The aesthetic appreciation of natural places is one of the most fundamental ways in which people relate to their environment. It provides wellbeing, an opportunity for recreation and reflection, a sense of place, and cultural enrichment. It also motivates people to take care of natural places and to conserve them for current and future appreciation. Aesthetically valuable places also support significant economic activity. However, there is little guidance available to assist environmental managers and policy-makers to consider and integrate aesthetic values into decision-making processes. In this study, we present an approach for developing robust and practical indicators of aesthetic value to enable environmental managers to consider, assess and report on aesthetic condition and trend. We demonstrate its utility using the case of the Great Barrier Reef, a region currently undergoing significant social, economic and environmental change and an area formally protected, in part, for its aesthetic values. A qualitative scoping study with 30 key informants identified over 180 potential qualities contributing to reef aesthetics. We tested five for their utility in capturing key aspects of the coral reef aesthetic: (i) coral cover, (ii) coral pattern, (iii) coral topography, (iv) fish abundance, and (v) visibility. We asked 1,417 online Australians to aesthetically rate 50 out of 181 underwater coral reef images that varied in relation to these five attributes. Coral topography, fish abundance, and visibility were significantly correlated with aesthetic ratings, whilst coral cover and coral pattern were not. We also tested for demographic patterns in aesthetic ratings. Our pilot study has demonstrated that readily measurable characteristics of coral reefs can provide useful indicators of aesthetic quality, opening up opportunities for coral reef managers and policymakers to assess and track changes in aesthetics in ways that are relevant to the public. There is considerable scope to further advance capacity for monitoring and managing aesthetic values of coral reefs through additional research that resolves nuances in the meanings associated with aesthetics in coral reef settings. The importance of protecting beauty in nature has also become acknowledged in the context of sustainable development: the aesthetic values of biodiversity are recognised by Member States at the Rio+20 Conference as a part of the “Future We Want”  of Australia is one of the world’s largest coral reef systems. It houses globally-significant biodiversity, a rich diversity of underwater habitats, and a scenic appeal that is universally recognised . These vAcademics have been assessing the aesthetic value of landscapes since the 1970s , 36. MosWithin terrestrial environments, researchers have found that several natural cues inspire higher aesthetic scores. Rare species, diversity, biodiversity and complexity within landscapes are important , 43–46. Typically, across a range of landscape types, people prefer ‘openness, light and a good view’. Openness within a scenic environment refers to the degree of visibility and spaces within the surroundings and provides a sense of accessibility and movement . This maAesthetic ratings have also been related to congruency and continuity, or people’s sense of place, where familiarity as well as novelty are joint predictors of aesthetic judgment , 59. TheAesthetics can also be experiential. The sound or smell that is associated with an image can be important when responding to visual cues. For example, the aesthetic of water might be influenced by the sound of its travel or by the flow of movement . SimilarIndicators of aesthetic preferences within landscapes and seascapes need to be relatively objective and able to be similarly assessed by people through time to be useful for monitoring. It is likely that GBR managers are concerned that this might not be possible or sufficiently robust to assess for trends. For example, while some may judge an image by the pleasantness to the eye, another may regard its aesthetic value according to the composition of the image or its colour or light, whilst another may be influenced by the subject and an associated childhood experience , 61. Datcsshrec@csiro.au.Our first aim was to scope the potential indicators of aesthetic value within the context of the Great Barrier Reef (GBR). Scoping was important to identify and compare the many ways in which people appreciated the GBR. Using our own established and local networks, we identified people who were generally recognised for their long-standing relationship with the GBR. They were 50% female, with a mean age of 52.8 years (range 42 to 72 years), and the participants included well-known local reef photographers, senior reef managers, famous reef experts , and senior tourism industry representatives. They were invited to participate in the study by the primary author via email and written consent was provided by return email by those interested in participating in the study. Most interviews occurred as face-to-face interviews and generally lasted less than 30 minutes. Two interviews were conducted by telephone. All interviews were conducted by the primary author and not audio recorded. Extensive notes were taken, instead. Ethics approval was received from CSIRO Human Ethics Committee (023/17). Due to potentially identifying information, data is available upon request due to ethical restrictions imposed by the authors’ institutional ethics committee. Requests may be sent to the chair of the ethics committee, Dr. Cathy Pitkin, at The interview questions were open-ended and broad in nature so as to capture the most comprehensive range of responses that we could. The questions included:What would you say are the important aspects of the GBR to protect, particularly those that relate to the aesthetic qualities of the Reef?What should we be monitoring in order to know whether these aspects are being protected?Key themes and attributes identified from the interviews were analysed in Word, and text was coded into thematic concepts that could potentially be used as indicators . All initial coding was undertaken by the primary author. This process generated an initial list of 184 potential indicators. Related themes and attributes were then merged through iterative analysis, and in conjunction with the authorship team. For example, words such as shape, texture, composition were merged together into a theme called ‘composition’. This process continued until a final list of 12 indicators emerged that were faithful to the interview transcripts.From the 12 indicators we selected five that reflected distinct attributes of coral reefs, were analogous to existing biophysical measures, could be easily recognised by reef managers, and could potentially be incorporated into existing monitoring programs. They were (i) coral cover, (ii) coral pattern, (iii) coral topography, (iv) fish abundance, and (v) visibility.‘Coral cover’ represents the proportion of live coral that covers the substrate in a coral reef scene (e.g. ranging from 0% to >80%). ‘Coral pattern’ is likened to ‘coherence’ within the field of landscape aesthetics where a harmony arrangement within a landscape composition, such as unity in colour, shape, or texture, exists , 62. ‘Cohttps://gbrmpa.dams.me/), we selected 181 images representing typical, unglamourised, coral reef scenes. Selection criteria also included the perspective of a person in the water (snorkelling or scuba diving), looking forward across a substrate from a position between 5–10 metres above the substrate, in approximate depths of ten metres or less. This perspective was considered similar to the visual monitoring surveys conducted by the Australian Institute of Marine Sciences, the Great Barrier Reef Marine Park Authority, and other agencies. The photos can be accessed here: https://eatlas.org.au/nesp-twq-3/defining-assessing-monitoring-gbr-aesthetics-3-2-4From a large pool of underwater photographs provided by co-authors and sourced from publicly available collections .Members of the research team independently rated the 181 images against the five key attributes, assigning a rating of low, medium or high for each attribute, for each image. We note that this is a researchers’ point of view , and that non-researchers may have rated each attribute within each image differently. There was a high level of agreement between researchers, and in the small number of cases with differing scores, the image was reviewed and a final ranking agreed by consensus. The ranked images were mapped to a matrix to ensure each attribute and ranking were represented by a sufficient number of replicates to enable statistical analyses (see the full range of photos in www.pollinate.com.au). A survey was constructed to collect respondents’ basic demographic information, their self-rated level of interest in coral reefs, and their aesthetic ratings of a series of photos, using a scale of 1–10 . Once an individual agreed to participate, by accepting to participate in the study, they were allocated a randomised selection of 50 photographs to rate from the pool of 181 photographs. Our pilot testing of the survey suggested that 50 photographs could be rated within a 10 minute period. The style of the survey was not dissimilar from some online games in which individuals are asked to rank their aesthetic preferences of fashion or interior design items .Photos were then delivered to an online service provider with the ability to reach a large number of random, representative Australians (csshrec@csiro.au).A total of 1,417 individuals completed the online survey. Each photo was thus rated on the ten-point scale at least 380 times. Twenty-nine percent of the sample came from Queensland, and 71% were distributed across the rest of Australia. Some 62.3% of people came from Metropolitan Australia, whilst 37.7 came from rural/regional Australia. Some 51.4% were female. Participants reported a range of previous experience with the GBR and level of interest in coral reefs generally, where 7.2% had never visited , and 7.9Aesthetic ratings were analysed in SPSS, version 20, where we obtained the mean and standard error of the aesthetics rating for each photo. An analysis of covariance was conducted where (i) coral cover, (ii) coral pattern, (iii) coral topography, (iv) fish abundance, and (v) visibility, were tested in combination for their influence on the dependent variable, i.e. the mean aesthetic rating of each photo and each of the factors. The influence of gender, experience, age and self-reported interest in coral reefs were also assessed.The interviews with 30 key informants identified over 180 potential elements or themes contributing to reef aesthetics, indicating the richness of meaning behind the concept of aesthetics, while also highlighting the challenge of distilling a complex characteristic into a set of robust and practicable indicators for monitoring purposes. The full list of qualitative indicators, and the relative frequency with which they were mentioned by individuals is represented in Naturalness. Respondents spoke of the importance of naturalness in describing the aesthetic of the GBR, and the need for it to remain natural in order for aesthetics values to remain intact. Whilst some respondents did mention that the presence of boats, divers or snorkelers on the water could be aesthetically pleasing (see ‘recreation’ below), for the most part any human-made construct was generally perceived as an impact on aesthetic quality. Jetties, marinas, harbours were described as potentially aesthetically interesting infrastructure, where one respondent suggested that if such infrastructure had to go into the World Heritage Area, then every effort should go into ensuring it was as aesthetically pleasing as possible.Composition. Some participants talked about the aesthetic qualities of the GBR in terms of the structural landscape (where interesting topography was rated very highly), texture, pattern, diversity, habitat-complexity, structural heterogeneity, design, size and/or shape. Some participants used words such as Feng Shui, balance, and symmetry to convey the appreciation of the visual experience. Others described how they associated their visual experience with the experience of adventure, excitement, mystery or interest. For example, swim-throughs were mentioned twice as inviting images to explore.Clear Water. The clarity of the water was mentioned by nearly all respondents as contributing to their aesthetic appreciation of the GBR. For some, the clear water itself was beautiful, particularly with the sun shining through it, and particularly if there were small fish reflecting the sunshine just below the surface. Two respondents mentioned that they were aware that clear water did not always mean safe water where a cocktail of various toxins could exist within the water but not be visually detectable. Other respondents mentioned that muddy water was sometimes the more natural situation in areas such as Magnetic Island, and that knowing that helped them to appreciate water that was not necessarily clear.Colour. Colour was identified by many respondents as contributing to their appreciation of the aesthetic value of the Great Barrier Reef. Colour could refer to the colour of coral, the colour of coral bleaching, the colour of fish or the combination of colour within a single scene. Participants frequently described the blues, greens or turquoises of the water and/or water/sky and of the whiteness of the sand to describe important aspects of the GBR to protect. White sand visualised through clear blue water on a sunny day was mentioned several times.Experience. Some participants wanted to describe aspects of the GBR that they appreciated with words like movement (of fish or waves), connection with nature, destination, and referred to smell, sound, touch, taste, and/or opportunity for adventure and experience. These respondents regarded the aesthetics of the Great Barrier Reef in its entire sensory definition. For example, several participants described nooks and crannies, or swim-throughs or interesting topography that fell away to the unknown, as exciting or appealing and full of invitation to explore further. Aerial images of the Great Barrier Reef, with its channels and sea cays and islands, also made some participants want to experience the image.Charismatic. Whales, turtles, nesting seabirds, dolphins, big fish, clown fish, seahorses, nudibranchs, and several other charismatic animals were highlighted during the interviews as beautiful. Encounters with charismatic animals were described as special interactions that were very much about the personal response to being in nature and interacting closely with wildlife. These experiences were described as a critical part of their aesthetic appreciation of the GBR. One respondent remarked that the intense emotional environment created when interacting with Nature might be misinterpreted as beautiful, when it fact it might not be, but be a form of ‘love’ or ‘infatuation’.Coral. Coral was described by most people as the ultimate Great Barrier Reef aesthetic experience. Coral epitomised what was so special about the Reef and what was so beautiful. Coral was described in many different ways. Some respondents described it in terms of its composition , whereas others described it in terms of abundance/cover and diversity of species and form. Two respondents made comment that coral reefs were like old growth forests, where massive Porites coral bommies represented the slow-growing ancient giants of the system, whilst the staghorns, whilst beautiful, represented the fast-growing weeds or undergrowth of the system, and that both forms together represented aesthetic values, and were together, related to coral reef health.Fish. Fish, like corals, were described either in terms of their physical presence , or in terms of their ecological value (fish abundance and fish diversity). Most respondents agreed that fish abundance and diversity in fish size were important contributions to their aesthetic experience below the water. One respondent described how she liked to imagine that there were large schools of various fish on the Reef.Tiny things. This aspect of aesthetic appreciation relates to below the water, and is closely related to the clusters, experience and charisma. Many respondents liked to describe how they enjoyed interacting with the Reef as part of their aesthetic enjoyment of the Reef. Inevitably, this involved aesthetically appreciating the small invertebrates existing amongst the coral such as the ascidians, nudibranchs, sessile organisms and worms. These tiny organisms were beautiful to look at in their own right, but were also beautiful in their contribution to the conglomerate of what a coral reef represented to them.Healthy system. Some respondents described what they thought was a beautiful experience both above and below the water in terms of words such as healthy, connectivity, whole-of-system, alive, vibrant. Respondents described how sometimes it wasn’t possible to tell whether a coral system or any other natural system was healthy or not, and that education played an important role in helping people understand what was healthy and what was not. One respondent suggested that it was possible to change one’s perspective on what was beautiful simply by informing them on whether it was healthy or not. Bleaching came up frequently as an example of a system that is unhealthy but beautiful. Another respondent wanted to point out that if the whole system was healthy , then through the channels of connectivity, the best indicator would be a healthy coral reef . No-one described the role of people in the visual healthy image of a whole of Great Barrier Reef system.Recreation. Only a few respondents mentioned the role of people on the aesthetic of the Great Barrier Reef. This indicator cluster is closely related to the cluster of experience. These people described the image of boats on the water, particularly yachts, as relaxing, inspiring or beautiful. Importantly, they described only a few boats at a time, and sometimes they were described as nestled into bays. People enjoying themselves such as playing on the beach, and snorkelling or diving was seen as contributing to the aesthetic experience of these respondents.Aerial view or spatial considerations. Whilst most respondents were happy to discuss the aesthetic values of the Reef below or above the water, some respondents described the beauty of the reef from an aerial perspective. Several respondents described the appreciation of ‘space’. Vast, open areas that are so typical of the region were regarded by some as particularly stunning. This inevitably led to discussions about the various different scales that ought to be considered when one considers the beauty of the Reef. As such, this indicator cluster is closely related to tiny things and experience. The wide open space provided by beaches (Whitehaven and Port Douglas in particular) and the broad vista of the sea from the beach were particularly recognised as part of the aesthetic of the region.The mean aesthetic rating for all 181 photos was 6.86, with means for individual images ranging between 4.35 and 8.34  see . The higResults from the ANOVA suggested that coral topography, fish abundance and visibility were significantly correlated with survey participants’ overall ratings of the images’ aesthetic quality . People Our pilot study has demonstrated that readily measurable characteristics of coral reefs can provide useful indicators of aesthetic quality, opening up opportunities for coral reef managers and policymakers to assess and track changes in aesthetics in ways that are relevant to the public. Results from this study suggest that there are numerous factors that could potentially be used to monitor aesthetic quality within the context of the Great Barrier Reef (GBR) Marine Park. However, not all factors are likely to be suitable indicators for monitoring, given their inherently subjective nature. ‘Reef structure’ and ‘reef health’, for instance, were mentioned by some interview respondents, and further work would be required to convert these into indicators that are similarly interpreted by all people. Our approach chose five indicators (attributes) that could potentially be used in objective terms for standardised monitoring and assessment of coral reef environments, and to quantitatively test them for their ability to predict a general observer’s assessment of coral reef aesthetics. We found that ‘coral cover’ and ‘coral pattern’, while important emergent attributes from our key informant interviews, were not reliably correlated with how people judged the aesthetic quality of coral reef images. It may be that elements contributing to ‘coral pattern’ are too numerous and/or subjectively appreciated, requiring additional interpretation. However, given that ‘coral cover’ is a clear, standardised and robust attribute, we were surprised that it did not correlate significantly with our respondents’ overall aesthetics ratings. We suggest that, in isolation, high levels of coral cover may be insufficient to appeal aesthetically to a general observer in the absence of other attributes . Coral cover (and pattern for that matter) may be appealing attributes in many situations, however other factors can combine to negate their overall attractive influence. Instances of reef flats with high coral cover that also coincided with low fish abundance and low visibility (or other cues) meant that people did not perceive them as particularly beautiful.Coral topography, fish abundance and visibility were found to be statistically reliable predictors of aesthetic quality in this study. Coral topography, which provides habitat for creatures to hide in, might be appealing to people’s sense of adventure, exploration, and intrigue . Such unThe presence and abundance of fish were a highly significant influence on how people rated the underwater images. Fish abundance and fish diversity were both mentioned by most interviewees in the qualitative study. Fish may represent ecological completeness and wholesomeness within a seascape. One of our interviewees remarked “a coral reef without fish is like a playground without the laughter”. Fish also provide important, dynamic elements to a reef scene and photo composition. Fish add movement and colour, reflection and sound, and offer diversity of size and shape. Photos with high schooling behaviour  did not significantly influence aesthetic judgement. Apart from significantly higher overall ratings given by female respondents, which we are unable to explain, the only social factor that we found to be an important co-variable was the self-reported ‘interest in coral reefs’, where people who identified as more interested in coral reefs were more likely to rate a photograph highly. This can be interpreted where people are more likely to reinforce their own identity of being interested by finding the images of their interest particularly appealing.There is considerable scope to further advance the capacity for monitoring and managing aesthetic values of coral reefs, through research that resolves additional nuances in other attributes associated with aesthetics in coral reef settings. Many more indicators can and should be tested if aesthetic consideration, reporting and monitoring are to occur, not only within underwater seascapes, but within the variety of landscapes within which environmental managers work. This process of further testing is likely to be extensive, but with significant benefits for decision-making and management of coral reefs. Opportunities to perform mathematical image analysis may enable more reliable, reproducible and automatic measurements.Within the Great Barrier Reef, we identify three steps that managers can take initially to manage for aesthetics within the region. Firstly, we suggest that aesthetic indicators can be trialled alongside established monitoring programs for the purposes of adaptive management. The indicators that we have tested are, to some extent, already being recorded in ecological monitoring programs, and they have the potential to provide additional insights into trend and condition of aesthetic value. However, it is unlikely that the three indicators that we have tested are alone sufficient for management agency reporting and assessment, and further work will be needed. We also suggest that by asking monitoring staff to include holistic ratings of the aesthetic quality of each site over time, it is likely that, whilst absolute measures will not be possible (given the low sample size of monitoring staff), aesthetic changes through time may be detectable, as other environmental values change. We have not considered issues of shifting baselines, nor have we tested for different cultural preferences of aesthetic quality (such as the extent to which indigenous residents have different aesthetic preferences in landscapes), but both are likely to be important, and will continue to be a source of concern in issues validity for those interested in implementing aesthetic monitoring. Understanding the influence of shifting baselines and diverse cultural preferences will impart greater confidence in monitoring processes. It is possible that as natural resources respond to the changing environment around them, the service of aesthetic quality is maintained (or the ecosystem service), whilst the indicators themselves  change.http://www.sensiphi.com/). Their research showed that mathematical approaches to assessing compositional features of a coral reef digital image can correlate with human assessments of the aesthetics of the image. As computational learning algorithms improve (e.g. through AI), such tools are likely to produce results increasingly aligned with human assessments. However, thus far the relationship between coral reef health and the aesthetic appeal of such imagery has not been established empirically.Secondly, we see that environmental managers may be able to improve decision-making processes around developments in the Great Barrier Reef by including checklists around aesthetic impacts in the permitting process. Hass et al. (2015) developed an online tool called Sensiphi for converting the aesthetic appearance of an ecosystem in simple numbers that potentially provides a cost efficient monitoring tool Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m7G-MaP-seq efficiently detects known m7G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m7G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m7G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m7G modifications.Methylation of guanosine on position N7 (m In the yeast small ribosomal subunit (SSU) rRNA, position 1575 is m7G modified by the Bud23-Trm112 heterodimer  RNA modification is an example of this development. The m1A modification causes stops or misincorporations during reverse transcription. Initially, m1A was detected on a global scale using specific antibodies to immunoprecipitate m1A modified RNA combined with detection of the reverse transcriptase stop events resulting from m1A , which has been exploited to create abasic sites at m7G modified RNA positions (7G specific antibody based method (m7G MeRIP-seq) to confidently map the m7G modifications on mouse tRNAs  based on pull-down of the biotinylated RNA fragments followed by sequencing and applied it to human miRNAs, which led to the discovery of several human miRNAs with m7G modifications, including Let-7e, which was further characterized  method for high throughput nucleotide resolution mapping of internal m7G modifications. Our method is based on NaBH4 treatment of RNA and direct detection of mutations resulting from misincorporation at abasic sites during reverse transcription. We show that m7G-MaP-seq detects known and novel m7G modifications in rRNA and tRNA with mutational rates of up to 25%. We find that arabidopsis SSU rRNA has an m7G modification at position 1581, which corresponds to the location of the SSU rRNA position modified both in human and yeast rRNA, suggesting that the SSU m7G modification is universally conserved in eukaryotes. Moreover, by applying m7G-MaP-seq to small RNA sequencing libraries, we detect tRNA m7G modifications from yeast, human and Arabidopsis. In contrast, m7G-MaP-seq analysis of other types of short RNAs and of E. coli and yeast mRNAs failed to identify any internally m7G modified positions.Here, we describe the mMATa strain BY4741 (cat. no. YSC1048) were obtained from GE Healthcare Dharmacon (Dharmacon Yeast Knockout collection). The yeast cells were grown at 30°C in YPD medium. At OD = 0.75, the cells were collected by centrifugation, and pellets were washed with ice-cold water and re-suspended in 400 μl ice-cold TES solution . RNA was extracted using a hot acid phenol protocol.Yeast E. coli Keio Knockout RsmG strain (OEC4987-213607518) and the parental BW25113 strain (OEC5042) were obtained from GE Healthcare Dharmacon (E. coli Keio Knockout panel). The strains were inoculated in Luria-Bertani (LB) media (containing 50 μg/ml kanamycin for the knockout strains). Overnight cultures were diluted 1:100 and at OD600 = 0.4, ice-cold EtOH/Phenol stop solution (5% saturated phenol (pH < 7.0) 95% ethanol) was added (6.25 μl per 25 ml cell culture). Cells from 25 ml culture were pelleted at 3000 g for 5 minutes at 4°C and resuspended in 800 μl TE buffer  with 0.5 mg/ml lysozyme, followed by the addition of 1/10 vol of 10% Sodium dodecyl sulphate (SDS). After incubation at 64°C for 2 min, 1/10 vol of 1 M NaOAc, pH 5.2 was added, followed by addition of an equal volume of water saturated phenol (pH < 7.0). After incubation at 64°C for 6 min and centrifugation at 14 000 g for 10 min at 4°C, the aqueous layer was transferred to a new tube containing an equal volume of chloroform, inverted repeatedly, and centrifuged at 14 000 g for 5 min at 4°C. The aqueous layer was ethanol precipitated, and the RNA pellet was dissolved in RNase-free water.The Arabidopsis Columbia-0 seeds were washed in 96% ethanol followed by 10% bleach, spread onto solid agar plates and incubated 48 h at 4°C in darkness. Next, the seeds were incubated under light and harvested at day 14. The plant tissue was grinded in liquid nitrogen, and 100 mg of the grinded tissue was mixed with 1 ml of Trizol , followed by addition of 200 μl chloroform. After vortexing, samples were centrifuged at 14 000 g at 4°C for 10 minutes and 500 μl of the supernatant was mixed with 500 μl isopropanol and incubated at room temperature for 30 minutes. RNA was pelleted by centrifugation at 14 000 g at 4°C for 15 min and washed with twice with 70% ethanol before resuspension in RNase-free water.l-glutamate, 1% penicillin/streptomycin. The cells were harvested at ∼80% confluency and RNA was isolated using Trizol reagent  according to the manufacturer's protocol.HeLa cells were cultured in DMEM medium containing 10% Foetal Bovine Serum, 1% DNA was depleted from the RNA using the RNase Free DNase Set  according to manufacturer's protocol. The RNA quality was assessed on an Agilent 2100 Bioanalyzer using a RNA pico assay .E. coli was rRNA depleted using the Ribo Zero rRNA removal Kit . Poly-A enriched or rRNA depleted RNA was purified with Agencourt Ampure XP RNAclean beads  in a sample:beads ratio of 1:1.2, to reduce the small RNA fraction. The purification was performed according to the manufacturer's protocol and the purified RNA was eluted in 10 μl RNase-free water.RNA from yeast were poly(A)-enriched, using the Poly(A) Purist MAG Kit , using the standard protocol. The enrichment procedure was performed twice. RNA from 2), and the samples were incubated at 95°C for 3 min and 20 s. The samples were then placed on ice and EDTA was added to a final concentration of 10 mM. The RNA was purified with Agencourt Ampure XP RNAclean beads (Beckman Coulter) in a sample:beads ratio of 1:3.6, and the purified RNA was eluted in 10 μl of 1 M Tris pH 8.2.The RNA was mixed with an equal volume of 2× fragmentation buffer (100 mM Tris–HCl (pH 8), 10 mM MgCl4 (NaBH4 (+) samples) or with RNase-free water (NaBH4 (–) samples). The pH of the 0.3 M NaBH4 solution was 9. The reactions were incubated on ice for 30 min in darkness, followed by ethanol precipitation of the RNA, which were used directly for reverse transcription to preserve abasic sites.Fragmented RNA was either incubated with 0.3 M freshly prepared NaBHReverse transcription was performed with the PrimeScript Reverse Transcriptase (Takara Bio) in 35 μl reactions using 10 μl of RNA as template. 100 μM reverse transcription primer  was added to RNA sample and the mixture was incubated at 65°C for 5 minutes and placed immediately on ice. Next 7.5 μl 5× Primescript buffer, 7.5 μl 3.3 M/0.6 M sorbitol–trehalose mix, 1.9 μl 10 mM dNTP, 7.6 μl water and 1.5 μl Primescript reverse transcriptase to each reaction and reverse transcription was carried out at 25°C for 30 s, 30°C for 10 min, 42°C for 30 min, 50°C for 10 min, 56°C for 10 min, 60°C for 10 min. The cDNA was purified with Agencourt Ampure XP beads (Beckman Coulter) in a sample:beads ratio of 1:1.8, and eluted in 35 μl water. The cDNA was then vacuum concentrated to 7 μl.2, 2 μl 50% PEG 6000, 2 μl betaine and 0.5 μl CircLigase enzyme. Reactions were transferred to a thermocycler and incubated 2 h at 60°C, 1 h at 68°C, and 10 min at 80°C to inactivate the enzyme. The cDNA was purified with Agencourt Ampure XP beads (Beckman Coulter) in a sample:beads ratio of 1:1.8, and the purified cDNA was eluted in 10 μl water.Ligation of the adapter to the 3′-end of the cDNA was performed using CircLigase ssDNA Ligase (Epicentre) in 10 μl reactions. Initially, 3 μl RNA/cDNA duplex were mixed with 0.5 μl 100 μM Ligation_adapter (see supplementary S1) on ice, followed by addition of 6.5 μl master mix consisting of 1 μl CircLigase buffer, 0.5 μl ATP, 0.5 μl MnCl® High-Fidelity DNA Polymerase  and custom Illumina-compatible primers (see supplementary S1). Reactions of 50 μl were prepared and consisted of 7 μl single stranded cDNA, 3 μl forward primer, 2.5 μl reverse index primer, 10 μl Phusion 5× HF buffer, 4 μl 2.5 mM dNTPs, 22.5 μl water and 1 μl Phusion Polymerase.The amplification of the library was conducted using PhusionThe reactions were denatured for 3 min at 98°C, followed by 4 cycles of 98°C for 80 s, 64°C for 15 s and 72°C for 1 min followed by 14–17 cycles of 98°C for 80 s and 72°C for 1 min and subsequently 72°C for 5 min.® iBase™ Power System (Thermo Fisher) with the ‘SizeSelect 2%’ program to remove primer-adapter contamination. Following collection of the 150–600 bp fraction, the pooled library was ethanol precipitated, and sequenced on the Illumina NextSeq platform.The samples were pooled and run on the E-Gel4 or mock treated as described in the ‘NaBH4 reduction’ section. Small RNA libraries of yeast, human and arabidopsis were prepared using ‘The NEBNext Multiplex Small RNA’ kit according to manufacturer's protocol. The pooled libraries were run on the E-Gel® iBase™ Power System (Thermo Fisher) with the ‘SizeSelect 2%’ program, to remove 5.8s rRNA and to selectively exclude the >250 bp fraction. The pooled libraries were ethanol precipitated and sequenced on the Illumina NextSeq platform.Total RNA (1 μg) with a RIN value of 9.8 or higher was either NaBHA. thaliana SSU rRNA sequence around G1581 was isolated for MALDI Time-of-Flight mass spectrometric analysis. Purified total RNA was hybridized with a complementary oligodeoxynucleotide Ara_LSU_1581 (m/z’ (Proteometrics). The observed digestion pattern was compared to a theoretical ditto calculated using GPMAW software . Tandem mass spectrometry to investigate exact positions of the observed modifications was performed on a Waters Q-TOF Premiere instrument  equipped with a MALDI ion source in positive ion mode as previously described  was used to remove the 7 base barcode from the sequencing read using the options ‘-b NNNNNNN -t 9’ and the barcode information was saved in a separate file. Reads having one or more Ns in the barcode sequence were discarded before mapping with bowtie2 using high sensitivity options ‘–local -N 1 -D 20 -R 3 -L 15’.All raw data files and the fines processed datafiles containing the data used for making figures are available at GEO under accession GSE121927. The analysis is described in detail at ftp://ftp.ensemblgenomes.org/pub/release-29/bacteria//fasta/bacteria_87_collection/escherichia_coli_bw25113/cdna/Escherichia_coli_bw25113.GCA_000750555.1.29.cdna.all.fa.gz. For yeast mRNA analysis, reads were mapped to the ensembl cDNA release 84 of S. cerevisiae ftp://ftp.ensembl.org/pub/release-84/fasta/saccharomyces_cerevisiae/cdna/Saccharomyces_cerevisiae.R64-1-1.cdna.all.fa.gz. Potential PCR duplicates among the sequencing reads were removed by discarding all, but one of the reads having identical barcode and mapping to the same position using the collapse.sh script (http://people.binf.ku.dk/jvinther/data/RNA-seq).For analysis of ribosomal rRNA, reads were mapped to ribosomal sequences obtained from the CRW database . For E. http://gtrnadb.ucsc.edu/index.html). Prior to mapping, redundant/identical RNA sequences from different genes were collapsed, tRNA introns were removed from the sequences and 3′ terminal CCA were added. For histidine tRNAs a 5′ G were added to the sequence. In the analysis of unspliced tRNA, both the spliced and unspliced tRNA sequences were included in the fasta sequence used for mapping. For the analysis of Arabidopsis and human miRNAs, reads were mapped to the miRbase miRNA precursor annotation ftp://mirbase.org/pub/mirbase/CURRENT/hairpin.fa.gz. Sequences for other sRNA were obtained using BioMart from the Ensembl human gene (GRCh38.p12) annotation with a filter restricting to genes annotated as ‘scaRNA, scRNA, snoRNA, snRNA, sRNA’ and Arabidopsis thaliana gene (TAIR10) annotation with a filter restricting to genes annotated as ‘snoRNA, snRNA, SRP_RNA’.Sequencing reads from small RNA-Seq experiments were processed and mapped using the settings described above but as these reads do not include the adapter barcode, no pre-processing and collapsing of PCR duplicates on barcodes were performed. For tRNA analysis of yeast, human and arabidopsis, reads were mapped to the high confidence tRNA fasta sequences obtained from the genomic tRNA database  within control or NaBH4 treated replicates were used for preparing figures. The Modomics database , which lead to formation of RNA abasic sites  -stops observed at the m7G modified positions  . NaBH4 t% Figure , respectd Figure . Next, wt Figure , whereast Figure  with 1D.troscopy . We thenperiment . As expeeletions . Both thositions , indicat7G-MaP-seq to total RNA isolated from yeast (S. cerevisiae), arabidopsis  and human (H. sapiens) cells in a single replicate. For both human and yeast rRNA, we detect the known small subunit (LSU) rRNA m7G modification at positions G1639 and G1575, respectively  and tandem mass spectrometry. The mass spectrometry analysis demonstrates two methylations in the RNase A product G1578-U1584, m/z 2375.42—and a spontaneous truncation of this, G1578-A1582, m/z 1722.28—close to 100% modified  shows that the methylation of position 1581 is on the guanine. This demonstrates that the SSU G1581 methylation is conserved in arabidopsis . Some tRNAs, such as Ile-TAT-2, Pro-AGG-1 and Thr-TGT-2 showed highly significant mutation rate differences specifically at position 46, whereas others, such as tRNA-Gly-TCC-1 did not .To detect ms Figure . Using as Figure  10). Th. Th7G mof Figure . For twod Figure . Moreove7G positions, some positions known to be N1-methyladenosine (m1A) and Dihydrouridine (D) modified also pass our filter and have increased relative mutation frequency and low P-values  and N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G), resulting in relatively fewer tRNAs having sufficient sequencing depth for analysis. Nevertheless, among the tRNAs expressed in HeLa cells, m7G-MaP-seq identifies eight different tRNA isodecoder families modified at position 46 . Wh. Wh7G moA Figure . For LetA Figure  but we d% Figure . If we cd Figure . The fac7G modified positions were recently reported, suggesting that mRNA in other species may also be m7G modified. We applied m7G-MaP-seq to mRNA from E. coli and yeast in biological triplicates. In our analysis, 193590 E. coli mRNA , which allows sensitive and high throughput detection of internal m7G modifications in any type of RNA. Mutational profiling has previously been applied to obtain nucleotide resolution mapping of RNA-protein interactions, m1A modifications and for Selective 2-hydroxyl acylation analysed by primer extension (SHAPE) based probing of RNA structure, and here we demonstrate that this strategy also can be successfully applied to detect internal m7G modifications. Our method is based on the treatment of RNA with NaBH4, leading to the formation of abasic sites at m7G positions. Abasic sites in RNA are relatively stable, and during the subsequent reverse transcription m7G positions are permanently recorded as mutations in the cDNA sequence. m7G-MaP-seq can be implemented in any sequencing protocol, including regular RNA-seq experiments, and small RNA-seq by addition of NaBH4 treated samples in the experimental setup and application of our pipeline for data analysis. Compared to methods based on the mapping of reverse transcription stops, mutational profiling makes better use of the sequencing reads and has improved sensitivity because bias from RNA fragmentation, adapter ligation and secondary structures is reduced. In global RNA modification mapping experiments, it becomes very important to establish the background detection rates and account for these when modifications are mapped , dihydrouridine (D) and m1A. This is in agreement with a recent publication using NaBH4 treatment for detection of ac4C  and Protoscript II  for m7G-MaP-seq, however, the use of thermostable group II intron reverse transcriptase (TGIRT) could potentially allow higher read-through rates and thereby increase the signal. Another possibility could be inclusion of Mn2+ in the reverse transcription buffer, which in SHAPE-MaP-seq has been shown to increase RT read-through rates of 2′ acetylated RNA positions . By. By7G-Maiced out , similarications . Among hs Figure . Their serodimer . These ret al., it was reported that a subset of human miRNAs are m7G modified, including Let-7e, for which the m7G modification was validated and localized to position 18 using mass spectroscopy of a RNase A fragment of the gel purified miRNA fraction . In. Inet alt Figure . The LSUt Figure , which it Figure . LSU rRNt Figure , meaninget al. used mass spectroscopy on purified and decapped mRNA fractions from different human mouse cell types and found that the mRNAs contained from 0.02 to 0.05% m7G when compared to the amount of G  gives us around 90% power to detect a m7G modification present at a frequency of 0.2, whereas a sequencing depth of 3000 reads per group gives 90% power to detect a modification present at a frequency of 0.05 (7G modifications are not widespread in E. coli and S. cerevisiae mRNA. Future application of m7G-seq or m7G-MaP-seq with higher sequencing depth and potentially combined with antibody mediated m7G pull-down will allow detection of potential low frequency m7G modifications in highly expressed mRNA and high frequency m7G modifications in lowly expressed mRNA.Zhang unt of G . They al of 0.05 . We ther7G-MaP-seq method, which allows high throughput detection of m7G RNA modifications. The method can be implemented with existing RNA-sequencing protocols and we provide an efficient computational pipeline for data analysis. In this study, we identify known and novel m7G modifications in rRNA and tRNA and find that the human Let-7e is not m7G modified in HeLa cells. We expect that the m7G-MaP-seq method will facilitate future studies on internal m7G RNA modifications.In conclusion, we have developed the mgkz736_Supplemental_FileClick here for additional data file.


As neurons are one of the most highly polarized cells in our body, they require sophisticated cellular mechanisms to maintain protein homeostasis in their subcellular compartments such as axons and dendrites. When neuronal protein homeostasis is disturbed due to genetic mutations or deletions, this often results in degeneration of neurons leading to devastating outcome such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and fragile X syndrome (FXS). Ribonucleoprotein (RNP) complexes are macromolecular complexes composed of RNA binding proteins (RBPs) and their target RNAs. RBPs contain RNA binding domains and bind to RNA molecules via specific sequence motifs. RNP complexes have various functions in gene expression including messenger RNA (mRNA) trafficking, RNA processing and silencing. In neurons, RBPs deliver specific sets of mRNAs to subcellular compartments such as axons and dendrites to be locally translated. Mutations or deletions in genes coding for RNPs have been reported as causes for neurological disorders such as SMA, ALS, and FXS. As RBPs determine axonal or dendritic mRNA repertoires as well as proteomes by trafficking selective mRNAs and regulating local protein synthesis, they play a crucial role for neuronal function. In this review, we summarize the role of well-known RBPs, SMN, TDP-43, FUS, and FMRP, and review their function for local protein synthesis in neurons. Furthermore, we discuss their pathological contribution to the neurological disorders. Dysfunctional RNA processing in neuronal tissue is often observed in neurodegenerative diseases and plays a crucial role in neuronal pathology. This implies the essential part of ribonucleoprotein (RNP) complexes, that play an important role in RNA metabolism. RNP complexes contain RNA molecules with specific sequences/motifs bound to RNA-binding proteins (RBP). RNPs regulate eukaryotic gene expression in various cellular mechanisms including formation of ribosomes, spliceosomes and RNA-induced silencing complexes (RISCs) , and fragile X syndrome (FXS). Protein synthesis is an essential cellular process for function, development and survival. In this process, a coordinated interplay between proteins and RNA is crucial to produce functional proteins. Mutations in genes involved in RNA metabolism lead to various diseases. Interestingly, many of these mutations are associated with neurodegenerative, neurodevelopmental and neuromuscular disorders. Because neurons are post-mitotic, highly polarized and larger in size compared to other types of cells, less severe mutations in genes involved in RNA metabolism may cause dysfunction in neurons, but other types of cells might tolerate these mutations. Dysfunction of RNA-related processes including tRNA synthesis, ribosome biogenesis, sequestration and mislocalization of mRNAs has been reported as pathological mechanisms in neurological disorders  to neuronal projections is often located in the 3′-untranslated region (UTR) of the mRNA  and tRNP granule  proteins can be synthesized in mature axons SMN1 gene encoding the full length SMN protein. Humans possess a duplication of the SMN1 gene, SMN2, which produces a transcript lacking exon 7 due to a single point mutation located in exon 7  is mis-spliced in motor neurons of SMA mice, and notably it is important for organizing acetylcholine receptors at the NMJ  has been developed to restore SMN protein levels. Briefly, ASO binds to an intronic silencer in intron 7 and thereby it enhances the inclusion of exon 7 of SMN2 , annexin 2 (Anxa2), and neuritin/cpg15 , TDP-43, FUS and a hexanucleotide expansion repeat in Chromosome 9 Open Reading Frame 72 (C9orf72)  and around 1% sALS  via RAN translation. These dipeptide proteins form aggregates in neurons and cause cellular toxicity , which is characterized by insoluble inclusions containing TDP-43 aggregates  is a DNA/RNA-binding protein and mutations in FUS are implicated in ALS (about 4% of fALS and <1% of sALS) and FTLD  in FUS  Da Cruz. Due to Interestingly, it has been shown that motor and cognitive deficits in ALS are caused by increase of mutant FUS in axonal compartments, that lead to reduced intra-axonal translation and therefore to synaptic dysfunction   is a genetic disorder with intellectual disability caused by loss/deficiency of fragile X mental retardation protein, FMRP. FMRP is a polyribosome-associated RNA binding protein, which is encoded by the fragile X mental retardation 1 gene  and one RGG domain, which can bind to poly-riboguanylic acid . This process is important to NGF induced axonal outgrowth , and Mmp-9  (Schaeffer et al., in vivo studies are required, this data unveiled a new cellular mechanism underlying neuronal activity induced FMRP-mediated protein synthesis at the synaptic site. Taken together, FMRP regulates local mRNA translation in neurons via multiple layers of cellular mechanisms.The cellular mechanism of dendritic local translation has been intensively studied as well as the role of FMRP in postsynaptic protein synthesis. FMRP participates in dendritic local translation in various steps . First, Here, we have reviewed the current knowledge about the role of RNA binding proteins in local protein synthesis in subcellular compartments of neurons. While there are many more RNA binding proteins in neurons and their roles are equally important and interesting, we have only covered the most well-known four proteins, SMN, TDP-43, FUS, and FMRP. Importantly, dysregulation of these proteins caused by mutations and deletions is often associated with human neurological disorders. As neurons are highly polarized, often large in size, and their function is transient, neuronal protein homeostasis needs to be regulated timely and spacially. Neurons solve this issue by quick production of proteins at local sites when new proteins are required. For this process, RNA binding proteins traffic mRNAs and store them in mRNP granules in synaptic compartments, and upon stimulus, mRNA can be released to produce new proteins. As this process is important for neuronal function, dysfunction of RNA binding proteins often cause neurological disorders, in the worst case, leading to organismal death. While many underyling cellular mechanisms have been characterized, the molecular functions and biophysical properties of RNA binding proteins are still not fully understood. Furthermore, it will be important to understand how dysfunctional RNA binding proteins and impaired local translation contribute to pathology of human diseases.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


PLOR is a powerful tool to improve the efficiency of obtaining RNA structures de novo by X-ray crystallography.Due to the paucity of known RNA structures, experimental phasing is crucial for obtaining three-dimensional structures of RNAs by X-ray crystallography. Covalent attachment of heavy atoms to RNAs is one of the most useful strategies to facilitate phase determination. However, this approach is limited by the inefficiency or inability to synthesize large RNAs (>60 nucleotides) site-specifically labeled with heavy atoms using traditional methods. Here, we applied our recently reported method, PLOR (position-selective labeling of RNA) to incorporate 5-iodouridine at specific positions in the adenine riboswitch RNA aptamer domain, which was then used for crystallization and subsequent  In vitro transcription can produce RNAs with lengths varying from tens to thousands of nucleotides (nt), but it is usually applied for producing RNAs labeled specifically by nucleotide type rather than at selected positions. Solid-phase chemical synthesis is the most widely used method for preparing site-specifically labeled RNAs. However, heavy-atom labeled phosphoramidites are not always commercially available, and it is challenging to incorporate with adequate efficiency heavy atoms at specific positions of RNAs larger than 60 nt in length using step-wise chemical synthesis [The ongoing discovery of numerous biologically important RNAs demands the determination of their structures to better understand their functions. X-ray crystallography contributes the majority of RNA three-dimensional structures in the PDB . Solvingivatives –5. Heavyivatives , 2, 6. Synthesis . This siynthesis . Howeverde novo by X-ray crystallography, we applied the position-specific labeling of RNA (PLOR) method to incorporate heavy atoms at desired positions in RNA. PLOR is a solid-liquid hybrid transcription method for preparing position-specifically labeled RNAs [in vitro transcription; it also allows for incorporation of functional groups, including bulky ones such as fluorophores, into RNAs as long as they can be tolerated by RNA polymerase. Due to the high costs of synthesizing heavy-atom labeled RNAs, initial crystallization trials are typically performed using their unmodified counterparts. However, in many cases, introducing heavy-atoms can alter crystallization conditions, thus requiring additional screening with the modified RNA sample. The capability of PLOR to generate large amounts of site-specifically labeled RNAs at low cost and high efficiency saves time, effort, and materials required for such crystallographic studies. We reported a general strategy for heavy-atom derivatization of RNA using PLOR to selectively incorporate 5-iodouridine (IU) at two positions in the adenine riboswitch aptamer domain (rA71) [de novo structure determination, while minimizing RNA structural perturbations and effects on crystal quality.To overcome limitations in phasing RNA structures led RNAs –11. All n (rA71) . Here weI×En, where I and E are the percent efficiencies of initiation and elongation, respectively, and n is the total number of cycles used for synthesis [in vitro transcription) showed major m/z peaks of 23,162.50 Da  and termination (100~200 μM NTPs) stages, which are much lower than the millimolar concentrations used routinely in conventional transcription. Such low usage of NTPs, especially expensive modified NTPs such as I-UTP, greatly reduces the costs of synthesizing selectively labeled RNAs. The overall yield for PLOR-generated U28-U31-I-riboA71 was approximately 37.5%. The expected yield for this synthesis is 35.5% . This s62.50 Da , bottom,62.50 Da , top, reHKL2000 [P21212, with unit-cell dimensions very similar to the non-labeled rA71 crystals  >1.8) to ~2.7 Å . PHENIX xtriage  was usedELXC/D/E  through [HKL2MAP . SHELXC o ~2.7 Å . The topes > 0.9 , and ini  compared to 0.224/0.257 (non-labeled). Importantly, this indicates that the introduction of iodines at the two positions has little, if any, effect on crystal quality.Crystals of U28-U31-I-rA71 proved resilient to radiation damage after an average dose of photons for a routine SAD dataset. This is particularly important since a high redundancy of measurements is often required for anomalous phasing of crystallographic data, which can be problematic for heavy-atom derivatives that are more susceptible to radiation damage. However, with only two iodines per ~23 kDa RNA molecule, and an 8.5-fold overall data redundancy, the anomalous signal was sufficiently measurable and complete for almost the entire resolution range. The anomalous difference Fourier , generatANODE  revealedANODE  demonstra = 48.99, b = 151.58, c = 24.74, corresponding to fractional differences relative to the native crystal [Rint) [SHELXD using the SIRAS method correctly identified the heavy-atom sites, but only when the resolution was limited to 3.5 Å. However, phasing in SHELXE using the correctly identified sites and phase extension to 2.01 Å with the native data failed to generate an interpretable electron density map. In this case, therefore, non-isomorphism precluded de novo phasing by SIRAS.For comparison, experimental phasing was also attempted using the single isomorphous replacement with anomalous scattering (SIRAS) method. The unit cell dimensions for the derivative crystal are  crystal  of 1.3%,de novo phasing. It is straightforward to extend the strategy to synthesize RNAs containing other heavy-atoms, including Cl, Br, Se, etc. by PLOR. This application of PLOR will hasten the rate of novel RNA structure determination by X-ray crystallography.The process for obtaining heavy-atom modified RNAs is a key bottleneck in solving unique biologically important RNA structures by X-ray crystallography. The introduction of heavy-atoms into RNA can be routinely achieved by PLOR, with the aim of breaking the phase ambiguity of crystallographic data. Such an application for PLOR is especially feasible for large RNAs that cannot be synthesized by solid-phase synthesis with high-enough efficiency. The easy scale-up of PLOR also makes it practical to carry out initial crystallization screening with heavy-atom containing RNAs. This avoids the potential pitfalls of non-isomorphism between native and derivative crystals. Covalent incorporation of heavy atoms into RNA at desired positions using PLOR offers a general strategy for SAD/MAD phasing of novel RNA crystal structures. This application of PLOR can be extended to structure determination of RNA/protein complexes by derivatizing RNA instead of, or in addition to, the protein, thereby increasing the chances for successful 2, 1 mM spermine and 5 mM adenine. The incorporation of the iodine atoms was verified by mass spectrometry  Polyethylene Glycol 400 (PEG 400). The RNA stock concentration used for crystallization was 0.5 mM and the volume ratio between RNA and reservoir for the sitting drops was 1:2. U28-U31-I-rA71 crystals appeared as rectangular plates in ~2–3 days, and grew to maximum dimensions of ~100×25×15 μm3. Crystals for data collection were flash-frozen directly in liquid nitrogen, without additional cryoprotection. Diffraction data were collected at beamline 19BM, Advanced Photon Source (APS), Argonne National Laboratory, using a photon energy of 8 keV (1.54 Å) and an exposure time of 1 s per 0.5°, for a total of 240° (480 frames). The crystals diffracted to a maximum resolution of ~2 Å and exhibited no signs of radiation damage. Crystallographic data and refinement statistics are summarized in PyMOL [Crystals of U28-U31-I-rA71 were grown at 4°C by sitting-drop vapor diffusion using as the reservoir 50 mM Tris-HCl (pH:8.5), 100 mM KCl, 10 mM MgClin PyMOL .


Over the past 35 years, RNA has become a molecule of utmost interest for researchers in the life sciences. The many functions that RNA fulfills in the cellular machinery have been elucidated with constant progress, revealing a complex network of RNA-mediated regulation of key processes in the cellular life cycle. First examples of RNA catalysis were discovered in the early 1980s, and it was nothing less than the Nobel prize that was awarded for this discovery . Since tTwo review articles highlight important aspects of ribozyme structure and function. Lau and Ferré-D’Amaré discuss the functional plasticity of ribozyme folds in their review . The funDidymium iridis to form full-length circular RNAs and highlight the structural aspects explaining their relative stability [In addition to the ongoing research into the structure and mechanism of ribozymes, RNA catalysis has also inspired a large number of efforts into RNA engineering and design. This is the topic of the review of Müller on trans-splicing group I Intron ribozymes . It summtability .The discovery of ribozymes has revitalized the RNA world theory, and ever since this discovery, much effort has gone into the development of ribozymes with useful activities in a scenario of life based on RNA functioning as both a genome and genome-encoded catalyst. Along this line, the paper by Agmon follows the interesting hypothesis that a proto-ribosome, comprised of a dimer of tRNA-like molecules, is still embedded in the contemporary ribosome and provides a computational analysis of crystal structures to confirm it . SatterwOverall, research into ribozymes and RNA catalysis has remained a very exciting field with unchanged potential for new discoveries. This Special Issue illuminates just a small window in this broad field. Ribozyme research has not lost the intriguing and highly motivating flair of the early days, when the first reports of ribozymes occurring in nature appeared. There is still much to be learned: from the discovery of novel catalytic RNA motifs in nature and novel genetic locations of known ribozymes, over the elucidation of ribozyme structure and its catalytic mechanism, up to engineering and design towards application in diverse fields. Ribozymes and RNA catalysis are of ongoing interest to the research community.


Sus scrofa RNA editome. Our study presents the first profile of the dynamic A-to-I editome in developing animal skeletal muscle and provides evidences that RNA editing is a vital regulator of myogenesis.Adenosine-to-inosine (A-to-I) RNA editing meditated by adenosine deaminases acting on RNA (ADARs) enzymes is a widespread post-transcriptional event in mammals. However, A-to-I editing in skeletal muscle remains poorly understood. By integrating strand-specific RNA-seq, whole genome bisulphite sequencing, and genome sequencing data, we comprehensively profiled the A-to-I editome in developing skeletal muscles across 27 prenatal and postnatal stages in pig, an important farm animal and biomedical model. We detected 198,892 A-to-I editing sites and found that they occurred more frequently at prenatal stages and showed low conservation among pig, human, and mouse. Both the editing level and frequency decreased during development and were positively correlated with ADAR enzymes expression. The hyper-edited genes were functionally related to the cell cycle and cell division. A co-editing module associated with myogenesis was identified. The developmentally differential editing sites were functionally enriched in genes associated with muscle development, their editing levels were highly correlated with expression of their host mRNAs, and they potentially influenced the gain/loss of miRNA binding sites. Finally, we developed a database to visualize the  To our knowledge, this is the first systematic study of RNA editing in skeletal muscle. The findings of this study suggest that RNA editing is a vital, but underappreciated, mechanism involved in regulating skeletal muscle development.Here, we constructed a comprehensive dynamic atlas of the longissimus dorsi) samples were collected from Landrace (L) pigs at 27 developmental stages, including embryonic days 33, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, and 105  and postnatal days 0, 9, 20, 30, 40, 60, 80, 100, 120, 140, 160, and 180 . The experimental pigs were allowed access to feed and water ad libitum and were housed under identical conditions before slaughtering. After copulation with the boar, the sows and piglets were sacrificed at a commercial slaughter house at the selected stages. At each stage, skeletal muscle samples from three pigs were harvested as biological replicates. All samples were stored immediately in liquid nitrogen. All animal procedures were performed according to the protocols of the Chinese Academy of Agricultural Sciences and the Institutional Animal Care and Use Committee.Skeletal muscle , followed by rRNA depletion and DNaseI treatment (Qiagen). Strand-specific RNA-seq libraries for paired-end sequencing were prepared using the Illumina Ribo-Zero protocol. For each stage, three independent biological libraries were constructed. The libraries were sequenced to generate paired-end reads with 150-bp read lengths using the Illumina HiSeq X Ten platform. Approximately 100 million reads were acquired for each sample.For WGS, genomic DNA was isolated from the ear tissue of a Landrace male pig with the same genetic background as the animals from which the samples used for RNA-seq was collected. The DNeasy Blood & Tissue Kit (Qiagen) was used for isolation of DNA according to the manufacturer’s instructions. Qualified DNA was sonicated into 350-bp fragments using a Covaris S220 ultrasonicator (Covaris). Libraries were constructed according to the standard operating procedure provided by Illumina. Sequencing was performed on an Illumina HiSeq 4000 platform, and 125-bp paired-end reads were generated.The samples from the RNA-seq experiment were also used for WGBS. After DNA extraction, DNA was fragmented into 200–300 bp fragments with a Covaris S220 ultrasonicator (Covaris), followed by end repair and A-ligation. Cytosine-methylated barcodes were ligated to the sonicated DNA according to the manufacturer’s instructions. The DNA fragments were treated twice with bisulphite using the EZ DNA Methylation-Gold™ Kit (Zymo Research). The libraries were prepared according to the manufacturer’s instructions (Illumina). The libraries were sequenced on an Illumina HiSeq X Ten platform, and 150-bp paired-end reads were generated by Novogene .For each library, high-quality reads were obtained after removing reads containing adapters, reads containing poly-N sequences, and low-quality reads using in-house Perl scripts. The remaining clean, high-quality reads were used for subsequent analyses.S. scrofa reference genome  by BWA (v0.7.17). The paired reads were mapped separately using the commands ‘bwa aln’ and ‘bwa sampe’, with only four mismatches allowed. Reads that uniquely mapped (q > 10) to the reference genome were kept by Samtools (v1.6).The RNA and DNA reads were aligned to the S. scrofa reference genome was first transformed to a bisulphite-converted version (C-to-T and G-to-A conversion) and then indexed using bowtie2.For WGBS reads, the q > 25). Sites with two or more variants were discarded. At least three reads with high quality were required to support the variant form to eliminate false positives due to amplification bias or sequencing error.Reliable SNP sites were identified using stringent criteria, which required at least 10 sequencing reads at these sites and a high-quality score ; (iii) Intronic sites that occurred within 4 bp of splice junctions were discarded; (iv) Variants in homopolymer runs were discarded; (v) Because of the relatively high error rate of Illumina sequencing towards both ends of a read, variants that occurred within 6 bp of both ends of a read were removed; (vi) The remaining candidate sites were checked with BLAT alignment filtering, and sites in regions highly similar to other regions of the genome were discarded. The remaining variants were considered as candidate RNA editing sites and annotated by snpEff (v4.3t)In order to accurately identify RNA editing sites in pig skeletal muscle, a computational pipeline was developed to make reliable calls on RNA editing sites  as reporcmdscale’ function in R was used for the MDS analysis.For an RNA editing site in a given skeletal muscle sample, the A-I RNA editing level was quantified as the ratio of the number of G reads to the total number of A and G reads covering the site. The overall editing rate of each sample was determined as the total number of reads with G at all candidate editing positions compared with all A and G reads covering the editing positions.S. scrofa were obtained from miRBase (release 21).A cluster of editing sites was defined as the occurrence of three or more candidate sites within a 100-bp window. Genes that contained at least five editing sites and one clustered region were defined as hyper-edited genes. The sequence spanning 200 bp upstream and downstream of the editing site or cluster was used to predict the secondary structure and to calculate the minimum free energy using the RNAfold programme in the ViennaRNA package.http://david.abcc.ncifcrf.gov/).For gene expression analysis, RNA-seq reads were aligned to the reference genome using Hisat2 (v2.0.4) with the parameter –rna-strandness RF, while the other parameters were set to their default values. The expression levels of genes were quantified by calculating fragments per kilobase of exon per million fragments (FPKM) values using StringTie (v1.3.1) in a reference-based approach.https://genome.ucsc.edu). The known human and mouse A-to-I editing sites were downloaded from the RADAR http://srv00.recas.ba.infn.it/atlas/)For conservation analysis, we converted the coordinates of all pig A-to-G RNA editing sites to positions on the human and mouse reference genomes using liftOver tools and the chain files (susScr11ToHg38.over.chain.gz and susScr11ToMm10.over.chain .gz) provided by the UCSC Genome Browser . Modules with eigengenes that were highly correlated (above 0.8) were merged to assess their relationship with skeletal muscle development. We identified 13 co-editing modules with minModuleSize = 30. The correlations between module eigengenes and phenotypic traits (development stage and sex) were used to identify modules associated with development or sex.Weighted gene co-edited network analysis (WGCNA)http://echarts.baidu.com/index.html) are used to exhibit the editing level of editing sites at 27 developmental stages of skeletal muscle. The web services were built using nginx (v2.10.0), an HTTP and reverse proxy server. JBrowse (version 1.14.2) genome browser is used to display the positional relationships between genes and genome annotations.The database was composed of a web interface and a MariaDB database engine, which is used to store and manage all data. The web interface is composed of native HTML elements. The data processing and customer/service interaction programmes were written in PHP (v5.4.16) and JavaScript (v1.10.2). The search results are shown by asynchronous JavaScript and XML (AJAX) serves belonging to JavaScript. Baidu ECharts  according to the manufacturer’s protocols. The selected regions were amplified from the gDNA and cDNA samples using the primers listed in Total protein was isolated from pig skeletal muscle using a Protein Extraction Kit . Western blotting was performed as previously described.AVPR1A, TMX4, and ACTN2 genes  were randomly selected to verify whether A-to-I editing can affect miRNA binding. The 3′-UTR fragments flanking the miRNA binding sites of these three genes  were amplified by PCR. The PCR products were cloned into the pmirGLO Dual-Luciferase Vector using the SacI and XhoI restriction sites. The mutant types corresponding to the RNA editing sites (3′-UTR-edt) in these three genes were made by site-directed mutagenesis (Takara) and confirmed by Sanger sequencing. The primer sequences are listed in Three editing sites in the 3′-UTRs of the 2 at 37 °C. The negative control duplexes and miR-21, miR-378 and miR-133b mimics (double-stranded RNA oligonucleotides) were synthesized by GenePharma. HEK293 cells were co-transfected with pmirGLO-3′UTR-wt plasmid/3′ UTR-edt and the miRNA mimic/negative control. Co-transfection assays were performed in 12-well plates with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After transfection for 24 h, cells were harvested. Finally, renilla luciferase and firefly luciferase were detected with the dual-luciferase assay system (Promega).HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) with 5% COMIB1 gene on MIB1 expression was evaluated. The mutation in the MIB1 3′-UTR was made by site-directed mutagenesis (Takara) and confirmed by Sanger sequencing. The edited and wild-type MIB1 3′-UTRs were cloned into the pGL3-basic vector. Luciferase activity and the expression level of firefly luciferase were detected to evaluate the effect of this editing site on MIB1 mRNA stability.The effect of a selected A-to-I editing site  in the 3′-UTR of the To profile the dynamic patterns of RNA editing during skeletal muscle development, we carried out strand-specific RNA-seq in skeletal muscle across 27 developmental stages (SRA accession number SRP158448), including 15 prenatal and 12 postnatal stages, ranging from 33 days after conception to 180 days after birth. A total of 8.2 billion paired-end reads (150 bp × 2) were obtained from 81 transcriptome libraries. A WGS library (SRA accession number SRP157242), which was based on DNA isolated from the ear tissue of an adult male pig with the same genetic background as the pigs from which the samples were collected for RNA-seq, was sequenced. Additionally, our group previously performed WGBS (SRA accession number SRP160645) on the same skeletal muscle samples used for RNA-seq to understand the dynamic regulation of DNA methylation during skeletal muscle development (data not shown). Because differences in DNA and RNA sequence were found to be mainly caused by RNA editing, the WGS and WGBS data were also used in the process of filtering out false-positive DNA-RNA differences caused by heterozygous SNPs. A total of 56.88 billion paired-end reads (150 bp × 2) were obtained from 81 methylome libraries, and 909.92 million paired-end reads (125 bp × 2) were obtained from the WGS library, representing a genome-wide coverage depth of approximately 35× and 47×, respectively. These multi-omic datasets with relatively deep coverage provided an extensive dataset for profiling the RNA editome of developing skeletal muscle.,r = 0.78, Based on the multi-omic dataset mentioned above, we identified 236,569 putative RNA editing sites in skeletal muscle using a pipeline similar to those used in previous studiesAmong A-to-I editing sites, 79.6% of sites were located in intronic regions, followed by intergenic (16.3%) and 3′-UTR (3.6%) regions, while only 379 (0.19%) sites overlapped with protein coding sequences (CDS) . A-to-I ,,,LAMP2, which harboured 70 and 45 editing sites, respectively, in their 3′-UTRs . The MDS analysis showed that the first dimension explained approximately 10% of the editing variation across all samples, and the prenatal skeletal muscle samples were clearly segregated from the postnatal samples . Notably, the ‘darkmagenta’ module was also positively correlated with the overall editing rate   to assess the relationship of each module with skeletal muscle development and sex . The two= 1e−21) . Eight en = 42.5, P = 0.002) and chromosome 3 , respectively (P = 8.58e−72) and 5′-UTR . In contrast, dDESs were underrepresented in intronic regions   across skeletal muscle development. A total of 1,608 sites  showed significant editing rate differences . These dectively . Additio.05e−07) , Table 2.05e−07) . Of noteDUSP11, DACT3, SACS, and CDK13 genes. DUSP11 negatively regulates members of the mitogen-activated protein (MAP) kinase superfamily associated with cellular proliferation and differentiation, and DACT3 is a key regulator of canonical and/or non-canonical Wnt signalling pathways during development.CDK13 were decreased during skeletal muscle development . Our experiments validated that both luciferase activity and the expression level of firefly luciferase for the editing type were significantly lower than those associated with the wild type, confirming that this editing site significantly affected the expression of its host gene   . To expl2.2e−16) . Three es effect . In compectively . These fhttp://www.rnanet.org/editing/home.html). Users can use the query interface and web interface of DREP to search RNA editing annotations, including their locations in the genome, genes, genic components , repetitive elements , repetitive element family membership ] that allows researchers to retrieve information about the A-to-I editing sites identified in the present study . We found that both the frequency and level of RNA editing were decreased across skeletal muscle development, which is contrary to observations in the brain.in vitro. These experiments confirm the functionality of these editing sites in skeletal muscle development. Previous studies have suggested that RNA editing at 3′-UTRs is developmentally regulated,,,,We detected thousands of dDESs and noted that the editing levels of most dDESs were decreased during skeletal muscle development. Additionally, most dDESs were significantly correlated with host mRNA levels and functionally associated with muscle organ development. Moreover, we experimentally validated the effects of RNA editing on host gene expression S. scrofa RNA editome in skeletal muscle across 27 developmental stages. To our knowledge, this is the first genome-wide atlas of the RNA editome of developing animal skeletal muscle. This work identified a series of RNA editing sites associated with skeletal muscle development and revealed their potential roles in myogenesis. This study presents new insight into skeletal muscle development and provides abundant information regarding regulation of RNA editing. This knowledge may be helpful to animal breeders, as well as to biomedical researchers studying muscle-related diseases.In summary, we created a comprehensive and dynamic atlas of the Sequencing data have been deposited to Sequence Read Archive (SRA) at the National Center for Biotechnology Information (NCBI) under accession numbers SRP157242, SRP160645, SRP158448 and SRP159202.dsz006_Supplementary_DataClick here for additional data file.


The development of RNA-encoded regulatory circuits relying on RNA-binding proteins (RBPs) has enhanced the applicability and prospects of post-transcriptional synthetic network for reprogramming cellular functions. However, the construction of RNA-encoded multilayer networks is still limited by the availability of composable and orthogonal regulatory devices. Here, we report on control of mRNA translation with newly engineered RBPs regulated by viral proteases in mammalian cells. By combining post-transcriptional and post-translational control, we expand the operational landscape of RNA-encoded genetic circuits with a set of regulatory devices including: i) RBP-protease, ii) protease-RBP, iii) protease–protease, iv) protein sensor protease-RBP, and v) miRNA-protease/RBP interactions. The rational design of protease-regulated proteins provides a diverse toolbox for synthetic circuit regulation that enhances multi-input information processing-actuation of cellular responses. Our approach enables design of artificial circuits that can reprogram cellular function with potential benefits as research tools and for future in vivo therapeutics and biotechnological applications. RNA-encoded regulatory circuits are desirable because they do not integrate in the host and are less immunogenic, but the availability of regulatory devices is limited. Here the authors develop viral protease RNA-binding proteins and protease–protease genetic circuits that ultimately regulate mRNA translation. To date, most devices used in synthetic biology are encoded by DNA, with associated risks of insertional mutagenesis and immunogenicity3. RNA-based circuits overcome these limitations while still providing control over strength, timing, and location of gene expression5. In addition, RNA does not need to cross the nuclear barrier and can be delivered into non-dividing, as well as dividing cells3, with advantages for broad biomedical, industrial, and pharmaceutical applications including cancer immunotherapy6, genome engineering, and genetic reprogramming7. The development of foundational tools that control RNA activity is thus paramount to engineering RNA-encoded synthetic networks to program cellular functions.Mammalian synthetic biology is developing research tools and programmable therapeutics that are based on sophisticated genetic circuit design11. These can modulate the translation of associated output, but cannot currently be interconnected to implement complex circuits. The ability to compose parts into circuits has recently become possible with the use of RNA-binding proteins (RBPs) such as L7Ae and MS2-cNOT7. These RBPs have been successfully interconnected to create post-transcriptional circuits in which they function both as inputs and outputs of the regulatory devices12. Here, we propose to further expand the available toolset of post-transcriptional regulators using novel protein–protein interaction-based devices through re-engineering of RBPs and proteases.Initial efforts on the design and characterization of new genetic modules to implement RNA-based networks focused on building parts such as aptamers, ribozymes, and riboswitches13. Proteolysis devices present a possible platform for multi-element circuit engineering, but today are largely used in synthetic biology to modulate single device protein half-life, or to trigger selected cellular responses17. Proteases are broadly expressed in all organisms and function by cleaving short, specific amino acid sequences. Proteases are involved in all areas of metabolism and constitute key effectors of several cellular pathways, modulating protein–protein interaction, protein localization, and ultimately regulating cellular fate19. They are classified on the basis of cleavage site21 , evolution, substrate specificity, and reaction catalyzed23. Proteases can be adapted to operate in several environmental conditions and optimized to function in different organisms. Viral proteases such as tobacco etch virus protease (TEVp) are widely used in biotechnology for endoproteolytic removal of affinity tags from recombinant proteins24, and in synthetic biology to help convert extracellular or intracellular ligand sensing into transcriptional output25. They are also used to control protein levels via an N-terminal degron fused to the target protein with a protease-responsive cleavage site, as demonstrated in bacteria14. To date there are no off-targets substrates for TEVp reported in the human proteome26 and in other organisms where they are in fact well tolerated29. The use of TEVp has enabled innovative studies of biological processes. For example, engineering of a TEVp responsive Rad21 in a cell-type-specific and temporally controlled manner unveiled cohesin function in post-mitotic cells like Drosophila neurons27. We thus envisioned that proteases represent an attractive tool for engineering more sophisticated post-transcriptional and translational regulatory networks, and that protease-RBP and protease–protease regulatory devices could lay the foundation for circuits that are solely RNA-encoded and are based on protein–protein interactions.Protein systems engineering using well-characterized standardized protein modules offers several useful features such as enabling information processing and actuation in target cells and in vitro optimization before investigating their behavior in vivoToward this goal we inserted protease cleavage sites into RBP and proteases sequences, linking their activity to a set of upstream proteases. The devices were genetically encoded on plasmid DNA to allow rapid testing in human embryonic kidney (HEK293FT) cells via transient transfections.30 (TVMVp), turnip mosaic virus protease31 (TUMVp), and sunflower mild mosaic virus protease (SuMMVp), and implemented post-transcriptional regulatory cascades. Finally, we functionally connected proteases to create multi-layered cascades.As discussed below, we first engineered L7Ae to harbor a TEVp cleavage site (TCS), and implemented a set of circuits  that operate solely with protein:RNA (L7Ae:2Kt) and protein:protein (TEVp:L7Ae) interactions. Next, we inserted into MS2-cNOT7 specific cleavage sites for any of four proteases: TEVp, tobacco vein mottling virus proteaseOur work expands the toolbox of parts available for post-translation control, and provides an important proof of concept study that enables new means to control post-transcriptional regulatory circuit behavior with endogenous or exogenous protein inputs that respond to cellular and environmental changes.33 in the 5’UTR of target mRNAs, inhibiting their translation35. As a first step to link L7Ae activity to TEVp, we identified possible insertion points for the TEV cleavage site (TCS) based on the crystal structure of L7Ae bound to its native RNA recognition sequence. We had two main objectives in choosing the sites: (i) TCS insertions should minimally affect L7Ae structure or RNA-binding, and (ii) TEVp cleavage should render L7Ae non-functional. We thus chose three candidate insertion sites for TCS in loop regions away from the K-turn binding domain and closer to the center of the L7Ae sequence  motifnce Fig. . Accordince Fig. , we obsence Fig. , 3, 18. nce Fig.  and in a36. We used two single-chain fragment intrabodies scFv35 and scFv162 specific for two distinct epitopes of NS3 previously reported to interfere with its viral activity37. We have recently shown that these intrabodies can be efficiently wired into a transcriptionally based protein-sensing-actuation device to detect NS3 expression in mammalian cells16. Here, we designed a new device in which the sensor is based on translational regulation using L7Ae-CS3 and TEVp fused to scFv35 and scFv162, respectively  or L7AeCS3. We observed efficient dEGFP repression by all chimeric proteins  to enable regulation by TEVp  with different affinities for the cognate protease . Based on this information we designed a structurally modified TUMVp with TEVp cleavage site insertion (TCS)  TVMVp-TUCS1 between amino acid residues D26-G27, (ii) TVMVp-TUCS2 between amino acid residues Q119-K120, and (iii) TVMVp-TUCS3 between amino acid residues T173-N174  Fig. . Since t43. Recently, self-replicating RNA viruses such as Sindbis virus have garnered significant interest for a wide range of medical applications including vaccine delivery, gene therapy, and cellular reprogramming45. A main advantage of RNA replicons is that since they self-replicate, they can generate high levels of gene products even when starting from a relatively low dose, while the risk of undesired chromosomal integration is minimal because they do not reverse transcribe. Sindbis replicons with reduced cytopathicity and longer-term expression have been recently developed, making them an attractive platform for engineering RNA-encoded synthetic devices48.Synthetic devices that can be encoded by RNA are a promising framework to reprograming cellular function. These have potential applications both as research tools and for future in vivo therapeutics due to their transient expression and lack of chromosomal integration hazards, overcoming safety issues associated with DNA based circuit delivery. Indeed, a key aspect to consider when using RNA-encoded devices for biomedical applications is mRNA stability; chemical and structural modifications of in vitro transcribed mRNA have enabled production of significant levels of target proteins for durations longer than a weekPotyvirus proteases. This class of proteins is attractive for many applications since to our knowledge they have no reported substrates in the human proteome26 and in a broad range of other organisms that tolerate their expression29. Furthermore, the remarkable specificity of proteases to the 7-mer cleavage sequence  minimizes the likelihood of unwanted knockdown of RBP function by endogenous proteases, thus creating an effective orthogonal system.In this proof-of-concept study we lay the foundation for protein-driven synthetic circuits that enable construction of tunable RNA-encoded networks. Our approach relies on 16. Tuning gene expression post-transcriptionally could be achieved in a variety of ways, for example by adding a degradation domain to the native protein that responds to small molecules. These domains, which are stabilized by externally provided small molecules, enable fine tuning of protein stability and thus control of abundance54. Alternatively, destabilizing elements can be added in the 3’UTR of the mRNA encoding the protein of interest such as AU-rich motifs56. Engineered L7Ae based devices was programmable both in HEL293FT and HeLa cell lines, although the level of repression was reduced in HeLa cells. This confirm that HEK293FT cells are the optimal cell line for design and test synthetic biology-based devices for high transfection rate, and production capacity. Different behavior is perhaps due to different cellular composition and resourced distribution that may feedback on circuits performances. Identifying the bottle neck of circuits functionality could perhaps help to optimize genetic circuits design.We demonstrated a set of regulatory networks including cascades, switch and protein sensor-actuator, showing that they operate in an orthogonal fashion combining post-transcriptional and post-translational regulation. We first engineered RBPs L7Ae and MS2-cNOT7 to include cleavage sites for cognate proteases, which exhibit robust and long-term protease-dependent regulation of target mRNA. With MS2-cNOT7 fusion protein, we created a protease-dependent system via cleavage sites inserted at the junction of the two modules. The design of L7Ae was based on analysis of the crystal structure of the RBP bound to native recognition RNA sequence, with aims to minimize alteration of wild-type L7Ae structure upon insertion, to minimize interference with L7Ae’s ability to bind RNA, and maximize cleavage in presence of TEVp that render L7Ae non-functional. The L7Ae proteins with integrated cleavage sites were used to compose three different devices, including NS3 protein sensor. We used an inducible promoter to control TEVp-scFv162 expression to minimize the likelihood of random cleavage41. For adding new regulatory elements, computational protein design methods are becoming increasingly accurate at predicting folding and functionality of newly design proteins60, which should facilitate the construction of new protein-based synthetic networks. We envision that our platform could also be connected to intracellular protease inputs  without requiring modifications to endogenous pathways to link changes of intracellular state to output regulation. It has been recently demonstrated that synthetic mRNAs harboring cell-type-specific microRNAs can be used to efficiently purify human pluripotent stem cell (hPSC)-derived populations. Similarly, we envision that protease-responsive RBPs may represent an effective means to link detection of specific cellular states such as infection or cancer to conditional expression of output genes. For example, the SUMO1-/sentrin-specific protease SENP1 has a pro-oncogenic role in several types of cancer , prostate cancer, hepatocellular carcinoma, and metastatic neuroblastoma tissues), correlating with poor prognosis63. Circuits such as ours that sense SENP1 can be used to reprogram cancer cells to express relevant immunomodulators, or trigger specific cell killing by modifying circuit output in an application-tailored fashion. Overall, our system provides a strategy for composable and scalable circuit design encoded post-transcriptionally in mammalian cells, offering a useful research toolbox with potential benefits for RNA delivered in vivo applications such as cell-based therapies, vaccination, regenerative medicine, and biotechnological applications.Similar to L7Ae, engineering of the proteases was based on crystal structure analysis of TEVp and TVMVp, and we observed that the best insertion site was in a similar region of these two proteases. We confirmed the same result for TUMVp, whose crystal structure has not been resolved yet, but was inferred with a homology-based modell-Glutamine (Sigma-Aldrich) and 1% non-essential amino acids (HyClone) at 37 °C and 5% CO2.HEK293FT (Invitrogen) and HeLa cells used in this study were maintained in Dulbecco's modified Eagle medium  supplemented with 10% FBS (Atlanta BIO), 1% penicillin/streptomycin/5 cells seeded in 500 μl of complete culture medium in 24-well plate. Transfection complexes were added dropwise to the freshly seeded cells, followed by gentle mixing. Cells were supplemented with 1 ml of fresh growth medium 24 h post transfection and analyzed by flow cytometry after 48 h.Transfections were carried out in 24-well plate format. L7Ae-CS-based devices transfections were carried out with Attractene (Qiagen). An aliquot of 400 ng total DNA was mixed with DMEM base medium (Cellgro) without supplements to a final volume of 60 μl. A total of 1.5 μl attractene was added to the dilutions and the samples were promptly vortexed to mix. The complexes were incubated for 20–25 min During the incubation time, cells were harvested by trypsinization and 2 × 105 cells were harvested by trypsinization and seeded in 500 μl of complete culture medium in 24-well plate. Transfection complexes were added dropwise to the freshly seeded cells followed by gentle mixing. Transfection tables that list components and relative concentrations used in this study are available in the Supplementary MS2-cNOT7 devices, L7AeCS3 experiment in HeLa cells and 96 h experiments were performed with Lipofectamine 3000 following manufacturer instructions. Total of up to 400 ng DNA was mixed with Opti-MEM I reduced serum medium (Life Technologies) to a final volume of 100 μl followed by addition of 0.5 μl P3000 reagent. After 5 min, 1.5 μl Lipofectamine 3000 was added, the samples were briefly vortexed and incubated for 30 min at room temperature. During the incubation time, 2 × 1064. For L7AeCS-based devices the plotted data represent the ratio between reporter Geo-mean and frequency and mkate Geo-mean and frequency: EGFP values (EGFP Geo-Mean*%EGFP + ) normalized by the transfection marker (mKate Geo-Mean*%mKate + ) for all mKate-positive cells. For MS2-CS-cNOT7-based devices, data represent the ratio between EGFP Geo-mean and mKate Geo-mean.Cells were analyzed with LSR Fortessa flow cytometer, equipped with 405, 488, and 561 nm lasers (BD Biosciences). We collected 30,000–100,000 events per sample and fluorescence data were acquired with the following cytometer settings: 488 nm laser and 530/30 nm bandpass filter for EYFP/EGFP, 561 nm laser and 610/20 nm filter for mKate, and 405 nm laser, 450/50 filter for EBFP. Data of 96 h experiment for L7AeCS3 and MS2-CS-cNOT7 devices and L7AeCS3 test in HeLa cells were collected with BD Accuri C6 (BD Biosciences). Population of live cells was selected according to FCS/SSC parameters. Data analysis was performed with Flowjo. Red fluorescent protein (mKate) was used in all experiments as transfection marker. For each sample, we gated the population of live cells and then the mKate-positive cells including the frequency. Whitin the mKate-positive population we calculated the geometric mean (Geo-Mean) of mKate, and the Geo-mean and frequency of EGFP, EYFP, and EBFP positive cellsPlots in Figs. Plasmid vectors carrying gene cassettes were created using In-Fusion HD cloning kit (Clonetch), Gateway system (Life technology), or Golden Gate system.Reaction included 1:2 molar ratio of plasmid backbone:gene insert starting with 100 ng of vector backbone digested with selected restriction enzymes.12 was used as destination vector for L7AeCS, MS2-CS-cNOT7 and protease engineering. L7Ae cleavage sites were synthesized as gblocks (IDT). MS2 and cNOT7 and proteases were amplified with primers including proteases cleavage sites Supplementary InformationPeer Review File