We experimentally study competitive markets with socially responsible production. Our main focus is on the producers’ decision whether or not to reveal the degree of social responsibility of their product. Compared to two benchmark cases where either full transparency is enforced or no disclosure is possible, we show that voluntary and costless disclosure comes close to the full transparency benchmark. However, when the informational content of disclosure is imperfect, social responsibility in the market is significantly lower than under full transparency. Our results highlight an important role for transparent and standardized information about social externalities.The online version contains supplementary material available at 10.1007/s10683-022-09752-z. The social responsibility (SR) of products and their production processes is a central topic of many public controversies. Prime examples are the accusation of deficiencies in working conditions , damaging environmental effects  to be transferred to unicef in case he sells the product to a consumer. Donations do not influence the consumer’s monetary benefit from the product, but the vast majority of participants in our experiment consider unicef a charity worth supporting. Higher donations incur higher monetary costs for the seller.4In this paper, we experimentally study the emergence of SR in competitive markets where SR is not a directly visible characteristic of the product, but information about the SR of the product can be voluntary provided by its seller. In the experiment, our proxy for SR is a monetary transfer to the charity No Info) or fully informed (Full Info) about the donation associated with the product (i.e. the SR of the product). The degree of information (either none or full) is externally enforced and neither producers nor customers can change this. The Full Info case mirrors a situation in which, for example, regulation requires an unambiguous declaration of the SR characteristic of the product, e.g. the specification of the energy efficiency class of a light bulb or a fridge.To get a nuanced picture we study various scenarios in our experimental treatments. Our benchmark cases are that customers are either completely ignorant , but revelation is potentially imperfect. The Choice treatments vary the degree of the imperfection of this revelation. In treatment Choice-100 customers unambiguously see the information whenever a producer decides to reveal the SR information. This resembles the case of a (standardized) label and customers that are aware of what this label certifies . In treatments Choice-85 and Choice-60 the producer also decides whether or not to reveal the SR information, but - in case of a revelation decision - both the revelation decision and the revealed SR information may not reach the consumer (in Choice-85 there is a 85 percent chance that the revelation decision and SR information reaches consumers and there is a 60 percent chance in Choice-60). These two treatments are designed to map situations in which the producer faces an uncertainty whether his SR information reaches the consumers. Reasons for this uncertainty may be that consumers do not get the informational content of the provided information, e.g., due to a lack of standardization or limited attention of consumers. Hence, in treatments Choice-85 and Choice-60, a consumer facing non-disclosed information does not know whether the producer chose not to disclose or whether he disclosed, but the information was not transmitted. This means that while strategically sophisticated buyers should interpret undisclosed donations in Choice-100 as zero donations, this is no longer the case in Choice-85 and Choice-60. The unreliability of disclosure provides strategic incentives to producers to offer products with a lower level of social responsibility compared to Choice-100 where buyers can rely on non-disclosure being fully reliable.Three further No Info). We find that when voluntary disclosure provides unambiguous information for customers (Choice-100), the level of social responsibility comes close to the outcome under full and externally enforced disclosure (Full Info). While this is, in general, positive news when trying to establish more socially responsible markets, our results for the cases where disclosure is imperfect show the limitations of such an approach. When producers cannot be sure that the provided SR information has full informational content for consumers (Choice-85 and Choice-60), SR in production is significantly lower compared to Full Info. Yet, since our data does not provide an unambiguous statistical difference between the three choice treatments, the sensitivity of social responsibility to changes in the disclosure probability cannot be precisely determined. Furthermore, we show that producers benefit from more opaque information environments via higher profits. They produce products at lower cost, while selling them with a higher mark-up, taking advantage of the reduced competition in the market.In a nutshell, our results show that disclosure of SR information has large and positive effects compared to a regime where producers are unable to disclose the social responsibility of their products to consumers , yet higher donations incur higher monetary costs c(d) for the seller, as shown by the following table:7Participants in our experiment interact on markets with four sellers and two buyers. Each participant receives an endowment of unicef is attractive for at least two reasons. First, the donation to unicef is—as confirmed through a questionnaire at the end of the experiment  observe the donation decisions and levels of all products as well as the products’ prices.We implement five treatments which vary the information about the donation. There are two benchmark treatments, one where information on each product’s associated donation is fully disclosed and one where the information is fully hidden: In the Choice treatments where information can be disclosed by sellers voluntarily. The three treatments differ in the informational content provided. In treatment Choice-100, whenever sellers decide to reveal the level of donation d, customers unambiguously see the production information, i.e., d is revealed with probability 100%. In the treatments Choice-85 and Choice-60, the revelation decision of the seller is implemented with probability 85% and 60%, respectively. Thus, when a seller in a given period decides to reveal his donation to the buyers, a random draw by the computer determines whether the decision to reveal and the donation associated with the product are visible to the buyers (and the other sellers) or not. In all three Choice treatments, the decision not to reveal is always implemented with certainty.9In addition, we run three The markets in which buyers and sellers interact retain the same composition throughout, that is, there is no re-matching across periods, and participants are aware of this. After all decisions within a round have been made, all participants are informed about their earnings. In addition, throughout the experiment, every subject has access to the full information of his/her past behavior, i.e., prices, earnings and (if known) generated donations. Also, all buyers and sellers are identified through letters and behavior can thus be traced across rounds.10unicef at the day of the experiment. Since donations are both the result of market interaction and sometimes private information (and we wanted to keep this private), we provide all participants with a session average of the donations. This strikes a balance between not revealing much about the individual behavior but at the same time maintaining credibility about our procedures. We furthermore send proof of the donation to all participants (unless they told us they preferred not to receive such an email).To calculate the total earnings of the participants, the earnings from all periods are added up and converted into Euros at the rate of 130 points = 1 EUR. The same conversion rate applies to donations. These donations are transferred to unicef were 68.15 EUR per session.We implemented 12 markets per treatment, leading to 360 participants overall. After the 30 periods of the main experiment, subjects answered a short questionnaire which elicited perceptions about unicef and donation behavior outside the lab , as well as demographics such as age, gender and subject of study. The experiments were run at the Cologne Experimental Economics Laboratory (CLER) between May 2017 and October 2018. Recruiting was done via ORSEE Greiner  and the Full Info and Choice-100. For a detailed description, including those aspects in which the replication study differs from the initial study, see Online Appendix A.5.In this experiment we are powered to detect effects of around one standard deviation (or larger) with 12 markets per treatment condition  than in No Info.In this section, we derive the predictions for our experiment. We start by considering treatments No Info vs. Full Info) Forcing disclosure of the donation leads to a higher level of social responsibility compared to the case where donations are not visible for buyers. Due to the increased production cost, market prices will be higher when donations are disclosed. Making disclosure of donations voluntary leads to the same market outcome as when sellers are forced to reveal this information. Donations and market prices are identical in treatments Choice-100 and Full Info. choose a low donation and to not disclose. As a countervailing force to this mechanism, however, it also does not seem a profitable strategy for sellers to always choose to not disclose. Since the probability of disclosure is fairly high in both treatments, a seller who decides to disclose a high donation - and this decision actually reaches the buyers - is likely to have a competitive advantage, especially if competing offers have their donation hidden.Intuitively, we thus predict that such an unreliability in disclosure allows sellers to strategically exploit this more opaque environment. As buyers have to entertain the possibility that an undisclosed donation may in fact also be a high donation, they should, compared to Full Info benchmark. Furthermore, since, as outlined in the previous paragraph, sellers can use the option to disclose strategically in order to exploit the inherent unreliability, we expect that this softens competition between sellers and allows them to charge higher prices, compared to treatment Full Info. We do not make predictions as to whether these effects are larger in treatment Choice-85 or in Choice-60 as this would require a formal model which is beyond the scope of this paper.Taking these effects together, we predict that the possibilities for sellers to exploit the limited informational value of non-disclosure lead to lower donations compared to the Choice-85/Choice-60 vs. Full Info/Choice-100) When donations are voluntary but donation decisions of sellers have limited informational value, donations are lower and prices are higher compared to when there is perfect information.(Choice treatments) the disclosure choices. In Sect. We divide the presentation of the results in several parts. First, in Sect. Table Full Info are almost five times larger than those in No Info: When donations are always observed by buyers, their purchase decisions generate donations of 62.1 points, compared to 11.5 points when donation levels are private information of an individual seller . In Full Info), donations are almost five times larger than when buyers do not observe it (No Info). This confirms Prediction When the social dimension of products is visible to buyers , whereas the share of accepted offers with the maximum donation level of 100 is 17.6 percentage points lower (44.2% vs. 26.6%).Mirroring the comparison between Full Info and Choice-100 with 27 markets per treatment condition . In this replication average donations in the two treatments turn out to be very similar and we find no significant difference between the two conditions , donations are not significantly different compared to the case where sellers are forced to do so (Full Info). This supports Prediction When sellers can choose whether to reveal the social impact of their products to buyers and disclosure is perfect  and -27.0 (Choice-60).Prediction Choice-85, 11.9% of realized donations yield the highest donation, while 39.9% of accepted offers do not generate any donation at all. These shares are about halfway between No Info and Choice-100. For Choice-60, the share of realized donations of zero is at 36.0% and therefore comparable to behavior in Choice-85. Perhaps surprisingly, the share of maximal donations is substantially higher, at 23.3%. We discuss possible reasons for this in Sect. We can furthermore see a similar bimodality in donations as in the other treatments. In Choice-85 and by 35.5% in treatment Choice-60, compared to when sellers are forced to do so and there is full transparency (Full Info). This supports Prediction When sellers can choose to reveal the social impact of their product, but the informational content of the choice to disclose is limited, donations decrease by 56.1% in treatment Choice-100 and in the two imperfect disclosure treatments we do not establish a significant difference when using the non-parametric test. Yet, the regression approach in Table A1 in the Online Appendix indicates a significant treatment effect of -19.3  compared to the other two , though this difference is not statistically significant. Of course, in these two treatments, the intentions of the sellers do not correspond to the share of disclosed donations in the market. In Choice-85, 55.6% of offers feature a visible donation, for Choice-60 this number drops to 39.2%.Next, we analyze the disclosure behavior of sellers. Across the three As discussed in Sect. Full Info and 99.7% of cases in No Info). Remarkably, however, there is no significant difference in market prices between the two treatments. While in treatment Full Info a product is sold at an average price of 40.6, in No Info, buyers pay on average 39.5 .Market prices in No Info, followed by Choice-60 and Choice-85, which, in turn, slightly, but not significantly, outperforms the other two cases where disclosure leads to perfect information about the level of social responsibility associated with the product.18Comparing profits across all five treatments, it is noteworthy that they can be ranked according to their informational content. In particular, seller profits are highest in The results from the previous two subsections provide evidence that the disclosure (and its informativeness) of the social responsibility of products plays a crucial role both for the amount of social responsibility obtained in the market as well as for the profitability of firms competing. The goal of this subsection is to analyze the mechanisms behind the results and to highlight the implications for policy interventions, such as regulation. The discussion is guided by four questions opened up by the results.No Info higher than in Full Info?Why are seller profits in Our two treatments where disclosure of social responsibility is exogenously determined reveal a meaningful difference both in the degree of social responsibility in the market as well as in seller profits. While the first is in line with the hypothesis, and indeed previous evidence  increment implies a 2.6-point increase in the price, Full Info only in 7.3% of cases buyers choose offers which are dominated by another offer with both a lower price and a higher observed donation, indicates that choosing offers with higher prices in No Info is not caused by erroneous behavior but stems from conscious decisions of buyers.19Strikingly, we find that in 30.6% of decisions, a buyer does not buy from the cheapest seller. Instead, she seems to deliberately favor a seller with a higher price, suggesting that she expects this seller to have chosen a higher donation. While it is indeed the case that in No Info sometimes buy higher-priced products because they firmly believe that they are able to correctly predict undisclosed donations, or whether other types of reasoning, such as motivated beliefs  play an important role as well. Yet, it is clear that this buyer behavior has consequences for the competitiveness of the market: that in No Info in almost one third of the cases a seller can sell his product although it is not the cheapest (while dominated choices are rare in Full Info) yields a reduced intensity of competition in No Info. This explains why market prices in No Info display no decreasing trend, while in Full Info the steady decline of market prices suggests that sellers actively try to undercut each other.It remains speculative whether buyers in Full Info, buyers who hold a positive view about the charity generate almost three times more average donations than those who do not informative labeling and its effect on market outcomes, we predict that if there are no possibilities for sellers to inform consumers about the social responsibility of their products, e.g., because this dimension is too complex to convey, this has additional anti-competitive effects. Since consumers would have to make inferences about product characteristics that they care about but have no way of finding out, they mistakenly infer too much from prices, hampering competition and leading to higher prices.How does limited transparency affect donations when disclosure is voluntary?Choice-100 come close to Full Info (Result 2) and that donations in Choice-85 and Choice-60 decrease compared to Full Info (Result 3) suggest that the decrease in donations is not caused by the fact that disclosure is voluntary, but by the limited informational content of disclosure. However, as described above, our data remains inconclusive when comparing donations in Choice-85 and Choice-60 to donations in Choice-100. In particular, we cannot say by how much the observed decline in social responsibility between Full Info and Choice-60 and Choice-85, respectively, can be attributed to the effect of making disclosure voluntary and by how much to the additional effect of a decrease in the reliability of information. One reason for this is that our initial data, as becomes clear when looking at Fig. Choice-100. Therefore, in order to shed more light on whether donations in Choice-100 are indeed different from Full Info - which would indicate that it is voluntary disclosure per se which decreases donations - we conducted a high powered replication of treatments Choice-100 and Full Info. As already described in Sect.Our findings that donations in Full Info and Choice-100. The effect of reducing the disclosure probability from 100% (Choice-100) to 85% (Choice-85) is estimated to cause a reduction in donations of 26.5 points (Choice-100) to 60% (Choice-60) is estimated to decrease donations by 16.0 points  and sellers earn significantly more in Choice-60 than in treatments Choice-100 and Full Info. Seller earnings in Choice-85, however, are not significantly different from those in Full Info and Choice-100, respectivelyA more detailed analysis of the two imperfect disclosure treatments can provide further insights into how conditions which make it hard for firms to provide easily-interpretable and unambiguous information about the social responsibility of their products to consumers, affect market outcomes. As discussed above, prices in Choice-60 do consumers know the donation level. In these cases, the decision which product to buy bears some resemblance to the No Info treatment in the sense that buyers might engage in inferences about the donation level based on the prices. Corroborating our earlier finding for the No Info treatment, we now find that in 13.8% of decisions in Choice-60, a buyer buys a product with a hidden donation which is not the cheapest among those with a hidden donation. In comparison, in Choice-85 this only happens in 4.3% of cases.To make sense of these results, note that given the disclosure decisions of the sellers and the imperfect implementation probability of 60%, only for 39% of offers in Choice-60 is similar to No Info in the sense that buyers try to predict donation levels for those donations they do not observe. The difference between the two treatments is that in Choice-60 buyers do sometimes get information about seller behavior. This might allow sellers to create a reputation for being a socially responsible seller (remember that sellers’ identities are known to buyers and do not change over time). This may explain why there are relatively many donations of 100 in this treatment, compared to Choice-85 (23.3% vs. 11.9%). At the same time, having developed a reputation for being socially responsible could create leeway for sellers to charge higher prices, consistent with what we observe in the data. We discuss the role that reputation plays in the Choice treatments in more detail below.This suggests that behavior in Choice-treatments?What role does reputation play in the Choice-treatments a buyer will regularly be confronted with offers with hidden donations. But, since sellers are identified through letters A to D in the experiment, buyers have the possibility to track seller behavior across periods. This might allow them to make inferences about hidden donations based on sellers’ past behavior. At the same time, sellers might therefore be able to develop a reputation for being, for example, a socially responsible seller. Buyers might then be more likely to also buy from such a seller even if the donation is hidden in some of the periods.In the three p and a donation d is given by In order to analyze whether reputation plays a role in our markets, we estimate, similar to Bartling et al. , a condiOur main interest, however, lies in using this conditional choice model for quantifying a potential effect of reputation. To do that, we include an additional variable into this model which captures how often a buyer previously bought from a specific seller. More precisely, for each alternative available to a buyer we calculate the share of offers she accepted from this specific seller in the past. Hence, controlling for the price and donation of a given offer, we check whether a buyer is more likely to buy from a seller he already bought more from in the past.Choice-60 treatment. To quantify this effect, a ten percentage points increase in the share of offers bought from a seller is comparable in magnitude to a price increase of about one point. In Choice-85 and Choice-100, the effect is not significant.Table entclass1pt{minimaChoice-treatments where disclosure is imperfect. However, we note that our experiment was not designed to specifically analyze the role of reputation. It would be an interesting avenue for further research to analyze the role of reputation more thoroughly.Hence, corroborating our results on the dominated choices presented above, we do indeed find evidence that some form of reputation plays a role in those No Info). We find that when voluntary disclosure provides unambiguous information for customers (Choice-100), the level of social responsibility comes close to the outcome under full and externally enforced disclosure (Full Info). Yet, in the cases where producers cannot be sure that the provided SR information has full informational content for consumers (Choice-85 and Choice-60), SR in production is significantly lower, compared to Full Info. Yet, since our data does not provide an unambiguous statistical difference between the three choice treatments, the sensitivity of social responsibility to changes in the disclosure probability cannot be precisely determined.In this paper, we have analyzed the role of social responsibility (SR) in markets. We show that disclosure of SR information has large and positive effects compared to a regime where producers are unable to disclose the social responsibility of their products to consumers Supplementary file1 (PDF 289 kb)Below is the link to the electronic supplementary material.


Two N-terminal motifs, including a glutamine residue, are essential for conferring ACS activity to ACS-like proteins. Motif and activity analyses of ACS-like proteins from plants at different evolutionary stages suggest that the ACC-dependent pathway is uniquely developed in seed plants. A putative catalytic mechanism for the dual activities of ACSs is proposed on the basis of the crystal structure and biochemical data. These findings not only expand our current understanding of ACS functions but also provide novel insights into the evolutionary origin of ACS genes.Ethylene plays profound roles in plant development. The rate-limiting enzyme of ethylene biosynthesis is 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), which is generally believed to be a single-activity enzyme evolving from aspartate aminotransferases. Here, we demonstrate that, in addition to catalyzing the conversion of  During the evolution from aquatic algae to terrestrial gymnosperms and angiosperms, plants underwent tremendous and extremely complex changes to adapt to the challenges of the terrestrial environment. To survive and reproduce, plants must change their growth, development, and metabolism accordingly. Gaseous ethylene is a very ancient phytohormone that exerts profound effects on many aspects of plant growth and development. It is also indispensable for plants to deal with a range of biotic and abiotic stresses (S-adenosyl-methionine (SAM), which is further converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS), and finally, ACC is oxidized to ethylene by ACC oxidase. Among these, the conversion from SAM to ACC represents the first committed and rate-limiting step of ethylene biosynthesis. The ethylene biosynthesis pathway of seed plants is often called the ACC-dependent pathway. The ACS proteins in seed plants are encoded by a multigene family. All ACS proteins contain a conserved aspartate aminotransferase (AAT)–like domain and belong to the α superfamily of pyridoxal-5′-phosphate (PLP)–dependent enzymes  in the moss Physcomitrella patens genome and found that neither of their encoded proteins had ACS activity  (Arabidopsis ACS7 (AtACS7), we found that, in addition to catalyzing the formation of the ethylene precursor ACC, genuine ACSs widely have Cβ-S lyase activity. Two N-terminal motifs and a glutamine residue were found to be essential for conferring ACS activity to ACS-like proteins during the evolutionary process.Here, starting with Arabidopsis, catalyzing the conversion of SAM to ACC through α,γ-elimination  tags. Neither the Cβ-S lyase nor the ACS activity of AtACS7 was affected by the type or position of the epitope tags , a competitive inhibitor of PLP-dependent enzymes, on the C of 5 μM .l-cystine as a substrate, AtACS7 achieved its maximal Cβ-S lyase activity at approximately 30°C  at 2.20-Å resolution . There are four AtACS7-PPG complexes that make up two dimers in each asymmetric unit. The PPG molecules showed good electron density, except for the flexible aminoethoxyvinyl groups (fig. S2A). The two dimers (chain A and B dimer and chain C and D dimer) exhibit almost identical structures, with a root mean square deviation (RMSD) of 0.76 Å, and both have the classic AAT-like folding  of 0.83 Å in the overall structure comparison and 0.176 Å in the active-site residue comparison. In particular, the side-chain atoms of the above-mentioned AtACS7 active-site residues, including Q98, nearly completely overlap with their corresponding MdACS1 residues.Since apple MdACS1 has been suggested to have ACS and very low aminotransferase dual activities , with thβ-S lyase activity. As expected, MdACS1 indeed had both ACS and Cβ-S lyase activities in vitro, although both were lower than AtACS7  . The asymmetric unit contains one AtACS7-R6 dimer, and each AtACS7-R6 monomer forms a complex with the PPG molecule. The structures of these two subunits are nearly the same, with an RMSD of 0.12 Å. Compared with the wild-type AtACS7 dimer, the structure of the AtACS7-R6 mutant overall does not change much (with an RMSD of only 0.59 Å), except that the region replaced by PpACL1 BOX2 and adjacent region (residues 91 to 104) become disordered . Such a e AtACS7 . When trArabidopsis, tomato, and apple (table S2). The results showed that only functional ACSs had the glutamine residue corresponding to Q98, further supporting the importance of this residue for ACS activity  under the mediation of the lysine residue corresponding to K285 of AtACS7, thereby generating an external aldimine  analysis using ACSs from urselves  with Cβ-terminus .β-S lyase PpACL1 has seven C-terminal ACS motifs but lacks the two N-terminal ones  and determined their ACS activities by measuring ACC contents in the supernatants of the transformed bacterial cultures as described in Materials and Methods  Genuine ACSs with aminotransferase activity are very rare in seed plants. So far, the only example of ACC synthase with aminotransferase activity is apple MdACS1 (β-S lyase activity. (ii) Neither of the two ACS-like proteins in the moss P. patens exhibits ACS activity, and PpACL1 functions as a Cβ-S lyase . Furthermore, both ACS-like proteins in Marchantia lack the key glutamine residue corresponding to the Q98 of AtACS7  profile. Subsequently, HMMER (http://hmmer.org/download.html) searches against the above-mentioned plant proteomes (table S4) were performed. All putative ACS proteins sequences obtained were evaluated and confirmed using the NCBI Conserved Domain Database (https://ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Transcript sequences of the confirmed ACS protein sequences were retrieved on the basis of the locus numbers.To identify ACS homologs from the sequenced genomes of land plants, lycophyte and ferns, liverworts, charophytes, and chlorophytes listed in ACS homologous genes were either cloned from the cDNA of the corresponding plant species or chemically synthesized at the Beijing Genomics Institute. Fragments in TA cloning vectors were then transferred into pET-28a or pGEX-6p-1 expression vector by the routine digestion and ligation method. The recombinant plasmids were then sequenced for confirmation and transformed into expression host E. coli BL21 [Rosetta 2 (DE3) plysS]. Construction details for each gene are presented in table S5. Recombinant proteins were purified by affinity chromatography using HisTrap FF columns or Glutathione Sepharose 4 Fast Flow  columns according to the manufacturer’s instructions.HisTrap affinity chromatography–purified AtACS7 or AtACS7-R6 mutant protein was further purified with ion exchange (Hitrap Q HP) and gel filtration (Superdex 200) chromatography. The purified protein was concentrated to 10 mg/ml in 20 mM tris (pH 8.0) and 150 mM NaCl. Before crystallization, 1 mM PLP and AVG were supplemented to the protein solutions. Using the hanging drop vapor diffusion method, AtACS7 was crystallized in 0.1 M tris (pH 8.6), 23% (w/v) polyethylene glycol (PEG)–3350, and the AtACS7-R6 mutant was crystallized in 0.1 M Hepes (pH 7.6), 6% (w/v) PEG-10000. Diffraction data were collected at SSRF (Shanghai Synchrotron Radiation Facility) synchrotron and processed with the HKL2000 . After induction for 20 hours at 16°C, accumulations of these proteins were visualized on SDS–polyacrylamide gel electrophoresis gel. The cell cultures containing ACS-like protein with similar accumulation levels of AtACS7 were centrifuged at 10,000g. Aliquots of the supernatants were transferred to 20-ml GC vials and sealed with parafilm. ACC contents were indirectly determined by measuring ethylene product, as described in  leaves, Agrobacterium tumefaciens strain GV3101 harboring 35S:eYFP, Q98A-eYFP35S:AtACS7, or 35S:AtACS7-eYFP fusion gene was mixed with the strain containing P19 silencing suppressor and injected into the epidermis of the leaves of 5-week-old tobacco plants, as described previously (eYFP-only control culture (35S:eYFP) and the other half with an AtACS7 construct culture Q98A-eYFP(35S:AtACS7 or 35S:AtACS7-eYFP). After being incubated in the dark for 48 hours, five to six infiltrated leaves were pooled. ACC contents were measured as described in  medium and grown in plant growth chamber for 24 hours (22/19°C) in darkness after being sealed with caps. Ethylene accumulated in the vials were measured by GC (Agilent 7890A), and the rate of ethylene production was expressed as nanoliter per gram of seedlings (fresh weight) per 24 hour. All experiments were performed in three biological replicates.Ethylene emissions from 3-day-old etiolated β-S lyase activity was assayed using purified ACS proteins with a His or GST tag as described . The mixture was incubated at 30°C for 30 min before chloroform was added to denature proteins. The mixture was then centrifuged at 12,000 rpm for 10 min at 4°C. To quantify the pyruvate product, the supernatant recovered after centrifugation was mixed with 2,4-dinitrophenylhydrazine [0.1% (w/v) in 2 M HCl], and the reaction was stopped by adding 1.5 M NaOH. Then, the pyruvate content was determined by measuring the absorbance at 520 nm and comparing to a standard curve. To measure the reduction of l-cystine and production of NH4+, supernatant of the reaction mix mentioned above was purified by suction filtration  0.22 μm; syringe 2.5 ml) and analyzed using an amino acid analyzer  following the manufacturer’s instructions. Data shown for the in vitro Cβ-S lyase activity of purified GmACS7-like, OsACS5, OsACS1, SlACS4, and AtACS6 in Q98A, AtACS7N217A, and AtACS7D245N in β-S lyase activity of purified ACS7 with different tags in β-S lyase activity of purified AtACS8 were from five biological replicates. Data shown for the in vitro Cβ-S lyase activity of purified OsACS6 in In vitro CKm and Vmax of AtACS7 Cβ-S lyase were calculated using the Michaelis-Menten equation. Data presented in β-S lyase activities of these proteins using crude protein extracts as described in  leaves. The same infiltration extracts prepared for ACC content measurements were used to determine the pyruvate level in each treatment. Measurements of pyruvate contents were performed following the manufacturer’s instructions . Data shown here were based on the results of six biological replicates. For 35S:AtACS7-eGFP stable transgenic lines and their wild-type control, 3-day-old etiolated seedlings were harvested, and pyruvate contents were quantified using the same kit . The results presented were from three biological replicates.In planta Cβ-S lyases, and ACSs from a wide variety of organisms such as human, mouse, yeast, bacteria, protozoa, mosquito, barley, Arabidopsis, tomato, and apple (table S2) were aligned using MUSCLE . The phylogenetic tree was generated using the neighbor-joining method in MEGA X software  (http://meme-suite.org/tools/mast) (Motifs of the ACS proteins were identified statistically by the web-based MEME motif finder (


The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented, streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome, named ancient Northwest territories cripavirus (aNCV), was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. The aNCV IGR sequence adopts a secondary structure similar to contemporary IGR IRES structures, however, there are subtle differences including 105 nucleotides upstream of the IRES of unknown function. Using filter binding assays, we showed that the aNCV IRES could bind to purified ribosomes, and toeprinting analysis pinpointed the start site at a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR can direct translation in vitro in a PKI-dependent manner. Lastly, a chimeric infectious clone swapping in the aNCV IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework for the importance of this viral translational mechanism. Due to their limited genomes, viruses do not encode ribosomes nor a full complement of translation factors and as a result, they must hijack the host translation machinery to drive viral protein synthesis . Most eucis-acting elements, called internal ribosome entry sites (IRESs), which enable mRNAs to recruit ribosome internally and initiate translation  UTP (3000 Ci/mmol). The integrity and purity of RNAs were confirmed by agarose gel analysis. Uncapped bicistronic RNAs were first pre-folded by heating at 65 °C for 3 min, followed by the addition of 1xbuffer E  and slowly cooled at room temperature for 10 min. The pre-folded RNAs (20–40 ng/μL) were incubated in RRL containing 8 U Ribolock inhibitor , 20 μM amino acid mix minus methionine, 0.3 μL [35S]-methionine/cysteine , and 75 mM KOAc pH 7.5 at 30 °C for 1 hr. For the infectious clones, 2 μg RNA was incubated at 30 °C for 2 hr in Spodoptera frugiperda (Sf21) extract  in the presence of [35S]-methionine/cysteine (PerkinElmer) and an additional 40 mM KOAc and 0.5 mM MgCl2. The translated proteins were resolved using SDS-PAGE and analyzed by phosphor-imager analysis .Mono-cistronic and bicistronic reporters were linearized with NcoI and BamHI, respectively. pCrPV-3 and chimeric CrPV-aNCV clones were linearized with Eco53kI. RNAs were in vitro transcribed using a bacteriophage T7 RNA polymerase reaction and RNA was purified using a RNeasy Kit . Radiolabeled RNAs were bulk labelled by incorporating α-  is the concentration of RNAs, [B] is the concentration of ribosomes, [AB] is the concentration of RNAs bound to the ribosomes, fmax is the saturation point, and DK is the dissociation constant.RNAs  were preheated at 65 °C for 3 min, followed by the addition of 1xbuffer E and slowed cooling in a water bath preheated to 60 °C for 20 min. The pre-folded RNAs, with 50 ng/μL of non-competitor RNA, were incubated with an increasing amount of 40S subunits from 0.1 nM to 100 nM, and a 1.5-fold excess of 60S subunits for 20 min at room temperature. Non-competitor RNAs were in vitro transcribed from the pcDNA3 vector 880–948 nucleotides. Reactions were then loaded onto a Bio-Dot filtration apparatus  including a double membrane of nitrocellulose and nylon pre-washed with buffer E. Membranes were then washed three times with buffer E, dried, and the radioactivity was imaged and quantified by phosphor-imager analysis. The dissociation constant is determined by the formula,S] is 80S ribosome concentration, [R] is radiolabelled RNA concentration, θ is fraction of radiolabelled RNAs bound to ribosomes, [C] is competitor RNA concentration, DK and CK are dissociation constants of labelled RNAs and competitor RNAs, respectively.Bulk-labeled RNAs , IRES competitors, and non-competitor RNAs (50 ng/μL) were pre-folded in buffer E. Unlabeled IRES competitors were added in increasing concentrations from 2 nM to 250 nM. RNAs were then incubated with 6 nM 40S and 9 nM 60S subunits at room temperature for 20 min. Reactions were then loaded onto the Bio-Dot filtration apparatus, and data were fitted to the Linn-Riggs equation that describes competitive ligand binding to the target: 32P]-labeled RNAs  were pre-folded in buffer E and incubated with 0.6 μM 40S and 0.9 μM 60S at room temperature for 20 min. 1 μL of 1 U/μL of RNase I  was added to the mixture and incubated at 20 °C for 1 hr. RNAs without RNase I treatment were incubated at 20 °C with the same time length. RNAs from mixtures with or without RNase I treatment were TRIZOL-extracted and loaded onto 6% (w/v) polyacrylamide/8M urea gels to separate them. RNA Ladders (Thermo Fisher Scientific) were synthesized per manufacturer’s instruction. Gels were dried and imaged by phosphor-imager analysis. [32P] dATP , 8 mM MgOAc, in the final reaction volume. The reverse transcription reaction was incubated at 30 °C for 1 h, after which it was quenched by the addition of STOP solution . Following the reverse transcription reaction, the samples were extracted by phenol/chloroform (twice), chloroform alone (once), and ethanol precipitated. The cDNA was analyzed under denaturing conditions on 6% (w/v) polyacrylamide/8M urea gels, which were dried and subjected to phosphor-imager analysis.Toeprinting analysis of ribosomal complexes in RRL was performed as previously described . 0.4 µg 6 S2 cells using lipofectamine 2000 reagent  per the manufacturer’s instructions. 3 μg of in vitro-transcribed RNA derived from the pCrPV-3 or chimeric clones was transfected into 3 × 10CTATGGATCCATGGGAGAAGATCAGCAAAT-3′; tag is underlined) was used. Primer (5′-CTATGGATCCATGGGAGAAG-3′) and primer (5′-GTGGCTGAAATACTATCTCTGG-3′) were used for PCR amplification of the negative-sense strand of the CrPV genome. Rps6 was amplified using primers (5′-CGATATCCTCGGTGACGAGT-3′) and (5′-CCCTTCTTCAAGACGACCAG-3′). Viruses were passaged three times after transfection of viral RNAs in S2 cells. Total RNA was isolated from S2 cells using TRIzol reagent. RT was performed using 1 μg of RNA using LunaScript™ RT SuperMix Kit  per manufacturer’s instructions. For reverse transcription of the negative-sense CrPV viral RNA to detect replication, tagged primer . Equal amounts of protein lysate were resolved by SDS-PAGE and subsequently transferred onto polyvinylidene difluoride Immobilon-FL membrane . Following transfer, the membrane was blocked for 30 min at room temperature in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated for 1 h with rabbit polyclonal antibody raised against CrPV 1A  or CrPV VP2 . Membranes were washed three times with TBST and subsequently incubated with IRDye 800CW goat anti-rabbit IgG  for 1 h at room temperature.Nine representative untrimmed Type I IRES were aligned with muscle  and theiUsing computational and compensatory base-paring analysis, a secondary structure model of the aNCV IGR was predicted A. The aNBased on a maximum-likelihood phylogeny constructed from a nucleotide alignment informed by RNA structure C, the aND 0.4 nM) -methionine/cysteine to monitor protein expression level. Wild type but not mutant (mPKI) aNCV IGR IRES was active in RRL in vitro . In RRL, both wild type and mutant (Δ1-99) aNCV can direct IRES activity to similar levels B, indicaHaving demonstrated that the aNCV IGR can bind to ribosomes directly and drive IRES activity from a non-AUG codon, we next examined whether the aNCV can support infection in a more physiological system. Because only a partial sequence of the aNCV genome was recovered in the 700-year-old caribou feces , we usedTo determine whether the CrPV-aNCV clone is infectious, we transfected in vitro transcribed RNA into S2 cells and monitored viral protein expression by immunoblotting. Transfection of the wild-type CrPV and CrPV-aNCV containing the full-length aNCV IGR RNA resulted in expression of the CrPV non-structural protein 1A and the structural protein VP2 at 120 h post-transfection (h.p.i) C, which We attempted to propagate the CrPV-aNCV virus from transfected cells by reinfecting and passaging in naïve S2 cells. Despite multiple attempts, the CrPV-aNCV (Δ1-99) did not lead to productive virus as measured by viral titres. However, the CrPV-aNCV yielded productive virus (1.15 X 10^10 FFU/mL). We sequence verified that the aNCV IGR was intact in viral RNA isolated from the propagated CrPV-aNCV (data not shown). To further validate virus production, we infected S2 cells with CrPV-aNCV and monitored viral expression by immunoblotting. Similarly to that observed with CrPV infection, the CrPV 1A protein produced from CrPV-aNCV was detected at 2-10 h.p.i., albeit VP2 expression was reproducibly detected until 4 h.p.i., thus likely reflecting the decreased IRES activity of aNCV compared to CrPV E. In sumTracking back and identifying ancient RNA viruses may provide insights into the evolution and origin of present-day viruses including the mechanisms that permit virus translation, replication and host virus interactions. In this study, we have examined the IGR of an ancient dicistrovirus that was discovered from 700-year-old ice-preserved caribou feces. To our knowledge, the aNCV genome has not be identified in RNA metagenomic analysis; thus, this study is the first to characterize the oldest IRES to date and provides insights into the origins of this type of IRES. Using molecular and biochemical approaches, we demonstrate that the aNCV IGR possesses IRES activity using a mechanism that is similar to that found in present day Type IV dicistrovirus IRESs. The aNCV IGR can direct ribosome assembly directly and initiates translation from a non-AUG codon. The aNCV IGR IRES can support virus infection in a heterologous infectious clone; thus, a functional aNCV IGR IRES has been resurrected from an ancient RNA virus. The secondary structure model of the core aNCV IGR resembles classic Type I IGR IRESs containing all three PKs. However, there are subtle differences, including the L1.1 bulge region, responsible for 60S recruitment, which is comprised of a mixture of Type I and II IRESs elements. Additionally, aNCV IGR contains 105 nucleotides upstream of the core IGR IRES, within which several stem-loops are predicted. We showed that this upstream sequence is not required for aNCV IGR IRES translation  but is rTo date, there are increasing numbers of dicistrovirus-like genomes identified via metagenomic studies ,23. FromGiven that the aNCV IGR IRES structure resembles an IGR IRES found in contemporary dicistrovirus genomes, it was not surprising that it functions by a similar mechanism. Relatively speaking, the IGR IRES from a 700-year-old RNA viral genome is far from the presumed primordial IRES. However, this study provides proof-in-concept that this viral RNA translation mechanism can be resurrected and investigated to provide context in the evolution of viral mechanisms. The challenge in identifying more ancient RNA viral genomes and mechanisms has been and will always be in capturing intact RNA viral genomes as RNA, compared to DNA virus counterparts, are unstable and apt to degrade over time. The remarkable discoveries by Ng et al. and others of ancient RNA viral genomes ,27,28 pr


The correct repair of DNA double-strand breaks is essential for maintaining the stability of the genome, thus ensuring the survival and fitness of any living organism. Indeed, the repair of these lesions is a complicated affair, in which several pathways compete for the DNA ends in a complex balance. Thus, the fine-tuning of the DNA double-strand break repair pathway choice relies on the different regulatory layers that respond to environmental cues. Among those different tiers of regulation, RNA modifications have just emerged as a promising field. When DNA is damaged either physically or chemically, the integrity of the genomic information might be compromised. Therefore, living organisms have many repair pathways that try reverting the damaged DNA to the original DNA sequence or, at least, to minimize the impact of such changes. Particularly difficult-to-manage DNA lesions are DNA double-strand breaks (DSBs), which are produced when both DNA strands break at the same time in close proximity. Such DNA alteration triggers a very complex response, commonly known as the DNA damage response (DDR), that not only initiates the repair of the lesion but causes a complete upheaval of the cellular metabolism . In the Many different aspects of cellular biology impact the outcome of the DSB repair. Among them, it has become clear in recent years that the RNA itself and RNA-related proteins play some essential roles  or by the presence of DNA:RNA hybrids at the site of DNA breaks.There is extensive literature on the regulatory effects of different RNAs in DNA repair through terature . But addThe first piece of evidence in this regard was shown by de novo transcription around the DSB (de novo long lncRNAs. The production of these damage-induced non-coding RNAs (dincRNAs) due to de novo transcription at the sites of the breaks is supposed to be essential for the proper activation of the DDR , the ones that remove the modifications (erasers), and the ones that bind specifically to these modifications (readers) have been identified. The most prominent examples of RNA modifications, their writers, readers, and erasers, and their connection with DNA repair can be found in Ribosomal RNA (rRNA) and transfer RNA (tRNA) are heavily modified RNA molecules , but in writer complex modifies the RNA substrate. In the case of m6A, it is mostly dependent on the methyltransferase complex formed by METTL3 and METTL14  and ALKBH5 (reader. Five components of the YTH domain family (YTHDF1-3 and YTHDC1-2), insulin-like growth factor 2 mRNA-binding protein (IGF2BP), heterogeneous nuclear ribonucleoproteins A2/B1(HNRNPA2B1), and proline-rich and coiled-coil containing protein 2A (PRRC2A) can bind to RNAs bearing the m6A modification , arguably the best studied of them. First, a  METTL14  although METTL14 . In agred ALKBH5 . On the fication  and elicwriter factor, TRDMT1, at sites of DNA damage. Importantly, in the absence of TRDMT1, repair by HR is hampered (Recently, it has become clear that RNA modifications also affect DNA repair  Table 1Table 1. hampered  (Figure hampered .2O2 treatment, which causes DNA oxidation, causes specific changes in the patterns of N1-methyladenosine on RNA (eraser ALKBH3, an enzyme that has been involved in DNA repair (Less clear are the connections of other RNA modifications with the repair of the DNA molecule. He on RNA . Additioe on RNA . StrikinA repair . FurtherA repair , with RNA repair . Albeit A repair . The facA repair  opens thA repair .Human ALKBH8, which catalyzes methylation of tRNA to form mcm5 U, has also been connected with the DDR. Not only its expression is upregulated upon exposure to DNA damaging agents in an ATM0 dependent fashion but also its depletion sensitizes cells to MMS and bleomycin treatments . Interesin vitro by several ADP-ribosylhydrolases (Additionally, it has been recently demonstrated that RNA can be a target of reversible mono-ADP-ribosylation. Indeed, PARP10, PARP11, and PARP15, as well as a PARP homolog TRPT1, are able to ADP-ribosylate RNA-phosphorylated ends. This ADP-ribosylation of RNA is efficiently reversed drolases . PARylatdrolases . So, theAnother form of RNA modification is RNA editing, caused by the deamination of either C or A, to change the RNA sequence to either U or I, respectively. Unlike other RNA modifications, these are not reversible. However, on the other hand, it can elicit many different responses, such as changes in RNA stability, splicing, or even altering the coding sequence. In principle, every mammalian transcript can be subjected to RNA editing . InteresWhen RNA acts as a donor of information to fulfill HR, a DNA:RNA hybrid has to be formed. These DNA–RNA structures also form as a consequence of damage-induced transcription at DSBs sites. Thus, the formation, stability, and regulation of these DSBs-related R-loops play a central role in many aspects of the DNA repair process . As mentOne way to reconcile these observations is that R-loops might vary their impact on DNA end resection and HR, depending on modifications on the RNA moiety. Thus, RNA modifications of R-loops will bring an extra epitranscriptomic layer of control to DSB repair, affecting the balance between resection-mediated repair, such as HR, and those repair pathways that are resection-independent like NHEJ. This new level of regulation might rely on the stabilization/destabilization of R-loops at DSBs as a response to different cellular cues, such as DNA chromatin structure, cell cycle position, time after the break formation, etc. In this model, R-loops will initially block DNA end resection, providing an initial window of opportunity for NHEJ to act, but if the break cannot be repaired or in the presence of pro-HR signals, the modification of the RNA will facilitate its removal, allowing resection to proceed. Moreover, prior to DNA:RNA dissolution, such modifications could enhance the recruitment of HR proteins, thus explaining why some authors have seen a positive effect of R-loop formation in recombination. So, modifications such as the aforementioned m6A, m5C, or A-to-I editing will facilitate HR  Figure . To add Defective DNA repair is recognized as a hallmark of cancer cells, as it will increase the mutation rate associated with tumorigenesis . Strikinwriter METTL3 has been found to be overexpressed in several cancer types (For example, the m6A er types . METTL3-er types . But as The ADAR-mediated adenosine-to-inosine editing has also been linked to several types of cancers. High levels of ADAR1 suppress the immune response and promote cell growth in cancer cells. Therefore, targeting ADAR1 has been proposed as a potential strategy to prevent resistance to immunotherapy . On the SJ with the help of FB browsed the literature and collected the source data. SJ with PH wrote the manuscript. 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.


Arabidopsis endogenous genes, JMJ14, but not IBM1, counteracts DNA methylation of expressed transgenes. Additionally, JMJ14-mediated specific attenuation of transgene DNA methylation enhances the production of aberrant RNAs that readily induce systemic post-transcriptional transgene silencing (PTGS). Thus, the JMJ14 chromatin modifying complex maintains expressed transgenes in a probationary state of susceptibility to PTGS, suggesting that the host plant genome does not immediately accept expressed transgenes as being epigenetically the same as endogenous genes.Transgenes that are stably expressed in plant genomes over many generations could be assumed to behave epigenetically the same as endogenous genes. Here, we report that whereas the histone H3K9me2 demethylase IBM1, but not the histone H3K4me3 demethylase JMJ14, counteracts DNA methylation of  Accumulating evidences point to a discrepancy in the epigenetic behaviour of transgenes and endogenous genes. Here, via characterization of mutants impaired in histone demethylases JMJ14 and IBM1, the authors show that transgenes and endogenous genes are regulated by different epigenetic mechanisms in Arabidopsis. It occurs when double-stranded RNA (dsRNA) is formed by transcription of an inverted repeat, the copying of single-stranded RNA (ssRNA) by viral and host RNA-dependent RNA polymerases (RDRs), or the simultaneous transcription of both the positive and negative strands of DNA. DICER-like (DCL) proteins convert the dsRNA into microRNAs (miRNAs) or small-interfering RNAs (siRNAs), which then guide ARGONAUTE-like (AGO) proteins to mediate sequence-specific gene silencing.RNA-mediated gene silencing is a highly conserved eukaryotic mechanism that regulates endogenous gene expression and acts as a defense mechanism against viruses and transposons6. The process of de novo RdDM also requires the cytosine methyltransferase DRM27. Following the establishment of DNA methylation and through subsequent rounds of DNA replication, cytosine methylation at CG and CHG sites can be maintained in the absence of siRNA by the cytosine methyltransferases MET1, and CMT2 and CMT3, respectively, whereas maintenance of CHH methylation requires the continual presence of 24 nt siRNAs and DRM28.In plants, 24 nucleotide (nt) siRNAs guide transcriptional gene silencing (TGS) and RNA-directed DNA methylation (RdDM)9. Once PTGS is triggered, the biogenesis of 21 and 22 nt siRNAs usually extends along the entire length of the mRNA in a process known as transitivity12. Furthermore, once induced, PTGS moves systemically throughout the plant14.Abundant siRNAs are also derived from viral RNA and RNA Polymerase II (Pol II) transcripts that have been converted into dsRNA by viral or host RDRs. In plants, RDR6 plays the major role in producing these dsRNAs, which are processed by DCL4 and DCL2 into 21 nt and 22 nt siRNAs, respectively. These siRNAs form a complex with AGO1 and guide silencing of complementary RNA viruses and post-transcriptional gene silencing (PTGS) of complementary mRNAs16 or poly(A) tail17 induces RDR6-dependent PTGS in plants. The transcription of aberrant RNAs is a by-product of RNA Pol II transcription in all eukaryotes, but normally, aberrant RNAs are intercepted and removed by the highly conserved RNA Quality Control (RQC) pathway. In addition to removing aberrant RNAs, the RQC pathway modulates gene expression via mRNA turnover. The mechanism of mRNA turnover has been studied in much more detail than the fate of aberrantly transcribed RNA, and the first step in mRNA decay is the removal of the polyA tail, followed by the removal of the 5′ cap by the decapping complex, which is composed of DCP1, DCP2 and VARICOSE (VCS) in plants18. Once the stabilizing 5′ and 3′ modifications have been removed from the mRNA, the exposed RNA is degraded 5′-3′ by XRN exoribonucleases, and 3′-5′ by the exosome in all eukaryotes19. In Arabidopsis, dpc1, dcp2, vcs, xrn, and exosome mutations that compromise RQC allow aberrant RNA to accumulate and result in spontaneous RDR6-dependent PTGS of both transgenes24 and endogenous genes25.It is well established that over-expression of aberrant mRNAs lacking a 5′ cap27. Molecular analysis of jmj14 plants compared to wild-type (WT) plants revealed a decrease in transgene siRNA accumulation, a decrease in transgene H3K4m3 and an increase in CHG methylation at the transgene promoter28. Thusfar, DNA methylation has only been mechanistically implicated in TGS of promoters in most eukaryotic lineages7, inhibition of transcriptional elongation in fungi29, and most recently, inhibition of aberrant transcription from the gene body, i.e., the intragenic regions, of plants31 and mammalian embryonic stem cells32. Therefore, JMJ14 could link DNA methylation to aberrant RNAs and PTGS. However, the increase of transgene DNA methylation in jmj14 mutants contrasted the decreased methylation of CHH and CHG sites observed in two endogenous transposons in jmj14 mutants27, and resembled more the effect of ibm1 mutations on endogenous sequences. IBM1 encodes a histone H3K9me2 demethylase, and ibm1 mutants have been reported to cause increased CHG and CHH methylation in the gene body of endogenous genes35. Therefore, the effect of jmj14 and ibm1 mutations on the epigenetic features of transgenic versus endogenous sequences required further investigation.Mutants impaired in the histone H3K4me3 demethylase JMJ14 have been identified in two independent forward genetic screens for PTGS-deficient mutantsjmj14 mutant and show that very few endogenous loci exhibit a change in DNA methylation in the mutant compared to wild type, and the DNA methylation changes in the jmj14 mutant are restricted to genomic sequences that normally exhibit highly variable DNA methylation levels37. In contrast, we detect increased methylation at all sequence contexts  in transgenic sequences of the jmj14 mutant, but not the ibm1 mutant. We also show that JMJ14 promotes and IBM1 prevents transgene PTGS, respectively, and that the susceptibility of transgenes to PTGS correlates with low levels of DNA methylation in the transgene and high levels of transgene aberrant RNA, indicating that JMJ14 in combination with other chromatin modifying proteins plays a crucial role in establishing and maintaining stably expressed transgenes in an epigenetic state that is distinct from endogenous genes.In this work, we perform genome-wide DNA methylation analysis of a 10027-338, a genetic screen recovered three jmj14 mutants, #38, #90 and #148, that showed defects in root-to-shoot PTGS transmission  RNA11. Although it is not certain that this abSUG RNA is the aberrant RNA that triggers PTGS, its abundance correlates perfectly with the efficiency of PTGS in the two p35S:GUS reference lines 6b4 and L1. Indeed, it is detected at very low levels in 6b4 plants, which do not trigger PTGS spontaneously. It is also detected at only low levels in 6b4 xrn3 xrn4 and L1 plants, which trigger PTGS spontaneously with 100% efficiency, most likely because the abSUG RNA is degraded by PTGS, similar to GUS mRNA. However, abSUG RNA accumulates to higher levels in 6b4 xrn3 xrn4 rdr6 and L1 rdr6 plants, but remains low in 6b4 rdr6 plants. Given that the p35S:GUS transgene at the L1 locus is transcribed at a higher level than its identical transgene counterpart at the 6b4 locus, these results suggest that the production of abSUG at a high level in L1 plants or the absence of its degradation in 6b4 xrn3 xrn4 plants could explain the capacity of these plants to trigger PTGS11.We previously identified an uncapped RNA, antisense to the jmj14 mutations limits the production of aberrant RNAs, the abundance of abSUG RNA was measured in L1, L1 rdr6 and L1 jmj14 rdr6 plants. To reliably assess the effect of JMJ14 on the levels of the abSUG RNA, the rdr6 mutant background was used to avoid RDR6-dependent PTGS of the abSUG RNA. The amount of abSUG RNA was very low in L1 WT plants compared to L1 rdr6 plants, thereby confirming that abSUG RNA is degraded by PTGS in L1 WT plants  analysis was performed on plants carrying the 6b4 locus. WGBS revealed that the transgenic line 6b4 carried a single T-DNA insertion. However, the left border did not delineate the 6b4 T-DNA insertion and the entire binary vector was also found to have inserted adjacent to the left border  in the nts Fig. , and we nts Fig. , and indnts Fig. . These r6b4 WT and 6b4 jmj14-4 plants revealed that loss of JMJ14 had a limited effect on the extent of DNA methylation at endogenous loci  exhibited hyper CG, hyper CHG and hyper CHH methylation  of JMJ14 was performed on the 6b4 jmj14-4 line complemented with a pJMJ14:3xFlag-JMJ14 transgene43. ChIP was conducted using Flag antibodies and followed by quantitative PCR (qPCR) using primer pairs located in the 35S promoter, and in the 5′ and 3′ regions within the GUS transgene body. Quantitative ChIP-PCR revealed a significant enrichment of JMJ14 primarily at the 35S promoter . Moreover, transgenic sequences not only exhibited hyper CHG methylation but also hyper CG and hyper CHH methylation  experiments were performed on plants resulting from a cross between a pJMJ14:JMJ14-Myc line46 and the 6b4 + pUBQ10:NAC52-GFP line. As expected, JMJ14-Myc and NAC52-GFP proteins co-precipitated in pull-down assays using a GFP antibody  mutation  pathways. In plants, this is essential to prevent the conversion of aberrant RNAs into dsRNA by RDRs, which would result in the production of siRNAs that could destroy homologous and essential mRNAs. Indeed, RQC impairment causes lethality in Agrobacterium proteins, and once integrated into the chromosome, must associate with histones to form chromatin. At present, the type of eukaryotic histones that associate with naked or newly integrated foreign DNA is not known. One could imagine that the insertion of extrachromosomal DNA into the chromosome attracts particular histone marks to label this DNA as ‘new or foreign’. It seems likely that what happens for transgenes also occurs when a new copy of a retrotransposon integrates into the genome. Supporting this hypothesis, it has been shown that activation of retrotransposons triggers a PTGS response, just like transgenes do58. Although this hypothesis is attractive, our findings suggest that there are striking epigenetic differences between transgenes and endogenous loci, including genes and stabilized transposons. Here, we showed that transgenes and endogenous loci (genes and stabilized transposons) behave differently when the histone H3K4me3 demethylase JMJ14, and two cooperating transcription factors that interact with JMJ14, NAC50, and NAC52, are impaired. Indeed, in jmj14 or nac50 nac52 mutants, an increase in CHG methylation, and CHH methylation to a lesser extent, is observed at the 6b4 transgene locus , vernalized at 4 °C for 2 days, and then placed in a culture chamber at 23 °C, 70% humidity, 120 µE m−2 light with a 16 h light/8 h dark (long-days) or 8 h light/16 h dark (short-days) photoperiod. Seedlings grown under long-day conditions were transferred to soil after 2 weeks. Seedlings grown under short-day conditions were used for grafting expertiments as described in ref. 63. Briefly, 6 days after gemination, seedlings were cut transversely across the hypocotyl using a razor blade (90° butt graft). Then, scions and rootstocks were placed on a nitrocellulose filter (Hybond). Hypocotyls of scions and rootstock were introduced into a silicon microtube (2 mm long) to connect them to each other, and incubated under short-day conditions  for 7 to 14 days. Grafted seedlings that did not show adventitious roots were transferred to soil and grown under a short-days photoperiod.64.GUS protein was extracted from plant leaves, and GUS activity was quantified by monitoring the quantity of 4-methylumbelliferone produced by cleavage of the substrate 4-methylumbelliferyl-b-D-glucuronide (Duchefa) on a fluorometer (Thermo Scientific fluoroskan ascent)GUS, U6 and 25S probes16. For the reverse transcription, RNA was treated with DNaseI (Invitrogen) and 1 μg of DNA‐free RNA was reverse transcribed with the primer called RT_ASGUS_Linker using the RevertAid H Minus Reverse Transcriptase  Amplification was performed by using the LK and RbcS1Rev primers11, and qPCR results were normalized with EiF1a11.RNA extraction and HMW or LMW RNA gel blot analyses were performed using 5–10 µg of total RNA and 6b4-1, 6b4 jmj14, 6b4 jmj14 cmt3 drm2, 6b4 cmt3 drm2) or eight weeks . Genomic DNA was isolated from leaf tissue from a mix of eight plants per genotype using the Nucleospin Plant II Maxi kit (Macharey Nagel), and library preparation and sequencing was performed by BGI Genomics (Hong Kong). Briefly, genomic DNA was fragmented to 100–300 bp by sonication, end-repaired, and ligated to methylated adaptors. Bisulfite treatment was then performed using the EZ DNA Methylation-Gold kit (Zymo), and the bisulfite-treated fragments were PCR amplified and sequenced as paired-end 100 bp reads (PE100) with DNBSEQ technology. Pre-processed high-quality reads were mapped to the TAIR10 genome using bismark with default settings for paired-end libraries65, and all downstream analysis were performed using custom R scripts. All figures presenting bisulfite data were generated using R version 4.0.2. A summary of all bisulfite sequencing data generated in this study is presented in Supplementary Table Plants were grown under short-day conditions for five weeks (experiment 1: 66. Chromatin was sonicated (30 s ‘’ON”/30 s ‘’OFF”) with a Bioruptor UCD200 (Diagenode). The chromatin solution was diluted 10 fold with ChiP dilution buffer. Fifty microliters of Dynabeads Protein G (invitrogen) and 25 µL of GFP-Trap Dynabeads (chromotek) was washed twice with ChiP dilution buffer. Nineteen micrograms of Flag (Sigma F3165) antibodies were added to G Protein and incubated at least 2 h at 4 °C with gentle rotation. Beads were washed three times with ChiP dilution buffer. Then, 1 mL of the diluted chromatin was added to the beads/antibodies and incubated overnight at 4 °C with gentle agitation. Beads were washed as decribed66. After the last TE wash, reverse crosslinking (at least 4 h at 65 °C) and elution were performed using an IPure kit (Diagenode (AL-100-0100)). The final elution was performed in 60 µL and the chromatin was stored at −20 °C until analysis.ChiP was performed on chromatin using 2 g of crosslinked in vitro shoots from 15-day-old seedlings67. Results are represented as fold change: normalized expression (ΔΔCq) given by the ratio of Relative Quantity of the sample (2(Cq 6b4-Cq sample) for each identical oligo with 100% efficiency) divided by the Relative Quantity of GAPDH. At least two biological replicates were analyzed each time. Results show the mean and SD of the independent biological replicates.Using the Biorad-CFX-Maestro Software, the ChIP was analyzed by qPCR using 2 µL of chromatin in triplicate. Primers are listed in Supplementary Table TM Protease Inhibitor Cocktail (Merck). After 30 min on ice, samples were centrifugated at 7650 × g at 4 °C and the supernatant quantified after miracloth filtration. Five micrograms of this crude extract was submitted to preclear on 50 μL of magnetic beads coated with protein A for 2 h at 4 °C. After magnetic decantation, 50 μL of GFP-Trap_M  was rinsed (2x PBS0.1% and 1x CoIP buffer) and added to the supernatant. This was incubated overnight at 4 °C with gentle agitation. The GFP beads were then rinsed 6x in CoIP buffer, and finally adjusted to 40 μL of CoIP buffer, 5 μL of 6x Laemmli buffer and incubated at 95 °C for 5 min. After vortexing, 40 μL of this final supernatant was loaded onto a 10% polyacrylamide gel. Following sodium dodecyl sulphate–polyacrylamide gel electrophoresis, western blotting was performed using anti myc , anti GFP (Rabbit Abcam-Ab290) or anti tubulin  antibodies. An ImageQuant-LAS4000  was used for detection.For each bulk of plants 15 days after germination, 2 g of fresh tissue was ground in CoIP Buffer (50 mM Tris pH 7.5, 100 mM EDTA, 15% Glycerol, 1% NP40, 1% Triton X100, 1x cOmpleteFurther information on research design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Description of Additional Supplementary FilesReporting Summary


RTR1 deletion alters RNA pol II assembly and increases the amount of RNA pol II associated with the chromatin that lacks Rpb4, decreasing Rpb4-mRNA imprinting and, consequently, increasing mRNA stability. Thus, Rtr1 interplays RNA pol II biogenesis and mRNA decay regulation. Our data also indicate that Rtr1 mediates mRNA decay regulation more broadly than previously proposed by cooperating with Rpb4. Interestingly, our data include new layers in the mechanisms of gene regulation and in the crosstalk between mRNA synthesis and decay by demonstrating how the association of Rpb4/7 to the RNA pol II influences mRNA decay.Rtr1 is an RNA polymerase II (RNA pol II) CTD-phosphatase that influences gene expression during the transition from transcription initiation to elongation and during transcription termination. Rtr1 interacts with the RNA pol II and this interaction depends on the phosphorylation state of the CTD of Rpb1, which may influence dissociation of the heterodimer Rpb4/7 during transcription. In addition, Rtr1 was proposed as an RNA pol II import factor in RNA pol II biogenesis and participates in mRNA decay by autoregulating the turnover of its own mRNA. Our work shows that Rtr1 acts in RNA pol II assembly by mediating the Rpb4/7 association with the rest of the enzyme.  Saccharomyces cerevisiae -1-propanesulfonate (CHAPS)) supplemented with 1X protease inhibitor cocktail , 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium orthovanadate, and 1 mM sodium fluoride. Cell disruption was carried out by vortexing  at 4 °C using 0.2 mL of glass beads . For the Rpb3 immunopurification, 1 µg of anti-Rpb3 antibody  was coupled to 40 µL of Dynabeads Sheep-anti-Mouse IgG  per sample, and 2 mg of a whole-cell protein extract were used for each immunoprecipitation. The affinity-purified proteins were released from the beads by boiling for 10 min and were analyzed by Western blotting with different antibodies.Whole-cell protein extracts and protein immunoprecipitation were performed as described . BrieflyProtein electrophoresis and Western blot were carried out as described in .S. cerevisiae Rpb5 in our lab), anti-Rpb6 (a gift from M. Werner), anti-Rpb7 (Rpb7 (yN-19); Santa Cruz Biotechnology, Dallas, TX, USA), antiphosphoglycerate kinase, Pgk1 , anti-H3 , antihemaglutinin  and PAP (Sigma) antibodies were used.For the Western blot analyzes, anti-CTD , anti-RpIntensities of immunoreactive bands on Western blots were quantified by densitometry using the software IMAGE STUDIO LITE from images acquired with an office scanner.600~0.5–0.7. For Rpb4 localization, strains were transformed with the centromeric vector expressing a Gfp-Rpb4 fusion protein [The cells expressing Rpb1-Gfp and Gfp-Rpb4 were grown at 30 °C in SD medium supplemented with the corresponding amino acids to reach an OD protein  (Supplem600~0.6–0.8). The chromatin-bound proteins were resuspended in 1X SDS-PAGE sample buffer, boiled for 10 min and analyzed by Western blotting with different antibodies.Preparation of chromatin-enriched fractions was carried out by the yChEFs procedure ,64 using600~0.6–0.8). The cytoplasm-enriched fraction corresponds to the S2 fraction [Preparation of cytoplasm-enriched fractions for immunoprecipitation experiments was carried out by the yChEFs procedure ,64 usingfraction . The P3 fraction .600~0.6–0.8 were exposed to 1200 mJ/cm2 of 254 nm UV in a UV crosslinker (Biolink Shortwave 254 nm), resuspended in 350 µL of lysis buffer  and 200 µL of glass beads  and broken by vortexing for 15 min at 4 °C. An aliquot of lysate was used as the INPUT control. Lysate was incubated with 150 µL of oligo (dT)25 cellulose beads  for 15 min at room temperature. PolyA-containing RNA isolation and elution were carried out as previously described [mRNA crosslinking was carried out as described elsewhere  with somescribed . The mRN600~0.5. Cells were treated with 5 μg/mL of thiolutin. Next 15-mL aliquots of cell samples were taken at different times after thiolutin addition (up to 100 min), then pelleted and frozen. Total RNA was isolated from these samples and cDNA was synthesized as previously described [mRNA stability analysis was performed as previously . Brieflyescribed . The mRN2 of 254 nm UV in a UV crosslinker (Biolink Shortwave 254 nm). After crosslinking, the membrane was incubated in prewarmed hybridisation buffer  at 56 °C for 45–60 min. Then, 50 pmol of oligodTCy3 were added and incubated overnight at the same temperature. The membrane was washed six times at room temperature using prewarmed washing buffer . Fluorescence was analyzed with a Molecular Imager VersaDoc™ MP system at 635 nm and the Quantity one software. The graphical quantifications of the dot blot are shown and represented on a natural logarithmic scale.For total mRNA half-live calculation, the previously obtained RNA was used for dot-blot analysis: 2 µg of total RNA dots were added to a nitrocellulose membrane previously washed with SSC 2X buffer . The membrane was incubated for 5 min at 65 °C and exposed to 400 mJ/cmGAL1 and GAL10 genes mRNA stability was performed as previously described [600~0.5–0.6) from SD-galactose to SD-glucose to stop transcription. Cell samples were collected at different time points after glucose addition . RNA extraction was performed as described above. mRNA stability was analyzed by RT-qPCR with specific primers for the corresponding genes . Reactions were performed using cDNA corresponding to 0.1 ng of total RNA in 5 µL of total volume. Each PCR reaction was performed at least three times with three independent biological replicates to obtain a representative average. The 18S rRNA gene was used as a normalizer. The employed oligonucleotides are listed in Real-time PCR was performed in a CFX-384 Real-Time PCR instrument  with the EvaGreen detection system ‘‘SsoFast™ EvaGreenThe chromatin immunoprecipitation experiments were performed using anti-Rpb3  or anti-Rpb4 (Pol II RPB4 (2Y14); Biolegend, San Diego, CA, USA) as previously described . For rea


We compare the electrostatic potential of the presented motif I major groove with previously published structures of tandem motifs I, II (5′GU/3′UG), and III (5′GG/3′UU). A local patch of a strongly negative electrostatic potential in the major groove of the presented structure forms the metal binding site with the contributions of three oxygen atoms from the tandem. These results give us a better understanding of the G•U tandem motif I as a divalent metal binder, a feature essential for RNA functions.G•U wobble base pair frequently occurs in RNA structures. The unique chemical, thermodynamic, and structural properties of the G•U pair are widely exploited in RNA biology. In several RNA molecules, the G•U pair plays key roles in folding, ribozyme catalysis, and interactions with proteins. G•U may occur as a single pair or in tandem motifs with different geometries, electrostatics, and thermodynamics, further extending its biological functions. The metal binding affinity, which is essential for RNA folding, catalysis, and other interactions, differs with respect to the tandem motif type due to the different electrostatic potentials of the major grooves. In this work, we present the crystal structure of an RNA 8-mer duplex r[UCGUGCGA] Watson–Crick (WC) base pairs shape the RNA double-helical landscape. However, multiple non-WC interactions  have beeLys wobble position of the codon/anticodon triplet) have been observed , the tautomeric G•U pairs , and the minor groove-shifted G•U pair  is iAla ; 1996; aAla . The majAla . The unbnthetase . The connthetase ; the G cnthetase . The nonactivity  and tRNAcylation . Moreovecylation .2+ cation, showing that G•U wobble motif I attracts metal ions and functions as their potent binding site. Structural analysis of the presented RNA duplex confirmed the A-form helix and indicated a few unique features of the G•U tandem.Here, we present the crystal structure of an RNA duplex containing motif I of the G•U wobble tandem. In the previous study, the major groove of G•U wobble motif I has been determined as less negative than the major groove of motifs II and III and accordingly suggested as the least favorable metal binding site among the G•U tandems . In this2 duplex was obtained even though an equimolar of its complementary strand UCGCACGA was used for crystallization as a hybrid duplex. The presence of a divalent cation (Ba2+), bound in the major groove (see below), might be the major driving force behind the formation of the r(UCGUGCGA)2 duplex. The crystals in the H32 space group, with one duplex in the asymmetric unit, diffracted X-rays to 2.2 Å resolution. The detailed diffraction data and model refinement statistics are listed in The crystal of the r(UCGUGCGA)2+ cation and two water molecules in its coordination sphere   may not be visible in the electron density maps due to the limited data resolution.Bachain A)  and 4. TThe G•U wobble pair is negatively charged with the group composition of guanosine N7, guanosine O6, and the unpaired O4 carbonyl of uridine, which are exposed to the major groove forming a region of strong negative electrostatic potential  McDowel. A compa2+ cation was located in the center of the 5′UG/3′GU tandem  indicates that the G•U pair major groove is the most common metal binding RNA motif . Divaleneactions .2+ cation binding site  affected the stacking interactions between the G•U pair and flanking base pairs . The thr2+ preferentially binds to G•U motif I, whereas complex ion [Co(NH3)6]3+ favorably binds to motifs II and III (1999). Thus, the binding affinity of specific RNAs is likely a composite of multiple factors involving geometry, electrostatic potential, hydration pattern, and some quantum effects  . For strendo sugar puckers and the displacement from the helical axis (X-displacement) had a negative value, on average −4.46 Å, which is close to the ideal A-RNA helix, approx. −4 Å. However, a large negative X-displacement of −10.3 Å were observed at the 5′UG/3′GU step  subtended at the glycosyl carbon atoms C1 , which is consistent with the asymmetric coordination sphere of the metal cation. It is also apparent that the distribution of the electrostatic potential within the major groove contributed to the formation of the metal binding site. The aforementioned features may provide elements for recognition by RNA binding proteins.The metal binding site in the r(UCGUGCGA)2 duplex was obtained in the presence of its complementary strand UCGCACGA. The crystals grew in the 12th condition of nucleic acid mini-screen from Hampton  in 2 weeks with size of ∼0.1 × 0.06 × 0.03 mm, using a hanging drop setting. They were cryoprotected by increasing the MPD concentration to 35%.The crystal of the r(UCGUGCGA)Diffraction data were collected at the SER-CAT beamline 22-BM at the Advanced Photon Source, Argonne National Laboratory . The diffraction data were processed with the XDS package ; the pro2 duplex was solved by molecular replacement in PHASER n PHASER  using anER  server 2 generated by COOT] ) server . The eleby COOT]  was calcby COOT]  with defsoftware .


Addressing intracellular targets is a challenging task that requires potent molecular transporters capable to deliver various cargos. Herein, we report the synthesis of hydrophobic macrocycles composed of both amino acids and peptoid monomers. The cyclic tetramers and hexamers were assembled in a modular approach using solid as well as solution phase techniques. To monitor their intracellular localization, the macrocycles were attached to the fluorophore Rhodamine B. Most molecular transporters were efficiently internalized by HeLa cells and revealed a specific accumulation in mitochondria without the need for cationic charges. The data will serve as a starting point for the design of further cyclic peptoid-peptide hybrids presenting a new class of highly efficient, versatile molecular transporters. Biological membranes envelop intracellular structures, protect them from harmful substances and enable diverse physiological processes. The unique function of these phospholipid bilayers is crucial for all processes of life but also hampers the intracellular delivery of therapeutics or other cargos. The efficient internalization of a certain compound is a key step for both pharmaceutical and basic research. Thus, molecules that overcome the physiological barrier, so-called molecular transporters, are constantly needed not only to resolve and understand the fascinating processes of life but also to provide treatments poor in side effects.N-substituted oligoglycines are readily accessible via well-established solid-phase synthesis techniques.  protein of the Human Immunodeficiency Virus (HIV) to overcome cellular barriers without any receptor interaction was discovered. . Therebyhniques.  Formallygen atom . Howevergen atom .α-carbon is lost when transforming peptides to peptoids. Interestingly, it has been shown that the absence of these features can even increase the efficiency of cellular uptake.  served as solid support (Macrocyclic tetramers and hexamers were synthesized in a multistep procedure using solid and solution phase methods. The synthetic approach involved the assembly of linear precursors on solid supports following the submonomer method published by Zuckermann  as well  support .7) or to the growing oligomer chain using diisopropylcarbodiimide (DIC) as a coupling reagent. A certain amine was added allowing for the assembly of an alkylated glycine monomer (→ 8). The possibility to incorporate any amine gives rise to various side chains and, thus, easily tunable properties of the resulting peptoids. For the design of molecular transporters, the substitution step was performed with four different amines that led to the assembly of the alkylated glycine monomers N1ph (2), N1phpCl (12), N3m (13), and N4am  served as conjugation site for the fluorophore Rhodamine B.The submonomer method requires an acetylation step using a haloacetic acid followed by a substitution implementing the desired side chain.  Thus, brN4am 14, . The forN4am 14,  The pept4) under basic conditions or to the growing linear precursor using DIC. To avoid racemization at the α-carbon, hydroxybenzotriazole was added for amino acid couplings. The temporary Fmoc-protecting group was cleaved with piperidine after coupling (→ 5). The combination of both solid-phase methods enabled the straightforward synthesis of linear hybrids composed of alkylated glycine monomers as well as amino acids. The linear precursors 11 were cleaved from the resin under mildly acidic conditions releasing a carboxylic acid at the C-terminus.The SPPS was performed using Fmoc-protected amino acids. They were analogously attached to the resin . Product formation was confirmed via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and subsequent nuclear magnetic resonance (NMR) spectroscopy. The synthetic protocol yielded the macrocyclic compounds 15–24 methylidene]-dimethylazanium hexafluorophosphate (HATU) as potent coupling agent was performed under high dilution conditions to avoid favored side reactions like dimerizations.  Thus, thds 15–24 .15–21) and three cyclic hexamers (22–24) were successfully synthesized. Every macrocycle is composed of several alkylated glycine monomers and up to two amino acids. The hybrids share aromatic as well as aliphatic residues and an aminobutyl side chain. In every case, an aromatic amino acid represents the N-terminus of the linear precursor. It was assumed that the higher nucleophilicity of a primary amine might be beneficial for the subsequent ring closure reaction.Seven cyclic tetramers  and satisfying purities (94% ± 5). For the assembly of hybrids ibility, , resultivial-phenylalanine (1). Due to the additional reaction steps, the yields of the crude linear hexamers were lowered (49% ± 13). Nevertheless, every linear precursor was synthesized in a sufficient quantity for cyclization. Depending on the amount of the linear precursor isolated, cyclization was carried out by the addition of a 3.25–7.50 mm solution of the respective linear hybrid. However, no effects on the efficiency of the ring closure were observed within this concentration range.As the attachment of an amino acid to the solid support proved to be more efficient, linear hexamers were immobilized via its carboxylic moiety to the aminobutyl side chains of the single macrocycles  and excellent purities .15–24, as well as their conjugates 15-Rhod to 24-Rhod, was examined using a standard MTT assay in human epithelial cervix carcinoma (HeLa) cells. This colorimetric assay displays the cell viability based on their capability to reduce the tetrazolium dye 3--2,5-diphenyltetrazolium bromide (MTT). (15–24 (0.50─50 µm) for 72 h, no significant cytotoxic effects were observed. Most hybrids resulted in IC50 > 50 µm. However, in most cases the attachment of the chromophore Rhodamine B slightly increased the cytotoxicity of the respective molecular transporters 15-Rhod–24-Rhod .  After tr–24-Rhod . As show23-Rhod is the only hybrid with an IC50 value in a single-digit micromolar range (IC50 = 6 µm). Its parent macrocycle 23 turned out to be the most cytotoxic hybrid tested (IC50 = 20 µm). Considering every cyclic hybrid, the incorporation of the alkylated glycine monomer N1phpCl (12) was decisive for the cytotoxicity of the respective macrocycles: the higher the amount of N1phpCl building blocks, the lower the viability of the HeLa cells after treatment. However, comparing the macrocyclic transporters 19-Rhod and 20-Rhod, the substitution of two N1ph (2) monomers by two N1phpCl building blocks led to a decrease of cytotoxicity.Conjugate 15-Rhod to 24-Rhod were not toxic at 5 µM the following transport experiments were carried out using this concentration. To study their cellular uptake and organellar localization, HeLa cells were treated with 5 µM of the cyclic conjugates 15-Rhod to 24-Rhod for 5 h and eventually imaged using confocal microscopy  by its halogenated relative N1phpCl (13), no conclusive effects were observed as well. Conjugates 15-Rhod and 16-Rhod showed a similar cellular uptake efficiency, while an exchange of two N1ph (2) side chains by N1phpCl (13) increased the uptake of the molecular transporter 20-Rhod compared to conjugate 19-Rhod. However, the halogenation of three N1ph (2) building blocks led to a significantly decreased cellular uptake efficiency which becomes evident by comparison of hexamers 22-Rhod and 23-Rhod.A high cellular uptake efficiency, as well as mitochondrial specificity, was observed for the molecular transporters 15-Rhod, 20-Rhod and 22-Rhod are characterized by low cytotoxicity, efficient cellular uptake, and high specificity. Thus, they represent the most promising cyclic hybrids capable of the transport of different cargos into mitochondria. The data obtained support previous studies on peptides and peptoids indicating that a certain amount of lipophilic residues may be crucial for mitochondrial targeting. (22-Rhod, the mitochondrial uptake is occurring at 37°C as well as at 4°C (see supplemental material, 22-Rhod might directly penetrate the cell membrane. However, the uptake mechanism of macrocyclic hybrids will be further examined.The molecular transporters rgeting.  Besides,rgeting.  It is asrgeting.  The lattrgeting.  As exempThe versatile substance class of peptide-peptoid macrocycles shows high potential as molecular transporters. Their specific targeting of mitochondria paves the way for the treatment of various diseases that are associated with these organelles.  StructurN-alkylated glycine monomers resulting in a hybrid structure increases their polarity and variability by allowing for the incorporation of plenty of different side chains and easily tunable properties. The hydrophobic molecular transporters presented selectively accumulated in mitochondria without the need for additional cationic charges. Thus, their uptake mechanism might differ from known CPPs and CPPos. Further studies on these versatile structures will reveal their potential as selective and highly efficient molecular transporters.Ten macrocyclic peptide-peptoid hybrids were synthesized in a straightforward approach using solid and liquid phase techniques. Their unique structure combines the advantages of both peptides and peptoids. The conformational constraints introduced by chiral amino acids as well as the cyclization of their backbone render them promising model structures for selective compounds. Their capability to form hydrogen bonds makes them soluble in an aqueous environment. Furthermore, the incorporation of