and NTF2L domains with NXT1. The resultant domain-swapped dimer generates a flat platform with 2-fold symmetry in which the three pairs of RNA-binding domains  are all arranged on one face that results in their being ideally placed to interact with a dimeric binding motif such as that found in the CTE. Structure-guided mutagenesis of residues identified to be important components of the dimerization interface impact on the CTE-dependent enhancement of mRNA export, consistent with the formation of this dimeric platform contributing to the binding CTE-RNA. Intriguingly, these engineered mutations do not impair bulk mRNA export indicating that NXF1:NXT1 appears to bind CTE-RNA differently from bulk transcripts.We present here the crystal structure of the complex formed between H. sapiens NXF1ΔNΔUBA (residues 96–555) and H. sapiens NXT1 were subcloned into the first and second multiple cloning site of the pETDuet-1 vector and co-expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells (Agilent Technologies) by Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction at 18°C for 16 h. Cells expressing NXF1ΔNΔUBA:NXT1 were lysed in 50 mM Tris–HCl pH 7.4, 500 mM NaCl, 20 mM imidazole pH 8.0. The lysate was clarified by centrifugation and the supernatant incubated with Ni-NTA beads for 1 h. The beads were pelleted by gravity flow filtration and washed with 60 ml of 50 mM Tris–HCl pH 7.4, 500 mM NaCl, 20 mM imidazole pH 8.0 after which the protein was eluted in 10 ml of the same buffer containing 250 mM imidazole pH 8.0. The His6 tag was removed by incubation overnight with TEV protease at 4°C. The NXF1ΔNΔUBA:NXT1 complex was purified to homogeneity by heparin affinity chromatography followed by gel filtration chromatography using a Superdex 200 column . Peak fractions containing pure stoichiometric NXF1ΔNΔUBA:NXT1 complex were pooled and concentrated and flash-frozen in liquid nitrogen and stored at −80°C in 20 mM Na–HEPES pH 8.0, 200 mM NaCl.PCR amplified in vitro transcription. 0.04 mg/ml T7 RNA polymerase was incubated with 100 ng/μl DNA template, 7.5 mM NTPs, 50 mM MgCl2, 1 mM spermidine, 80 mM Na–HEPES pH 7.5 at 37°C for 4 h. Hammerhead (HH) ribozyme and Hepatitis Delta Virus (HDV) ribozyme were introduced into the 5′ and 3′ of the target to ensure a homogenous RNA product. Full-length CTE-RNA was separated from the two ribozymes by preparative urea–PAGE followed by gel extraction by electroelution using an EluTrap system . Eluted RNA was concentrated and buffer exchanged using a Millipore spin concentrator. RNA was stored until required at −20°C.CTE-RNA was produced by ΔNΔUBA :NXT1 complex were grown at 18°C by sitting-drop vapor diffusion using 9% PEG 6000, 1 M LiCl, 0.1 M Na citrate buffer pH 5.1. Thin, blade-like crystals with P1 symmetry were harvested and cryo-cooled in mother liquor supplemented with 20% glycerol. Crystallographic data was collected at the Diamond Light Source  using beamline I04-1. Reflections were indexed and integrated using XDS  and the maximum sizes Dmax were computed using GNOM (I(0) of the samples obtained using Guinier analysis by AUTORG  was performed using an on-line HPLC system (Viscotek) equipped with Superdex 200 Increase 3.2/300 column  mounted on beamline BM29 at the European Synchrotron Radiation Facility . Data collection was conducted at 20°C, using a wavelength of 0.995 Å and a sample-to-detector distance of 1 m. Data were processed automatically by the on-line pipeline AUTOSUB . Pair ding GNOM  and moley AUTORG  with thag DAMMIF , withoutg DAMMIF . Envelopg DAMMIF .200 calorimeter . All proteins samples were buffer exchanged by dialysis into 20 mM Na–HEPES buffer pH 7.5, 200 mM KCl, 10 mM MgCl2. The RNA concentration in the injection syringe was 100 μM for CTE-RNA. The concentration of the NXF1ΔNΔUBA:NXT1 in the cell was 20 μM for the wild-type and 25 μM for the 3A mutant. The data were fitted using a one-site binding model using the Microcal ORIGIN software.ITC measurements were conducted using an Auto-ITCHEK293F cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection of plasmids was conducted as previously described . Luciferin situ hybridization  were constructed by introducing silent mutations to the siRNA target site (changing TCT ATC ATC ATC to agc ATt ATa ATt where lower case letters indicate the mutations introduced).HEK293F cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection of plasmids and siRNA were conducted as previously described, where the sequences of the siRNAs against dsRed and NXF1 are also given . Cells wdization  or HoechWestern blot analysis was conducted using standard methods using antibodies against NXF1 as previously described ,39. AntiΔNΔUBA:NXT1 was obtained from P1 symmetry crystals by molecular replacement using the structures of the LRR and NTF2L:NXT1 fragments  as search models. The asymmetric unit contained two copies of the complex that were arranged to form a domain-swapped dimer  and the multi-domain structure of S. cerevisiae Mex67ΔUBA:Mtr2 (PDB ID: 4WWU), was conserved in the present multi-domain structure. The interaction between the pre-α1 loop and the canonical copy of NXT1 was largely dominated by hydrophobic interactions centering around residue Leu370 of NXF1 with NXT1  that diIn addition to the interaction between the pre-α1 loop with the canonical NXT1 copy, the linker between the LRR and NTF2L domain also made contacts with the alternate non-canonical copy of NXT1 in a domain swapped manner Figure . Becausein vitro transcribed CTE-RNA and used to calculate a low-resolution envelope of the solution state of the molecule. Ab initio reconstructions using the DAMMIF package  and the NXF1 RRM and LRR domains ; PDB ID:. The sym package  produced package  that rou package . The env package .in vitro and also reduced the CTE-mediated enhancement of transcript export in dual-luciferase assays (accompanying manuscript). For simplicity, the region of the CTE linking the two NXF1-binding regions illustrated in Figure A striking feature of the model generated from the domain-swapped dimer was that the RNA-binding regions of the RRM, LRR and NTF2L domains were all arranged on the same face, opposite the regions that facilitate passage of the complex through the nuclear pores through interactions with the FG-nucleoporins that line the transport channel. Residues previously identified to form contacts with CTE-B in the RRM  and LRR domains  were found clustered on one face of the NXF1:NXT1 dimer . Alanine3A, NXF13D, NXF13R). These mutations were in regions not thought to be important with making direct contacts with RNA and so any impact they had on binding should instead reflect their altering the formation of the domain-swapped dimer. The expression levels of NXF1 mutants in HEK293F cells were comparable to that of wild-type and recombinantly purified protein from E. coli showed the ability to bind NXT1 was unaffected  were mutated to either alanine, aspartic acid or arginine  was fused with the CTE coding sequence and, in the presence of functional NXF1:NXT1, the transcript for RLuc would be exported preferentially over Firefly luciferase (FLuc) , the enhancement RLuc activity observed relative to GFP was less than a factor of 2  and the NXF1 RRM and LRR domains  indicateS. cerevisiae Mtr2 (E106G and R109G) disrupt pre-60S ribosomal subunit export but not bulk poly(A)+ RNA export. The observation that the disruption of NXF1:NXT1 dimerization inhibits CTE-mediated export, but not bulk poly(A)+ RNA may be an analogous situation in metazoans. The positioning of the LRR domain with respect to the NTF2L domain may be important for the export of a subset of highly structured RNAs such as the 5S rRNA in S. cerevisiae or the CTE-containing RNA for primates.It has been previously reported that mutations in in vivo.In summary, the 3.4 Å resolution crystal structure of NXF1:NXT1 has shown that this complex has the potential to form a novel domain-swapped dimer conformation that depends critically on interactions involving the pre-α1 loop and which generates a potential interaction platform in which all of the regions of the three RNA-binding domains of NXF1:NXT1 are located on one face. This arrangement is ideally suited to binding dimeric RNA motifs such as that of CTE-RNA and mutations engineered to impact on dimer formation reduce the nuclear export of CTE-RNA Coordinates and structure factors for the 3.4 Å resolution crystal structure of NXF1:NXT1 have been deposited in the Protein Data Bank with accession number: 4WYK.


Endometrial cancer is one of the most common malignancies in the female genital tract. Programmed cell death 5 (PDCD5) is a newly identified apoptosis related gene and plays an important role in the development of some human tumors. However, the expression and clinical significance of PDCD5 in endometrial cancer have not been fully elucidated. Here, we evaluated the expression of PDCD5 in endometrioid endometrial carcinoma and control endometrium by qRT-PCR, western blot and immunohistochemistry, and analyzed the associations of PDCD5 expression with clinicopathological parameters of patients. In addition, we detected the expression of PDCD5 in control endometrial glandular epithelial cells and endometrioid endometrial carcinoma-derived cell line KLE by immunocytochemistry. The results showed that PDCD5 protein mainly expressed in the cytoplasm of glandular epithelial cells and endometrial carcinoma cells, and there was a low level of PDCD5 expression in the nuclei of the above cells. Furthermore, PDCD5 protein level was significantly lower in endometrial carcinoma samples than that in control endometrium. The decreased PDCD5 expression was correlated with the tumor differentiation degree. It is clear that PDCD5 protein expression was lower in middle and low differentiated endometrial carcinoma compared with control endometrium and high differentiated endometrial carcinoma. However, there were no significant differences of PDCD5 expression between the proliferative phase and the secretory phase of control endometrium, as well as between high differentiated endometrial carcinoma and controls. The results were verified in control glandular epithelial cells and KLE cells by immunocytochemistry. Therefore, PDCD5 may play a key role in the pathogenesis of endometrial cancer and may be a novel target for diagnosis and treatment of endometrial cancer. Programmed cell death 5 (PDCD5) is a novel apoptosis accelerating gene which was first identified from the leukaemic cell clone TF-1 cells undergoing apoptosis in Human Disease Gene Center of Peking University. Therefore, it is also called TF-1 cell apoptosis-related gene 19 (TFAR19)   Fig. a, b.Fig.To verify the results of western blot, we next determine the levels of PDCD5 protein expression in 51 endometrioid endometrial carcinoma tissues and 53 control endometrium using IHC staining. In accordance with the results of western blot, the levels of PDCD5 protein were significantly decreased in endometrioid endometrial carcinoma tissues compared with control endometrium (P < 0.01) Figs. a, which The endometrial lining undergoes cyclic regeneration and displays menstrual cycle  under the influence of estrogen and progesterone. Among 53 cases of control endometrium, 30 cases were proliferate phase, 23 cases were secretory phase. However, our results showed that there were no significant differences in PDCD5 expression between proliferative phase and secretory phase of the control endometrium (P > 0.05) Figs. b.In addition, endometrioid endometrial carcinoma is divided into three types, such as high differentiation, middle differentiation and low differentiation according to their differentiation degree. Therefore, we further analyzed the differences in PDCD5 protein expression between control endometium and high differentiation or middle-low differentiation of endometrioid endometrial carcinoma samples. The results showed that PDCD5 protein expression was significantly decreased in middle-low differentiation of endometrioid endometrial carcinoma tissues compared with control endometrium (P < 0.001), but there was no obvious difference between high differentiation of endometrioid endometrial carcinoma tissues and control endometrium Figs. a–e, 4c.The results from IHC revealed PDCD5 protein was mainly located in the cytoplasm of the control endometrial glandular cells or endometrioid endometrial carcinoma cells, and there was also a low level of PDCD5 expression in the nuclei of these cells Fig. a–e. In oIn order to explore the clinical significance of PDCD5 expression in endometrioid endometrial carcinoma, we analyzed the relationships between PDCD5 expression levels and the clinicopathological parameters. As shown in Table PDCD5 was initially identified as a novel apoptosis-related gene  system (2009). Tumor differentiation degree, depth of myometrial invasion (MI), the expression of estrogen receptor (ER) and progestrone receptor (PR) were also evaluated. The basic characteristics of endometrioid endometrial carcinoma patients are listed in Table Total RNA was extracted from frozen tissues using a modified TRIzol  one-step extraction method. cDNA was synthesized using the FastQuant RT Kit  according to the manufacturer’s instructions. Quantitative real-time PCR was performed in 20 µL volume containing 10 µL of UltraSYBR Mixture , 0.2 µL of cDNA, and 1 µL of primer (PDCD5 and GADPH). The sequences of the primers are as follows: PDCD5: sense, 5′-GTT CTG GAT CAG TCG GCC C-3′, and antisense, 5′-TCG TCA TCT TCA TCA GAG TCC A-3′; GAPDH: sense, 5′-AAC GGA TTT GGT CGT ATT GGG-3′, and antisense, 5′-CCT GGA AGA TGG TGA TGG GAT-3′. Polymerase chain reaction was performed according to the following programs: denatured at 95 °C for 10 min, and then followed by 39 cycles of 95 °C for 15 s, 60 °C for 1 min and 65 °C for 5 s to stop the reaction. Each sample was conducted in triplicate. The results were analyzed using the 2 − ΔΔCt method.The proteins were extracted from freshly frozen endometrioid endometrial carcinoma and control endometrium tissues using Radio Immunoprecipitation Assay (RIPA) buffer containing 1 % Phenylmethanesulfonyl fluoride (PMSF) and 0.5 % phosphatase inhibitor (PI). Then the bicinchoninic acid protein assay kit  was used to measure the protein concentrations. Each sample (40 µg) was analyzed by 15 % sodium dodecyl sulfate–polyacrylamide gel and then transferred onto a PVDF membrane. Then the membrane was blocked by 5 % bovine serum albumin for 2 h. The membrane was incubated overnight at 4 °C with 1:1000 dillution of PDCD5  and β-actin  antibodies followed by horseradish peroxidase conjugated second antibodies  for 1 h at room temperature. After washing, the signals were visualized by an ECL  western blotting detection system according to the manufacturer’s instruction. Western blot was performed at least three times for each sample.The paraffin sections (4 µm) were dewaxed and rehydrated by infiltrating in dimethylbenzene twice and graded ethanols. After antigen microwave retrieval and endogenous peroxidase blocking, the slides were blocked by 10 % goat serum for 15 min at 37 °C. Then the sections were incubated with 1:1000 dillution of PDCD5 antibody  overnight in a wet chamber at 4 °C followed by secondary antibody conjugated with horseradish peroxidase  for 30 min at 37 °C. Then the expression of PDCD5 was visualized using diaminobenzidine (DAB) kit  and the nuclei were counterstained with hematoxylin. Equal volume of PBS was used instead of PDCD5 antibody and served as a negative control. Each sample was performed in duplicate.All IHC staining was analyzed by two experienced pathologists independently. The staining intensity was scored from 0 to 3 . Based on the percentage of positive cells, the staining extent was scored from 0 to 4 . For each slide, the two scores were combined to produce a final grade of PDCD5 expression: 0 (−), 1–3 (+), 4 and 6 (++), 8 and 9 (+++), 12 (++++). The expression of PDCD5 was defined as follows: −, + and ++ were classified as low and middle expression, whereas +++ and ++++ was graded as high expression.5 cells/well at 37 °C in 95 % air and 5 % CO2. To remove the non-attached cells, the culture medium was replaced with fresh medium 2 h later. The 38.5 mm monofilament cupreous mesh was washed thoroughly upside down with culture medium, then the cell suspension containing glandular epithelial cells was collected and centrifuged at 1000 rpm/min for 10 min. The pellet was resuspended and incubated in 24-well plates with cover slips at 5 × 104 cells/well at 37 °C in 95 % air and 5 % CO2.The glandular epithelial cells and stromal cells derived from secretory phase of control endometrium were isolated according to the method described previously  , cytokeratin (1:100)  and PDCD5 (1:1000)  overnight at 4 °C. The cells were washed with PBS and incubated with horseradish peroxidase-conjugated secondary antibodies  for 30 min at 37 °C. Then the protein expression was visualized using diaminobenzidine (DAB) kit  and the nuclei were counterstained with hematoxylin. Each sample was performed in duplicate.The human endometrioid endometrial carcinoma cells  were purchased from China Center for Type Culture Collection  and cultured in DMEM/F12 1:1  containing 10 % fetal bovine serum , 100 U/ml of penicillin and 100 U/ml of streptomycin. The cells were seeded in 24-well plates with cover slips at 5 × 102 test. And the GraphPad Prism 5  was used for statistical analysis. All data were expressed as mean ± SEM. P < 0.05 was considered statistically significant.The unpaired Student’s t test was used to analyze the expression of PDCD5 detected by qRT-PCR. The Gel-Pro analyzer was used to detect the grey value of the bands of PDCD5 expression detected by western blot, and then the unpaired Student’s t test was used for analysis. The immunohistochemical data was analyzed using chi


Choice effect is a robust phenomenon in which even “mere choice” that does not include actual choosing actions could result in more preference for the self-chosen objects over other-chosen objects. In the current research, we proposed that autonomy would impact the mere choice effect. We conducted two studies to examine the hypothesis. The results showed that the mere choice effect measured by Implicit Association Test (IAT) significantly decreased for participants with lower levels of trait autonomy (Study 1) and when participants were primed to experience autonomy deprivation (Study 2). The theoretical and practical implications are discussed. People make choices according to their preferences, indicating the important role that preferences play in choices. In addition, choice also has an impact on post-choice preferences. After the choice has been made, people’s liking of the chosen objects tends to increase while that of the rejected objects tends to decrease, known as the post-decisional spreading of alternatives . In otheOver the past decades, the cognitive dissonance theory and self-concept related theories have been widely used in explaining the mechanism underlying the choice-induced preferences. The main classical explanation is based on the cognitive dissonance theory , which aThe theory referring to the positive valence of self node helps to explain the choice effect as well . “Self nThe sense of autonomy refers to the extent to which people feel free to make their own decisions and experience a sense of volition in their actions . ChoosinWe speculate that the experience of autonomy may enhance the preference on self-chosen objects. Thus, we measured the relationship of trait autonomy and choice effect in Study 1 and propose the hypothesis: (1) trait autonomy is positively correlated with choice effect. We infer that autonomy may moderate the choice-preference link. When an individual takes a choosing action or is simply acknowledged that something has been chosen by himself/herself, the sense of autonomy is generated, which brings him/her positive feelings. These positive feelings in turn may enhance his/her positive evaluation to the surroundings. On the contrary, the lack of autonomy may reduce the preference for self-chosen objects. To our best knowledge, however, no study has provided empirical evidence for the role of autonomy in the choice effect. In Study 2, we investigated the influence of different levels of autonomy experience on the choice effect by using a priming task to set three conditions: the autonomy fulfillment condition, the autonomy deprivation condition, and the control condition. Here we propose the hypothesis: (2) the choice effect would occur in the autonomy fulfillment condition and in the control condition, but not in the autonomy deprivation; (3) autonomy deprivation would decrease or even eliminate the choice effect when compared with the autonomy fulfillment condition; (4) autonomy deprivation would decrease or even eliminate the choice effect when compared with the control condition; (5) autonomy fulfillment would increase the choice effect when compared with the control condition.In order to study the influence of autonomy on the choice effect while excluding the impact of cognitive dissonance, we employed a modified illusory choice paradigm, adapted from In measuring autonomy, we treated it as an individual difference variable . It can In Study 1, we used a scale to measure trait autonomy as an individual differential variable. Subsequently, we tested the mere choice effect using an IAT paradigm. We then calculated the relationship of trait autonomy and the mere choice effect.SD = 2.39, ranging from 19 to 25 years old) participated in the experiment for a cash reward (US$2). We asked all the participants to conduct an object chosen task in which they would see some texts in a computer screen and react by push some buttons on the keyboard. Each of them wrote informed written consent before the test. They were told there would be no any dangers while they were doing the experiment. They were told their rights and they can decide to or not to participate in this experiment, and they had the right to quit the experiment at any time of the experiment. This study was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Department of Psychology, Peking University.A total of 91 graduate and undergraduate students . The answers were coded such that lower scores indicated lower level of autonomy. Our data showed good internal consistency (Cronbach’s α = 0.697)The five-item Choicefullness Subscale of the Self Determination Scale  was usedTo evaluate the mere choice effect (an affect reflected the degree of preference on self-chosen objects over other-chosen objects), both of the two studies used a modified illusory choice paradigm developed by This part was completed on computers using the Inquisit laboratory software.In Step 1, participants read the following two-page scenario on the computer screen:Please visualize the following scenario. You and your friend (marked asthe otherin the experiment) bought six products in a supermarket for another friend: a mug, a small figurine, a piece of chocolate, a piece of candy, a pen, and a ruler. Please visualize and remember these products. They will be used in the following experiment (Page 1).Among these six products, three of them were chosen by you, and the other three were chosen by the friend (the other) shopping with you. You chose the mug, the chocolate, and the pen. Your friend chose the small figurine, the candy, and the ruler. Please spend 2 min to visualize and separately remember your choices and your friend’s choices. They will be used in the following experiment (Page 2).Half of the participants were shown the aforementioned scenario. The other half read similar instructions except that we swapped the products assigned to the self and the other.Then the participants began the modified illusory choice IAT . This IATable 1): attribute discrimination task , initial target-category discrimination task , initial combined task , reversed target-category discrimination task , reversed combined task .The IAT consisted of five classification tasks , participants were asked to press a left key (F) when a positive word appeared on the screen and a right key (J) for a negative word. Similarly, in the initial target-category discrimination task , objects chosen by the self (responding by pressing the left key) and objects chosen by the other (responding by pressing the right key) were discriminated. In the initial combined task , attribute and target discrimination trials were combined and participants had to press the left key when either a positive word or an object chosen by the self was presented and the right key when a negative word or an object chosen by the other was presented .We analyzed the data following the processes suggested by F = 4.023, p < 0.05, η2 = 0.046. Participants’ RT in the compatible condition  was faster than that in the incompatible condition . We suggest that participants preferred the self-chosen objects with positive descriptions and other-chosen objects with negative descriptions over other-chosen objects with positive descriptions and self-chosen objects with negative descriptions. In other words, compared with perceived other-chosen objects, perceived self-chosen objects were more strongly associated with positive than with negative words, indicating that people implicitly preferred self-chosen objects to other-chosen objects, despite their lack of actual experience of a choosing process, namely the mere choice effect. This result is consistent with the previous study of In the IAT task, the compatible condition was composed of self-chosen objects with positive descriptions and other-chosen objects with negative descriptions, while the incompatible condition was composed of self-chosen objects with negative descriptions and other-chosen objects with positive descriptions. The choicer-attitude valence compatible level (the compatible condition and the incompatible condition) was a within-subject variable. We conducted a one-way repeated ANOVA of choice-attitude valence compatibility level (compatible condition vs. incompatible condition), after controlling for gender and age. Results showed a significant main effect, Mean different RT = 129 ms, SD = 174 ms). Longer d-RT indicated a larger choice effect while shorter d-RT indicated a smaller choice effect. In the meantime, lower scorers on the five-item Choicefulness Subscale of the Self Determination Scale indicated lower level of trait autonomy, and higher scores indicated higher level of trait autonomy. The mean score of trait autonomy was 14.78, and standard deviation was 3.289.We used the difference response time (d-RT). It is the RT in the incompatible condition (other-chosen objects that were implicitly linked with positive words and self-chosen objects that were implicitly linked with negative words) minus the RT in the compatible condition (self-chosen objects that were implicitly linked with positive words and other-chosen objects that were implicitly linked with negative words) as the indicator of the choice effect , and the coefficient of determination change (delta R2) for each model are shown in Table  The results of regression analysis showed that after controlling for gender and age, the β of trait autonomy on choice effect represented by d-RT in the IAT task was 0.341, , which suggested a significant direct effect. Table 2 shows that trait autonomy explained incremental variance of d-RT in IAT (11.1%), p < 0.01, suggesting that people with a higher level of trait autonomy showed a larger choice effect. It should be noted that only the effect of trait autonomy on choice effect was obtained; the other two variables  were not significant predictors of the criteria. This above-mentioned result provides support for Hypothesis 1.Hypothesis 1 proposed that trait autonomy is positively correlated with choice effect. We examined the effect of trait autonomy on choice effect by controlling for gender and age in a hierarchical analysis. We conducted a hierarchical regression analysis by entering gender in a first block/model, age in a second block/model, and the trait autonomy as the independent variable in a third block/model. All variables were normalized as Study 2 is designed to extend the results of Study 1. According to the findings in Study 1, there is a positive correlation between trait autonomy and the choice effect. To further investigate the nature of this relationship, we examined whether the choice effect would remain when autonomy was deprived in a between-subject design. We aimed to test whether experimentally manipulated autonomy affects the choice effect. We repeated the steps of Study 1, except that we did not measure trait autonomy by questionnaire but manipulated the level of autonomy. In this study, the perceived autonomy was manipulated by a priming task, which comprised: an autonomy fulfillment condition, an autonomy deprivation recall condition, and a control condition.Mage = 22.3, SD = 1.9, range from 18 to 27 years old), all of them were university students. They were randomly assigned to the three experimental conditions. Twenty-two participants were assigned in the autonomy-fulfillment condition, 21 participants were assigned in the autonomy-deprived condition, and 21 participants were assigned in the control condition. All the participants were informed of conducting an object chosen task during the recruitment and before the experiment. Informed written consent was obtained from each participant before the test. They were told there would be no any dangers while they were doing the experiment in which they would see some texts in a computer screen and react by push some buttons on the keyboard. They were told their rights and they can decide to or not to participate in this experiment, and they had the right to quit the experiment at any time of the experiment. They were rewarded for about 2 US dollars for their participation. This study was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Department of Psychology, Peking University.Sixty-five participants “Now, please write down an event based on your real experience, in which your autonomy was satisfied. Please elaborate the details as much as possible, including the objective circumstances and your subjective feelings.”The first part of this session is story collection. Please describe an event about personal autonomy. Here, the autonomy is defined as when an individual is able to make their own choices freely, and experiences a sense of control over their decisions. “Now, please write down an event based on your real experience, in which your autonomy was deprived. That is to say, your behaviors were not completely controlled by yourself and some decisions were not self-decided. Please elaborate the details as much as possible, including the objective circumstances and your subjective feelings.”After completing the recall task, participants completed the Choicefulness subscale of the Self Determination Scale , as a maMage = 21.7, SD = 1.95, range from 18 to 26 years old).After applying the same data protocol used in Study 1, the final data analysis of Study 2 included 64 participants  = 3.190, p = 0.048, η2 = 0.044. After controlling for gender and age, the ANOVA showed that there was no significant difference of Choicefulness Scale scores between the autonomy fulfillment condition  and the control condition , F = 2.012, p = 0.164, η2 = 0.048. While participants scored significantly higher  on the Choicefulness Scale in the control condition than in the autonomy deprivation condition , F = 1.109, p = 0.299, η2 = 0.028. Participants scored significantly higher  in the autonomy fulfillment condition than those in the autonomy deprivation condition , F = 6.194, p = 0.017, η2 = 0.137. This result confirmed the validity of autonomy manipulation in autonomy fulfillment condition and autonomy deprivation condition. That is, compared with those in autonomy deprivation condition, participants in autonomy fulfillment condition experienced a higher level of autonomy (see Table 3).For the manipulation check of autonomy priming, a one-way ANOVA revealed that there was a significant main effect of the autonomy manipulation  on the Choicefulness Scale scores, Figure 1). The results showed a main effect: autonomy priming manipulation was significant, F = 5.341, p = 0.007, η2 = 0.15. The response time in the autonomy fulfillment group  was significantly longer than that in the control group , F = 7.406, p = 0.01, η2 = 0.155. The response time in the autonomy deprivation group  was also significantly longer than that in the control group , F = 8.477, p = 0.006, η2 = 0.174. The response time in the autonomy deprivation group  was not significantly longer than that in the autonomy fulfillment group , F = 0.142, p = 0.709, η2 = 0.003. These results indicated that autonomy priming (whether fulfillment or deprivation) led to slower participant RTs. A main effect of choice-attitude valence compatibility level on response time was significant, F = 11.877, p = 0.001, η2 = 0.15. The response time of incompatible trials  was significantly longer than that of compatible trials , indicating the conflict of objects and adjectives in the incompatible condition.To further identify how autonomy affects RT in compatible condition and incompatible condition, we analyzed a 3  by 2  mixed design, in which the autonomy priming manipulation was a between-subject variable and the choice-attitude valence compatible level was a within-subject variable. A two-way repeated ANOVA of autonomy priming manipulation and choicer-attitude valence compatible on response time was conducted, controlling for gender and age  = 4.550, p = 0.015, η2 = 0.11, indicating that the response time was modulated by the priming manipulation. Post hoc analyses showed that, in the autonomy fulfillment condition, participants responded significantly faster in compatible condition  than in the incompatible condition , F = 6.004, p = 0.024, η2 = 0.219, indicating the existence of the mere choice effect. For participants in the control condition, they responded significantly faster in the compatible condition  than in the incompatible condition , F = 8.787, p = 0.008, η2 = 0.255, which also indicates the presence of the mere choice effect. These two findings demonstrated that participants preferred the self-chosen objects with positive descriptions  and other-chosen objects with negative descriptions  over other-chosen objects with positive descriptions and self-chosen objects with negative descriptions. People implicitly preferred self-chosen objects to other-chosen objects even without owning those objects. The mere choice effect occurs, even without actually experiencing a choosing process.The interaction of priming manipulation and compatibility level was significant, M = 897 ms, SD = 259 ms) was not significantly different from that in the incompatible condition , F = 2.111, p = 0.163, η2 = 0.086, suggesting no mere choice effect. Here, the effect did not occur because there was no significant difference between compatible condition and the incompatible condition. The reference point is the participants’ RT in the compatible condition, and we compare this reference RT with RT in the incompatible condition. The choice effect appeared in the autonomy fulfillment condition and the control condition, but not in the autonomy deprivation condition. The result is consistent with our Hypothesis 2.For participants in the autonomy deprivation condition, their RT in the compatible condition  = 4.550, p = 0.015, η2 = 0.11.We also used the difference response time (d-RT) as the indicator of the choice effect. The longer d-RT represents the larger choice effect. We conducted a one-way ANOVA of priming perceived autonomy on choice effect indicated by d-RT in the IAT task, controlling for gender and age. As hypothesized, the main effect of perceived autonomy was significant, Md-RT = 145 ms, SD = 165 ms) significantly increased the choice effect compared to priming autonomy deprivation , t(61) = 2.698, p = 0.009, d = 0.843, indicating a significantly larger choice effect in the autonomy fulfillment condition than in the autonomy deprivation condition. Compared with autonomy fulfillment, autonomy deprivation decreased the choice effect. This result is consistent with Hypothesis 3. Participants primed in the control group  did not have a significantly larger choice effect compared to participants primed with autonomy deprivation , t(61) = 1.785, p = 0.075, d = 0.564, the trend did not reach significance. Here, we did not find supporting evidence for Hypothesis 4, which proposed that autonomy deprivation would decrease the choice effect when compared with the control condition. Participants primed with autonomy fulfillment  did not show a significantly larger choice effect compared to participants in the control group , t(61) = 0.892, p = 0.376, d = 0.279; the trend did not reach significance. Among the above effect sizes, the first one  is a fairly large effect. For Hypothesis 5, which stated that autonomy fulfillment would increase the choice effect when compared with the control condition, we neither found statistical support (see Table 4).We conducted the planned contrasts. By setting contrast coefficients, we can not only compare two means at once, but also combine multiple means from different levels to compute mean pair tests in these contrasts. Planned contrasts revealed that priming autonomy fulfillment , and the coefficient of determination change (delta R2) for each model are shown in Table  The results of regression analysis showed that after controlling for gender and age, the β of autonomy priming on choice effect represented by d-RT in the IAT task was.330, , which suggested a significant direct effect. Table 5 shows that autonomy priming explained incremental variance of d-RT in IAT (10.8%), p = 0.006, suggesting that participants with autonomy fulfillment showed a larger choice effect and supporting Hypothesis 2 (see Table 5).Similar to the data analysis procedure in Study 1, we examined the effect of autonomy priming on choice effect (indicated by the d-RT) in a regression model after controlling for gender and age. We entered gender in a first block/model, age in a second block/model, and the autonomy priming  as the independent variable in a third block/model. All variables were normalized as By using a modified illusory choice paradigm  and when it was experimentally manipulated (Study 2). In Study 1, the level of trait autonomy was positively related with the choice effect. In Study 2, when state autonomy was enhanced, participants displayed a larger choice effect. When primed by the autonomy fulfillment recalling task, participants rated their chosen objects as more favorably than the objects chosen by others. That is to say, the choice effect occurred after one’s state autonomy had been induced . Consistent with previous findings , we alsoChoice-induced preference has been a topic of longstanding interest in social psychology . In the The occurrence of the mere choice effect is possibly related to the many aspects of the self-concept, such as self-serving or self-protecting biases e.g., , self-enThe most intriguing and main finding in the current study is that this choice effect was affected by the sense of autonomy. As showed in the results, a lager choice effect was elicited in the participants that experienced state autonomy fulfillment rather than in those with no priming treatment, but the trend did not reach a significant level. In addition, the choice effect disappeared when participants experienced autonomy deprivation. In the perceived choice-preference link, people’s favorability on the self-chosen objects in the state autonomy fulfillment condition remains as high as in the control condition, whereas this favorability would be weakened and would even disappeared if they experienced autonomy deprivation. The evidence that trait autonomy is positively correlated with the choice effect is consistent with this finding. In a word, autonomy moderated the relationship between the perceived choice and the induced preference.The mere exercise of choice itself is assumed to provide a sense of autonomy e.g., . People One thing that needs to be pointed out in our objects evaluation IAT task is that participants were given the choosership and assigned to specific objects. That is, the perceived choice assigned to participants was not actually based on their free will. The autonomy induced by mere choice may be weaker than that induced by actual choice. Assuming that one’s trait autonomy is stable, although the subsequent object evaluation task may elicit autonomy, this level could be canceled out by the previously primed autonomy deprivation. When the sense of autonomy has been deprived, one’s intrinsic motivation and sense of control decreases  individual’s autonomy trait is positively correlated with the mere choice effect; (2) the experience of autonomy deprivation decreases the mere choice effect, which results in that people do not valuate self-chosen objects more favorably than other-chosen objects anymore. Our research provides good insights in the relationship between autonomy and the mere choice effect, and contributes to the theoretical understanding of the mechanism in choice-induced preferences.LW proposed the main research idea. TT, ZS, and LW made the research design. TT ran the experiments. TT and ZS did the statistic analysis. ZS and LW wrote 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.