Trypanosoma brucei TUTase, RET1, we identify TUTase inhibitors and lay the groundwork for the use of this new target class as a therapeutic opportunity for the under-served disease area of African Trypanosomiasis. In a broader sense this work demonstrates the therapeutic potential for targeting RNA post-transcriptional modifications with small molecules in human disease.Non-coding RNAs are crucial regulators for a vast array of cellular processes and have been implicated in human disease. These biological processes represent a hitherto untapped resource in our fight against disease. In this work we identify small molecule inhibitors of a non-coding RNA uridylylation pathway. The TUTase family of enzymes is important for modulating non-coding RNA pathways in both human cancer and pathogen systems. We demonstrate that this new class of drug target can be accessed with traditional drug discovery techniques. Using the  RNA plays a vital role in multiple cellular processes, and is involved in many disease states, yet drugs that directly target RNA are rare.One of the principal challenges of targeting a particular RNA with a small molecule drug is the lack of well-defined secondary and tertiary structures. A key requirement for a small molecule ligand to achieve good potency is a well-defined fit in a binding pocket, allowing for multiple interactions to be made between the drug and its target. RNA adopts many transient conformations that are generally short-lived and rarely fold to generate the cavities that are characteristic of the active sites of many druggable enzymes. Thus, a promising strategy to sidestep this difficulty would be to target upstream of the RNA transcript by inhibiting proteins that themselves modify and regulate RNA.To date, drugs that act either directly on ncRNA or upstream via RNA-modifying enzymes are few in number and unselective. For example 5-azacytidine inhibits the cytosine methylation of both DNA and RNA.T. brucei viability. In the absence of RET1, trypanosomes are unable to employ their unique RNA editing mechanism which is dependent on the efficient polyuridylation of gRNA by the enzyme. The resultant failure in RNA regulation results in complete arrest in cell division followed by massive parasitic cell death.One potential class of targets in this field is the RNA terminal uridylyl transferase (TUTase) family, which is comprised of enzymes that modulate the function of target RNA by the sequential addition of uridines to the 3′ terminus.T. brucei is complex with many developmental transitions. Two developmental forms can be grown in culture and are experimentally accessible, the mammalian bloodstream form and the procyclic form from the tsetse fly midgut. The two forms respond differently to chemical treatments. The bloodstream form is clearly of most interest and relevant for the treatment of human disease. Previously published results have demonstrated that RET1 is essential for the viability of T. brucei in its procyclic form.The lifecycle of RET1 is therefore a particularly relevant target in this disease context. In the present study, we develop a high-throughput assay that identifies inhibitors of uridylylation activity. We apply this methodology in a proof of concept screen using RET1 to demonstrate that TUTases represent a tractable target for drug discovery.T. brucei , we first required a robust, practical assay system with which we could detect and optimise enzyme activity. Previously reported detection of TUTase activity was achieved by measuring the increased length of RNA as the enzyme adds successive uridines. This was accomplished by radioactive labeling of the nucleotide substrate and quantification of the higher molecular weight products on a denaturing agarose gel.in vitro overcomes such challenges. Thus, to improve throughput we optimised a non-radioactive, gel-based protocol using Sybr Gold staining for vizualization of RET1 activity at RNA concentrations similar to those used in radioactive experiments.In order to develop and use an assay suitable for HTS screening for inhibitors of RET1 from E. coli as an N-terminal GST-tagged fusion protein and purified on glutathione sepharose beads as described in the Supplementary Information . Starting from the conditions reported by Aphasizheva and Aphasizhev (2010) the uridylylation reaction was optimized to meet our screening requirements of a medium-throughput robust assay and analyzed by gel electrophoresis.Experimental Section for detailed assay protocol). We used negative controls to validate this assay, including replacement of RET1 on glutathione sepharose beads with glutathione sepharose beads pre-incubated with lysed E. coli cells, beads purified with un-induced E. coli extract and a GST tagged N-terminus truncation of RET1 (300aa), which does not contain the enzyme active site.Full-length RET1 protein (975aa) was expressed in In order to screen a large panel of molecules, we developed a solution based high-throughput assay to monitor the activity of RET1. As RET1 catalyzes the ligation of terminal uridyl groups to the 3′ end of a target ncRNA, the enzyme consumes UTP and produces pyrophosphate (PPi) as a by-product. Thus we quantified the production of PPi to measure RET1 activity over time. While luciferase coupled PPi detection assays of this nature have previously been used to monitor phosphotransferase and other enzyme activities, their use in monitoring the post-translational modification of nucleic acids is recent, as discussed in the contemporary study on TUT4.An assay coupling the RET1-catalyzed RNA uridylylation with commercially available PPiLight™ reagents (Lonza) was developed. During the “reaction” step , poly-U m of both RNA and UTP. At these concentrations there is an optimal window for competitive inhibition to be observed. Measured Km values for RNA substrate (25 nM) and UTP (50 μM), were consistent with those previously reported.In order to identify substrate-competitive inhibitors, we optimised assay conditions at the KExperimental Section for detailed assay protocol). As a result, a robust and reliable assay for the detection of RET-1 activity in both 384 and 1536 well format was developed. In order to quantify the statistical power of the high-throughput formats, we calculated the Z’ factor for our assay. A value above 0.5 indicates that more than 12 standard deviations separate the positive and negative controls. In calibration runs for the 384-well format, with heat inactivated protein providing the positive inhibition control, the signal-to-background ratio was approximately 18 and the Z’ factor was 0.77, indicating a reproducible and robust screen.Finally, we miniaturized the assay volume, and simplified the liquid dispense steps to make them amenable to automated dispensing  and exifone (2) have both been entered into clinical trials for unrelated indications.3, 4 and 5), a structural class which has given rise to successful drugs such as Rosiglitazone,6 and 7).As is to be expected from a pilot screen of this size, the potency of these hits is unlikely to be sufficient for 6 and 8, despite the fact that fragment-like molecules were poorly represented in this screening deck. With the wealth of structural information in this area derived from a number of TUTase crystal structures, a fragment based approach would be suitable for this class of target.Additionally, we observed some activity in small fragment-like molecules, for example compounds 9, 10, 11) are reported here because compound 9 has been reported to have efficacy in the topical treatment of the related pathogen Leishmania.http://www.centerwatch.com/drug-information/fda-approved-drugs/drug/1311/impavido-miltefosine). Homology between RET1 in T. brucei and Leishmania infantum is high in the domains surrounding the active site  and it is plausible that the RET1 inhibition that we report here may indeed contribute to their mode of action.Although not normally considered drug-like, the aliphatic quaternary amines (1) and exifone (2) were among the most potent drug-like hits, and have been characterized previously in human subjects, we performed preliminary experiments to evaluate their effect on the trypanosome T. brucei. The lifecycle of T. brucei is split into distinct phases depending on whether the host is an insect or mammalian. The organism can respond differently to chemical treatments in the different forms and the blood form is clearly the most relevant for the treatment of human disease. We found that compounds 1 and 2 are toxic to both blood and insect forms at concentrations close to their IC50 values (70μM and 2μM respectively) . This is an important characteristic of these hit compounds, and is consistent with a RET1 driven mode of action. In the case of exifone, its potency is sufficient to rule out non-specific cytotoxicity, since it is not toxic to PC12 cells at concentrations of 10 μM.As ataciguat ,1). In RET1, both binding modes enable similar interactions at either end of the molecule. In configuration 1 (1) is rotated by 180 degrees and this pattern is reversed; the morpholine group lies in the small hydrophobic pocket and could stack with Phe296, the central benzene could stack with Tyr524, the intervening sulfonyl oxygen atoms form direct or water mediated H-bonds with Glu657/Arg664/Asp654. The central carbonyl oxygen may form H-bonds with nearby catalytic triad aspartates Asp473, Asp310, Asp312, and sulfonyl oxygen atoms adjacent to the thiophene ring would interact with Lys505/Lys509/Ser523. Either binding mode predicts a similar interaction, leading to a snug fit of the molecule into the elongated RET1 cavity.We observed two putative binding modes for ataciguat  also gave rise to two potential binding modes. In configuration 1, exifone is near to the phosphate-binding end of the site . Again focusing on the drug-like hit compounds that gave complete RET1 inhibition curves, we assessed each for selective inhibition of RET1 over CID1. Representative curves for compounds re shown ; and IC5provided .Figure 1). However, the closed NTD β-loop conformation of RET1, which as described above leads to a snug fit of inhibitor within its binding cavity, is not present in CID1 despite the conservation of the equivalent salt bridge residues (Arg137 and E333 in CID1). This is demonstrated in Finally, we used the existing crystal structure of CID1 (pdb 4E80) to build hypotheses for the observed selective inhibition of RET1 over CID1.Non-coding RNAs remain a little-explored area in drug discovery. The enzymatic machinery responsible for the post-translational modification and control of ncRNAs presents an enticing hub to exploit post translational modifications as drug targets. The TUTase family of enzymes is an integral part of multiple non-coding RNA regulatory pathways. In this study we have demonstrated the suitability of this class of enzymes as a druggable target. Using RET1 as an example we have developed a tractable route for the identification of tool compounds and drug-like inhibitors of TUTases, demonstrating one of the first effective and achievable means of manipulating disease-relevant regulatory RNAs therapeutically.Employing the screening protocol reported in this study, we identified the first inhibitors of RET1 from a library of 3,000 small molecules. Hit compounds included drug- and fragment-like molecules. We were also able to demonstrate cross-species selectivity between TUTase homologues. Using a homology model of RET1, putative binding modes were solved for the primary hit compounds. Importantly, this model was in agreement with our experimental results and rationalized the selectivity we observed over a related RNA processing enzyme. These results will help to inform further investigations to identify inhibitors with improved potency against RET1 that have the potential to provide a much needed new treatment for African trypanosomiasis.−1), UTP (Thermo Scientific R0471 100 mM) was added to a final concentration of 50 μM. RNA substrate (see SI text) was added to a final concentration of 200 nM. Inhibitors were added as required (0.5 μl in DMSO) to a final concentration of 50 μM. When no inhibitor was included 5 % (vol/vol) DMSO was added as control. The volume was made up to 10 μl in buffer D  and the reaction was incubated for 20 mins at 27°C, then inactivated at 65°C for 10 mins. RNA loading dye  was added and tubes were heated (5 mins at 70°C), 10 μl was loaded onto 15 % TBE urea pre-cast gel (Invitrogen EC68852) and run in 1 x TBE buffer for 1.5 h at 130 V. Gels were incubated with Sybr Gold nucleic acid stain (Life Technologies S11494) for 20 mins, then imaged on an ultraviolet trans-illuminator . Experiments were carried out in duplicate for each inhibitor.Individual reactions were made up in 0.2 ml PCR tubes (Peqlab 820264-A). RET1 enzyme (see SI text) was added , UTP (Thermo Scientific R0471 100 mM) and RNA substrate final concentrations were equal to their Km: 50 μM and 20 nM respectively.2 3.2 mM) and the reaction was incubated 20 mins at 25°C, then heat inactivated at 65°C for 10 mins. Test compounds were added in 0.5 μl of DMSO stock solution. The positive control included of 0.5 μl of DMSO. After a 10 min cooling period, 5 μl of each of detection and conversion reagents from PPiLight™ Inorganic Pyrophosphate Assay Kit  were added. Luminescence was read with a Pherastar Plus micro plate reader. IC50 determinations were carried out in triplicate using the luciferase assay protocol in a dilution series of 1 mM to 99 nM.Reactions were made up in in Corning® 384 Well Low Flange White Flat Bottom Polystyrene TC-Treated Micro plates (Product #3570). RET1 enzyme was added at 0.1 μl (0.2 mgml−1) in buffer 6 was dispensed into each well using a Bioraptr reagent dispenser (Beckman-Coulter). Columns 1 and 4 were reserved for negative controls and received buffer only. Second, the compound library and DMSO controls were dispensed at indicated concentrations in 23 nL using a Kalypsys 1536-pin tool (Wako). Finally, RNA substrate (100 nM), UTP (200 µM) and PPi light reagents were dispensed in 1µL on the Bioraptr. The plates were read for luminescence 20 mins after dispensing using a ViewLux high-throughput charge-coupled device (CCD) imager (Perkin-Elmer). Following this initial read, the reaction was spiked with PPi . The plate was read again in the same fashion to identify no responding wells, which represented off target molecules that inhibited the assay.Minor modifications were made to the large-format assay to adapt the protocol into high-throughput, 1536-well format. Dispense steps were simplified into three steps. First, RET1 (3 µl, 5 µgml


Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosomein vitro, but identifying the pathway followed by individual RNA speciesin vivo remains challenging.Methods: We attempted to address this question using RNase protection.In vivo RNA-protein crosslinking (CRAC) was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the “central channel” and “direct access” routes to the Rrp44 exonuclease active site observedin vitro.Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity.Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channelin vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported fromin vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads. Rrp44 is composed of an N-terminal PIN (PilT N terminus) domain with endonuclease activity, two continuous RNA-binding cold-shock domains (CSD domains), an RNB domain carrying the exonuclease active site, and an RNA-binding S1 domain . Initial (CRAC) . To alloin vivo is very challenging. These pathways involve distinct conformations of the exosome and would be expected to protect different lengths of the substrate RNA.In vitro analyses have confirmed the protection of the 3’ terminal 30-33 nt for RNAs threaded through the channel, whereas only ~9-10 nt might be expected to be protected on the direct access route. The aim of the work reported here was to use this distinction to identify RNA substrates for each pathway.RNAs that are targeted to the exonuclease domain of Rrp44 can follow at least two routes; threading through the central barrel of the exosome complex, or direct access to the active site. However, identifying the substrates that follow each of these pathways6 - TEV protease cleavage site – 2 copies of the Z-domain of protein A) , or lacked endonuclease activity, due to point mutations at each of the four conserved endonuclease active-site amino acids .CRAC was performed on a Rrp44 construct expressed from the endogenous locus and carrying a tripartite C-terminal HTP tag (Histein A) . OtherwiDuring CRAC analyses , bait prin vitro analyses, we expected two major length populations; around 30-33 nt from RNAs threaded through the central channel, and around 9-10 nt from RNAs that directly access the Rrp44 exonuclease site . Mapping of ~10 nt fragments therefore creates significant problems with false positive results. Despite this we were able to identify sites where the long and short read populations yielded very consistent mapping data. Prioritization of the data, such that ambiguous reads are first mapped to the most common exosome substrates, appeared to substantially improve the quality of mapping. This provided clear evidence that some substrates are strongly disfavored in the short reads. This was most marked for the pre-rRNAs, which are normally the predominant exosome substrate. Recent data indicated that the exosome-associated RNA helicase Mtr4 is actively and specifically recruited to pre-rRNAs  (RRID:SCR_005012). Rrp44 and Rrp44-exo CRAC datasets were previously published (GSE77863). Rrp44-exo split-CRAC and Rrp44-endo CRAC datasets were previously published in (GSE40046 andGSE94889).Most of the primary sequence data were previously published and deposited in NCBI Gene Expression Omnibus , grown in SD-medium to log phase and UV crosslinked  to covalently bind RNA to protein. Cells were lysed in buffer containing 50 mM Tris-HCl pH 7.8, 1.5 mM MgCl2, 150 mM NaCl, 0.1% NP-40 and 5 mM β-mercaptoethanol, and RNA-protein complexes were isolated by binding to an IgG column. Bound material was washed briefly in the same buffer, but with 1M NaCl  using Novoalign  with genome annotation from Ensembl (EF4.74) (EF4.74) , with paCounting overlaps with features and prioritization. Downstream analyses were performed using pyCRAC software  and normalized per reads per millions.GSE77863,GSE40046 andGSE94889.All sequence data are available from GEO (RRID:SCR_005012) under accession numbers The revised version has addressed most of the questions raised by the reviewers. Certainly new experiments can be performed in the future to reveal the exosome-RNA interactions more thoroughly.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The exosome complex is an important RNA degradation and processing machinery that is responsible to degrade RNAs from their 3’ ends. Previous works suggested the presence of at least two routes for RNA substrates to be recruited to the exosome complex’s exonuclease activity site in the Rrp44/Dis3 protein. The Rrp44 protein also has a weak endonuclease activity site in its N-terminal region. The activity of exosome is also regulated by a few co-factor proteins. These all lend the complex a rather complicated RNA degradation or processing mechanism that still awaits to be fully revealed. The authors in this work used CRAC technology in combination with the RNase protection to analyze the RNA substrates that are loaded to Rrp44 protein and protected by the exosome complex. Their results showed the accumulation of 10-nt RNA species bound to Rrp44’s C-terminal region and the absence of 30-nt RNA species. Instead, they observed a broad shallow peak around 20-nt. Interestingly, the lack of endonuclease activity caused the reduction of the 10-nt RNA species. Deep sequencing analysis of the bound RNAs showed a whole spectrum of RNA species bound to Rrp44, indicating a complex behavior of the exosome in RNA substrate recruitment in vivo. The authors proposed the presence of another pathway for RNA substrates to be recruited to the exosome and a potential role played by the endonuclease during the different pathways. This work provided new data to study the exosome-RNA transactions although the exact mechanism seemed more complicated than proposed previously or in this work. More data would be helpful if the authors can also perform similar CRAC of yeast strains lacking Rrp6 gene to understand the cytoplasmic exosome’s substrate more clearly. A minor issue is that the author didn’t explain what the Rrp44-exo-S1 mutant is.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. in vivo with the wild type exosome or with mutants that are defective for the exo- or endonucleolytic function.  They exploit the RNase protection step included in the CRAC protocol and assess the average length of protected fragments to infer the topology of RNA degradation by the exosome . Using a standard protocol for sequencing (50nt reads) they see a distribution of length with a clear peak at very short reads (6-8 in the wt and 9-10 in the exo- mutant). Formation of these short fragments requires the endonucleolytic function of the exosome. The authors propose that these fragments derive from endonucleolytic cleavage of stalled substrates in the central channel of the exosome.In this article the authors analyze the RNA fragments that associate Using a longer read sequencing protocol, they observed the presence of two additional peaks (39 and 44nt) that likely represent fragments derived from substrates that are being degraded after threading through the central channel. Mapping of these read classes allowed inferring on the substrate preferences for the mechanisms of degradation . This is a well-conducted study that provides many interesting details on the mechanism of RNA degradation by the exosome. The data convincingly support the model proposed. What follow are a few suggestions that could complement the study.The distribution of the mismatches due to crosslinking is not exploited in the study. This could provide useful information on the nature of the reads. For instance, the authors propose that the 39 and 44nt reads derive from protection either by the sole central channel, or by the central channel and the helicases associated (Mtr4 or Ski2). If this is true, the two families of reads should have an identical distribution of crosslinking sites when aligned to the 3’ end. Also, reads derived from threading or direct access should have a different distribution of mismatches.The authors propose that stalled substrates threaded through the central core are cleaved by the endo activity for further processing. This should imply endo cleavage of substrates within the central core. Is this consistent with current structural models ? The authors should comment on this.Use of a mutant that prevents threading might allow demonstrating that the 39nt and 44nt reads indeed derive from substrates being threaded through the central core  I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. in vitro experiments, one would predict that the exosome would protect RNAs of ~30 bases when they are threaded through the barrel of the exosome, and ~10 bases when the RNA accesses the active site directly.This manuscript addresses the paths by which RNAs enter the exonucleolytic and/or endonucleolytic active sites of the Rrp44, which is the catalytic subunit of the eukaryotic exosome. This is an interesting issue since the exosome has two main nucleolytic activities and their relative importance and how different RNAs access these different sites is not clear. Based onin vivo.  The approach is to cross link RNA to the exosomein vivo, purify the exosome and then the cross-linked RNAs are digested to protected fragments subjected to deep sequencing.  This work revealed the WT exosome protects a broad range of RNA sizes with three main classes: 1) an enrichment ~6-8 bases, which is dependent on the endo activity, and converts to ~10 bases in the absence of exo activity: 2) a broad size distribution from 12-34 that is largely independent of exo or endo nuclease activities, and 3) RNAs of 39 and 44, which are not dependent on the direct entry channel and are likely to reflect RNAs that are threaded through the exosome barrel, but more than 30 bases are protected possibly due to exosome co-factors. The main contribution of this work is to use deep sequencing to identify fragments of RNA protected by the exosome in vivo will suggest unanswered questions about how RNA molecules interact with the exosome and access the active sites. Taken together, this provides insight that the endonuclease activity may be quite important for releasing RNAs that are stalled during exonucleolytic degradation.  It also highlights that understanding the size of RNAs protected by the exosome The experiments are well done and the interpretations valid.  Some suggestions that could be considered to improve the work are given below:  Although not required for indexing, it would be interesting to know if the reads that map to mRNAs  map to distinct subsets of mRNAs. For example, the exosome would be predicted to preferentially target mRNAs with slow decapping rates, which might be revealed by a more granular analysis of the read distribution to different mRNAs.I might rephrase the summary of the work to highlight that there is a clear set of fragments of 39 & 44 bases, which would be consistent with RNAs threaded through the barrel of the exosome but protected by additional co-factors.  As written now, one is not aware of these fragments till deeper in the manuscript and the comments about co-factors in the conclusions come a bit out of the blue.Although not required for indexing, it would be interesting to see how the shorter reads are affected by the exo-S1 mutations.   I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Crassostrea virginica) growth to interannual variations in mean sea level (MSL) and improve long‐term forecasts of reef response to rising seas, we monitored the morphology of constructed and natural intertidal reefs over 5 years using terrestrial lidar. Timing of reef scans created distinct periods of high and low relative water level for decade‐old reefs (n = 3) constructed in 1997 and 2000, young reefs (n = 11) constructed in 2011 and one natural reef (approximately 100 years old). Changes in surface elevation were related to MSL trends. Decade‐old reefs achieved 2 cm/year growth, which occurred along higher elevations when MSL increased. Young reefs experienced peak growth (6.7 cm/year) at a lower elevation that coincided with a drop in MSL. The natural reef exhibited considerable loss during the low MSL of the first time step but grew substantially during higher MSL through the second time step, with growth peaking (4.3 cm/year) at MSL, reoccupying the elevations previously lost. Oyster reefs appear to be in dynamic equilibrium with short‐term (month‐to‐year) fluctuations in sea level, evidencing notable resilience to future changes to sea level that surpasses other coastal biogenic habitat types. These growth patterns support the presence of a previously defined optimal growth zone that shifts correspondingly with changes in MSL, which can help guide oyster‐reef conservation and restoration.Ecosystems at the land–sea interface are vulnerable to rising sea level. Intertidal habitats must maintain their surface elevations with respect to sea level to persist via vertical growth or landward retreat, but projected rates of sea‐level rise may exceed the accretion rates of many biogenic habitats. While considerable attention is focused on climate change over centennial timescales, relative sea level also fluctuates dramatically (10–30 cm) over month‐to‐year timescales due to interacting oceanic and atmospheric processes. To assess the response of oyster‐reef ( Changes to the environment along these boundaries could result in a variety of outcomes including species adaptations ; all of which vary in rate geographically and can interact to cause complex responses in ecological communities as populations react differently . These changes can range from 15 to 20 cm interannually with the most dramatic being greater than 30 cm  alter their surface elevations through passive trapping of sediment from the water column, accumulation of annual aboveground biomass, and by augmenting belowground biomass forcing the sediment surface upwards , Back Sound, North Carolina, USA . Tides are semidiurnal with a mean range of 0.9 m and mean sea level at −0.03 m . Oyster reefs are predominantly intertidal in areas around Back Sound, occurring along marsh shorelines (fringing) or isolated on sandflats (patch).This study was conducted using 2.2Our study included an assortment of reefs of different ages (grouped into three “generations”) ranging from 2 years to a century old Figure a. Growth2) by the total number of grid cells and average vertical change within a particular elevation bin. A portion of the centennial reef was excluded from the growth analysis due to signs of heavy disturbance from harvesting and boat impacts along the adjacent tidal channel.To examine fine‐scale growth across oyster reefs, terrestrial lidar (Riegl LMSZ210ii laser scanner) was used to image reefs Figure . Terrestt‐tests were run to compare monthly mean water temperatures and salinities between scan periods within each reef generation.Fluctuations in sea level from 2009 to 2015 were examined using monthly mean sea‐level (MSL) data from a NOAA tide gauge  located approximately 5 km northeast of the reefs. We used VDatum 3.6  to transform monthly MSL data into elevations in m NAVD88. Average sea level during scan periods was then calculated for each time step. Six‐minute water level data were also queried from the Beaufort tide gauge and used to construct elevation‐exposure histograms to predict the OGZ elevation range for each scan comparison. Local water salinity and temperature recorded in Back Sound by the North Carolina Coastal Reserves  during the study period were also examined by month to elucidate if patterns in reef growth were tied to changes in water quality. Using JMP v12 (SAS Institute Inc.), regression analyses were run on monthly mean water temperatures and salinity during the overall scan period (June 2010–June 2015) to determine any trends in water quality beyond regular seasonal fluctuations. Additionally, data were transformed (cube transformation) to meet assumptions for parametric analysis, and a series of 33.1SD)  Figure b. Prior ) Figure . The seaR2 = −0.02, F1,57 = 0.99, p = .33). Furthermore, all comparisons of water temperatures between scan periods within reef generations did not yield significant differences . Prior to the first scan period, the area experienced a distinct drop in salinity with high water associated with Nor'Ida (winter 2009–2010). Subsequent drops in salinity occurred periodically during the scan periods, with the most pronounced declines coinciding with high water in late 2014 and low water in early 2015  during the first time step, but regained it over the second (0.03 m3/year).For the decade‐old reefs, average sea level between the two periods increased approximately 4 cm from −0.053 m NAVD88 (averaged through Spring 2010–Spring 2012) to −0.016 m NAVD88 (average through Spring 2012–Spring 2015)  to −0.043 m NAVD88 (averaged through Winter 2013–Winter 2014)  just above MSL.With the centennial reef, mean sea levels over the two time periods increased 3.9 cm, from −0.021 m NAVD88 (averaged through June 2012–June 2014) to 0.018 m NAVD88 (averaged through June 2014–June 2015) Figure a. DuringZ Figure c. In con4Changes in reef morphology and reef‐wide growth were tightly aligned with month‐to‐year patterns in sea level Figures , Table 2C. virginica  is not outside of the Eastern oyster's tolerance , which shift correspondingly with sea level, reinforcing sea level as the primary control (and certainly the most parsimonious based on the available data).4.1Haematopus palliates), resulting in oyster mortality. Thus, it appears that while oysters cement together to create a solid reef matrix, oyster mortality within the taphonomically active zone (layer of living oysters) due to overexposure could compromise the outer reef structure, making it more susceptible to erosional forces. A similar process has been documented on coral reefs during extreme low tides (Anthony & Kerswell, Distinct patterns of growth were exhibited by the decade‐old reefs during the two sampling timeframes Figure  coincidiFor the decade‐old reefs, mean sea level increased 3.7 cm between the two time steps with prolonged higher water during the winter of 2014–2015, which corresponds to increased growth at higher elevations on the reef as both the OGZ and growth ceiling shifted upward Figure . While t4.2Elevations of growth maximums on young reefs also paralleled changes in sea levels Figure . Sea lev4.3Growth changes on the centennial reef over the study period behaved comparably to patterns displayed by the other study reefs Figure . Peak grGrowth on the centennial reef was measured at higher elevations than the other reefs included in this study. This reef is much larger than the constructed reefs, and we may be witnessing a certain degree of facilitation (Bruno, Stachowicz, & Bertness, As a larger reef spread across a greater exposure gradient, the degree to which the centennial reef would have been subject to forces impacting surface elevation may have been different than the constructed reefs. Having a much larger mass, the reef may be more prone to compaction in years of low water. Its size and proximity to sandy shoreline may have also fostered higher foraging by the American Oystercatcher during years when the reef was more exposed, although this is likely not as significant as desiccation mortality and reef compaction. Alternatively, the centennial reef is in a more sheltered environment and encompasses a much larger population of oysters compared to the constructed reefs and is adjacent to other reefs of similar size. This would provide a greater localized source of oyster larvae, helping maintain recruitment even during years of lower larval supply relative to the smaller, more exposed constructed reefs (O'Beirn, Heffernan, & Walker, 4.4Oyster reefs appear to be in dynamic equilibrium with sea level. Like other intertidal habitats, decadal or longer measurements of mature reef surface elevation changes would show they track RSLR (DeAlteris, The ubiquity of this response across oyster‐reef ages is a testament to their resilience to RSLR as well as their utility and longevity for stabilizing shorelines, likely reducing the potential impacts of the coastal squeeze Pontee, . This woThis study presents evidence that intertidal oyster reefs are highly responsive to short‐term fluctuations in local sea level even at maturation. When compared to other coastal habitats and their capacities for RSLR response, oyster reefs are unparalleled in their ability to maintain surface elevation with changing sea level. Greatest recorded rates of surface elevation change in intertidal and shallow subtidal systems such as marshes, mangroves, and corals are below 1–2 cm/year excluding storm‐related allochthonous sedimentation (Baustian et al., None declared.All authors participated in project conception, data collection and analysis, and the writing and editing of the manuscript. Click here for additional data file.


The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)–RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT–RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT–RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT–RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT–RAM is suppressed, indicating that RNMT–RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT–RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts. In eukaryotes, gene expression is dependent on the mRNA cap being added to RNA pol II transcripts –3. The cThe enzymes that catalyse mRNA cap synthesis are configured in a species-specific manner . In mammHere we demonstrate that RNMT–RAM controls ribosomal RNA (rRNA) production and present the mechanism involved.2. Cells (5 × 105) were transfected with 50–100 nM siRNA  using Lipofectamine RNAiMax (Thermo Fisher Scientific). Cells were infected with INI-based expression plasmids by retroviral transduction and selected using 0.5 mg/ml G418.HeLa cells were cultured in DMEM/10% FBS at 37°C and 5% CO2, 10% glycerol, 0.5% Triton X-100 (TX-100), 1 mM EGTA, 1 mM EDTA and 1 mM DTT] was used to extract cellular protein 24–48 h post-siRNA transfection. Western blots were performed to detect RNMT and RAM , c-Myc , GAPDH , TAFID , TAF1B , RNA Pol subunit RPA194  and RNA Pol subunit RPA135 .Lysis buffer -uridine, cells were incubated with pre-warmed media containing 2.5 µCi/ml -uridine for 30 min. Cells were washed with cold PBS and RNA was extracted using an RNeasy kit (Qiagen) or TRIzol (Thermo Fisher Scientific). RNA (2 μg) was resolved by denaturing electrophoresis, transferred to Hybond-NX membrane and analysed by autoradiography. Quantification of -uridine signal in 45S pre-rRNA was determined by Storm phospho-imager and analysed using the AIDA imager analyser software. -uridine incorporation into total RNA was quantified by scintillation counting using equal amounts (150–300 ng) of RNA. For chase experiments, cells were labelled as above, washed three times in pre-warmed medium and then incubated in pre-warmed media for 30 min, 1 and 2 h. RNA was extracted and analysed as above.For the labelling of nascent rRNA with -uridine incorporation . However, since RNMT–RAM is recruited to rDNA, it may assist RNA pol I-dependent transcription under certain conditions. As discussed above, in HeLa cells, under the conditions used here, the major mechanism by which RNMT–RAM controls rRNA transcription is by regulating c-Myc.In the course of this work, we observed RNMT–RAM recruitment to rDNA loci. The recruitment of RNMT–RAM to rDNA was equivalent to the recruitment to GAPDH, an RNA pol II-dependent gene. However, we did not find a function for RNMT–RAM in rRNA synthesis in HeLa cells. RNMT–RAM is not required for RNA pol I-dependent transcription


Shigella and its expression is required for the invasion of the human intestinal mucosa by pathogenic bacteria. VirF was shown to directly activate the transcription of virB and icsA, which encode two essential proteins involved in the pathogenicity process, by binding their promoter regions. In this study, we demonstrate by band shift, enzymatic probing and cross-linking experiments that VirF, in addition to DNA, can also bind the icsA transcript and RnaG, an antisense non-coding small RNA that promotes the premature termination of icsA mRNA through a transcriptional attenuation mechanism. Furthermore, we show that VirF binds in vitro also other species of RNAs, although with lower specificity. The existence of VirF–RnaG and VirF-icsA mRNA complexes is confirmed in a pulldown assay carried out under experimental conditions that very close reproduce the in vivo conditions and that allows immobilized VirF to “fish” out RnaG and icsA mRNA from a total RNA extract. The VirF binding sites identified on both icsA mRNA and RnaG contain a 13 nucleotides stretch (5′-UUUUaGYcUuUau-3′) that is the RNA-converted consensus sequence previously proposed for the VirF–DNA interaction. Band-shift assays with a synthetic RNA molecule whose sequence perfectly matches the consensus indicate that this signature plays a key role also in the VirF–RNA interaction, in particular when exposed in a stem–loop structure. To further explore the icsA-RnaG-VirF regulatory system, we developed an in vitro test (RNA–RNA Pairing Assay) in which pairing between icsA mRNA and synthetic RNAs that reproduce the individual stem–loop motifs of RnaG, was analyzed in the presence of VirF. This assay shows that this protein can prevent the formation of the kissing complex, defined as the initial nucleation points for RNA heteroduplex formation, between RnaG and icsA mRNA. Consistently, VirF alleviates the RnaG-mediated repression of icsA transcription in vitro. Therefore VirF, by hindering the icsA transcript-RnaG interaction, exhibits an activity opposed to that usually displayed by proteins, which generally assist the RNA–RNA interaction; this quite uncommon and new function and the regulatory implications of VirF as a potential RNA-binding protein are discussed.VirF is the master activator of virulence genes of  Shigella flexneri is the causative agent of human bacillary dysentery which causes about 1 million of deaths worldwide each year, the majority of which are children , is a member of the AraC family  was amplified by PCR using the oligonucleotides H238 and H239 and 10 ng of E. coli MRE600 chromosomal DNA as template. The DNA product was cloned downstream the T7RNA polymerase promoter into the EcoRI and BamHI sites of pSelectTM1 . The resulting pSelecthns was used to synthesize the hns mRNA. The virB DNA fragment (212 bp) used to synthesize the leader region of virB mRNA was amplified by PCR from plasmid pBN1 (virB and the oligonucleotide VB212 (reverse primer). VirF purification was performed as described by The mid pBN1  as templicsA370, mRNA (hns (this study), virB212 mRNA (this study), cspD mRNA (Met (Sigma). Each RNA was transcribed with T7 RNA polymerase as indicated by 32P]-ATP as described by 32P]-ATP. For M129/130 duplex formation, 5 pmol of M129 32P-labeled were mixed with 15 pmol of M130 in Annealing buffer . After incubating at 90–95°C for 1 min, the mixture (10 μl) was gradually cooled down to 49°C and kept at this temperature for 10 min before stopping on ice. The RNA duplex as formed was used in EMSA. Each 5′-end-labeled RNA (0.05 pmoles of mRNAs or 0.5 pmoles of oligonucleotides) was incubated for 10 min at 25°C in gel retardation buffer  with the indicated amounts of VirF and immediately loaded on a native 7% polyacrylamide gel electrophoresis (PAGE).Electrophoretic mobility shift assay (EMSA) was made using the following RNAs: RnaG120 , icsA37070, mRNA , hns (thspD mRNA  and tRNAE. coli HMG11 cells transformed with plasmid pGT1127 (Pulldown assay was performed using the Dynabeads His-Tag Isolation & Pulldown (Novex). 50 μl of Dynabeads were mixed to 80 μg of purified histidine-tagged VirF in Buffer B  and incubated on a roller for 30 min. A control sample containing only Dynabeads was processed in parallel. Then samples were washed two times with Buffer B and were incubated for 2 h at 4°C with 60 μg of total RNA extracted from  pGT1127 . SubsequicsA370 mRNA (∼1 pmol) denatured for 1 min at 90°C, were renatured for 5 min at 32°C and then incubated for 3 min without and with different concentrations of VirF in 15 μl of buffer A . RNA was treated with 0.075 U of RNase T1 (specific for unpaired G), 0.0025 U of RNase T2  or 0.05 U of RNase V1 (specific for double-stranded RNA) for 3 min in presence of 1 μg of tRNA as competitor. The reaction was stopped with an equal volume of a phenol–chloroform mixture (1:1), and RNA was precipitated with ethanol. The reaction products were analyzed on 10% PAGE-urea gel in parallel with ΔT1 and OH- ladders (Labeled RnaG or  ladders .32P]-labeled G+1H oligonucleotide (2 pmol) was heated at 90°C for 1 min to denature RNA. Annealing of the primer with RnaG120 was carried out at 32°C for 5 min in buffer A (see above) in the presence or absence of VirF. The mixture was transferred to an ice-cold plate and U.V. irradiated for 1 min using the GS Gene-linker BioRad . Finally the cross-linked RNA was primer-extended using the AMV Reverse Transcriptase as previously described (A mixture (8 μl) containing RnaG120 (0.5 pmol) and the [escribed .icsA mRNA (2.5 pmol) and the [32P]-labeled RNA oligonucleotide GR4-48 (0.5 pmol) were denatured separately at 85°C for 1 min and placed on ice. The GR4-48 oligonucleotide was renaturated at the indicated temperatures for 5 min in the presence or absence of VirF before adding the denatured icsA mRNA. Pairing between icsA mRNA and GR4-48 was obtained prolonging the incubation for further 10 min at the same temperature in gel retardation buffer (see above) and then samples were loaded on 7% PAGE under native conditions.The in vitro transcription assay was carried out by incorporating [α-32P]-UTP into mRNA using the E. coli RNA polymerase essentially as previously described (32P]-UTP. The icsA fragment (304 bp) to use as DNA template in the in vitro assay, was amplified by PCR with the oligonucleotides pair G-100/G+187 and plasmid pGT1129 (icsA transcription products (full-length and truncated transcripts), were analyzed on 7% PAGE-urea gel.The escribed . NTPs co pGT1129 . After picsA full transcription. For this reason, we tested whether VirF, in addition to DNA, could also bind RNA. To verify this hypothesis, we investigated the possible interaction of VirF with icsA mRNA and RnaG by EMSA. These RNAs have been chosen because they belong to the icsA–RnaG genetic system which is regulated by VirF itself. As can be seen in Figures 1A,B VirF produces retarded bands with both the icsA370 transcript, consisting of the whole leader region and part of the coding region, and RnaG120, carrying the entire antisense sequence (120 nt). About 50% of probe is recovered as RNA–protein complexes at 21 and 75 nM of VirF with RnaG120 and icsA mRNA, respectively. This result provides the first evidence that VirF is able to interact with RNA, displaying a ∼3.5-fold higher binding affinity for RnaG than that estimated for the icsA messenger RNA . This finding led us to probe the possible interaction of VirF with a series of RNAs available in our laboratory, several of which unrelated to the vir system. The selected transcripts were: hns mRNA coding the nucleoid proteins H-NS, virB mRNA coding a transcriptional activator of virulence genes of Shigella, cspD mRNA coding a member of the cold shock protein family of E. coli and the E. coli tRNAMet. As shown in Supplementary Figure virB, cspD, and hns transcripts is retarded at VirF concentrations ranging from 180 to 320 nM while tRNAMet is shifted only at elevated protein concentrations (500 nM) . These experiments demonstrate that, within the limit of accuracy of EMSA, VirF forms stable complexes with RnaG and icsA mRNA at a significant lower concentration than that required to bind the other control RNAs.VirF was shown, at least in few well-characterized genetic systems, to bind the promoter regions of its target genes and these interactions were required to affect their transcription . As explicsA mRNA from a total RNA extract, to mimic the RNA–protein recognition occurring in vivo. Pulldown assay shows that both RnaG and icsA mRNA are recovered when bulk RNA is incubated with the VirF-coated magnetic beads  while are undetected when empty beads (not complexed with the protein) are used as control . The different levels of the two recovered RNAs reasonably reflect the higher expression of RnaG with respect to icsA mRNA, already observed in a previous work , results from the transcriptional interference regulation controlling this genetic system , we performed RNA footprints using RNases T1, T2 and V1 to localize its potential binding sites on this target RNA. As seen in Figure 3A, VirF interaction causes several alterations of the sensitivity pattern of RnaG120 to RNases, most of which are localized on the GH1 domain (nucleotides 15–40). These changes include few positions protected against RNases T1 and T2 cleavages on single-stranded regions  and many positions exhibiting an increased hydrolysis by RNase V1 on stem structures . Overall, the GH2 stem–loop is generally less affected displaying only few nucleotides hyper-sensitive to RNase V1 cleavage at positions 54–56, 65, and 66. Even less pronounced effects are visible at GH3 hairpin where the susceptibility to RNases digestion of RnaG120 alone and that of the VirF–RNA complex does not significantly vary except for positions 80, 81, and 87 . Downstream positions were not analyzed due to technical limitations. Localization of VirF binding sites on RnaG120 was confirmed also by means of RNA–protein U.V. cross-linking studies. In this assay, irradiation of RNA–protein complexes with ultraviolet light causes the formation of covalent bonds between VirF and RnaG120. These contact points can be mapped on RNA as stops (more intense bands) of reverse transcriptase in a primer extension reaction. The result of this experiment is shown in Figures 4B. As expected, VirF remains tightly cross-linked to GH1 (nucleotides 28–33) whereas very weak interactions are observed at domains GH2 (nucleotides 66 and 67) and GH3 (nucleotides 79 and 80).Given that VirF tightly binds RnaG120  and pulled down  by VirF. As seen in Figures 4A, the two different techniques identify three regions as potential sites for the VirF–RNA interaction. These discrete sequences are: (i) the apical loop (nucleotides 30–36) of AH1 arm; (ii) the stem structure and the basal bulge (nucleotides 60–65) of AH1 arm; (iii) the basal bulge (nucleotides 77–79 and 99–103) and the apical loop (nucleotides 84–86) of AH2 arm. Therefore, the results of the U.V. cross-linking experiments, fully consistent with the enzymatic probing data, strongly suggest that the domains GH1 of RnaG and AH1 and AH2 of icsA mRNA are the preferential targets of VirF .In order to locate possible VirF binding sites on lcrGVH-yopBD operon, Figures 4C,D). The gel shown in Figure 5A and the relative quantification  clearly reveal that VirF binds M129, but not M130. However, a higher VirF concentration is required to shift the short M129 RNA compared to icsA mRNA and RnaG , indicating that the identified 13-bp consensus sequence is an element necessary but not fully sufficient for the interaction of VirF with the RNA. Notably, the sequences of M129 and M130 are complementary so that they could be annealed to yield a RNA duplex . Comparing the binding capacity of VirF with the M129 and M129/130 RNAs emerges that this protein, particularly at lower concentrations (<1 μM), interacts less efficiently with its target sequence when this is buried within a RNA duplex  partially exposed in internal or apical loops.By means of DNase I footprinting experiments on the icsA transcription resides in its antisense region (nucleotides 1–120) which hybridizes to the cognate messenger RNA. Complete pairing passes through the formation of an intermediate, the kissing complex, in which GH1 contacts AH2 whereas GH2 contacts AH1 whereas GH3 is dispensable . These interactions provide the initial nucleation points for RNA hetero-duplex propagation and are required for the establishment of icsA attenuation by RnaG (We have previously shown that the ability of the non-coding RnaG (450 nt) to prematurely terminate  by RnaG .Figure 5C, VirF retards both the GR4-48 and GR49-80 oligonucleotides indicating that its target sites are independent and binding occurs even when they are separated and individually assayed. Importantly, VirF exhibits, within the limit of accuracy of this type of analysis, a 2.5-fold higher capacity to interact with GR4-48 (∼50% bound RNA at 1 μM of protein) than with GR49-80 (∼45% bound RNA at 2.7 μM of protein) . This result, in agreement with RNA footprints and U.V. cross-linking assays, confirms that GH1 represents the primary anchorage point recognized by the protein.The evidence of a clear detectable VirF binding site on GH1 domain allowed to suppose that VirF, bound to RnaG, could interfere with the RNA–RNA interaction by hiding the sequences critical for the formation of kissing complex between sense and antisense RNAs. To verify this hypothesis and to study the interaction of VirF with the individual structural domains of RnaG, we designed two RNA oligonucleotides reproducing the GH1 (GR4-48) and GH2 (GR49-80) motifs. Initially, these synthetic RNAs were used to carry out EMSA experiments. As seen in icsA mRNA pairing. To this purpose, we developed an in vitro test called “RNA–RNA Pairing Assay” (RRPA), in which the heat-denatured icsA370 messenger was added to a reaction mixture containing the 5′-end-labeled GR4-48 and VirF. Then, the free GR4-48 oligonucleotide and that hybridized with the icsA mRNA were electrophoretically resolved under native conditions and radioactivity associated with the RNA hetero-duplex was quantified. RRPA was performed at two temperatures (37 and 42°C) and VirF concentrations . Increasing amounts (from 1 to 2 μM) of VirF cause a progressive reduction of bound GR4-48 suggesting that this protein blocks the interaction between the GH1 domain of RnaG and icsA mRNA. This effect is clearly detectable at 37°C and becomes very pronounced at 42°C .The outcomes so far presented prompted us to analyze the effect of the VirF–GH1 interaction on RnaG-in vitro assay, we investigated the transcriptional attenuation of icsA by RnaG120 as a function of increasing concentrations of VirF. According to icsA transcript due to the formation of an intrinsic terminator on the target mRNA. Under the experimental conditions used , termination products promoted by RnaG120 represented about 70% of all transcription events. VirF addition causes lower levels of transcriptional termination of icsA (30%), indicating that this protein is able to alleviate, albeit not completely, the repressive action of RnaG possibly by sequestering the RnaG itself and its target mRNA.Next, by means of an trans-encoded and cis-encoded RNAs. trans-encoded sRNAs have multiple target mRNAs and regulation is exerted via the interactions of short and imperfect complementary sequences. cis-encoded sRNAs, also known as antisense RNAs (asRNA), are transcribed from the opposite DNA strand of established coding sequences and hence share fully complementary with their targets. In the majority of cases, both trans- and cis-encoded small RNAs act at post-transcriptional level by inhibiting the function of target RNAs. Multiple aspects concerning small regulatory RNAs in bacteria are reviewed in In the last decade, there has been an explosion in the discovery of bacterial small regulatory RNAs (sRNAs) and in the identification of their targets. Most of characterized sRNAs function by base-pairing with their target mRNAs and fall into two categories: S. flexneri. This RNA, named RnaG, represses by a transcriptional attenuation mechanism, the expression of IcsA  H-NS-mediated inhibition of icsA mRNA synthesis; (iii) transcriptional interference, being icsA and RnaG promoters convergent and 120 nt apart.Few years ago, we have identified the first regulatory antisense RNA, encoded by the virulence plasmid pINV of  of IcsA , an invaShigella . In addichanisms : (i) diricsA–RnaG genetic system with a focus on the role of VirF, has led us to identify a new property for this protein. In fact, VirF known so far to solely bind DNA, can also interact with RNA as demonstrated in this work. Due to the complexity of the overall regulatory mechanism governing icsA expression and to the objective difficulties to evaluate the individual and combined contributions of each factor in vivo, we chose to study this new feature of VirF by means of an in vitro approach. Gel mobility shift assays show that VirF binds icsA mRNA and RnaG very tightly although it is able to associate also with other RNAs  . Furthermore, their primary role is validated by the fact that VirF binding capacity is strongly impaired with a RNA oligonucleotide (M130) that lacks this 13-bp conserved motif. Accordingly, VirF shows a higher binding affinity (∼2.5-fold) for domain GH1 of RnaG, which contains two overlapping sequences matching in 6/13 and 9/13 positions the consensus, than for domain GH2, that has only a highly degenerated motif (5/13 matching positions), as clearly proved by the EMSA experiment carried out with the separated domains . Finally, similar sequences, although with lower frequency and conservation, have been found also the other RNAs retarded by VirF  adjacent to a helix which places part of the recognized bases into a structural context that reproduces the natural condition occurring in DNA. This circumstance is particularly evident at the apical hairpins of GH1 and AH1 and at the internal bulge of AH2 . In this context, the unpaired U-stretch seems to play a key role. In fact, VirF binds better the target sequence arranged in a stem–loop structure with respect to the same sequence entirely paired in a RNA duplex . Thus, multiple elements  can concomitantly contribute to the recognition process of RNA by VirF.A deep investigation on the icsA mRNA (∼150 nt) and RnaG120, identifying those nucleotides required for establishing the kissing complex between the sense and antisense RNAs. In particular, a mutational analysis of RnaG revealed that: (i) the apical loop of GH2 pairs with the basal bulge of AH1; (ii) the apical loop and the internal bulge of GH1 anneal with structurally similar motifs present on AH2; (iii) the GH3 domain is not involved in icsA mRNA–RnaG pairing. The secondary structures of these cognates RNAs, including their initial contact points as determined by Figure 4. As mentioned above, our analysis of the VirF–RNA interaction, overall suggests that this protein has two primary target sites: the GH1 domain of RnaG and  its complementary region on icsA mRNA, the AH2 arm. These regions actively participate to the formation of kissing complex. The schematic model of Figure 8 shows that, by masking at least one of the pairing sequences (GH1 and AH2), independently of its location on RnaG or icsA mRNA, VirF could prevent the formation of a functional kissing complex thereby allowing the synthesis of the full-length icsA transcript. Furthermore, several points of hypersensitivity to V1 RNase cleavage (a double-stranded specific ribonuclease) induced by VirF interaction are found at GH1 and GH2 stems of RnaG . This observation suggests that VirF may contribute to stabilize the RNA duplex thus trapping RnaG into its native structure, a rigid state functionally unable to hybridize with icsA mRNA. Our conclusions are supported by three main lines of evidence. First of all, RnaG and icsA mRNA are selectively pulled down by VirF from a total RNA preparation under competitive conditions that close mimics those existing in vivo . Secondly, VirF is able to alleviate the termination of icsA transcription in vitro by targeting RnaG . Finally, the yield of the hetero-duplex formed between the GH1 domain of RnaG and icsA mRNA increases as a function of temperature from 32 to 42°C, as shown in the RRPA assay of Supplementary Figure icsA mRNA–RnaG complex, a condition that represses icsA transcription. However, VirF is able to hamper this sense-antisense pairing in a temperature-mediated manner, being its effect more pronounced at temperatures ≥ 37°C .RNA loop–loop interactions are frequently used to trigger the initial recognition between two RNA molecules. Such pairing, involving exposed nucleotides, originates the kissing complex . In a prpromoter  and by hicsA–RnaG system is properly regulated in absence of other RNA helper proteins and is likely Hfq-independent (traJ mRNA (icsA transcript. This is quite an uncommon function for a protein able to bind RNA. To our knowledge, the icsA-RnaG-VirF three components system is the first well-studied case in which this kind of interplay takes place, providing new mechanistic aspects for the overall comprehension of riboregulation in bacteria.Except for VirF, the ependent . In addiependent , FinO faraJ mRNA . Unlike in silico analysis using program RNAbindRplus (Unlike many other DNA binding proteins, VirF seems capable of recognizing either the DNA sequences and their RNA counterparts. Although this property is rather uncommon, it is worth mentioning that also two important DNA binding proteins, namely H-NS  and HU , were foindRplus , that isindRplus .E. coli and Shigella cells expressing or not VirF have led to the identification of a large set of genes common to both bacteria, that are activated either directly or indirectly by this regulator (Microarray experiments using egulator . These oMG, CT, AG, and MF designed and performed most of the experiments giving an important contribution also to the analysis and interpretation of data. AA and CM carried out some experiments. MF, AG, and MG have been dealing with the preparation of figures, drafting the work and revising it critically.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.


C9orf72 repeat expansion are lending further support to the notion of defects in RNA metabolism as a key factor underlying ALS. RNA binding proteins are involved in all aspects of RNA metabolism ranging from splicing, transcription, transport, storage into RNA/protein granules, and translation. How these processes are affected by disease-associated mutations is just beginning to be understood. Considerable work has gone into the identification of splicing and transcription defects resulting from mutations in RNA binding proteins associated with disease. More recently, defects in RNA transport and translation have been shown to be involved in the pathomechanism of ALS. A central hypothesis in the field is that disease causing mutations lead to the persistence of RNA/protein complexes known as stress granules. Under times of prolonged cellular stress these granules sequester specific mRNAs preventing them from translation, and are thought to evolve into pathological aggregates. Here we will review recent efforts directed at understanding how altered RNA metabolism contributes to ALS pathogenesis.Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease affecting both upper and lower motor neurons. The molecular mechanisms underlying disease pathogenesis remain largely unknown. Multiple genetic loci including genes involved in proteostasis and ribostasis have been linked to ALS providing key insights into the molecular mechanisms underlying disease. In particular, the identification of the RNA binding proteins TDP-43 and fused in sarcoma (FUS) as causative factors of ALS resulted in a paradigm shift centered on the study of RNA dysregulation as a major mechanism of disease. With wild-type TDP-43 pathology being found in ~97% of ALS cases and the identification of disease causing mutations within its sequence, TDP-43 has emerged as a prominent player in ALS. More recently, studies of the newly discovered  Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease. Affecting both upper and lower motor neurons, ALS culminates in the inability to coordinate muscle movements with death due to respiratory failure typically within 2–5 years after diagnosis . About 50% of ALS patients display deficits reminiscent of FTD and 15% of FTD patients have ALS symptoms  were the first to be linked to ALS pathogenesis  associate with stress granules under times of cellular duress , two RNA recognition motifs (RRM1-2), and a glycine rich C-terminal prion like domain  and LISTERIN  harboring TLS/FUS response elements located in the promoter region  protein. Mechanistically, both the loss of detyrosinated microtubules and the sequestration of kinesin-1 mRNA and protein into FUS granules contribute to the altered transport and localization of specific RNAs  as a protein partner of FUS  repeat expansions in the C9orf72 (C9) gene were found to be causative of ALS and FTD , which are thought to contribute to toxicity at least in part by sequestering other proteins and RNAs  for the indistinguishable clinical presentations across fALS and sALS. In addition, it would be interesting to determine common and divergent RNA targets in different models of ALS. This will assist in the development of therapeutic strategies that may be effective for all or defined subsets of ALS patients.ANC, BLZ and DCZ wrote the manuscript. ANC focused on TDP-43 and C9, and made the figure. BLZ focused on the FUS section.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.


We also found that half of editing sites in 3′ UTR of coding genes were microRNA targets and most of the sites in the coding regions could lead to non-synonymous amino acid changes. Functional analysis of genes which suffered damaging non-synonymous editing events in each cancer stage show the gradual expansion of cancer related pathways accompanied by an increasing malignant grade of the samples. Our study, for the first time to our knowledge, comprehensively profiled and compared the editomes across the different cancer stages and revealed the functional impacts of RNA editing events during cancer development and progression.RNA editing is a post-transcriptional event that leads to transcriptome diversity and has been shown to play important roles in tumorigenesis. However, dynamical changes and the functional significance of editing events during different cancer stages have not yet been characterized systematically. In this paper, we describe a comprehensive study of the RNA editome of four samples from different cancer stages for the same patient based on analysis of both whole-genome and transcriptome sequencing data. We identified 35,225 and 33,784 RNA editing events for poly(A) RNA editing refers to programmed alterations in transcripts catalyzed by the double-stranded RNA-specific ADAR family of proteins. The emergence and rapid progress of high-throughput sequencing technology offers the promise of analysis of transcriptome-scale RNA editing events in cancer . RecentlOur previous study showed that transcriptomic analysis corroborated genetic alterations identified at the genomic level and could suggest the aggressiveness and metastatic potential of multifocal hepatocellular carcinoma . As the In this study, we conducted a comprehensive study to characterize the RNA editomes in cancer development and progression based on the whole-genome and transcriptome sequencing data from four samples including: non-cancerous liver, primary liver tumor, an intrahepatic metastasis, and a portal vein tumor thrombus from the same patient and systematically analyzed dynamical changes and functional significance of editing events.TM 2000 with 90-bp paired-end reads. Next, we fractionated total RNA into poly(A)+ RNA and poly(A)- RNA and constructed whole-transcriptome sequencing libraries. We then performed strand-specific sequencing on both poly(A)+ and poly(A)- RNA libraries with 90-bp paired-end reads. In total, an average of 97.8 Gb (31.6× coverage) genome and 7.6 Gb transcriptome sequences were obtained for each sample. The sequencing data were deposited at the European Genome-phenome Archive1, which is hosted by the EBI, under the accession number EGAS00001002338. Such deep sequencing data of each sample provides an ideal source for efficient characterization of the RNA editome.The sequencing data used in the current work were from our previous study . In brieFor both genome and transcriptom sequencing data, low-quality reads were removed if more than 10% of the bases were unknown or if more than 50% of the bases had a base quality lower than 5. The remaining high-quality paired-end reads were aligned to the NCBI Human Reference Genome Build 37 (hg19) using BWA  for geno2 and dbSNP (version 132)   and VarSion 132) .p-value should be lower than 0.05. We then performed Enrichment Map analysis using Cytoscape to group and display the gene sets with similar functions  to enrich the Gene Ontology terms . The p-vunctions . Statist+ and poly(A)- RNA-Seq data respectively in all four samples. Next, we cross-referenced this data against known protein-coding gene models in RefSeq (+ and poly(A)- data respectively were located in sequences that were either unannotated in the database or corresponded to overlapping transcripts on both strands. The remaining 35,225 and 33,784 sites for poly(A)+ and poly(A)- data respectively were unambiguously mapped to known gene models, and thus were selected for further analysis. Then we compared A-to-I(G) editing sites with those in the DARNED database .To exhaustively analyze RNA editome during cancer development and progression, we first obtained whole-genome and transcriptome data from our previous study of four samples including: non-cancerous liver (NL), primary tumor (PT), an intrahepatic metastasis (IM), and a portal vein tumor thrombus (PTVV) from the same patient, who was a 49-year-old male diagnosed with a poorly-differentiated primary hepatocellular carcinoma (HCC) with multiple satellite lesions and PTVV . We usedn RefSeq  and longn RefSeq . This stdatabase  and idendatabase . The lowdatabase  and that+ and poly(A)- data across each pattern are displayed . The results show that 93 and 90% sites correspond to stage specific editing events. Next, we investigated the editing events in Alu and repetitive non-Alu regions . Consistent with the previous observations, A-to-G editing events was significantly enriched in repetitive Alu elements when compared with the other 11 types of variants. Taken together with the above results, during cancer development and progression, only a few editing sites were preserved across different stages. Most were stage specific events and that strongly suggests the distinct adaptiveness of editome in cancer evolutionary process.To systematically analyze RNA editomes during cancer development and progression, we divided the editing sites into seven patterns: omnipresent across all four samples (ALL); partially shared by both tumourous and non-cancerous liver samples (STN); partially shared by only tumourous samples (ST); remaining four patterns are sites unique to each sample . Distribution of editing events for both poly(A)Figure 4). Among sites in poly(A)+ RNA, most of those were located in 3′-untranslated region (UTR) of coding transcripts. In comparison, the equivalent editing sites of exons and introns were observed in long non-coding transcripts. Interestingly, almost all the conserved sites across four cancer stages were located in non-coding regions, particularly in 3′ UTR of coding genes, and less than 4% conserved sites were in coding regions (CDS). However, further analysis of the sites in poly(A)- RNAs showed distinct location attributes although conserved editing sites were also enriched in non-coding regions, of which editing events in the introns is predominant irrespective of coding or non-coding transcripts. Moreover, the portion of editing sites in the exons of non-coding genes is decreased in poly(A)- RNAs when compared with that in poly(A)+ RNAs.We next studied the location attributes of the identified RNA editing events in each stage of cancer development and progression . Specifically, 52 and 44% editing sites in 3′ UTR were microRNA targets which predicted by miRTar (+ and poly(A)- RNAs respectively . The top 10 miRNAs according to the number of edited targets were shown in Table 1. In addition, more than half of the sites in coding regions could led to non-synonymous amino acid changes . Then we compared these editing genes with those identified in previous study .To further evaluate the functional consequence of editing events, we analyzed the sites in 3′ UTR and coding regions as well as editing impacts on gene expression. First, we found that the editing sites in 3′ UTR were significantly enriched in microRNA targets (y miRTar  for polyus study . The res+ RNA (Supplementary Data Sheet S1), although there were distinct editing profiles in different stages of cancer, similar biological function impacts were observed. Furthermore, the gradual expansion of cancer related pathways was clearly shown with the increasing malignant grade of samples . Specifically, there were no cancer pathways presented in non-cancerous liver, and several malignant processes arise in primary tumor including cell migration, cell cycle and blood vessel development. In addition, the regulation of MAPKKK and JNK cascades, which were connected to responses to stress and phosphorylation modules in enrichment maps and play important roles in cell death, appeared in intrahepatic metastases. In the portal vein tumor thrombus, the predominant functional impacts of editing events occurred at the post-translational level, such as protein complex biogenesis and protein localization. Furthermore, regulation of biosynthetic and metabolic process modules was commonly deregulated in all intrahepatic samples but not in the portal vein tumor thrombus and that was consistent with the special metabolic function of liver tissue.To characterize the biological significant of stage-specific editomes in cancer development and progression, we tested for functional enrichment of gene sets which suffered damaging non-synonymous editing events in each sample. Among damaging non-synonymous variants of poly(A)- RNAs (Supplementary Data Sheet S2) showed that they have a similar, but still have exclusive functional impacts compared with those in the poly(A)+ RNAs . Consistent with above findings, the cell migration module also presented in the primary and metastatic tumors, from cellular component movement expanding to cell migration during tumor metastasis. Moreover, two modules, including regulation tumor necrosis factor production and Ras protein signal transduction module, which have important roles in cancer, were unique to the primary tumor and portal vein tumor thrombus respectively. Notably, differing poly(A)+ RNAs, biological synthesis and metabolism process were not present in all intrahepatic samples of poly(A)- RNAs and that may imply the different regulatory mechanism between the two types of RNAs. Taken together, editing events in both poly(A)+ and poly(A)- RNAs may have great impacts in cancer development and progression.Further analysis of the damaging non-synonymous sites in poly(A)Initial studies of RNA editing in cancer suggests that both genome and transcriptome sequencing experiments are required to capture all the editing events comprehensively. In the current study, we conducted a systematical analysis of cancer RNA editomes based on the whole-genome and transcriptome sequencing data from four samples including: non-cancerous liver, primary tumor, an intrahepatic metastasis and a portal vein tumor thrombus from the same patient, which represented the whole process of tumorigenesis and metastasis.+ or poly(A)- transcripts (+ transcripts and relatively little is known about poly(A)- transcripts, which is a significant portion of transcripts. Previous studies revealed that many mRNAs may lack poly(A) tails and such types of mRNA are overrepresented in specific functional categories (+ and poly(A)- RNAs from four stages of cancer samples, and derived a comprehensive RNA editing landscape.According to the presence or absence of a poly(A) tail at 3′ ends, RNAs can be physically classified into poly(A)nscripts . Most sttegories . In this+ non-coding genes compared with poly(A)- ones, suggesting that more functional elements are related to editing events enriched in these regions.Our work not only focused on the editing sites in coding transcripts, but also in long non-coding transcripts, which have emerged as a new class of functional RNAs and play important roles in cancer . The latAlthough nearly half of editing sites in 3′ UTR were microRNA targets or could led to non-synonymous amino acid changes, there were little effect on gene expressions. The results suggested RNA editing could occur via different mechanisms to influence gene function, but it may just one complex systems of gene regulation during post-transcriptional processes.In the functional enrichment study, we found distinct editing sites and accordant functional impacts across each stage of tumorigenesis and metastasis, which may reflect the pattern of implement function in the RNA editome. Our study suggested that the dysregulation of RNA editing events may contribute to the altered transcriptional program necessary to sustain carcinogenesis, and may lead to identification of novel diagnostic and prognostic markers in cancer.HL, ZL, and YZ designed the study. HL, SF, and LS performed analyses. All authors contributed to writing the manuscript and approved its final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nicotiana benthamiana with two different begomoviruses and snatches capped leader sequences from their mRNAs. By determining the 5′ termini of a single RSV mRNA with high-throughput sequencing, the 5′ ends of almost all the mRNAs of the co-infecting begomoviruses could be identified and mapped on their genomes. The findings in this study provide support for the using of the cap snatching of sNSVs as a tool to map viral TSSs.Identification of the transcription start sites (TSSs) of a virus is of great importance to understand and dissect the mechanism of viral genome transcription but this often requires costly and laborious experiments. Many segmented negative-sense RNA viruses (sNSVs) cleave capped leader sequences from a large variety of mRNAs and use these cleaved leaders as primers for transcription in a conserved process called cap snatching. The recent developments in high-throughput sequencing have made it possible to determine most, if not all, of the capped RNAs snatched by a sNSV. Here, we show that rice stripe tenuivirus (RSV), a plant-infecting sNSV, co-infects  To do this, Agrobacterium tumefaciens strain EHA105 carrying the plasmid of interest were grown to an optical density at 600 nm (OD600) of 0.8 at 28°C on LB liquid medium supplemented with rifampicin (50 μg/mL) and kanamycin (50 μg/mL). The cultures were centrifuged at 12,000 g for 1 min followed by resuspension with an induction medium . After virus inoculation, N. benthamiana plants were grown under standard greenhouse conditions A Jiangsu isolate of RSV  was used in this study. This virus was maintained in rice by transmission with viruliferous N. benthamiana 15 days after inoculation of begomoviruses using Triziol (Invitrogen). After being dephosphorylated using alkaline phosphatase (NEB), the total RNA was treated with RppH (NEB), which removes the cap structure of mRNAs and leaves a monophosphate at their 5′ ends. T4 RNA Ligase 1 (NEB) was used to ligate a RNA oligo (TCTACrArGrUrCrCrGrArCrGrArUrC) to the 5′-monophosphate-bearing mRNAs. SuperScript® III First-Strand Synthesis System (Invotrogen) was used to reverse transcribe the oligo-tagged RNAs with a primer named NCPR1, which is specific to NCP of RSV. Two primers, one designed according to the RNA oligo (GTTCTACAGTCCGACGATC) and another specific to NCP but 5′ to NCPR1, were used to amplify the cDNA. After 30 (Sample 3) or 34 (Sample 1 and 2) cycles of amplification, the PCR products were resolved by agarose gel electrophoresis. PCR bands about 200 bp in size were recovered and sent to Biomarker Technologies  for high throughput sequencing with a HiSeq 2500 platform.The high throughput sequencing was done with a procedure diagramed in Figure A RNA ligase mediated RACE (RLM-RACE) was used to verify the 5′ termini of begomoviral mRNAs (Liu and Gorovsky, WL and PQ conducted the research. JJ and SU helped in virus inoculation. SL, JZ, and JY helped in sequence library construction. RK helped in data analysis, ZD and ZW conceived and supervised the research.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.


In contrast, only the double-stranded AgoshRNA precursor associated with Ago2 in cells, correlating with efficient intracellular processing and reporter knockdown activity. These results suggest the presence of a cellular co-factor involved in AgoshRNA loading into Ago2 in vivo. We also demonstrate specific AgoshRNA loading in Ago2, but not Ago1/3/4, thus further reducing unwanted side effects.The RNA interference (RNAi) pathway was recently expanded by the discovery of multiple alternative pathways for processing of natural microRNA (miRNA) and man-made short hairpin RNA (shRNA) molecules. One non-canonical pathway bypasses Dicer cleavage and requires instead processing by Argonaute2 (Ago2), which also executes the subsequent silencing step. We named these molecules AgoshRNA, which generate only a single active RNA strand and thus avoid off-target effects that can be induced by the passenger strand of a regular shRNA. Previously, we characterized AgoshRNA processing by deep sequencing and demonstrated that—after Ago2 cleavage—AgoshRNAs acquire a short 3’ tail of 1–3 A-nucleotides and are subsequently trimmed, likely by the poly(A)-specific ribonuclease (PARN). As a result, the mature single-stranded AgoshRNA may dock more stably into Ago2. Here we set out to analyze the activity of different synthetic AgoshRNA processing intermediates. Ago2 was found to bind preferentially to partially single-stranded AgoshRNA  MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are small, non-coding RNAs (∼21 nucleotides (nt) in size) that have important post-transcriptional regulatory roles by targeting messenger RNAs (mRNAs) for cleavage or translational repression . This prIt was always assumed that depending on the thermodynamic properties of the miRNA duplex, one strand will be preferentially selected as mature miRNA and guide the RISC complex to complementary mRNA targets , 12. HowThe preferential loading based on thermodynamic properties of the duplex means that both strands of the siRNA duplex can be loaded into Ago to form an active RISC, possibly leading to off-target effects. Recently, we described shRNA molecules that skip the Dicer processing step and instead are processed by Ago2. The unique slicing ability of Ago2, results in a single active siRNA strand , bottom.th and 11th bp, yielding a ~30 nt partially dsRNA (cleave). We show this product in A) ATP  according to manufacturer’s instructions. To remove unincorporated nucleotides, the LNA probes were purified using Sephadex G-25 spin columns  according to manufacturer’s protocol. Hybridization was performed overnight at 42°C with labeled LNA oligonucleotides in 10 ml ULTRAhyb hybridization buffer (Ambion) according to the manufacturer's instructions. To detect the 5’ strand the Gag5 sense probe was used (LNA positions are underlined) TGCTGTCATCATTTCTTC-3’5’-A and to detect the 3’ strand the Gag5 antisense probe was used (LNA positions are underlined) AAGAAATGATGACAGCAT-3’5’-G. After overnight hybridization the membranes were washed 3–4 times for 5 minutes at 42°C in 10 ml 2 × SSC/0.1% SDS (low stringency) and twice for 15 minutes at 42°C in 10 ml 0.1 × SSC/0.1% SDS (high stringency). Signals were detected and quantified using a phosphorimager (Typhoon Phosphorimager).Isolated RNA (5 μg), synthetic AgoshRNA (100 pmol) and Ago-FLAG immunoprecipitated RNA (1 μg) were analyzed by northern blotting. The RNA samples were denatured for 5 minutes at 95°C before electrophoresis on a 15% denaturing polyacrylamide gel . To check for equal sample loading, the 5.8S rRNA and tRNA bands were visualized by ethidium bromide staining (2 μg/ml) for 20 minutes. The RNA samples were electrotransferred to a positively charged nylon membrane  and crosslinked to the membrane using UV light at a wavelength of 254 nm (1200 μJ × 100) in the Stratalinker UV Crosslinker 1800 (Agilent Technologies). LNA oligonucleotide probes were 5’ end labeled with the KinaseMax™kit  in the presence of 1 μL [γ-2O. The Western Lightning ECL system (PerkinElmer Life Sciences) was used for luminometric detection with the LAS4000.For SDS-PAGE western blot the samples obtained in the Ago2 pull down assays were mixed with an appropriate amount of loading dye . The samples were denatured at 95°C for 5 minutes before electrophoresis on a Novex 6% Tris-glycine gel (Invitrogen). The samples were transferred to an Immobilin-P Transfer Membrane at 25V for 50 minutes. The membrane was then blocked for 60 minutes in 5% milk powder in PBS. The membrane was washed with 0.1% milk-PBS and incubated overnight at 4°C in 10 ml 0.1% milk-PBS with anti-FLAG primary antibody (1:5000) or alpha-actin (1:5000) with slow orbital mixing. After overnight incubation, the membrane was washed 3–4 times for 5 minutes with 0.1% milk-PBS and then incubated for at least one hour with slow orbital mixing in 10 ml 0.1% milk-PBS with goat anti-mouse secondary antibody (1:5000). The membrane was washed 2 times for 5 minutes in PBS-Tween, 2 times for 5 minutes in PBS and 2 times for 5 minutes in deionized HThe pre-Agosh RNA samples obtained from the Ago2 immunoprecipitation were cloned and sequenced. For this purpose, the small RNAs were 3’ polyadenylated using the A-Plus Poly(A) Polymerase Tailing Kit (Epicentre Biotechnologies) according to manufacturer’s protocol. The reaction was stopped by acid-phenol:chloroform extraction followed by ethanol precipitation. Subsequently, the polyadenylated RNAs were reverse transcribed into cDNA, using the ThermoScript RT-PCR System for first-strand cDNA synthesis (ThermoFisher Scientific), followed by PCR amplification. PCR products were separated on size via agarose gel electrophoresis, purified and subsequently used for TOPO-TA cloning . TA clones were sequenced with the T7 and m13RP primer using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).HEK 293T were co-transfected with 100 ng of firefly luciferase expression plasmid (Gag5 sense or antisense), 0.5 ng of renilla luciferase expression plasmid (pRL-CMV) and 5 μL of AgoshRNA expression construct of different dilutions (10–0.001 μM). The pRL plasmid served as an internal control for cell viability and transfection efficiency and pBluescript SK- (pBS) (Promega) was added to obtain equal DNA concentrations. Two days post-transfection, firefly and renilla luciferase activities were assessed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol.cleave, AgoshA and Agoshtrim. These AgoshRNA-forms were first tested for their ability to interact with recombinant human Ago2 (Ago2) in vitro by means of equilibrium fluorescence titration [A was the best competitor, followed by Agoshtrim, pre-Agosh and Agoshcleave.Synthetic AgoshRNA molecules were developed based on the distinct AgoshRNA processing intermediates shown in itration . We pre-itration . All forcleave variant with a 3’ U-extension (AgoshU) to resemble the miR-451 processing intermediate [cleave, 34 nt for AgoshA and AgoshU, and 25 nt for Agoshtrim. Cellular RNA was blotted and we used probes to detect either the 5’ or 3’ side of the hairpin  show the unaffected input RNA with the 5’ probe. This exclusive processing of pre-Agosh yielded multiple products, which appeared as a smear of differently sized RNA molecules. The 3’ probe showed a similar array of shortened RNA products. This pattern was reproducibly observed and is not due to RNA degradation in the sample, as was confirmed by ethidium bromide staining of intact cellular tRNAs and 5.8S rRNA. As expected, the 3’ probe was not able to detect the processed AgoshRNA forms due to insufficient sequence complementary with the truncated 3’ arm.Next, we initiated cell culture based tests for the set of synthetic AgoshRNA molecules. Stability and potential processing of the synthetic AgoshRNAs were assessed by RNA-transfection into 293T cells. We also included an Agoshrmediate . To do sThe silencing activity of the AgoshRNA set was tested on a luciferase reporter that contains a perfectly complementary target sequence. We co-transfected the luciferase construct with increasing amounts of the synthetic AgoshRNA (0.1–10 pmol) into HEK 293T cells. Co-transfection with the pBluescript plasmid (pBS) or an unrelated shRNA expressing plasmid (shRT5) served as negative controls. We scored high luciferase knockdown activity for the pre-Agosh . All procleave, AgoshA or AgoshU, and Agoshtrim) were not enriched in the Ago2-bound fraction and only weak signals were present in the unbound fractions. The weak RNA signal in the unbound fraction could relate to the RNA isolation method as these RNAs are abundantly present in total RNA  were separated from the unbound fraction. The bound and unbound fractions were analyzed by northern blot with an LNA probe complementary to the 5’ side of the hairpin . A promi RNA see . CaptureA (1x), Agoshtrim (7x), or intermediates (3x) that suggest gradual trimming from the 3’ end of AgoshA  in cells, which explains why it is exclusively processed inside the cell and active in reporter mRNA knockdown.Novel AgoshRNA designs form a promising class of RNAi-based therapeutics that do not require Dicer cleavage and instead use Ago2-slicer activity to generate a guide RNA that targets a complementary mRNA for degradation. The major benefit of the AgoshRNA design over regular shRNAs is that off-target effects induced by the passenger strand are avoided. Earlier studies pinpointed to the stem length as a critical element for Ago2-mediated processing to avoid Dicer-processing . Recentlin vitro results imply that Ago2 favorably loads partially ds/ss AgoshA and ss Agoshtrim intermediates over the ds pre-Agosh precursor  bind much better than pre-Agosh. We thus conclude that the AgoshRNA hairpin likely requires co-factors for Ago2 loading in vivo. Ago2-mediated processing of the ds RNA into a ss species subsequently facilitates a more stable and functional interaction.In this study we investigated the processing intermediates of the novel AgoshRNA design in detail. We demonstrate that the ds precursor AgoshRNA (pre-Agosh) is selected for loading into Ago2 in cells to form an active RISC, while the partially-ds/ss intermediates are not functional. We also show that this selection step is circumvented by recombinant Ago2 S1 Fig32P). Samples were collected at 0’, 10’, 30’, 60’ and 120’. Reactions were analyzed by 8% (w/v) PAGE.Cleavage of hAgo2 (3.7 μM) was assayed using the following radiolabeled substrates: no guide RNA (negative control), 100 nM guide (p-as2b or p-as2b-FAM) and 2,5 nM target (s2b-(TIF)Click here for additional data file.