Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration.Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope.The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3’UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope.By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair. Axons do not spontaneously regenerate in the injured CNS. This is in part due to diminished intrinsic capability of adult CNS neurons to regenerate axons and to extrinsic inhibition at the injury site . SupraspThe goal of this study was to define axonal mRNA profiles at the site of injury after SCI and identify the pathways modulated by differentially regulated axonal mRNAs to understand how SCI induced differentially expressed genes (DEGs) regulate axonal regeneration.Rims2 encodes a presynaptic protein that is important for neurotransmitter release, but the function of circRims2 is still unknown  accredited.All animal experiments were performed according to ethical regulations and protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Icahn School of Medicine at Mount Sinai and at the James J. Peters Department of Veterans Affairs Medical Center. Both institutes are Female Sprague Dawley rats 3 months of age were utilized, we utilized two animals per condition. A moderate to severe SCI at thoracic level 9 (T9) was performed using an Infinite Horizons (IH) impactor  was added . Cold active protease from All supernatant fraction from the spinal cord were checked for RNA integrity by Agilent 2100 Bioanalyzer and all samples had RIN value ≥9 . RNA libTo reduce artifacts caused by read imbalances during upper quartile normalization, we downsampled the sequencing reads of each sample to the number of reads that were detected in the sample with the lowest read counts, as described previously . Downsamhttps://docs.gdc.cancer.gov/Encyclopedia/pages/FPKM/#:~:text=Fragments%20Per%20Kilobase%20of%20transcript,total%20number%20of%20mapped%20reads. FPKM are a simple expression level normalization method. It is normalized based on the read counts based on gene length and the total number of mapped reads (50 million). Differentially expressed genes (DEGs) were identified with cufflinks 1.3.0 [2(fold change) = +/−log2(1.3)]. Up- and downregulated genes were subjected separately and combined to pathway enrichment analysis using Wikipathways 2016 . We followed the manufacturers protocol with minor modifications . Spinal For Ribonuclease R digestion, after the Protease step and 3 washes with PBS, we permeabilized the tissue with 0.1% Tween-20 in PBS for 10 min at RT. Then washed the samples with PBS three times. All RNAscope experiments were performed a minimum of 3–4 independent times.g for 15 min at room temperature, with the brakes decreased to the lowest setting to not disrupt the gradients. The layer closest to the bottom between 1 to 2mls of a 15 ml conical tube is the enriched fraction of neurons. The enriched neuronal layer was then pelleted at 1000 x g for 5 min and counted.Rat and mouse primary cortical neuronal cultures were prepared as previously described with minor modifications , 2021 weSquare microfluidic chambers  were purchased from Xona microfluidics. The chambers were sterilized under UV for 15 min and soaked in 70% ethanol for 2 min and allowed to air dry under a sterile TC hood. We used MatTek dishes  that we pre-coated with PLL overnight and then rinse 3 times with sterile water and air dried overnight in a TC BSL2 hood. Using autoclaved forceps we carefully place the microfluidic chamber on the glass area of the MatTek dish, and gently apply pressure to ensure the chambers are sitting on the glass. Primary cortical neurons were diluted to 5x10E6 cells/ml in NB supplemented with B27, L-glutamine and antibiotics, and in a final volume of 200μls. Approximately 15μls of the cell suspension was carefully inserted into the top well of one side of microfluidic chamber. We then placed the Mat-tek dishes with the microfluidic chambers inside a 37°C incubator for 20 min to allow the neurons to adhere. All wells were filled with 150μls of supplemented NB. Once we observe neurites growing across the microgroove (3-4 days), we then performed an axotomy.To quantify the outgrowth we immunostained using a monoclonal antiβIII tubulin antibody  and Alexa Fluor −405- (Blue) or 488- (Green) conjugated anti-mouse IgG (Invitrogen). For quantification, images were taken, and the length of the longest neurite for each neuron was measured using MetaMorph software (Molecular Devices). All imaging was performed on a Zeiss LSM 880 confocal microscope.All siRNA were purchased from Accell smartpool siRNA (Thermo Scientific), following the manufacturers protocol. We targeted ADAR1 for rats (Rat Accession number XM_006232778.3) and mice (Mouse Accession number NC_000069.7), scrambled non-targeting siRNA was also utilized for controls. Cortical neurons were plated on one side of the microfluidic chambers. We waited 2–4 h after plating to allow the cells to adhere. Utilizing 1 μM siRNA in 150μls of Accell Delivery Media was added to the neuronal cell bodies and incubated overnight at 37°C incubator. All siRNA experiments were performed a minimum of 3–4 independent times. The next morning, we added 150μls of supplemented NB. We incubated for a further 72 h before stopping the experiment with 4% paraformaldehyde and 4% sucrose. We then stained the chambers with β-III tubulin (Alexa-488) and in individual neurons panel, we also used Actin-Red staining (Thermo Scientific) and quantified the neurite length. For RT-qPCR, at the 72 h timepoint the wells were washed with ice cold PBS and lysed with 1 ml Trizol and immediately flash frozen in liquid nitrogen.t tests to compare two groups, and one-way ANOVAs with Bonferroni’s post hoc tests to compare between three or more groups.All analyses were performed using GraphPad Prism software, and data are represented as mean ± SEM. Statistical significance was assessed using paired one-tailed Student’s We adapted a tissue dissociation protocol used for single cell RNAseq (scRNAseq) that uses cold active proteases to gently dissociate tissue and isolate cells . Low speIn We also looked for known markers of Spinal Interneurons . Only a N = 4). With axotomy, more MAP4 and MACROH2A1 RNA are detected further along the axon when comparing to no axotomy control.We validated the expression of selected DEGs in axons of cultured primary rat cortical neurons plated in microfluidic chambers using RNA-scope. RNAscope enables us to visualize single RNA molecules within a cell using confocal laser microscopy. One novel DEG detected was GDA which can promote microtubule assembly in neurons, a necessary step for axonal extension, which is up-regulated after SCI . In FiguWe examined localization of selected DEGs in rat spinal cord sections using RNAscope. The RNA for MACROH2A1 (red) and MAP4 (green) by RNAscope , localizhttps://ngdc.cncb.ac.cn/circatlas/, amongst others (One DEG decreased after SCI was Rims2. Rims2 is reported to be predominately a circular RNA (circRNA) in the rodent brain . We thert others , 2022. Wt others . SupportN = 4). ADAR1 knockdown resulted in longer (1.2 mm from the microgroove) and more abundant outgrowth  compared to scrambled controls (white).To determine if circRNAs are involved in axonal regeneration, we knocked down the RNA editing enzyme ADAR1 by siRNA in primary rat cortical neurons . ADAR1 autgrowth ,C at 800utgrowth . We alsoutgrowth . There iWe confirmed the presence of circRims2 in rat spinal cord . AdjacenSince the known roles for circRNA are to serve as miRNA ‘sponges’ or to bind to RNA-binding proteins RBP; , we tookIdentifying the axonal RNAs transported to the site of axotomy in the injured spinal cord is critical to determining which of these may be required for promoting axonal repair. A reliable technique for determining specific axonal RNA without contaminating glial cell or spinal neuronal cell bodies RNA has been lacking. Here, we report a simple procedure for enriching axonal fractions of spinal cord incorporating cold active proteases that function at 6°C that helps reduce artifacts from transcription or translation of RNA that can occur with enzymatic digestion at 37°C . Our anaAn important consideration was the detection of MAP4 in our primary neuronal cultures isolated from pup brains compared to adult spinal cord see , 5. ThouOne DEG in the axon, Rims2, has been found to be predominately a circRNA in the rodent brain . We deteTo identify potential mechanisms underlying circRNA on axon growth, we used bioinformatic analyses to predict which miR and RBPs could be binding to circRims2. We found multiple likely miRNAs that could bind to circRims2 that have also been reported to either target known mRNAs that modulates axonal regeneration or has been shown to mediate SCI-injury see . CirculaYayon et al. utilize a method called Patch-Seq analysis which is single-cell RNA-seq from fixed tissues using iDISCO and advanced microscopy (NuNeX) to help spatially select VChIs neurons and detect changes in dendritic regions of these neurons after whisker deprivation model . By doinThese observations predict a RNA-based regulatory pathway from circRNA Rims2 → miRNA (miR-192 or −377) → GAP-43 mRNA to control axonal levels of GAP-43 protein and regulate axonal outgrowth . The dechttps://www.ncbi.nlm.nih.gov/, GSE230066.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The animal study was reviewed and approved by the IACUC at Icahn School of Medicine at Mount Sinai and James J. Peters VA Hospital.in vivo experiments, RT-qPCR, and preparation of RNA for RNA-seq. NJ assisted with all in vitro experiments, RNA-scope, preparation of primary neuronal cultures, and blinded quantification of data. JH performed all RNA-seq data analysis. YX assisted with circular RNA Interactome analysis. WM assisted with all RNA analysis. RT assisted with all in vitro experiments, preparation of primary neuronal cultures and RNA-scope. KJ assisted with in vivo experiments and RT-qPCR. GJ assisted with RNA-scope. ZS performed all blinded analysis of RNA-scope experiments. LH assisted with in vivo experiments. JD assisted with mouse primary neuronal cultures and RT-qPCR. KB performed RNA-seq experiments. RS performed RNA-seq experiments. DW assisted with RNA analysis and critical editing of the manuscript. CC assisted with developing the conceptual ideas for the experiments and critically reviewed the manuscript. RI developed the conceptual ideas for the experiments and critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.MS developed all conceptual ideas, analyzed and assisted with all experiments, microscopy, and wrote the manuscript. CT assisted with all This work was supported by NIH grant GM 137056 and NYS Spinal Cord Injury Research Program Contract number #C34460GG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Design strategies for DNA and RNA nanostructures have developed along parallel lines for the past 30 years, from small structural motifs derived from biology to large ‘origami’ structures with thousands to tens of thousands of bases. With the recent publication of numerous RNA origami structures and improved design methods-even permitting co-transcriptional folding of kilobase-sized structures – the RNA nanotechnolgy field is at an inflection point. Here, we review the key achievements which inspired and enabled RNA origami design and draw comparisons with the development and applications of DNA origami structures. We further present the available computational tools for the design and the simulation, which will be key to the growth of the RNA origami community. Finally, we portray the transition from RNA origami structure to function. Several functional RNA origami structures exist already, their expression in cells has been demonstrated and first applications in cell biology have already been realized. Overall, we foresee that the fast-paced RNA origami field will provide new molecular hardware for biophysics, synthetic biology and biomedicine, complementing the DNA origami toolbox. Tetrahymena group I intron ..121].More recently, a functional octahedral RNA origami was designed with intrinsic substrates for Dicer in order to produce multiple siRNAs that can be released with precise stoichiometric control . The oriS. cerevisiae yeast [A more complex example of scaffolding for cell behaviour control took inspiration from Cas9–mediated gene activation/inactivation. Catalytically inactive Cas9 (dCas9) is a popular tool for targeted gene modulated through the single-guide RNA (sgRNA)-directed, site specific recruitment of activating or repressing protein domains in yeast and mammalian cells. SgRNAs were fused with an RNA origami structure (termed sgRNAOs) containing multiple functional motifs which controlled the expression of genetic targets in ae yeast .2+ making them suitable for in vivo applications. The origami were then injected intraperitoneally into mouse models of peritoneal metastasis and peritoneal carcinomatosis. The RNA origami halted tumour growth or even caused tumour regression by stimulating the innate immune response through the Toll-like receptor 3 (TLR3) pathway. They were less toxic compared to polyinosinic/polycytidylic acid (PolyIC). RNA origami induced long-lasting antitumor immunity in presence of CD8 T and NK cells, which could potentially be explored as an effective and safe immunotherapeutic for treating cancer.Several RNA nanoarchitectures have been described to function as immune stimulants and therefore a promising platform for immunotherapy applications. In a study by Qi et al. , single-In another study by the same group on RNA origami as a TLR3 agonist, RNA origami decorated with antigens were used as a hybrid antigen/adjuvant for initiation of T-cell mediated immunity. These RNA origami-based vaccines were reported to induce anti-tumour immune response that contributed to long-term tumour immunity in the B16-OVA  murine melanoma tumour model .RNA origami has made considerable progress towards a technology which may soon be as widespread as DNA origami already is. The use of RNA has several advantages compared to DNA origami due to its greater structural and functional versatility, for example concerning the integration of catalytic activity. This makes it an exciting tool for bottom-up as well as top-down synthetic biology. As we progress further in the production of functional RNA origami, we may see a rise in RNA origami-based hardware for synthetic cells. Simultaneously, it will be exciting to explore the therapeutic potential, which goes far beyond current RNA therapeutics thanks to the possibilities that come with structural versatility and spatial organization of secondary components.The need for a more robust suite of design tools which offer friendly UI/UX experiences, enabling non-experts to leverage the power of designed RNA structures for their areas of interest rather than relying on domain experts who know the quirks and limitations of their favourite piece of software. Furthermore, better kinetic folding and MD simulation methods which are able to accurately model the complex, (pseudo)knotted folding pathways of RNA nanostructures are necessary to avoid costly and frustrating experimental re-engineering.A deeper understanding of how RNA structures interact with living systems, especially the immune system. There has been some progress in this direction ,125, layImproved production and purification methods. In order for RNA structures to be viable, especially as therapeutics, researchers need to achieve faster, cheaper, and higher fidelity production of structures. Based on the breakthroughs in DNA synthesis over the past 20 years ,127, theThere are many challenges that the RNA nanotechnology field must overcome to become a mature part of the biochemical toolbox. These can be broadly broken down into three categories:Looking at the momentum that the RNA origami field is gaining, it is interesting to ask: Will RNA origami replace DNA origami? Or will both technologies coexist? We believe that both technologies will continue to grow and find new applications in their respective niches. DNA origami will likely remain superior as a stable molecular breadboard to position secondary components. RNA origami will conquer the field of synthetic biology as the method of choice to build functional hardware co-transcriptionally. We are also likely to see additional approaches similar to that demonstrated in Parsons et al.  where lo


Physcomitrium patens is an RNA-binding pentatricopeptide repeat protein equipped with a terminal DYW-type cytidine deaminase domain. Transferred into Escherichia coli, PPR56 works faithfully on its two native RNA editing targets, nad3eU230SL and nad4eU272SL, and also converts cytidines into uridines at over 100 off-targets in the bacterial transcriptome. Accordingly, PPR56 is attractive for detailed mechanistic studies in the heterologous bacterial setup, allowing for scoring differential RNA editing activities of many target and protein variants in reasonable time. Here, we report (i) on the effects of numerous individual and combined PPR56 protein and target modifications, (ii) on the spectrum of off-target C-to-U editing in the bacterial background transcriptome for PPR56 and two variants engineered for target re-direction and (iii) on combinations of targets in tandem or separately at the 5’- and 3’-ends of large mRNAs. The latter experimentation finds enhancement of RNA editing at weak targets in many cases, including cox3eU290SF as a new candidate mitogenome target. We conclude that C-to-U RNA editing can be much enhanced by transcript features also outside the region ultimately targeted by PPRs of a plant editing factor, possibly facilitated by its enrichment or scanning along transcripts.The mitochondrial C-to-U RNA editing factor PPR56 of the moss  nad3 and nad4 transcripts of the moss Physcomitrium patens. Transferred into Escherichia coli, PPR56 edits its co-delivered native targets, but also more than 100 specific cytidines in endogenous bacterial transcripts. We have used the E. coli editing system to intensively study the editing properties of PPR56 as a prime example of a plant-type C-to-U RNA editing factor. We confirm that the selection of editing sites depends on the PPR region of the editing factor, which recognizes the RNA target upstream of the C to be edited. Some single amino acid modifications within this region can re-direct the editing factor to new targets, but others reduce or prevent editing completely. Modifications on the target side even outside of the RNA region recognized by the editing factor also affect editing efficiencies significantly. The combination of targets, for example, enhances the editing of the weaker target in many cases. Thus, not only the direct target-protein interaction, but also other transcript features influence the final selection of editing sites.RNA-binding pentatricopeptide repeat (PPR) proteins convert specific cytidines to uridines in mitochondrial and plastid transcripts of land plants to correct genetic information. PPR protein PPR56 is assigned to two editing sites in  PLS-type PPR arrays typical for RNA editing factors are characterized by “long” and “short” PPR variants with characteristic consensus profiles along with the canonical P-type PPRs of 35 amino acids. The E1 and E2 motifs of 34 amino acids are distantly related to TPRs (Tetratricopeptide Repeats) existing in other proteins, where they have been shown to mediate protein-protein rather than protein-RNA interactions. The more recent research on RNA editing and other processes of RNA maturation in the two endosymbiotic organelles of plant cells has clearly profited from parallel approaches taken not only with model flowering plants like Arabidopsis, maize or rice but also with bryophyte model organisms [The recent years have seen much progress towards understanding the molecular machinery behind cytidine-to-uridine RNA editing in plant chloroplasts and mitochondria –4. Amongleotides –14. The thaliana ,16. Suchrganisms . Flowerirganisms –20.Physcomitrium patens holds a key role as a model organism [Physcomitrium combine a stretch of pentatricopeptide repeats (PPRs) responsible for sequence-specific RNA recognition with a terminal DYW-type cytidine deaminase carrying out the site-specific C-to-U conversion.In contrast, a much simpler scenario has emerged for C-to-U RNA editing in “early-branching” land plants among which the moss organism ,21,22. Ain trans [Arabidopsis and relies on helper proteins but features an orthologue with a terminal DYW domain in the early-branching flowering plant Macadamia that was able to complement an RNA editing KO in Physcomitrium [To a large part, the complex editosomes of angiosperms seem to be the result of frequent separation of RNA target recognition and the catalytic DYW domain, now relying on protein-protein interaction including various helper proteins interacting in trans –31. Thisomitrium . Single Physcomitrium patens has a prominent role with its only 13 C-to-U RNA editing sites assigned to nine site-specific RNA editing factors [Physcomitrium is in no way representative for other bryophytes, which feature the full spectrum of RNA editing being entirely absent in the marchantiid liverworts, with massive C-to-U RNA editing in the early-branching moss Takakia lepidozioides [Anthoceros agrestis [Physcomitrium only nine are RNA editing factors and all of them, including PPR56 investigated here, are characterized by a PLS-type PPR array linked to a terminal DYW cytidine deaminase domain via the E1 and E2 domains [ factors ,22,33. Hozioides  or with agrestis . Among a domains .Physcomitrium could be functionally transferred into heterologous systems like the bacterium Escherichia coli [It is likely no surprise that the simple one-protein RNA editing setup of hia coli  and, morhia coli . The bacPhyscomitrium patens has been functionally characterized some years ago [Physcomitrium. Editing efficiency at its second target, nad3eU230SL, is more variable and may depend on environmental conditions but is generally above 70% in planta [nad subunits of respiratory chain complex 1, the NADH ubiquinone oxidoreductase), the RNA editing event towards uridine (eU), the transcript position counting from the first nucleotide of the AUG start codon and the resulting codon change, here serine to leucine in both cases [The mitochondrial RNA editing factor PPR56 of ears ago  and appen planta ,39. The th cases .th and the last (L) amino acid within the two antiparallel α-helices constituting an individual PPR are key to recognizing individual ribonucleotides with position ‘5’ distinguishing purines (adenosines or guanosines) from pyrimidines (cytidines or uridines) and position ‘L’ defining preferences for amino (A or C) or keto nucleobases (G or U). However, the situation is notably more complex for PPR proteins acting as RNA editing factors, which not only feature canonical ‘P-type’ PPRs of 35 amino acids but also variants with different consensus profiles and slightly variable lengths. Most widely distributed are the variants ‘L’  and ‘S’  contributing to PLS-type PPR arrays in most plant RNA editing factors. Yet more PPR variants such as ‘SS’ and ‘LL’ have recently been identified in the growing amount of genomic data for the huge PPR gene families in land plants, now also including hornworts, lycophytes and ferns [Defining a PPR-RNA recognition code has been a tremendous step forward in understanding the operation of pentatricopeptide repeat proteins –44. At tnd ferns .E. coli RNA editing assay system [The PPR-RNA code outlined above can be applied only to P- and S-type but not to L-type PPRs and the functional role of the latter remained mysterious. Notably, despite a conceptually slightly better overall fit of the nad3eU230SL target to the P- and S-type PPRs of PPR56 , the nady system . Hence, E. coli transcriptome as well as cox3eU290SF as a predicted further candidate plant mitogenomic target of PPR56.Here, we explored the impact of PPR56 protein mutations and of modified, extended, combined and differently placed RNA targets in the easily amenable bacterial system to identify the relevant elements contributing to efficient RNA editing. Most importantly, we found that sequences further upstream of the region ultimately bound by the PPR array contribute to high RNA editing efficiency and that tandem combinations of target sequences can significantly enhance RNA editing at previously less efficiently edited downstream targets. The latter include both selected off-targets in the Moreover, we observed that placing the otherwise moderately edited nad3eU230SL target of PPR56 in the 5’- vs. the 3’-UTR of a long mRNA can enhance RNA editing even above the level observed in its native plant mitochondrial environment. Hence, the wider environment of the core RNA target sequence as defined by the PPR array contributes notably to the observed RNA editing efficiencies. Altogether, we conclude that the operation of PLS-type RNA editing factors like PPR56 relies not only on the defined code for P- and S-type PPRs but also on the hitherto enigmatic L-type PPRs and on the wider transcript environment.PPR56 is a plant C-to-U RNA editing factor equipped with a highly conserved carboxyterminal DYW-type cytidine deaminase domain linked to an upstream PLS-type PPR array via the E1 and E2 extension motifs . For claFor mutations on the RNA target side, we will use small letters to label nucleotide changes and indicate positions relative to the editing site, which are added behind the respective RNA editing site labels after pipe symbols. For example, nad4eU272SL|u-4g will indicate the U-to-G exchange introduced four nucleotides upstream of the RNA editing site, which is assumed to be juxtaposed with the terminal S2-type PPR of PPR56 .Escherichia coli based on vector pET41Kmod [6-MBP tag behind an IPTG-inducible T7 promoter controlled by the lac operator and the respective target sequences are inserted in the 3’-UTR followed by a T7 terminator sequence. For further experimentation allowing to place target sequences alternatively also in the 5’-UTR, we equipped pET41Kmod with an additional MCS upstream of the protein coding sequence, giving rise to pET41Kmod2 . Replacing proline with alanine in the eponymous PG box at the N-terminus of the DYW domain (PPR56|DYW:P2A) has a much stronger effect than the corresponding replacement of the following glycine residue (G3A), despite 100% conservation of the latter in all nine Physcomitrium RNA editing factors -like E1 and E2 motifs linking the DYW domain and the upstream PPR arrays of plant RNA editing factors is still unclear. Given their location in the proteins and the distant similarity of TPRs to PPRs, E1 and E2 may contribute to binding to nucleotide positions -3 and -2 upstream of the cytidine target, but with a matching code different from the one for the P- and S-type PPRs. In the E2 motifs of RNA editing factors, the corresponding positions ‘5’ and ‘L’ (i.e. position 34 in the TPR-like E motifs) are strongly dominated by valine (V) and lysine (K), respectively, suggesting a structural rather than nucleotide-specific role. In contrast, position ‘5’ of E1 shows no significant conservation at all, but position ‘L’ (34) of the E1 motif has a resemblance to PPRs with aspartate (D) or asparagine (N) dominating in the conservation profiles. Given the unique opportunity to test impacts on the two different targets of PPR56 we created mutants PPR56|E1:N34D and PPR56|E2:K34A and tested them on both targets . While mnad3 target: nad3eU230SL|c-6u, which improves the conceptual fit to PPR P-3ND, hence fitting expectations. In the majority of mutants, we observe that effects are much stronger for the nad3eU230SL than for the nad4eU272SL target  and could not be rescued by corresponding mutations in either target , we found that RNA editing of the native targets was likewise significantly decreased  and higher editing of the adapted one , while editing of the original nad3 target and in the nad3eU230SL|g-13a mutant is abolished completely. The inverse mutation in the directly neighboring PPR P-9TN>TD again has only moderate effects on the nad4 target  had moderate consequences or could be rescued to a significant amount by corresponding changes in the targets . The S-14 target . HoweverGiven the striking outcome of completely abolished RNA editing for the S-7TD>TN mutant that could not even be partially rescued by the corresponding g>a exchanges in the two targets , we combnad3 target but only reduced to 89% for the generally more robust nad4 target , L-8VD (c vs. a) and L-5LD (u vs. c). Hence, we mutated these positions to check whether they could contribute to the different RNA editing efficiencies observed for nad4eU272SL and nad3eU230SL . In a set to 76% . This, anad3 target is again more affected, even by identical nucleotide exchanges in the same positions as in the nad4 target. For example, this is clearly seen for target mutations both immediately downstream of the respective edits, i.e. nad4eU272SL|a+1u (>99%) vs. nad3eU230SL|a+1u (49%) and nad4eU272SL|u+2g (>99%) vs. nad3eU230SL|u+2g (61%) as well as upstream of the respective edits: nad4eU272SL|c-3u (>99%) vs. nad3eU230SL|u-3c (22%) or nad4eU272SL|u-2g (31%) vs. nad3eU230SL|u-2g (0%).The general avoidance of a guanosine in position -1 immediately upstream of a cytidine to be edited has been recognized since long and is unequivocally supported by large editome data sets . Moreovenad4 target that had proven to be significantly more tolerant against variations. Indeed, all three possible stop codons  could be efficiently created by editing after mutations in positions +1 and/or +2 with >99% editing efficiencies  but also in position -5 opposite of PPR L2-VD  and moderate (nad3) efficiencies offered an interesting test case allowing to check upon RNA editing activities at targets of PPR56 in varying combinations . Notably, the enhancing effect on the downstream nad3 target remained unaffected, still resulting in >99% conversion at the nad3eU230SL target  into the upstream nad4 sequence was used  that showed variable editing efficiencies at different RNA read coverages and different matches to the PPR array of PPR56 , but rather exist in a pre-edited state with thymidines in the genomic positions. Using the TargetScan option of PREPACT [Physcomitrium. Indeed we could find cox3eU290SF as such as potential target matching excellently to the RNA binding properties of PPR56  using RNA oligonucleotides representing the region bound by the PPR array. Such experimentation has contributed tremendously to understand their mode of binding and may be entirely sufficient for the study of P-type PPR proteins, which largely stabilize transcript ends by tight binding to an RNA, for example. However, scenarios may differ for the PLS-type PPR proteins like RNA editing factors, which are expected to bind only temporarily to allow for cytidine deamination. The in vivo experimentation in E. coli reported here strongly suggests that the wider transcript environments and the placements of targets matter significantly for the ultimately detected RNA editing frequencies.PPR proteins are frequently investigated by th or last amino acid appears to very much rely on their respective position and/or the overall structure of the PPR array. Hence, any future experimentation with native RNA editing factors or those based on artificial “designer” PPR arrays [We here report that several circumstances affect RNA editing efficiencies even for “simple” single-polypeptide RNA editing factors like PPR56, including (i) the enigmatic L-type PPRs, (ii) the RNA sequences further upstream of the region ultimately bound by the PPR array, (iii) the tandem combination of targets or (iv) their respective placement in long transcripts as here exemplarily shown for the 5’- and 3’-UTRs flanking the PPR56 coding region with our modified vector setup. Whether binding preferences of individual PPRs in plant editing factors can be simply changed via modification of the 5R arrays ,67,73–80Physcomitrium patens PPR56 variants and targets in Escherichia coli was based on vector pET41Kmod as outlined earlier [6 tag and the maltose-binding protein (MBP) for improved protein solubility [XbaI site to create a NotI-EcoRI-PacI-PstI multiple cloning site (MCS) upstream of the ribosome binding site (RBS) in pET41Kmod. Target sequences including flanking restriction sites were generated with synthesized oligonucleotides for both DNA strands  and ligated into dephosphorylated vectors after hybridization and phosphorylation. All oligonucleotides used in the course of this work are listed in Cloning for expression of  earlier . PPR56 clubility  behind aE. coli system and the downstream analysis of RNA editing was done as outlined previously [E. coli Rosetta 2 (DE3) cultures were pre-grown in 100 mL Erlenmeyer flasks with baffles in LB medium supplemented with 50 μM kanamycin, 17 μM chloramphenicol and 0.4 mM ZnSO4 at 37°C until reaching an OD600 of ca. 0.5. The bacterial cultures were then cooled on ice for 5 min. before adding 0.4 mM IPTG for induction of expression and incubation for 20 h at 16°C and 180 rpm. To further explore the expression system, we here also tested elevated incubation temperatures of 24°C instead of the routinely used 16°C for incubation after induction of expression  and secondary antibody Rabbit anti-Mouse IgG (H+L) (Invitrogen).The setup for the expression of different constructs in the heterologous eviously . Brieflypression  and shorpression . These eE. coli transcriptome, total RNA was prepared from individual experiments by using the Nucleo-Spin RNA kit (Macherey-Nagel), followed by DNase I treatment (Thermo Fisher Scientific). Library preparation was done after rRNA depletion , followed by Illumina sequencing (150 bp paired-end with NovaSeq 6000) done by either Novogene or Macrogen. To generate construct-specific DNA reference reads, the simulated reads (by ART MountRainier version 2016-06-05) of pET41Kmod with PPR56 and respective target sequences were merged with genomic DNA reads (WTDNA_SRR941832) of BL21(DE3) cells [E. coli BL21 genome (CP010816.1). The datasets obtained are summarized in https://www.bioinformatics.babraham.ac.uk/ projects/ fastqc/), the transcriptome reads were aligned with construct-specific DNA reads against the construct-specific reference by GSNAP v2020/04/08 [To identify off-targets in the 3) cells . The con20/04/08  with pro20/04/08 . The SNP20/04/08 . The SNP20/04/08 . Final RS1 FigPhyscomitrium patens has been obtained with WebLogo [The conservation plot based on the alignment of the DYW domains of nine functionally characterized RNA editing factors of  WebLogo . Highlig WebLogo . The “ga WebLogo  is highl(TIF)Click here for additional data file.S2 Fig6 tag. Transcription is driven from a T7 promoter controlled by a lac operator and translation is initiated by a ribosome binding site (RBS). PPR56 is cloned with an N-terminal extension of 14 native amino acids upstream from its N-terminal PPR L-14. Target sequences were designed with hybridized oligonucleotides inserted by classic cloning into a multiple cloning site  in the 3’-UTR between attB2 and a T7 terminator. A new vector variant pET41Kmod2 has been created which also allows for cloning target sequences alternatively upstream into the 5’-UTR in a second MCS (NotI-EcoRI-PacI-PstI) inserted into a previous XbaI site. The vector map was created with SnapGene Viewer 6.2.1 (https://www.snapgene.com).Vector pET41Kmod for expression of RNA editing factors and their targets has been reported previously . Coding (TIF)Click here for additional data file.S3 Fighttps://varna.lri.fr).Artificial sequences have been added upstream (yellow) or downstream (green) to embed the cytidine targeted for RNA editing (red) into secondary structures. The sequence upstream of the cytidine editing target that is supposedly juxtaposed with the PPR array of PPR56 see  is shown(TIF)Click here for additional data file.S4 FigE. coli Rosetta 2 (DE3) arctic express system for a selection of altogether twelve constructs. The elevated temperature of 24°C generally disfavors RNA editing compared to incubation at 16° both on nad4 and on nad3 targets with the interesting exception of the PPR56|DYW:P2A mutant. B. RNA editing was checked for eight selected constructs also at shorter incubation times of only 4 h or 8 h, respectively, instead of the routinely used 20 h of incubation at 16°C after induction of expression. A reduction of RNA editing is seen in all cases of shorter incubation times except for the efficiently edited nad4 target, which already shows >99% editing after 8 h of incubation.A. RNA editing was checked at an elevated temperature of 24° (orange bars) instead of the routinely used 16°C (blue bars) for heterologous protein expression in the (TIF)Click here for additional data file.S1 DataE. coli RNA editing assays including standard deviations. C-to-U RNA editing frequencies are given as 100% when no remaining cytidine signal was detectable upon sequencing of RT-PCR products.Full table of results for all individual (XLSX)Click here for additional data file.S2 DataE. coli wild-type background control for reference, native PPR56 without co-delivered targets (2 replicates), with co-delivered nad3eU230SL target, nad4eU272SL target (2 replicates) and combined nad4-nad3 target, PPR56|S-10TD>TN without or with co-delivered target nad4eU272SL or nad4eU272SL|g-13a, and PPR56|S-4TN>TD without or with co-delivered target nad4eU272SL or nad4eU272SL|a-7g) analyzed in the course of this study.RNA-seq datasets analyzed for C-to-U RNA editing off-targets. Separate tabs for the summary off-target lists for PPR56, PPR56|S-4TN>TD and PPR56|S-10TD>TN and 13 individual data sets for JACUSA variant calls ((XLSX)Click here for additional data file.S3 DataOligonucleotides used in this study. All oligonucleotides were synthesized by IDT .(XLSX)Click here for additional data file. 24 May 2023Dear Drs. Knoop and Schallenberg-Rüdinger,Thank you very much for submitting your Research Article entitled 'Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56' to PLOS Genetics.The manuscript was fully evaluated at the editorial level and by three independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript.We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.In addition we ask that you:1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.archive. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.Please let us know if you have any questions while making these revisions.Yours sincerely,Bao-Cai TanGuest EditorPLOS GeneticsLi-Jia QuSection EditorPLOS GeneticsDear Drs. Knoop and Schallenberg-Rüdinger,Thanks for submitting your manuscript to PLoS Genetics.This manuscript by Yang et al. used a heterologous system in E. coli to dissect the determinants in the P. patens PPR56 recognition of its targets by introducing various mutations to both the RNA and the PPR protein. The findings, as pointed by the three reviewers in this field, have a significant contribution to the understanding of the molecular basis of RNA metabolism / RNA editing. Although all the reviewers recognize the high quality and value of this work, they also made some comments and suggestions, which I think could improve this work. In particular,1) if the authors could analyze function of the E1 and E2 motif in target site recognition, it will provide a complete story.2) Also, it would strengthen the conclusion if the authors could test the mutation u-16c that improves the conceptual fit to PPR S-13NS.3) Reviewers also suggested a revision of the text, to make it more clear, logical, and easier to the reader.4) The parts focusing on “Exploring novel candidate targets” in the Results and Discussion parts are not entirely clear. This should be explained in detail.Considering these comments and suggestions, a minor revision of this manuscript is required before it can be accepted for publication in PLoS Genetics.Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: In this report, the authors tested the RNA editing efficiency of the mutations of moss DYW-type RNA editing factor PPR56 on its target variants in the heterologous bacterial system. They found that (1) the mutations in DYW domain of PPR56 show different effects on the editing of two native targets; (2) the two native targets of PPR56 display different nucleotides opposite of its three central L-type PPR motifs; (3) several transcript features beyond the region ultimately targeted by the PLS-type PPR array likely contribute to enhance the editing activity. RNA secondary structures inhibit RNA editing, whereas native sequences further upstream enhance RNA editing. This work expands our understanding of how PPR proteins recognize and edit substrate RNA targets. The paper is well written, easy to read with experiments supporting most of the conclusions. I have only minor comments.1. The N-terminal PPR motifs of PPR65 appear to have a significant impact on the editing of nad3eU230SL target, as the mutation u-15c abolishes editing completely. It would be nice if the authors test the mutation u-16c that improves the conceptual fit to PPR S-13NS. Increased editing efficiency is a stronger support for the conclusion.2. It is not clear from the Materials and methods whether the transit peptide was removed from the proteins assayed in bacterial experiments.3. The authors should explain why the corresponding triple-mutations converting positions -14, -11 and -8 to the identities in the respective other target decrease editing at the nad4 target to 26% but improve editing at the nad3 target to 76%.4. In Fig.8, a negative control should be included to avoid that the reason for the increase in editing efficiency at nad3eU230SL site is due to the addition of a nucleic acid sequence before the target.5. P492. “of” should be “or”.Reviewer #2: see uploaded reviewReviewer #3: The expression of genes in plant organelles requires coordination of multiple RNA processing steps at the post-transcriptional level. This is run by a multitude of nucleus-encoded RNA-binding proteins (RBPs) that control RNA stability, processing, and degradation. PPR proteins play a pivotal role in these critical processing events in land plant mitochondria. In their MS, Yingying Yang et al analyzed the PPR56 factor from Physcomitrium patens in RNA editing. The authors provide with further details, following their progress made by using a heterologous system for RNA editing in the bacterium E. coli system, regarding the RNA editing properties of PPR56 to its two native transcripts in P. patens, nad3eU230SL and nad4eU272SL. Taking the advantage of this powerful system, the authors introduces various mutations to both the RNA binding PPR module, as well as to the deaminase (DYW) domain, to study the basis of RNA recognition and C-to-U substitutions mediated by the PPR56 cofactor. Undoubtedly, the MS and data therein are of high quality and provides with an excellent contribution to our understanding of these important cellular processes. Noteworthy, the authors are also leading experts in these field, where they have a significant contribution to the understanding of the molecular basis of RNA metabolism / RNA editing and the roles played by nuclear-encoded organellar-localized factors in mitochondrial  RNA maturation. I mostly have minor comments to the authors.Specific comments:1. The text may benefit from some revisions to make the MS, data and points raised therein easier to understand and read.2. Introduction part: Although referred later in this part, it would be easier for the reader if the authors provide with some details about PPR proteins as modular RBPs (often site-specific) as well to their "extra" related domains  that are associated with substrate RNA "editing to the beginning of the Intro.3. Mutating the PPR and DYW domains: One way to identify and characterize a domain is to find the part of a target protein that has sequence or structural similarities with a template through homology alignment. This is nicely demonstrated in the seq alignment of the nine DYW domains of the P. patens  but should be also presented by a phylogenetic illustration as well . An interesting question is whether swapping different DYW domains (e.g. from PPR65 into PPR56) may influences the editing, assuming that the sequence specificity resides mainly within the PPR repeats…4. Regarding the effects of aa's substitutions on editing activity: This is done in a heterologous system and thus the effects should be taken with some cautious. Also, the fact that an amino acid was found to be conserved at the nine DYW domains in P. patens doesn't necessarily indicate that it has any importance for the activity or folding of the protein/domain . The authors may want to comment on possible effects (predictions) of these substitutions on the folding of the DYW domain or the PPR arrays.5. The parts focusing on “Exploring novel candidate targets” in the Results and Discussion parts are not entirely clear. The expression, folding and activity of the recombinant P.s.PPR56 in E. coli can affect its specificity and activity resulting with various off-targets in E. coli that may not occur under the native condition in the plant mitochondria.6. Intro line 47: To a large part, the complex editosomes of angiosperms. Please elaborate on (plant mito) editosome(s).7. Intro lines 56-57: “Physcomitrium patens has a prominent role with its only 13 C-to-U RNA editing sites assigned to nine site-specific RNA editing factors. Please provide with REFs.8. Line 63: To readers which are not familiar with PPRs, please explain what are PLS-PPRs.9. Lines 111-115: This part is unclear and thus could be simplified.10. Results line 118: “ a typical “complete”, and likely evolutionarily ancestral,” looks to me as an unnecessary comment.11. Line 137: The coding sequence of PPR56 is cloned in fusion with…, consider in-frame to12. Line 406: Exploring novel candidate targets – “It is important to keep in mind that orthologues of a functionally characterized”. That's probably depends on the homology, and these might rather represent paralogs in such cases?!13. The discussion part can be shortened and more focused on the data (see comments 3 to 5).14. Also, and wityh regard to lines 537-541 is PPR56 is expected to be part of an editosome in P. patens, and whether this factor could be associated with additional RBPs in E. coli?! How these situations influence the activities seen in vivo, in vitro or in the heterologous E.coli systems?**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesNo: The authors indicate that RNAseq data have been deposited in the SRA archive but don't provide with BioProject REF data.Reviewer #3: **********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  16 Aug 2023PGENETICS-D-23-00376R1 Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56 Dear Dr Schallenberg-Rüdinger, We are pleased to inform you that your manuscript entitled "Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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Here we demonstrate that the first 160 amino acids of PYM (PYM1–160) are intrinsically disordered. PYM1–160 binds RNA independently of its nucleotide sequence, forming a fuzzy protein–RNA complex that is incompatible with PYM’s function as an EJC recycling factor. We propose that the role of RNA binding consists in down-regulating PYM activity by blocking the EJC interaction surface of PYM until localization has been accomplished. We suggest that the largely unstructured character of PYM may act to enable binding to a variety of diverse interaction partners, such as multiple RNA sequences and the EJC proteins Y14 and Mago.The discovery of several functional interactions where one or even both partners remain disordered has demonstrated that specific interactions do not necessarily require well-defined intermolecular interfaces. Here we describe a fuzzy protein–RNA complex formed by the intrinsically unfolded protein PYM and RNA. PYM is a cytosolic protein, which has been reported to bind the exon junction complex (EJC). In the process of  The localization of mRNA is an evolutionarily conserved process needed for spatial and temporal control of protein expression within the cell. For example, mRNA localization mediates confined protein expression during embryonic development, helping to sustain cell polarization and promote further differentiation. mRNA localization is essential for all developmental processes, such as cell movement, cell specialization and asymmetric cell division . The absDrosophila melanogaster (Dm), oskar mRNA localizes to the posterior pole  approximately 20–24 nucleotides (nt) upstream of the exon-1–exon-2 junction is essential for oskar localization  RNA—which consists of 18 nt from exon-1 and 10 nt from exon-2  is important for localization  . This exDrosophila, PYM does not interact with the ribosome and its function as an EJC dissociation factor is independent of translation  data, we demonstrate that PYM1–160 binds the SOLE RNA, as well as many other RNAs with both double-stranded and single-stranded structures. The interaction appears to be largely independent of the RNA sequence. Critically, the PYM–RNA complex is incompatible with the binding of PYM to the Mago–Y14 dimer, which engages the same PYM surface as RNA. We propose that the PYM–RNA complex serves to sequester PYM from fulfilling its role in EJC disassembly by blocking its canonical interaction with Mago–Y14, thereby preserving the EJC until oskar localization is complete. At the same time, the interaction of PYM with the RNA may contribute to ensure that PYM is localized together with the mRNA and the EJC. Our model implies that at end of the localization process PYM is released from the RNA to bind the Mago–Y14 heterodimer. This may happen through a conformational change of the mRNA and/or the EJC triggered by an as yet unknown event. While we started this investigation with the hypothesis that the SOLE RNA element is critical for PYM recruitment, we have found that the SOLE RNA is not a unique RNA binding partner of PYM but other double-stranded RNAs bind PYM with similar affinities. Thus, the role of the specific SOLE RNA element remains to be determined.Here, we determine the structure of PYM1–160 was cloned into the pET-M11 vector (EMBL plasmid library) with a cleavable His6-tag at the N-terminal end. The DNA was transformed into BL21 (DE3) cells and grown in LB media at 37°C. Protein expression was induced by adding 1 mM of IPTG at an O.D. of 0.8; the cell were allowed to grow at 16°C overnight and were harvested by centrifugation at 5,000 g for 20 min at 4°C.PYMThe bacterial cell pellet was resuspended in lysis buffer , sonicated on ice (for 15 min at 50% amplitude with 5 sec-on and 8 sec-off pulses) (Bandelin sonicator with KE76 probe) and centrifuged at 18,500 rpm using an ultracentrifuge (Beckmann Allegra). The supernatant was collected, loaded on a Ni-NTA column  and washed at least 3 times with 3 column volumes (CVs) of 500 mM LiCl and lysis buffer, alternately, to remove potentially bound nucleic acids. The protein was then eluted with a gradient of 0–500 mM imidazole over 4 CVs. The absence of contaminating nucleic acids was confirmed by evaluating the ratio of the protein sample absorption at 280 and 260 nm. In the next steps, the protein was desalted using a desalting column 26/10 (Cytiva), loaded on an heparin column  and eluted with buffer containing 50 mM Tris, pH 7.0, 2.5 mM β-mercaptoethanol, 50 mM glycerol and 1 M NaCl, followed by desalting and overnight cleavage with TEV protease (1:25 ratio) in the presence of 2 mM dithiothreitol (DTT). The protein was then purified by reverse His-trap, and further by size-exclusion chromatography in buffer containing 50 mM Tris, pH 7.0, 50 mM NaCl, 2.5 mM β-mercaptoethanol, 50 mM glycerol, using an S75 10/300 column (Cytiva). The protein was tested for RNAse contamination before further experiments using the RNAse Alert test kit (Applied Biosystems).in-vitro transcription using T7 polymerase produced in-house and a DNA template cloned in pUC19. Before large-scale RNA transcription, the concentrations of DNA, nucleoside triphosphates (NTPs) (Carl Roth), MgCl2 and T7 polymerase were optimized to maximize the yield. The RNA was purified by denaturing 12% polyacrylamide gel electrophoresis. To obtain homogeneous 3′ ends, trans-acting hammerhead RNA was used to cleave the SOLE RNA at a well-defined position (tris(2-carboxyethyl)phosphine (TCEP).SOLE RNA was prepared by position . The RNAThe following RNA constructs were designed and obtained from Sigma for NMR titrations and EMSAs (electrophoretic mobility shift assays). The purity of the Cy5-labelled RNAs varied slightly from batch to batch with some RNA preparations showing the presence of shorter oligonucleotides .SOLE RNA (32mer):5′-Cy5-GACGAUAUCGAGCAUCAAGAGUGAAUAUCGUC-3′5′-GACGAUAUCGAGCAUCAAGAGUGAAUAUCGUC-3′26mer SOLE RNA:5′-Cy5-GAUAUCGAGCAUCAAGAGUGAAUAUC-3′24mer SOLE RNA:5′-AUAUCGAGCAUCAAGAGUGAAUAU-3′22mer SOLE RNA:5′-Cy5-GAUCGAGCAUCAAGAGUGAAUC-3′20mer SOLE RNA:5′-GAUCGAGCAUCAAGAGUGAAUC-3′16mer SOLE RNA:5′-Cy5-CGAGCAUCAAGAGUGA-3′5′-CGAGCAUCAAGAGUGA-3′22mer PS RNA (PS stem of SOLE):5′-Cy5-GACGAUAUCUUCGGAUAUCGUC-3′SOLE RNA MSL mutant (32mer).5′-Cy5-GACGAUAUCACACAUCAAACACGAAUAUCGUC-3′dsRNA_1:5′-Cy5-GCGACCUGAGG-3′5′-GCGACCUGAGG-3′dsRNA_2:5′-Cy5-GCAUCGAAG-3′dsRNA_1 (complementary strand):5′-CCUCAGGUCGC-3′dsRNA_2 (complementary strand):5′-CUUCGGGGC-3′ssRNA_1:5′-Cy5-GCAUAAGAAAUU-3′ssRNA_2:5′-Cy5-GCAUAAGAAAGG-3′5′- GCAUAAGAAAGG-3′1–160 were added to reach RNA:PYM1–160 concentration ratios of 1:0, 1:1, 1:2 and 1:5 . As the efficiency of Cy5-coupling was different for each RNA, we used a different amount of labelled RNA for each RNA construct, to reach similar intensities of the fluorescence signal. We then complemented the amount of labelled RNA with unlabelled RNA to reach a final amount of 65 pmol. The RNAs were annealed by heating at 94°C for 3 min followed by gradual cooling to room temperature. Increasing amounts of PYM and 1:5 . The fin and 1:5 . The gel and 1:5 .15N-labelled PYM1–160 in buffer containing 50 mM Bis-Tris, pH 6.0, 50 mM MES, 50 mM NaCl, and 2.5 mM TCEP were monitored in 15N-HSQC spectra in the presence of increasing concentrations of RNA. The chemical-shift perturbations (CSPs) were calculated according to the formula:1H and 15N dimensions, respectively.The backbone amide resonances of 30 μM of 2-cooled triple-resonance probe-head. The spectra were processed in Topspin 3.2 (Bruker) and analysed in CcpNmr v2.4  and running Topspin 3.2 software (Bruker).Uniformly 15N-HSQC spectra were recorded using States-TPPI for frequency discrimination, with water suppression achieved via a combination of WATERGATE and water flip-back pulses to preserve the water magnetization CACB experiments were recorded with constant-time chemical-shift evolution in the 13C dimension (constant-time period = 28.6 ms). Aliphatic side-chain assignments were obtained from 3D H(CCCO)NH CO, HNCACB, HN(CO)CACB experiments , recorde(CCCO)NH  and H(C)(CCCO)NH  experime(CCCO)NH . Approxi13C-HSQC and NOESY–15N-HSQC spectra (mixing time = 150 ms)  .http://gwagner.med.harvard.edu/intranet/hmsIST/gensched_new.html). The time-domain data matrices were reconstructed using the hmsIST software with the assistance of the GNU program “parallel” (All 3D NMR spectra were recorded with non-uniform sampling (NUS) to maximize resolution in the respective indirect dimensions. NUS schedules were generated with Poisson-Gap sampling using the on-line Schedule Generator from the Wagner group  were calculated with TALOS-N from the backbone chemical shifts . The Che15N- and 13C-edited NOESY spectra were analysed in CcpNmr and the intensities of cross-peaks were used to quantify the inter-proton distances. In the NOESY spectra we picked a total of 3,146 peaks, of which we could assign 3,106. Only manually assigned peaks were used in the structure calculations by ARIA 2.3/CNS1.2 . The RNAs tested consisted of : 1) the SOLE RNA PS capped by a tetraloop (22mer PS RNA); 2) SOLE RNA constructs with progressively shortened PS stem by removal of nucleotides at 5′ and 3′ ends to yield 26mer, 24mer, 22mer , 20mer and 16mer RNAs; 3) two 12mer single-stranded (ss) RNAs with random sequences (ssRNA_1 and ssRNA_2); 4) a double-stranded (ds) RNA of 11 canonical base pairs (dsRNA_1); 5) a dsRNA of 9 base pairs, including one non-canonical G–A and one non-canonical G–U base pair (dsRNA_2).To find out whether PYM binds specifically to the SOLE RNA and if so, which of its structural elements are responsible for the binding, we designed several other RNAs and tested their interactions with PYM1–160–RNA complex, both because the strong positive charge of the protein (pI = 9.7) impaired the penetration of the complexes into the gel, and because the fast dissociation rate of some of the complexes  led to a smeared band. Because of the lack of a clearly-defined bound-RNA band, we refrained from quantification of the EMSA data. However, PYM1–160 shifted all RNAs, demonstrating that the SOLE RNA is not a specific target of PYM  68 and 86 with a possible discontinuity at aa 83 . We conclude that PYM15–35 transiently forms a strand-turn-strand β-domain. To confirm this, we calculated the structure of PYM1–160 using ARIA 2.3/CNS1.2  revealed additional features of PYM RNA recognition. At 0.5 equivalents of 32mer SOLE RNA, the peaks of PYM1–160 were shifted nearly all the way to their final position  peaks behaved as for the 32mer SOLE RNA while the minor (less-intense) peaks corresponding to the second species were weak and not always detectable. Assuming that PYM1–160 exchanges rapidly between each of the RNA-bound states and the unbound state, but that the two RNA-bound states exchange only slowly  with each other . Because of the weak intensities of the minor peaks, we were unable to determine the value(s) of x. Only with the RNA constructs shorter than the 32mer SOLE RNA could the peaks corresponding to the PYM–RNAx species be clearly detected at 0.5 equivalents of RNA  impact the intensity of the unbound RNA band (When we compared the amount of unbound RNA detected in EMSA assays with the size of the chemical-shift perturbations across the different RNA constructs, we observed that the values do not correlate. For example, while in the NMR experiments, double-stranded RNA constructs, such the 22mer PS RNA and dsRNA_1, are nearly fully bound to PYMRNA band . Because1–160 identically to the 32mer wild-type SOLE RNA (Finally, a construct of the 32mer SOLE RNA with a mutated MSL incapable of forming base pairs bound PYMSOLE RNA , indicat1–160 is an intrinsically unfolded protein with the exception of an α-helical region comprising aa 68–81. In the structure of PYM1–58 bound to the EJC proteins Y14 and Mago, the first 35 residues form a three-stranded β-sheet and a contiguous β-hairpin (1–160, despite not being properly folded, populates only a sub-space of the fully unfolded conformational landscape and shows a significant preference for more compact, globular-like conformations.Our work reveals that PYM-hairpin  (Bono et-hairpin . While wThe formation of a globular-like region results in a co-localization of positive charges that build an RNA-binding surface. Binding to RNA does not induce folding of PYM into a rigid structure but instead largely preserves its disordered character, thereby maintaining the conformational entropy of the protein in the complex. PYM engages amino acids in the stretch 22–34 in RNA binding. These residues, such as the positively charged side-chains of R22, R28, K29, R31 and K34, are also involved in contacts with the Mago–Y14 dimer  Bono et, suggestDm PYM were first reported in (The RNA binding properties of orted in . Howeverorted in  is longeoskar mRNA. The formation of the PYM–RNA complex may serve the purpose of preventing PYM from acting on the EJC before mRNA localization has been accomplished. Whether the SOLE RNA participates in this process remains to be determined, since our studies suggest that PYM1–160 does not bind the SOLE RNA specifically, at least in vitro. However, the PS element, which is essential for localization (2–RNA complex relative to complexes with different stoichiometry. Similarly, the sequence of the MS element, which is irrelevant for localization (in vivo, it links the role of the SOLE RNA in localization with its PYM recognition features, making a role of the SOLE RNA in both mRNA localization and PYM binding at least plausible. The lack of sequence specificity in RNA recognition by PYM may be dictated by the function of PYM in different cellular contexts. The proposed role of the mRNA in down-regulating PYM activity before it reaches the target location is endorsed by the finding that over-expression of PYM inhibits oskar mRNA localization in a manner that is dependent on the relative amount of PYM and mRNA (The EJC is essential for localization, as demonstrated by . In the lization , appearslization , also aplization . Thus, tand mRNA .D. melanogaster, PYM acts to facilitate EJC recycling in mRNA localization, in humans PYM interacts with the ribosome, where it facilitates mRNA translation by stripping off the EJC (Dm PYM, with the human protein featuring an insertion of 8 amino acids as well as several proline and glycine residues. Our data show that this region of Dm PYM, and the disordered stretch following it, are involved in RNA binding. Whether the divergent sequences in this region, and potentially, their different RNA binding properties, recapitulate the different functions of PYM in the two organisms, remains to be determined.Interestingly, the RNA binding surface of PYM in the globular-like domain is conserved from flies to humans , suggest the EJC . The C-t the EJC , but theUsing a combination of NMR spectroscopy and EMSAs we have demonstrated that RNA helical elements recognize the unfolded PYM protein in a manner that is principally dependent on the overall structural fold of the RNA rather than the nucleotide sequence, and which does not result in well-structured protein–RNA interfaces. The driving force for the interaction is charge complementarity, together with preservation of conformational entropy. Notably, such interactions have been previously observed between two intrinsically unfolded proteins or unfolded protein stretches and have been associated with specific cellular functions . We sugg


Affiliation 1. Instead of “Huangpu Branch, Shanghai Ninth People’s Hospital affiliated to Shanghai Jiaotong University, Shanghai, China”, it should be “Huangpu Branch, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China”.In the published article, there was an error in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.