O-methylation is certainly one of the most common RNA modifications found in almost any type of cellular RNA. It decorates transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs) , as well as regulatory RNAs like microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), and finally, eukaryotic messenger RNAs (mRNAs). Due to this exceptional widespread of RNA 2′-O-methylation, considerable efforts were made in order to precisely map these numerous modifications. Extensive studies of RNA 2′-O-methylation were also stimulated by the discovery of C/D-box snoRNA-guided machinery, which insures site-specific modification of hundreds 2′-O-methylated residues in archaeal and eukaryotic rRNAs and some other RNAs. In this brief review we discussed both traditional approaches of RNA biochemistry and also modern deep sequencing-based methods, used for detection/mapping and quantification of RNA 2′-O-methylations.Ribose 2′- O-methylation is a highly common modification in different cellular RNAs, present in transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nuclear/small nucleolar RNAs (snRNAs/snoRNAs), as well as in microRNAs (miRNAs)/Piwi-interacting RNAs (piRNAs) and some messenger RNAs (mRNAs) (for review  opened a new way for specific detection  b. AbortiO-methylated 3′-terminal riboses to periodate cleavage (IO4−). RNA is first randomly fragmented by a nuclease (benzonase for RibOxi-Seq or fragmentation reagent followed by end repair for Nm-Seq) leaving 5′-phosphates and 3′-OH extremities and these RNA fragments are subjected to periodate oxidation , considerably increasing the loss of material in these treatment steps. Therefore, substantial amount of input RNA is generally required for these applications. In addition, the presence of 2′-O-Me at the 3′-extremity is known to reduce the efficiency of 3′-adapter ligation (see above), thus further reducing the library yield and representativity.Two independently published protocols RibOxi-Seq  and Nm-Sxidation c. Protecydes see b. These O-methylation analysis were developed only recently, some of these approaches are already extensively used for RNA modification profiling under different physiological conditions. For the moment, the most popular application undoubtedly remains profiling of 2′-O-methylations in eukaryotic rRNAs by different versions of RiboMethSeq  are less widespread, certainly due to excessive amount of input RNA required for analysis (see below), precluding their massive application in biomedical research.S. cerevisiae and human rRNAs) and, in addition, using different subsets of ‘previously validated’ modification sites. Since eukaryotic rRNA may also change its methylation status depending on the cell line used and even upon cultivation conditions and media composition, false negative hits may be also explained by such undermethylation. Moreover, different metrics for performance measurements were used, like Receiver Operating Characteristic (ROC) curve parameters , as well as more simpler threshold level-based models defining validation of sites as candidates.Direct comparison of these high-throughput methods for the performance in ab initio discovery of modified residues is not easy, since their validation was generally performed with different model RNAs , where biological material is precious and extremely limited.O-methylations [4−-based oxidation require the highest amount of input material, original publications used 7.5 µg (RibOxi-Seq) [The best sensitivity was reported so far for variants of RiboMethSeq. Depending on the library preparation protocol this approach requires as low as 10 ng of total human RNA for complete analysis of rRNA 2′-ylations . Routineylations ,60. As aylations . FinallyOxi-Seq)  or 10 µgOxi-Seq) ,59.At the first glance, RiboMethSeq analysis requires the highest sequencing coverage since the signal is defined as a protection of a given phosphodiester bond against cleavage, compared to surrounding RNA positions. In principle, average coverage of 5′-/3′-ends of about 100 would be sufficient for reliable analysis, which is about 750,000 reads for human rRNA. However, this reasoning does not take into account irregularity of cleavage due to highly structured rRNA regions. In practice, about 20 times more raw reads are required for a reliable coverage of all rRNA positions. Thus we routinely use the coverage of 12–15 mln of raw reads for the analysis of human rRNA or tRNAs by RiboMethSeq ,64. SimiDespite the expected enrichment of the signal due to specific detection of RT-stops (2′-OMe-Seq) or protected methylated RNA 3′-end (RibOxi-Seq and Nm-Seq), the reported sequencing depth for analysis appears to be quite similar, ranging from 10–15 mln of raw reads for 2′-OMe-Seq to 15–40 mln of raw reads for RibOxi-Seq and Nm-Seq ,58.O-methylation level and thus analysis of modification dynamics in RNA is possible only with RiboMethSeq, since the protection signal linearly depends on the methylation level [O-methylation sites is close to 5% and only very few sites show higher dispersion. In practice >10% of difference of calculated MethScore (ScoreC in previous publications) can be considered as statistically significant. Absolute quantification of methylation level can be achieved if the exact values of MethScore in the absence of modification are known (unmodified transcripts or RNA from knock-out (KO) strains). In vitro transcripts were used for calibration of yeast and human rRNA modification levels, while yeast and Escherichia coli strains deleted for the corresponding RNA modification enzymes are useful for tRNA analysis [Precise quantification of 2′-on level ,66. Techanalysis ,66.In addition to RiboMethSeq, 2′-OMeSeq can also provide some relative quantification with appropriate spike-in of in vitro RNA transcripts, even if the absolute quantification remains impossible . In contDifferent technological platforms can be used for sequencing of generated amplicons (libraries). Illumina sequencing  remains the most popular in the field, though PGM/Ion Proton devices are also suitable. Standard RiboMethSeq requires only single-read 50 nt sequencing mode (SR50), paired-end sequencing does not substantially improve the results. Similarly, only single read sequencing mode is in principle required for 2′-OMe-Seq, since only 3′-cDNA ends are of interest see b. In conData treatment and analysis steps are similar in all approaches, reads’ processing generally begins with trimming, followed by alignment to the reference sequence and counting of 5′- or 3′- (or both) ends of mapped reads. A special care should also be taken at the mapping step to avoid multiply mapped sequences of unknown origin.O-methylation analysis now opens the way for an exhaustive identification of novel modified sites in diverse cellular RNAs as well as careful investigations of RNA 2′-O-methylation dynamics under various physiological conditions and in human pathologies related to RNA modifications.Combination of traditional and deep sequencing-based approaches for RNA 2′-


Small non‐coding RNAs repress or activate genes in consonance to environmental or developmental cues and they can modulate life and death of the cell. RNAs can change shapes to adapt to different temperatures (thermosensors), and this can modify their functions; for instance, changing to 37°C can lead to structural changes that trigger the translation of virulence factors  actively process, degrade and monitor the quality control of all types of RNA  is a universal feature of eukaryotes as well as prokaryotes. These extracellular vesicles have also been referred to as exosomes and microvesicles. They are small vesicles with phospholipid membrane carrying proteins DNA and RNAs and can serve as mediators of information in intercellular communication, inter‐ and intraspecies communication and molecular mimicry. The vesicles make selective sorting of cargo, but all major RNA classes can be present and delivered to bacterial and/or eukaryotic cells. Therefore, RNA carried by these EVs can be considered as a universal mediator of communication  are far more relevant than previously suspected (Nachtergaele and He, In conclusion, our understanding of intra and interkingdom communication is still in its infancy but brings great promises. It can be used to produce a new form of antibiotics that restrict and tame pathogens and can also be used to ‘motivate’ microbes to produce many powerful and useful molecules, some of which have yet to be discovered. Therefore, my Crystal Ball is that RNA and Intra/Interkingdom communication will drive the most exciting progress in microbiology over the next few years.


Wheat yellow mosaic virus (WYMV), which has been reported to contain two genomic RNAs with 3' poly(A) tails and caused severe disease on wheat in East Asia region.Polyadenylation influences many aspects of mRNA as well as viral RNA. variable polyadenylation at the 3' end have been reported in RNA viruses. It is interesting to identify the characteristic and potential role of 3' polyadenylation of 3' RACE was used to identify sequences of the 3′ end in WYMV RNAs from naturally infected wheat by WYMV. In vitro translation assay was performed to analyze effect of UTRs of WYMV with or without 3’polyadenylation on translation. In vitro replication mediated by WYMV NIb protein were performed to evaluate effect of variable polyadenylation on replication.Fluc) gene in in vitro WGE system, whereas additional adenylates had an oppositive effect on this enhancement on translation mediated by UTRs of WYMV. Additional adenylates remarkably inhibited the synthesis of complementary strand from viral genome RNA during the in vitro replication mediated by WYMV NIb protein.Variable polyadenylation in WYMV RNAs was identified via 3′ RACE. WYMV RNAs in naturally infected wheat in China simultaneously present with regions of long, short, or no adenylation at the 3' ends. The effects of variable polyadenylation on translation and replication of WYMV RNAs were evaluated. 5’UTR and 3’UTR of WYMV RNA1 or RNA2 synergistically enhanced the translation of the firefly luciferase (3' end of WYMV RNAs present variable polyadenylation even no polyadenylation. 3' polyadenylation have opposite effect on translation mediated by UTRs of WYMV RNA1 or RNA2. 3' polyadenylation have negative effect on minus-strand synthesis of WYMV RNA in vitro. Variable polyadenylation of WYMV RNAs may provide sufficient selection on the template for translation and replication.The online version of this article (10.1186/s12985-019-1130-z) contains supplementary material, which is available to authorized users. Polyadenylation at the mature 3′ end of eukaryotic mRNAs plays a fundamental role in mRNA export to cytoplasm, effective translation and mRNA stability –3. The cViruses are obligate parasites and rely on living cells to complete their life cycles. RNAs of many DNA viruses and RNA viruses also have a 3′ poly(A) tail that is synthesized in a post-transcripitional manner, similar to mRNAs, or derived from a poly(U) sequence on the template strand –11. The Wheat yellow mosaic virus (WYMV) causes yellow mosaic and dwarf symptoms on wheat and yield losses of 20–40% [Bymovirus in the family Potyviridae, whose members are 3′ polyadenylated and contain a 5′ genome-linked protein VPg [f 20–40% , 24. WYMf 20–40%  and has f 20–40% , 27. WYMtein VPg . WYMV hatein VPg . For mRNtein VPg . For pottein VPg  However,In this study, variable polyadenylation including the absence of poly(A) tail at the 3′ end of WYMV RNAs was found from wheat naturally infected by WYMV in China. In vitro assays showed that variable polyadenylation differentially regulated the translation and minus strand synthesis of WYMV. It is implied that the variable polyadenylation may regulate the template selection for translation and replication or the molecular transition between translation and replication of WYMV.0 (5-ccagtgagcagagtgacg-3), which complements with the terminal sequence of the adaptor. RT products were then used as templates to clone the 3′ terminal sequences using LA-Taq polymerase (Takara) with primers Q0 and WYM-R1–7264-F (5-gctgctttaggcacaggtac-3). RT-PCR products were cloned into pMD18-T (Takara) and sequenced using the primer WYM-R1–7264-F.Total RNA was extracted from wheat leaves showing yellow mosaic and dwarf symptoms at Linyi, Shandong province, China in 2015. Firstly, the adaptor (5-aaaaaaaaaaaaaaaaagcttgagctcgagtcctcgtcactctgctcactgg-3) was linked with the 3′ end of total RNA using T4 RNA ligase (New England Biolabs). Reverse transcription on the adapted total RNAwas performed using M-MLV reverse transcriptase (Takara) and primer QFluc) gene in the pT7-F-3’UTRssp vector using BamH I and Sma I; the in vitro transcript from this plasmid was designated F-WY-R1-5 U. pFluc-WY-R1-3 U and pFluc-WY-R1-3 U·15A, with or without 15 adenylates, respectively, were separately constructed by inserting the 3’UTR (258 nt) of WYMV RNA1 (GenBank: AF067124) downstream of the Fluc gene in the pT7-F-3’UTRssp vector using Nru I and Ssp I; the in vitro transcript from these plasmids were designated F-WY-R1-3 U and F-WY-R1-3 U·15A. pFluc-WY-R1-5 U-3 U and pFluc-WY-R1-5 U-3 U·15A, without and with 15 adenylates, respectively, were separately constructed by inserting 3’UTR of WYMV RNA1 (GenBank: AF067124) downstream of the Fluc gene in pFluc-WY-R1-5 U. The 5’UTR (171 nt) and 3’UTR(768 nt) of WYMV RNA2 (GenBank: AF041041) with or without 15 adenylates were inserted at the same sites as that of RNA1 in pT7-F-3’UTRssp vector to make pFluc-WY-R2-5 U, pFluc-WY-R2–3 U, pFluc-WY-R2–3 U·15A, pFluc-WY-R2-5 U-3 U and pFluc-WY-R2-5 U-3 U·15A.All plasmids were constructed based on the firefly luciferase reporter construct pT7-F-3’UTRssp vector  and confpfu Taq polymerase (Takara) and corresponding primers  by affinity column chromatography according to the manufacturer’s instructions.NIb coding sequences of WYMV HC isolate (GenBank. AF067124) was amplified by primers WYM-R1–4921-BamHI-F (5-atggatccatggcctccgacactctcagc-3) and WYM-R1–6504-SalI-R (5-atgtcgacgatagtttggagctcaatgctgct-3). PCR products were digested by BamH I and Sal I and inserted into pMAL-C2X (NEB) with T4 DNA ligase (Takara). The positive plasmid of pMAL-WY-NIb was transformed into 2, 0.01 M DTT, 0.11 M KCl, 17.5 μg yeast tRNA, 1 mM ATP, 1 mM GTP, 1 mM CTP, 0.01 mM UTP, 5 UCI α-32P-UTP (PerlinElmer), 12.5 μg of purified WYMV NIb protein and ddH2O. The in vitro replication reaction was performed at 20 °C for 1.5 h. After the reaction, 70 μL ddH2O and 120 μL of phenol/chloroform were added, followed by centrifugation at 13,000 rpm. The supernatant was precipitated by adding 2.4 volumes of NH4Ac / isopropanol (5 M NH4Ac: isopropanol = 1: 5) and placed at − 20 °C for at least 2 h. The precipitate was resuspended using 10 μL RNA loading buffer and separated in 5% PAGE gel containing 8 M urea with 1500 mA for 1.5 h. The gel was dried and exposed to a phosphorimager screen, followed by detection with the Typhoon FLA-7000 .The 50 μL reaction system consisted of 8 pmol of RNA templates, 0.1 M Tris-Cl (pH 8.2), 0.01 M MgClIn this study, status of the 3′ polyadenylation in WYMV RNA1 was analyzed for the first time. Total RNAs were extracted from wheat showing yellow mosaic and dwarf symptoms in fields at Shandong province, China in 2015. RT-PCR was performed to confirm the existence of WYMV (data not shown). An aliquot of the same total RNAs was used in 3’RACE to clone the 3′ terminal and potential poly(A) sequences. Sequencing data of four clones showed the differential polyadenylation at the 3′ end of WYMV RNA1. One clone (GenBank: KU254085) lacked adenylation at the 3′ end, two clones  had three adenylates at the 3′ end, and one clone (GenBank: KU254088) had 30 adenylates at the 3′ end  accounted for 27, 46% of clones A1-A5 had 1 to 5 adenylates, and 20% of clones A6-A10 had 6 to 10 adenylates (Table In order to estimate the ratio of clones with different lengths of poly(A), an additional 96 clones were randomly selected and the length of 3′ poly(A) of WYMV RNA1 was analyzed. The 3′ poly(A) in these 100 clones ranged from 0 to 33 adenylates in length Table . Clones Polyadenylation plays an essential role the translation of mRNA through the cyclization of mRNA, a process which also involves the 5′ cap . HoweverFluc reporter gene was tested. Compared with the mock vector, the presence of only the 5′ UTR of WYMV RNA1 or RNA2 enhanced translation of Fluc to 8 fold or 10 fold, respectively  on translation of corresponding constructs was tested. An additional 15 adenylates on F-WY-R1-5 U + 3 U decreased the translation of Fluc to 9.1% of F-WY-R1-5 U + 3 U  activity . WYMV NIAs small infectious entities replicating only in the living cell, viruses must evolve some properties so that they can exploit the translation machinery of the host to survive. mRNAs in eukaryotes are co-transcribed to obtain a non-templated poly(A) tail of about 250–300 adenosine residues, which contributed to the regulation of the stability, transport and translation of mature transcripts , 30. ManAlternative polyadenylation in RNA viruses should have more complex roles than in mRNAs since RNA from RNA viruses can serve as the template for both translation and replication . In thisAdditional poly(A) showed opposite effects on translation mediated by WYMV RNA1 or RNA2 UTRs Fig. . HoweverIn addition to being the template for translation, RNAs of RNA viruses also serve as the templates of their complementary strands during the replication of viral genomes. Previous studies on genome replication of RNA viruses were focused on characteristics of the core promoters and other cis-elements –44. The The detailed mechanism of alternative polyadenylation of RNA viruses remains unknown. If alternative polyadenylation tails are the direct transcriptional products from different poly(U) segments in the template strands, this alternative polyadenylation on WYMV RNAs could provide suitable templates for the different requirements of translation and replication. If alternative polyadenylation is caused by RNA decay, as is the case with mRNA, the change of polyadenylation of viral RNAs may be responsible for the transition between translation and replication. The transition from translation to replication for RNA viruses without polyadenylation may be regulated by the structural change of cis-elements at the 3′ end of viral RNA due to the binding of RdRp . In summ3′ end of WYMV RNAs present variable polyadenylation even no polyadenylation. 3′ polyadenylation have opposite effect on translation mediated by UTRs of WYMV RNA1 or RNA2. 3′ polyadenylation have negative effect on minus-strand synthesis of WYMV RNA in vitro. Variable polyadenylation of WYMV RNAs may provide sufficient selection on the template for translation and replication.Additional file 1:Table S1. The primers used in this study. (DOCX 15 kb)


Many non-coding and regulatory RNA elements have evolved to exploit transient or metastable structures that emerge during transcription to control complex folding pathways or to encode dynamic functions. However, efforts to engineer synthetic RNA devices have mostly focused on the thermodynamically stable structures. Consequently, significant challenges and opportunities exist in engineering functional RNAs that explicitly take advantage of cotranscriptionally generated transient or metastable structures. In this work, we designed a short RNA sequence that adopts a robust metastable structure when transcribed by an RNA polymerase. Although the metastable structure persists for hours at low temperature, it refolds almost completely into the thermodynamically stable structure upon heat denaturation followed by cooling. The synthetic RNA was also equipped with the Broccoli aptamer so that it can bind its ligand and become fluorescent only in the thermodynamically stable structure. We further demonstrated that the relaxation to the thermodynamically stable and fluorescent structure can be catalyzed by a short trigger RNA in a sequence-specific manner. Finally, the RNA architecture was redesigned to sense and respond to microRNA sequences. In summary, we designed RNA nanomachines that can detect an RNA sequence, amplify signal and produce an optical output, all encoded in a single RNA transcript, self-powered by a metastable structure. As RNA is transcribed in the 5′ to 3′ direction, local secondary structures may form cotranscriptionally which can affect the global folding pathway and kinetics . While translation is still suppressed in plasmid-containing cells due to the Sok-RNA, those cells that lack the plasmid  express Hok and are killed.In fact, natural RNA elements that actively exploit such cotranscriptionally generated structures have been reported. For example, the splicing . In anotsplicing . This issplicing . Similar Sok-RNA ,7. As thTetrahymena ribozyme), or sophisticated and dynamic regulation of RNA functions (as in the cases of MS2 and hok/sok systems).These and other observations ,8,9 suggin vitro , but after heat denaturation and cooling, the RNAs equilibrate equally to the two structures with similar thermodynamic stability ..in vitroAnother interesting example from the Sugimoto group studied the relaxation of a cotranscriptionally formed hairpin structure to a G-quadruplex (G4) using fluorescent probes Figure  11). Th. Th11). In this work, we sought to design more sophisticated functions that fully exploit the metastability of cotranscriptionally generated RNA structures. First, we aimed to design a robust metastable structure that can be generated simply by transcription. Second, we wanted the system to fully relax to a thermodynamically stable structure upon appropriate triggers. Finally, the designed RNA should exhibit a prototypical function beyond structural transformation.2, 1 mM dithiothreitol (DTT), 2 mM spermidine, 100 mM KCl.Enzymes, ribonucleotide triphosphates (NTPs) and gel loading buffers were purchased from New England Biolabs (NEB) unless noted otherwise. DFHBI-1T was purchased from Lucerna. All other chemicals used were of molecular biology grade or equivalent, or of the highest quality available, and purchased from Nacalai Tesque or Wako Pure Chemical Co. The 1 × reaction buffer used in this work has the following composition: 40 mM Tris–HCl, pH 7.9, 10 mM MgClIn vitro transcription was performed in a volume of 20 μl containing the 10 × supplied buffer (2 μl), 12.5 mM (each) NTP mix (3.2 μl), 40 mM MgCl2 (1 μl), RNase Inhibitor, Murine (0.8 μl), DNA template (400 ng) and T7 RNA polymerase (1 μl). The reactions were kept at 37°C for 3 h. SK-miR-21 was prepared similarly except with 200 ng DNA template, without additional MgCl2, and 1 h reaction time. After in vitro transcription, DNase I (2.5 μl) was directly added and incubated at 37°C for 15 min to remove DNA template. RNAs were then purified by column , and concentrations were calculated based on absorbance at 260 nm.Linear dsDNA templates that encode the T7 promoter sequence followed by the desired transcripts  were prewMT8 RNA samples  were mixed with RNA Loading Dye, (2×) and heated to 95°C for 3 min followed by cooling on ice. The samples were separated on an 8% polyacrylamide gel (acrylamide: bis-acrylamide 19: 1) in Tris-borate-EDTA (TBE) and 8 M urea at 40 V/cm for 25 min. The gel was stained with SYBR Gold (Invitrogen) and imaged by LuminoGraph II (ATTO).2O, the samples were diluted with 1 × reaction buffer to 50, 100 or 200 ng in 4.6 μl. The samples were mixed with 4.6 μl of 2× native gel loading buffer , no sodium dodecyl sulphate (NEB) and 50% sucrose) and gently loaded onto the precooled gel. The RNAs were separated at 38 V/cm for 25 min at 4°C and was stained with SYBR Gold and imaged by LuminoGraph II.An electrophoresis cell, native 8% acrylamide gel (acrylamide: bis-acrylamide 19: 1), and 1 × TBE buffer were cooled at 4°C for at least 1 h before sample loading. wMT8 RNA was transcribed and column purified as described above. Two 9 μl aliquots of wMT8 RNA (430 ng) diluted in 1.11 × reaction buffer were prepared. One aliquot was heated to 75°C for 5 min and slowly cooled to 37°C using a thermal cycler while the other aliquot was kept on ice. After adding 1 μl HColumn purified metastable RNAs (0.5 μM) and DFHBI-1T (5 μM) were diluted in the reaction buffer. An aliquot of each solution was renatured by heating to 75°C for 5 min and slowly cooling to 37°C in a thermal cycler. RNA samples (10 μl) were transferred to a 386-well black microplate and fluorescence was measured by an Infinite M1000 PRO microplate reader (Tecan) with excitation at 469 nm, emission at 501 nm and 5 nm bandwidth. The samples were stored at 4°C for 17 h and measured similarly.Column purified metastable RNA (1.11 μM) was diluted in 1.11 × reaction buffer with DFHBI-1T (11.1 μM). Nine microliters of the RNA/DFHBI-1T mixture was mixed with 1 μl of a 10 × trigger RNA solution (0–1 μM) in a PCR tube. For the positive controls representing the 1 μM fully thermodynamically stable structures, the metastable RNA (1 μM) was mixed with 1.5 μM each of the stem III binding oligonucleotides  and renawMT8 DNA template solution , 2.5 × reaction buffer (4 μl), 25 mM (each) NTP mix (0.8 μl), 50 μM DFHBI-1T (2 μl), RNase Inhibitor, Murine (0.4 μl), and T7 RNA polymerase (0.5 μl) were mixed on ice . To this solution was added 1 μl of 500 nM c4+8 or c4+8sc solution. Water (1 μl) was added for the uncatalyzed reaction. Broccoli-DFHBI-1T fluorescence of these samples were monitored at 37°C using a real-time PCR instrument .We based our initial design Figure  on the bWe started with a design shown in Figure in vitro transcription from appropriate DNA templates using T7 RNA polymerase. The reactions were stopped after 3 h and treated with DNase I. The RNAs were then purified by silica columns and diluted to 500 nM in the reaction buffer supplemented with 5 μM DFHBI-1T. An aliquot from each sample was renatured by heat to convert the metastable RNA to the thermodynamically stable structure. The Broccoli fluorescence measurements of the renatured and untreated RNAs showed higher fluorescence in the renatured samples in all RNA designs  showed a major band of the expected size (129 nt) with few minor bands attributable to prematurely truncated products . The untHaving established that L+U, wMT8 and L+U-mm6 adopt robust metastable structures after transcription and that the thermodynamically stable structure activates the Broccoli aptamer, we set out to explore means other than heating/renaturation to trigger the structural transformation. An intriguing possibility was to use an RNA trigger to transiently destabilize a stem in the branched structure to create a toehold from which an intramolecular strand exchange reaction can be initiated. For example, the short trigger RNA (c4+10) shown in Figure First, the three robust metastable RNAs (1 μM) were mixed with DFHBI-1T (10 μM) and the trigger RNA c4+10 (100 nM) in the reaction buffer, and the Broccoli fluorescence was monitored over time at 37°C . InteresNtoe+Ninv where Ntoe is the number of bases that hybridize with the loop sequence (toehold), and Ninv is the number of bases that hybridize with (invade) the 3′ side of stem I. Fixing Ninv to 10 bases, the size of the toehold (Ntoe) was varied from 0 to 9. Metastable wMT8 (1 μM) was mixed with DFHBI-1T (10 μM) and a trigger RNA (100 nM), and the Broccoli fluorescence was monitored over time at 37°C  probably due to spontaneous ‘breathing’ of the stem near the loop region. While relaxation of the metastable wMT8 to the thermodynamically stable structure was mostly complete within 300 min (for Ntoe = 2–5), slower catalysis and lower turnover was observed for Ntoe = 8 and 9 likely due to slower release of the trigger RNA. Overall, toehold size between 3 and 5 showed faster initial rate and higher conversion among the tested trigger RNAs.Interestingly, slow catalysis was observed even without the toehold  and c4+8 (50 nM). The results  with a built-in energy source into single RNA molecules opens a new path for design of biomolecular machines. Future goals include designing self-powered RNA nanomachines with more than one metastable state, with multiple sensors, or with other computational or output functions.gkz364_Supplemental_FileClick here for additional data file.


However, fluxes of nutrients vary by orders of magnitude among oyster reefs and methods. This study compares two methods of measuring nutrient and metabolic fluxes on restored oyster reefs: incubations including intact segments of oyster reef and incubations containing oyster clumps without underlying sediments. Fluxes of oxygen (O2), dissolved inorganic carbon (DIC), ammonium (NH4+), combined nitrate and nitrite (NO2/3-), di-nitrogen (N2), and soluble reactive phosphorus (SRP) were determined in June and August in Harris Creek, a tributary of the Chesapeake Bay, Maryland, USA. Regression of fluxes measured from clumps alone against those measured from intact reef segments showed significant positive relationships for O2, DIC, NH4+, and SRP . Regression of clump fluxes against the oyster tissue biomass indicates significant positive relationships for O2 and NH4+, marginally significant and positive relationships for DIC and N2, and no significant relationship for NO2/3- or SRP. Although these results demonstrate that the incubation of oyster clumps without underlying sediments does not accurately represent biogeochemical fluxes measured from the whole oyster and sediment community, this work supports the need to understand the balance between the metabolism of oysters and local sediments to correctly estimate biogeochemical rates.Oyster reef restoration can significantly increase benthic denitrification rates. Methods applied to measure nutrient fluxes and denitrification from oyster reefs in previous studies include incubations of sediment cores collected adjacent to oyster clumps, benthic chambers filled with intact reef segments that have undergone  Crassostrea virginica have experienced substantial decline due to overfishing, disease, and habitat loss [In Chesapeake Bay, populations of the native oyster tat loss . Oyster tat loss , 3. Oysttat loss  where nitat loss , 6. Chrotat loss –9. Oystetat loss  is follotat loss .2/3-) to N2 gas. Although the remineralization of biodeposits can result in fluxes of NH4+ from reefs [4+ to NO3-. Under aerobic conditions, attenuation of NH4+ concentrations via nitrification is associated both with living oysters and oysters shell [2 of surface area per square meter of reef [Oysters and other reef organisms appear to create optimal conditions for N removal via denitrification. Oysters remove oxygen through respiration , re-oxidom reefs ,14, thisrs shell  and withrs shell . Given t of reef , it is lin situ experimental chambers that encompass intact reef segments [in situ equilibration of intact reef segments with ex situ incubation and measurement [Although observations show that denitrification associated with oysters effectively removes nitrogen –21, methsegments , and in surement . Many ofsurement , 29. Alt2/3-) to N2 gas by denitrifying bacteria [Much of the work on oyster-associated denitrification focuses on the filtration of organic matter from the water column and its deposition on sediments as feces and/or pseudofeces . Biodepbacteria , 30–32. bacteria , 29, 33.2- to N2 is not controlled by the abundance of complete denitrifiers in the sediment but rather by complex interactions within the microbial community [Contrary to studies that have focused on the impact of biodeposits on sediment denitrification, recent literature suggests live oysters are denitrification hot spots , 21, 24.2/3- and NH4+) measured from oysters alone may differ from whole reef incubations, due to the exposure of new surface area and absence of sediment microbial communities. This prediction was made following knowledge that denitrification was consistently higher from restored reef sites compared to control sediments [We combined field sampling and replicated laboratory flux chamber incubations with and without sediments to assess whether the denitrification and nutrient fluxes occurring on oyster reefs are associated with oyster clumps. We hypothesized that oysters alone would provide accurate estimates of denitrification compared to those measured from reef segments containing oysters and sediment, whereas oxygen demand and inorganic nitrogen dynamics , a tributary to the Choptank River, Maryland, USA  was chos-2 .in situ equilibration of intact segments of oyster reef (hereafter “reef segments”) with ex situ incubation approach [-2. After trays had equilibrated in the field for at least one month, divers capped the base trays with a pvc pipe midsection fitted with a lid and intact reef segments were returned to Horn Point Laboratory, Cambridge, MD where reef segments were placed in filtered Choptank River water with the temperature adjusted to Harris Creek conditions (23.3°C in June to 28°C in August). An empty tray filled with only filtered river water served as a “blank”. Prior to each set of incubations, trays were aerated for ~1 h to bring oxygen concentration to saturation. At the start of each incubation, chambers were capped with a stirring lid containing ports for sample collection, but sealed the sample from the surrounding water bath and stirred the water column with a motor-driven impeller [Experiments were conducted in early and late summer (June and August) 2017 using the approach . This woimpeller . As part2) was then used to calculate the oyster biomass density (g DW m-2).Once incubations were complete, samples were rinsed over a 12.5-mm mesh sieve and all material ≥12.5 mm was frozen for later analyses. For each sample, all live oysters were counted and measured to the nearest mm. Shell lengths were converted to grams of oyster tissue dry weight from seasonal length to biomass regressions based upon samples collected from Harris Creek, MD 2015 . The area of an individual tray  and dissolved gases  were collected approximately every 45 minutes  during each set of incubations from a sampling tube fitted in the lid, while a water replacement tube pulled water from the water bath. Sampling intervals were determined based upon changes in dissolved oxygen (FireSting O2-Mini oxygen meter), and incubations of reef segments and oyster clumps alone had the same incubation duration. Final oxygen concentrations for reef segments and oyster clumps ranged from 0.14 ± 0.06 mmol l-1 (mean ≥ SD) in June to 0.15 ± 0.04 mmol l-1 in August. Dissolved gases were preserved with 10 μl of 50% saturated HgCl2, tightly sealed, submerged in water, and held at or slightly below incubation temperature until analysis. Solute samples were filtered through a 0.45 μm pore-size filter and kept frozen for analysis. N2, O2, and Ar concentrations were measured on a membrane-inlet mass spectrometer [-1. Phenol/hypochlorite colorimetry was used to determine NH4+ concentrations [2/3- was analyzed spectrophotometrically following Doane and Howarth [2≥0.80 and the change in concentrations over the incubation were greater than the precision of the analyses [-2 hr-1), ΔC/Δt is the slope (mmol L-1 hr-1), V is the volume of overlying water (L), and A is the area of the incubated tray segment (m-2). The overlying water volume was determined by subtracting the volume of sediment measured in the tray and the displacement volume of the live oysters from the total incubation chamber volume.Solutes (NHtrometer  within 2trometer ) with a trations . NO2/3-  Howarth , while a Howarth . Concentanalyses . When it2≥0.80), have correlation coefficients that did not differ significantly from one, and have intercepts that did not differ significantly from zero. All statistical analyses were performed in R 3.1 using its “base” and “stats” packages [We used a series of criteria to determine whether clump fluxes were representative of reef segment fluxes at this specific site. If the clump fluxes accurately estimate reef segment biogeochemistry, we would expect the two approaches to be highly correlated  positive relationship. However, the regression coefficient from the comparison of both methods was significantly greater than the predicted slope of one  with clumps generally overestimating oxygen demand, especially at high flux rates. The relationship between clump oxygen demand and oyster clump biomass density was significant and positive  as oxygen-based respiration rates. The relationship between the two methods was not significantly different from the predicted slope of one  and the intercept was not significantly different from the predicted value of zero . DIC fluxes tended to increase with oyster biomass, but were only marginally significant . In addition, the regression coefficient from the measured relationship was not significantly different from the expected relationship  and the intercept was not significantly different from zero . However, NH4+ fluxes measured from oyster clumps consistently overestimated rates measured from reef segments. NH4+ fluxes from oyster clumps alone had a significant positive relationship with oyster clump biomass . For the five clump fluxes that were significant, regression analyses found no relationship  between NO2/3- fluxes from clumps versus those measured from reef segments  similar to those for DIC and NH4+ . Similar to denitrification, incubations of clumps alone overestimated SRP fluxes at the lower end and underestimated fluxes at the higher end of reef segments. SRP fluxes were not significantly correlated with oyster biomass  with a degree of correlation  variables, correlation coefficients that did not differ significantly from one, and intercepts that did not differ significantly from zero.Overall, fluxes of DIC, NH2 and DIC-based respiration rates, oyster clumps overestimated O2 fluxes at the higher end of reef segments rates. The increase in O2 demand was likely due to the reoxidation of reduced chemical species occurring on the newly exposed surface area on the bottom of the clumps, considering previous studies have observed the development of anoxic sediments and increases in reduced chemical species below oysters during warmer months [2 fluxes, DIC-based respiration from oysters alone were the only fluxes that met the criteria of having a slope that was not significantly different from one and an intercept not significantly different from zero, indicating that remineralization rates were minimally affected the by the removal and placement of clumps.Although both measurement approaches were correlated for Or months , 41. In 4+ fluxes demonstrated that incubation of clumps alone produces a consistent error in measurements across fluxes that results in an overestimate of reef segment fluxes. It was uncertain why we observed higher NH4+ fluxes when oysters were incubated alone  concentrations in adhered sediment are high [4+. We speculate that DNRA was stimulated when oysters clumps were incubated alone by concentrations of reduced sulfides that were exposed on the bottom of the oyster clumps.Regression of NHed alone  but thesare high –44. Anot2 and NO2/3- fluxes from clumps were not significantly correlated with fluxes from reef segments, N2 fluxes tended to increase with increasing reef segment rate while NO2/3- showed the opposite trend. As noted above, the increase in NH4+ fluxes and decrease in NO2/3- fluxes measured from oysters alone may have allowed denitrification rates from oysters alone to have a positive relationship with whole tray estimates. Alternatively, rates of nitrification could have been reduced in our oyster only incubation simply through loss of surface area. The bottom of the ex situ incubation trays contain a large amount of shell and shell hash with high surfaces which could support high rates of nitrification. Interestingly, the underlying shell and shell hash does not appear to support high mineralization rates given minimal loss in DIC flux when the oyster clumps are incubated separately.Even though N2:SRP or ΣN:SRP ratios suggest that remineralized P was retained.Regression of SRP fluxes from the two measurement approaches found that clump fluxes underestimated higher rates, suggesting strong binding of P to oyster shell. In addition, high CO2 and NH4+ showed a significant positive correlation with oyster biomass and fluxes of DIC and N2 showed marginally significant positive correlations. Similarly, work by Green et al. [Crassostrea gigas densities are positively correlated with CO2 and CH4 fluxes, while Smyth et al. [C. virginica) densities. Given the spatial limitations of this work, future research should assess whether the observed relationships hold up at other sites with different sediment characteristics and hydrological conditions. Although the results from this subset of work are only from one site and season, this research is in agreement with previous observations that oysters stimulate denitrification and other biogeochemical transformations in reef environments [Positive relationships between several fluxes and oyster tissue biomass within the clump samples provides further evidence that much of the biogeochemical activity in this system occurs within oyster clumps rather than in underlying sediments. Fluxes of both On et al.  has showh et al.  have repronments , 21, 24.In situ approaches for measuring oyster denitrification and other biogeochemical fluxes are logistically challenging [llenging , 28, butllenging ). Nitrifllenging , this woThe data herein point to an important consideration in the determination of whether oyster-associated N removal should be a BMP for overcoming eutrophication. Much of the data available for BMP consideration focuses on the role that oysters play in stimulating denitrification in the surrounding sediments, rather than within the reef and associated organisms. As demonstrated by the high rates of N transformations from clumps of oysters alone, the coupled processes occurring on oyster reefs are complex, and require an improved understanding of the environmental drivers stimulating oyster-associated denitrification to establish oysters as a BMP.


Sus scrofa RNA editing sites showed high enrichment in repetitive regions with a median editing level as 15.38%. Expectedly, 96.3% of the editing sites located in non-coding regions including intron, 3′ UTRs, intergenic, and gene proximal regions. There were 2233 editing sites located in the coding regions and 980 of them caused missense mutation. Our results indicated that to an A-to-I editing site, the adjacent four nucleotides, two before it and two after it, have a high impact on the editing occurrences. A commonly observed editing motif is CCAGG. We found that 4552 A-to-I RNA editing sites could disturb the original binding efficiencies of miRNAs and 4176 A-to-I RNA editing sites created new potential miRNA target sites. In addition, we performed edQTL analysis and found that 1134 edQTLs that significantly affected the editing levels of 137 RNA editing sites. Finally, we constructed PRESDB, the first pig RNA editing sites database. The site provides necessary functions associated with Sus scrofa RNA editing study.Recently, the prevalence and importance of RNA editing have been illuminated in mammals. However, studies on RNA editing of pigs, a widely used biomedical model animal, are rare. Here we collected RNA sequencing data across 11 tissues and identified more than 490,000 RNA editing sites. We annotated their biological features, detected flank sequence characteristics of A-to-I editing sites and the impact of A-to-I editing on miRNA–mRNA interactions, and identified RNA editing quantitative trait loci (edQTL).  RNA editing is a posttranscriptional regulatory RNA-processing event (excluding RNA splicing) that increases the diversity of transcriptome by changing nucleotides of RNAs. In mammals, the most common form of RNA editing is A-to-I, catalyzed by the adenosine deaminase that acts on RNA (ADAR) family enzymes which contain three members  . In miceA-to-I RNA editing sites in coding regions that cause synonymous or nonsynonymous mutations have been found within many RNAs, including glutamate receptor subunits ,8,9, theWith the RNA-Seq technology, RNA editing events have been identified in pigs ,23. A spSus scrofa RNA editing sites. The domestic pig (Sus scrofa) is a non-rodent animal model used in biomedical research [Sus scrofa. We collected 211 RNA-seq datasets across 11 tissues  from the National Center for Biotechnology Information (NCBI). Then, we identified Sus scrofa RNA editing sites and performed statistical analysis. Moreover, we performed molecular properties analyses, including genomic feature annotations and flank sequence characteristics investigation, and potential functional effects, including the impact of A-to-I editing on miRNA–mRNA interactions and RNA editing quantitative trait loci (edQTL) identification.Although an increasing number of mammal RNA editing databases have been published ,27,28,29research ,31,32. Iresearch ,34,35. TSus scrofa (Sscrofa11.1) were downloaded from the ENSEMBL database [The RNA-seq data were downloaded from the National Center for Biotechnology Information (NCBI) SRA database. The sequence read archive (SRA) ID number is available in database . The readatabase  with defdatabase  with defdatabase  with defTo improve the accuracy of identification of tissue-specific editing sites, for each tissue, we removed the sites that were missing editing value measurements in more than one-third of the samples. The ROKU R package  was applp < 0.01, t-test). Adenosine sites randomly chosen from reference were used as the negative control.Two Sample Logo  were usehttps://genome.ucsc.edu/cgi-bin/hgLiftOver) to convert the genome position from human reference to pig reference. We also converted the genome position from pig reference to human reference. The RNA editing sites successfully converted on both turns (from the human to the pig and from the pig to the human) were reserved as conserved RNA editing sites. Pig quantitative trait locus (QTL) was downloaded from Pig QTLdb [Human RNA editing sites were download from the RADAR database , then weig QTLdb  and RNA For each A-to-G editing site located at 3′UTR, we extracted the editing site and the flanking 25 bps from reference  as an unedited-type (UT) sequence and changed the editing site from A-to-G (or T-to-C) as an edited-type (ET) sequence. We then used RNAhybrid  and miRa−7 (Bonferroni method).Variant calling using Samtools (v1.2) was performed and we selected variant positions from each sample in which the mismatches were supported by at least two reads while at least 10 reads covering this site and both base and mapping quality scores were at least 20. For QTL mapping, we examined 630 RNA editing sites with editing level measured in at least one-third of the samples. For each of the 630 RNA editing sites we normalized the editing level using Z-score method across all samples. The following protocol was performed to map edQTLs genome-wide: (1) For each editing site, we fit linear models without any covariates between normalized editing levels and genotypes of each variant in the genome using Plink . We onlyhttps://www.djangoproject.com) coupled with MySQL database. The front-end interface was developed based on Bootstrap open source toolkit (https://getbootstrap.com) and jQuery JavaScript library (https://jquery.com). PRESDB was published using Apache Http server and is accessible at https://presdb.deepomics.org.PRESDB was built using Django Python Web framework . We performed reads mapping and RNA-editing sites identification with Histat2 and SPRINT (see Methods). The genome-wide screening of RNA editing yielded a total of over 490,000 RNA editing events in all eleven tissues. The percentage of A-to-G mismatch, which is consistent with A-to-I editing, was 94.6% in repetitive region and 77.4% in non-repetitive  and G enrichment one nucleotide downstream (+1) RNA editing sites located in the repetitive region a,c. MeanSimilar to Single Nucleotide Polymorphism sites (SNPs), editing sites also have the potential to influence the miRNA–mRNA interactions. In this work, we selected all A-to-G editing sites  that located at 3′UTR region and predicted miRNAs that can target on these editing sites using RNAhybrid and miRanda. After that, we identified editing sites potentially to disturb original (loss) miRNA target sites and/or to create new potential (gain) miRNA target sites (see Methods). The results showed that 4552 RNA editing sites disturbed 5189 miRNAs target sites and 4,176 RNA editing sites created 5729 new potential miRNA target sites. Together, we found 1525 mRNA were impacted, and gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that biological function of these genes was enriched in metabolic pathways including small molecule metabolic process, organic acid metabolic process and cellular metabolic process a,b. SincQuantitative trait loci (QTL) mapping has been successfully applied to identify the regulatory mechanisms of many molecular phenotypes such as gene expression (eQTLs)  and spliIn order to provide users an easy access to pig RNA editing data, we built a database of pig RNA editing sites (PRESDB), containing more than 490,000 RNA editing sites across 11 tissues. PRESDB has four functional pages: An RNA editing site search page, a flanking sequence extraction page, a miRNA–mRNA interaction page, and an RNA editing QTL (edQTL) page . The seaSus scrofa RNA editing sites and explore their patterns, we collected RNA sequencing data across 11 tissues . We identified more than 490,000 Sus scrofa RNA editing sites, which represent a significant addition to the growing catalog of annotated mammalian RNA editing sites. Moreover, similar to results that have been reported in human and mouse RNA [Sus scrofa RNA editing sites showed high enrichment in repeated regions and low editing level. Our data indicated that the pig transcriptomes are highly editable among PRE-1 SINE retrotransposons which have similar features to the primate Alu. Since Alu elements enable substantial RNA editing among primate genomes, RNA editing in pigs may be achieved through a similar mechanism.Recently, an increasing number of A-to-I RNA editing sites have been published in mice and human. As RNA editing sites appeared in a tissue-specific manner, it is essential that studies of RNA editing in mammals assess various tissues. The pig is not only an important farm animal that provides protein for humans but an important non-rodent animal model that is widely used in biomedical research. Thus, to perform genome-wide identification of ouse RNA , Sus scrConsistent with previous studies, our data showed that the majority of RNA editing sites are located in non-coding regions, including intron, intergenic, and gene proximal regions, while little is known about the mechanism of the average effect of RNA editing in these non-coding regions. An instance in human RNA demonstrated that intronic A-to-G editing events contribute to alternative splicing of nuclear prelamin A. Thus, these editing sites are useful to understand the extent to which this phenomenon enhances pig genetic variation. Furthermore, we also identified 980 RNA editing sites that could result in missense mutation in 560 genes. Among these 560 genes, GRIA2 and GRIK2, both found to be edited in brain tissue, were previously identified as genes edited during porcine neural tissue development. ACOX1, CPT1B, CPT2, CYP27A1, and ACSL1, etc. participate in Peroxisome proliferator-activated receptor (PPAR) signaling pathway which plays a critical physiological role in the regulation of numerous biological processes, including lipid and glucose metabolism, and overall energy homeostasis. Therefore, these missense mutations may play important roles in the development and energy metabolism of the pig.Sus scrofa RNA editing sites affect miRNA and mRNA interactions, we predicted miRNA-originating targets and miRNA-edited targets by two computational methods. Moreover, we found editing sites with the potential to disturb original miRNA target sites and to create new potential miRNA target sites. The function of these target genes was enriched in metabolic pathways including a small molecule metabolic process and an organic acid metabolic process, indicating the importance of RNA editing events in metabolic processes. Moreover, as most RNA editing sites reside in intergenic and intron regions, RNA editing sites are considered to have impacts on lncRNA secondary structures. Because of the incomplete annotation of lncRNA in the Sus scrofa reference, we did not explore the effects of RNA editing sites on lncRNA secondary structure, which can be done in a future study. Although the molecular functions of RNA editing sites are mostly unclear, functional consequences of A-to-I RNA editing sites in coding and non-coding regions have been found, including synonymous or nonsynonymous within many RNAs, miRNA biogenesis, and editing of miRNA seed regions. To assess whether Sus scrofa, we tested the correlations between RNA editing sites and variants within 200 kb of the RNA editing site. We observed that variants within 1 kb of editing sites were more likely to have significant associations. Therefore, in a future study, these edQTLs can be used to bridge the gap in our understanding between RNA editing level and their respective susceptibility loci. Although edQTLs have been proved to affect editing levels through changes in the local secondary structure for edited dsRNA in human beings, the mechanism by which edQTLs affect editing levels in the pig remain to be studied.In order to investigate the cis variation of RNA editing in Sus scrofa. Exploring the pig genome can illuminate facets of human culture, organic evolution, biomedical research, and animal breeding. Therefore, we constructed the database of Sus scrofa RNA editing sites based on our data. To the best of our knowledge, this database is the first public resource containing information about the RNA editing sites of Sus scrofa. Although these identified editing sites in our database, especially in the coding regions, remain to be experimentally validated, our database will enhance pig genetic variation and provide explanations for a variety of traits of interest to both biomedical and agricultural communities.At present, RNA editing sites have been discovered in plants, animals, and humans, but are rare in


Lys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNALys3 were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2′-O-methylthymidine (m5Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2’O, as shown by the fact that neither Um54 nor m5U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2′-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNA A limited set of germline-encoded pattern recognition receptors is capable of detecting the broad variety of invading pathogens due to the recognition of highly conserved pathogen-associated molecular patterns (PAMPs). Besides cell wall components of bacteria, nucleic acids serve as an important PAMP of viruses as well as of bacteria to activate the innate immune system within minutes. DNA and RNA can either be recognized by endosomal Toll-like receptors (TLR) or cytosolic receptors including retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and activation of the cGAS –STING axis. Endosomal TLRs like TLR3 and TLR7 are known to sense double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA), respectively . Upon sta priori single-stranded. Yet, it is still unclear if recognition of Gm18 by TLR7 is based on an intact tRNA which would imply tRNA unfolding and presumably base-flipping or Gm18 might exhibit its antagonism only after degradation.Previous studies described activation of TLR7 by tRNAs although those RNAs are intrinsically folded and therefore show double-stranded structures. For example, the D-loop and T-loop of canonical tRNAs form very stable tertiary interactions which are reinforced by post-transcriptional modification of key nucleotides . Interesvia the continuous chain of a bound RNA  and a tri- or dinucleotide at binding site 2 (second) ,13. So found RNA  in turn ound RNA ,18 at a ound RNA , which iound RNA . The actound RNA  and highin vitro transcribed RNA reduced immune-stimulation. Yet, those approaches were done in artificial RNA and considered neither frequency nor position of endogenous, native RNA modifications  or Buffer C  and 0.8 μg Colicin D  for 1 hHybridization buffer  and an equimolar amount of complementary DNA oligonucleotide (listed below) were added to the fragment mixture. The solution was then incubated for 20 min at room temperature and after that, the DNA/RNA hybrid, as well as the remaining unhybridized RNA fragment were separated via 10% native polyacrylamide gel electrophoresis (PAGE). Bands containing hybrid and fragment were cut out of the gel and agitated in 0.3 M NaOAc overnight at room temperature. In the next step, the RNA was precipitated after addition of two volumes absolute EtOH and glycogen. Followed by resuspension in RNase-free water.via phenol-chloroform extraction and EtOH precipitation with 0.3 M NaOAc. The concentration of the received fragment in RNase-free water was then checked by UV absorbance.To obtain the purified, dephosphorylated native fragment, the DNA/RNA hybrid solution was incubated with 0.1 U/μl FastAP (Thermo Scientific) and 1.25 U/μl DNaseI (Thermo Scientific) for 1 h at 37°C. In the next step, the RNA was isolated 2), 5 mM DTT, 5 mM adenosine triphosphate (ATP) and 0.75 U/μl T4-Polynucleotide kinase (Thermo Scientific) for 1 h at 37°C. The reaction mixture was used directly for the subsequent splint ligation.To remove remaining 2′3′-cyclic phosphates and to get ligation-competent RNA strands (5′ phosphate necessary), 200 pmoles of the purified native fragments or unmodified commercial RNAs were treated with KL-Buffer  or an equimolar amount (Tm modivariant synthesis) of the remaining tRNA parts and an equimolar amount of DNA splint in KL-Buffer. After that, the resulting solution was heated for 4 min to 75°C and then slowly cooled down to room temperature for 15 min. In the next step, 1.5 U/μl T4 DNA-Ligase (Thermo Scientific) and 70 ng/μl RNA Ligase 2 (e.g. Thermo Scientific) were added and the mixture was incubated for 24 h at room temperature. At last, the DNA splint was digested  and the ligation product was purified using 10% denaturing PAGE . The concentration was again checked by UV absorbance.Unmodified RNAs (see below) were ligated to the corresponding native fragment (Colicin D treatment) or the unmodified 5′ tRNA fragment (Tm modivariant synthesis) to receive a complete tRNAvia thin layer chromatography (mobile phase: IPA:conc. NH3·H2O 1:1). In the next step, the bisphosphate was eluted in 0.5 M NH4OAc and used as a minimal substrate  for 30 min at 37°C. Conversion of the bisphosphate to the 5′ monophosphate indicated the presence of pTmp (verified by UV shadowing).To assure the production of the bisphosphate, an aliquot of pTmp (50 pmoles) was treated with 0.3 U Nuclease P1 (Sigma Aldrich) in 20 μl reaction solution  were incubated together with a 10× molar excess of pTmp and 1.6 U/μl RNA Ligase 1 (Thermo Scientific) in the corresponding buffer overnight at 37°C. After that, 1 U FastAP and FAP Buffer were added and the resulting solution (volume: 40 μl) was treated for 1 h at 37°C. At last, the resulting MH847pTm RNA strand was PAGE purified (see above).Ligation-competent MH847pTm, as well as MH848, were obtained by 5′ phosphorylation using T4-Polynucleotide kinase (see phosphorylation/dephosphorylation of RNA). The received phosphorylated RNA strands were used without further purification for the Tm modivariant synthesis (see Splint ligations using tRNA subfragments).Permutated modivariants were synthesized by using the RNA strands listed below. Here, RNA subfragments were 5′ phosphorylated and then subjected to splint ligations as previously described.Lys3 were mixed with bacterial RNA prior encapsulation with DOTAP. As a positive control, PBMCs were stimulated with bacterial RNA (see below) and TLR7/8-agonist R848 (1 μg/ml) . All stimulations were performed in duplicate wells per individual donor at a density of 2 × 105 cells/well PBMCs in a 96-well flat bottom plate. Cells were incubated in a humidified 5% CO2 atmosphere at 37°C for 20 h. Cell-free supernatants were analyzed by sandwich ELISA for secretion of IFN-α  according to the manufacturer’s protocol. Where indicated, values were normalized to cytokine production induced by stimulation with bacterial RNA to account for donor variation.Human PBMCs were isolated from heparinized blood of healthy donors upon informed consent and approval by the local ethics committee by standard Ficoll-Hypaque density gradient centrifugation (Ficoll 1.078 g/ml). PBMCs were resuspended in complete medium prepared of RPMI 1640  supplemented with 2% heat-inactivated human serum . For stimulation experiments, RNA was encapsulated with DOTAP propyl]-N, N, N-trimethylammonium-205 methylsulfate)  at a ratio of 3 μl DOTAP per 1 μg of RNA in Opti-MEM reduced serum medium (Life Technologies) and incubation for 10 min at room temperature. DOTAP encapsulation is necessary to deliver RNA into PBMCs. For transfection experiments, cells were stimulated with RNA at final concentrations of 500, 250 and 125 ng/ml. Where indicated, methylated or unmethylated oligoribonucleotides (ORNs) and native tRNAStaphylococcus aureus ATCC 25923 was grown in Luria-Bertani (LB) Medium  at 37°C until mid-log phase. Bacteria were harvested and treated with lysozyme  before RNA isolation using TRIzol reagent  according to the manufacturer’s protocol. RNA underwent further purification using RNeasy mini kit  including an on-column DNase treatment. The purity and amount of bacterial RNA preparations were validated by NanoDrop measurement. Only RNA preparations with ratios 260/280 and 260/230 > 1.8 were used for stimulation.P-values are indicated by ns , * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). The dose-response model was calculated by R software 3.4.1 with best fitting model for the corresponding RNA. Unmodified GG ORN: log-logistic function (three parameters), native Lys3: Weibull function (two to four parameters), modified GmG ORN: log-logistic function (two parameters).Data were analyzed using GraphPad Prism 6.05 (GraphPad Software Inc.). Mean plus SEM were plotted and significant differences were assessed by two-way ANOVA including multiple comparison tests or linear regression model. Tyr  and the small molecule agonist R848 served as positive controls for TLR7 activation and non-treated PBMCs as negative control to exclude auto-activation of pDCs.As an in-depth follow-up analysis of our initial work on bacterial tRNATyr  a side-bThe IFN-α emission from PBMCs is a commonly used readout for TLR7 activation, which is validated by a chain of evidence, including the following elements. Depletion of pDCs from PBMCs results in complete loss of IFN-α secretion from the latter , and isoS. aureus. A synthetic unmodified tRNALys3 showed nearly similar efficiency, whereas the fully modified (native) tRNALys3 induced only minor IFN-α levels  was analyzed: whereas pure bRNA induced IFN-α secretion, the addition of the GmG ORN containing a single modified Gm led to complete suppression of TLR7 activation even at moderate concentrations  effect.Importantly, the addition of tRNAs Figure  but not A Figure . These fLys3, we synthesized tRNALys3 -modivariants using a molecular surgery procedure  displayed clearly reduced immunostimulatory activity. Taken together, these results indicate that a single modified residue or the combination of multiple modifications in the 3′ part of native tRNALys3 caused decreased immunostimulation.As depicted in Figure Lys3, which harbors the following RNA modifications .Based on this latter observation, we further inspected the underlying sequence context of the 3′ part of tRNA sensors ,37,38. B5U) modification, also featured a 2′-O-methylated ribose moiety. The latter also occurs in Gm, which had been described as an inhibitory modification in recent studies  without ribose methylation did only slightly affect stimulation, similar to Um Figure . While sLys3 can only partially explain the observed low stimulatory potential on PBMC. Indeed, our results also imply a contribution of the remainder of the modifications shown in Figure As a final observation, this remarkable synergistic effect of Tm within tRNALys3, the object of the present work. Given that eukaryotic tRNAs may contain up to 25% modified nucleotides , suggesting that in vitro conditions for controlled cleavage of for any tRNA shaped molecule can be developed.As indicated by our recent studies , ‘self’ leotides , the neeleotides . Howeverleotides , as a neColicin D-based molecular surgery gave access to two modivariants whose testing identified a major effect caused by the modifications situated in the 3′-part. Among those modifications present, several occur in bacterial and other microbial tRNAs that had previously been found to be stimulatory. These modifications were considered as highly unlikely candidates for the observed effect.1A58 and Tm54. Given the previous findings on ribose-methylated modifications, we turned our attention to the synthesis of a modivariant containing Tm54 as sole modified nucleotide. In this context, Tm decreased the interferon response by 50%. While this is a pronounced effect, it is considerably weaker than what was typically observed for Gm. Of note, since the fully modified tRNALys3 evokes almost no response, it is clear that the remainder of modifications, i.e. the ensemble except Tm, also has a contributing effect. Further studies may identify additional contributions of single modifications, but we would like to point out some of our results with synthetic modifications in this context. We recently reported a generic ‘shielding’ effect of a number of synthetic RNA modifications with no discernible common denominator in terms of chemical structure (Lys3 would be shielded by two bulky modifications (ms2t6A and mcm5s2U). Furthermore, modifications in the anticodon stem as well as in the D-loop are so numerous that they approach a density previously investigated in in vitro transcripts, which contained high levels of, e.g. Ψ or m5C (The remainder consist of mtructure . The conΨ or m5C ,37,38. WLys3. Contrary to Gm18, tRNALys3 reveals no dominant inhibitory effect when delivered in trans with a stimulatory RNA. In fact, our findings imply some kind of ‘RNA muting’ and the data indicate that some 50% of this ‘muting’ is due to the presence of Tm. As already discussed, this implies that other modifications within the RNA are also necessary for the full effect of tRNALys3. Of note, Tm therefore classifies differently from previously identified immune-modulatory Gm and Am modifications, the latter also suppressing in trans. In a previous study, we identified a minimal tri-nucleotide motif within RNA which is necessary and sufficient to antagonize TLR7 and TLR8 (Lys3, Tm is followed by pseudouridine whereas in the described modivariants Xm was followed by a uridine. The effect of Tm might potentially be modulated by synergistic effects with further modifications in the defined sequence context of the native tRNA.Our results indicate that Tm at position 54 is responsible for much of the interesting effect observed in tRNAand TLR8 . A key fTLR7 reportedly responds strongly to single-stranded RNA rich in guanosine and uridine residues . SupportPure biochemical data of the type presented here cannot unambiguously determine the number and structure of effective binding sites. However, we have added an interesting piece of data by comparing ribose-modified nucleosides Cm, Um, Tm, Am and Gm at the precisely same position of a tRNA scaffold (n.b.: the effects of Am and Gm do not differ significantly in Figure Phe and mammalian tRNALys3 had previously been subject to structural comparison with their fully modified counterparts (Tyr, and similarly, low TLR7 response for Tm containing fragments compared to unmodified fragments. These observations argue for the overall tRNA structure to be irrelevant in TLR/ recognition. Indeed, the concept that TLR7 inspects small unstructured stretches of RNA is supported by TLR7 inhibtion of Gm-containing RNA oligomers as short as 9 nt (Several lines of evidence disencourage the notion, that unmodifed tRNAs as used in this study, are recognized because of their failure to properly fold into the typical threedimensional L-shape of tRNAs. In particular, unmodified yeast tRNAterparts . Based o as 9 nt .Lys3 (Figure 5s2U34 is involved in the annealing process of the tRNA as primer for HIV reverse transcriptase (1A58 causes an arrest of reverse transcription that is mandatory for HIV replication (Lys3 is selectively packaged into the HIV-virion (From the biological point of view, it might be possible that Tm serves as one of several modifications that differentiate between foreign- and self-RNAs. Whereas ample literature is available concerning the T54 modification ,48, litt3 Figure  is involcriptase ,50, and lication . Given tV-virion ,53, the E. coli, yeast and mammalian mitochondria (Part of our future research will be aimed at the contribution to immunoevasion of modifications other than Tm. We note that, while tRNA modifications in chondria ,55 have Supplementary DataClick here for additional data file.


Mutagenesis studies revealed that the stemloop II (SLII) at the 3’ UTR is required for the accumulation of sfRNA. According to the results of an in vitro RNA-dependent RNA polymerase (RdRp) assay, the (-)10431-10566 RNA fragment, containing the putative promoter on the antigenome for the sfRNA transcription, binds to RdRp protein and exhibits a strong promoter activity. Taken together, our results indicate that the JEV sfRNA could be transcribed initially and then be trimmed by XRN1 or other unidentified exoribonucleases.Flaviviruses accumulate abundant subgenomic RNA (sfRNA) in infected cells. It has been reported that sfRNA results from stalling of host 5’-to-3’ exoribonuclease XRN1 at the highly structured RNA of the 3’ untranslated region (UTR). Although XRN1 digestion of a 3’-terminal 800-nt RNA could stall at a position to generate the sfRNA  Flaviviridae and is a mosquito-borne zoonotic pathogen that causes human encephalitis and meningitis. The 10,976 nucleotides (nts) genome contains a single open reading frame (ORF) encoding a polyprotein which is subsequently processed into three viral structural proteins and seven nonstructural proteins [cis-acting elements involved in the regulation of viral translation, replication, and pathogenesis [Japanese encephalitis virus (JEV) belongs to the family ogenesis –5.During virus infection, all arthropod-flaviviruses generate abundant amounts of a noncoding subgenomic RNA (sfRNA) representing the 3’-terminal highly conserved region of the 3’ UTR –9. The sin vitro, and there were no intermediate byproducts during the exoribonuclease degradation process of the viral genome to the sfRNA in the virus-infected cells, which indicated that the degradation process is extremely efficient or that another biogenesis mechanism might be involved. The reason for these findings is not clear because degradation from the full-length genome seems uneconomic for viruses from an evolutionary point of view. Furthermore, being infected with RNA viruses induces double membrane vesicles to form replication/transcription complexes that are protease- and nuclease-resistant [How the sfRNA is made in the infected cells is intriguing. Several studies have shown that the sfRNA is a product of the incomplete degradation of the viral genome by cellular 5’-to-3’ exoribonuclease XRN1; a pseudoknot or a three-helix junction structure in the 3’ UTR is responsible for stalling XRN1 degradation , 23–26. esistant –29. The in vitro RdRp assay gave rise to synthetic products, suggesting that the JEV sfRNA could be initially transcribed from the antigenome and may be further trimmed by XRN1 or other unidentified exoribonucleases.In this study, we found that either knocking down or knocking out of exoribonuclease XRN1 had no effect on the accumulation of sfRNA in JEV-infected cells. In contrast, mutation of GDD motif in RdRp and disruption of the SLII structure halted accumulation of the sfRNA. Furthermore, the minus-strand templates covering the putative promoter region used for an BHK-21 and A549 cells were grown at 37°C in RPMI 1640 medium supplemented with 2% and 10% fetal bovine serum , respectively. A549 XRN1 knock-out cells were kindly provided by Dr. Bernard Moss  and were propagated as described previously . Human eDetailed methods for the construction of clones are described in the Supporting Information 296739) targeting human XRN1 gene (GenBank accession no. NM_019001.3) or a negative control shRNA targeting GFP (shGFP) were obtained from the Taiwan National RNAi Core Facility. To knockdown XRN1 expression, 5 × 105 HEK293T and A549 cells were transduced with the shXRN1 or shGFP lentiviruses, respectively, and selected with puromycin (1 μg/ml). At one-week post-transduction, cells were seeded on 60-mm plates (approximately 2 × 106 cells per plate) and infected with JEV RP9 (or DENV-2 as a control) at an MOI of 5 (for HEK293T) or 10 (for A549). The cells were harvested with PBS at 48 h post-infection, and the precipitates were then divided into two parts. One part was analyzed for Western blotting and the other part was used for Northern analysis. Equivalent amounts of proteins (20 μg/sample) were resolved by 8% SDS-PAGE, followed by Western blotting using anti-XRN1  or anti-β actin  antibodies. RNA extraction and Northern analyses were conducted as described previously [in vitro transcription  and then visualized using luminescent image analyzer , as described in a previous study [The lentiviral vectors expressing the short hairpin RNA (shRNA) TRCN-26739 targeviously . In someus study . The relSal I and transcribed in vitro using T7 RNA polymerase (Promega) according to the manufacturer’s protocol. DNA template used for run-off transcription to generate 3’-terminal 800 nts of DENV was amplified from pTight-DENV plasmid [Plasmid pGEMT-JEV-800 was linearized with  plasmid  using ol4 cells/well) in 12-well plates were transfected with 0.25 μg of pTight-JEV (or mutant) plasmid, 0.25 μg of pTET-OFF (BD Bioscience), and 1 μl of Lipofectamine 2000 (Invitrogen) in 200 μl of Opti-MEM medium per well at 37°C for 4 hours, and the cells were refed with fresh medium. Total RNAs were extracted at 5 days post-transfection (dpt), and the supernatant fluids were collected for measuring the virus titers. The transfected cells were passaged every 5 days if no apparent cytopathic effect (CPE) was observed. RNAs were extracted at the time when the cells showed a severe CPE.The procedure for DNA-based infectious clone transfection has been described previously . BrieflyThe titration of infectious virus was performed as previously described . Brieflyin vitro transcription with T7 RNA polymerase (Promega) and DIG-UTPs . The DIG-labeled RNAs (0.25 pmol) were denatured at 80°C for 10 min and then quick cooling on ice. Binding reactions were set at room temperature in binding buffer  with purified recombinant RdRp protein or bovine serum albumin (BSA) at different concentrations  in a volume of 20 μl for 30 min, followed by adding 2 μl heparin (25 mg/ml) for 10 min. The RNA-protein complexes were resolved by non-denaturing 5% polyacrylamide gel at 4°C, transferred to a positively charged nylon membrane (Amersham), and then cross-linked using UV Stratalinker (Stratagene). Detection of DIG-labeled RNA was performed using DIG Luminescent Detection Kit (Roche) according to the manufacturer’s instructions. To demonstrate the specificity of RNA-protein complex formation, competition assays were performed using various amounts of unlabeled RNAs added to the binding reaction.Riboprobe was made by E. coli Rosetta (Novagen). Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM and induced culture was grown at 22°C for 15 hours. Expressed RdRp was purified by metal affinity chromatography on a nickel-chelate column according to the manufacturer’s instructions (Invitrogen). The RNA templates used for the RdRp assay and the RNA size markers were synthesized by in vitro transcription (Promega) in the absence or presence of α-32P-CTP, respectively. The in vitro RdRp assay was performed with 500 ng of the recombinant RdRp protein and 200 ng of RNA templates or as indicated in a total volume of 25 μl of the reaction buffer containing 35 mM Tris-HCl (pH 7.9), 50 mM NaCl, 25 mM potassium glutamate, 5 mM MgCl2, 1 mM DTT, 5 mM MnCl2, 10% glycerol, 20 units of RNase inhibitor (Promega), 5 mM (each) ATP, UTP, and GTP, 0.5 mM CTP, and 10 mCi of α-32P-CTP . The reaction mixture was incubated at 30°C for 2 hours. RdRp reaction product was denatured in 2X loading buffer  and resolved on 6% polyacrylamide gel containing 6M urea. The gel was dried and visualized by autoradiography.A recombinant JEV RdRp was expressed in in vivo.To investigate whether the JEV sfRNA is an exoribonuclease XRN1 cleavage product, we analyzed the effect of XRN1 depletion caused by RNA interference on sfRNA generation in virus-infected cells. Knocking-down (KD) the XRN1 expression by up to 95% in HEK293T cells continued to increase the abundance of sfRNA throughout the 48-h infection period. More sfRNA (121%) was observed in XRN1 knocked-down cells than in the wild-type (WT) cells . It shouin vitro, a 3’-terminal 800-nt monophosphate RNA derived from genome of JEV or DENV was incubated with 0.01, 0.1, or 1 unit of XRN1, respectively, and analyzed by Northern blot analysis for sfRNA production. The DENV sfRNA is readily formed when treated with XRN1 at concentration as low as 0.01 units  for the production of sfRNA. To investigate the structure requirements for the production of JEV sfRNA e in the . The stre in the . We thene in the , lane 3,e in the .CAGCAUAUUGA) at 10863–10873 that is complementary to 5’CS (UCAAUAUGCUG) at nt 136–146, we found another motif located at the initiation site of JEV sfRNA that is very similar to the 3’CYC motif (named CYC-like motif), the sequence of which is UGCAUAUGGACA, where the mismatches with the 3’CYC motif are underlined). However, deletion of the CYC-like motif had no effect on the synthesis of the sfRNA and the genome , was compared to the wild-type (WT) infectious clone. The sfRNA in WT DNA-transfected cells were visible at 24 hpt and continued to increase to a very high level at 72 hpt  retarded the migration of the (-)10431-10566 RNA in a dose-dependent manner, whereas no band shift was observed with non-specific RNA or BSA control . It shouin vitro RdRp assay. The result showed that the (+)1-160 RNA is a functional template for RNA synthesis whereas the 5’ 226-nt RNA from bovine coronavirus (as a nonspecific template control) could not direct RNA synthesis, nor the reaction without adding the RdRp 10454-10566 RNA  by XRN1 revealed that XRN1 could stall at a position to generate the sfRNA  , 25. Preet al. [in vivo . A promet al. . However[in vivo , lane 6.Although viruses in the same family share many common strategies for completion of their life cycles, some differences in those strategies make certain viruses unique within the given family. In the present study, we provide evidence that the JEV sfRNA is likely synthesized by transcription and could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes . The SLIS1 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


The strength of SOX binding dictates cleavage efficiency, providing an explanation for the breadth of mRNA susceptibility observed in cells. Importantly, we establish that cleavage site specificity does not require additional cellular cofactors, as had been previously proposed. Thus, viral endonucleases may use a combination of RNA sequence and structure to capture a broad set of mRNA targets while still preserving selectivity.A number of viruses remodel the cellular gene expression landscape by globally accelerating messenger RNA (mRNA) degradation. Unlike the mammalian basal mRNA decay enzymes, which largely target mRNA from the 5′ and 3′ end, viruses instead use endonucleases that cleave their targets internally. This is hypothesized to more rapidly inactivate mRNA while maintaining selective power, potentially though the use of a targeting motif(s). Yet, how mRNA endonuclease specificity is achieved in mammalian cells remains largely unresolved. Here, we reveal key features underlying the biochemical mechanism of target recognition and cleavage by the SOX endonuclease encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). Using purified KSHV SOX protein, we reconstituted the cleavage reaction  Viral infection dramatically reshapes the gene expression landscape of the host cell. By changing overall messenger RNA (mRNA) abundance or translation, viruses can redirect host machinery towards viral gene expression while simultaneously dampening immune stimulatory signals . SuppresVirally encoded host shutoff endonucleases are usually specific for mRNA, yet broad-acting in that they target the majority of the mRNA population. This is exemplified by herpesviral nucleases, including the SOX endonuclease encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic human gammaherpesvirus that causes Kaposi's sarcoma and B cell lymphoproliferative diseases ,7. KSHV Sequencing data indicate that within the mRNA pool there appears to be a range of SOX targeting efficiencies; some transcripts are efficiently cleaved in cells, while others are partially or fully refractory to cleavage . Additioin vitro using purified components. Using an RNA substrate that is efficiently cleaved by SOX in cells, we revealed that specific RNA sequences within and outside of the cleavage site significantly contribute to SOX binding efficiency and target processing. In particular, we found that the polyadenosine stretch adjacent to the cleavage site is critical for SOX binding, and we experimentally verified the importance of an open loop structure surrounding the cleavage site. Finally, we demonstrated that this in vitro system faithfully recapitulates the initial endonucleolytic cleavage event that is an essential component of mRNA target specificity in vivo. Collectively, our data reveal that specific sequence features potently impact SOX binding, and thus provide key insight into the breadth of SOX targeting efficiency observed across the transcriptome. More broadly, this information provides a framework for better understanding the target specificity of endonucleases, which play central roles in mammalian quality control processes and viral infection outcomes.Thus, a major outstanding question is how RNA sequence and/or structure contribute to SOX target recognition. In this context, it is unclear how sequence features surrounding the RNA cleavage site might impact SOX targeting, for example by changing its affinity for a given RNA or the efficiency with which cleavage occurs. To address these questions, we sought to reconstitute the SOX cleavage reaction ® Baculovirus Expression System (Thermo Fisher Scientific) manual. After transfection, Sf9 cells (Thermo Fisher Scientific) were grown for 96 h at 22°C using SF-900 SMF media (Gibco) substituted with 5% fetal bovine serum (FBS) and 1% antibiotic antimycotic (AA). Supernatant was transferred to a six-well tissue culture plate containing 1 ml of 2 × 10∧6 cells/well. Cells were incubated for 96hr to generate passage 1 (P1). The P1 supernatant was transferred to a flask containing 50 ml of 1 × 106 cells/ml and incubated for 96 h, a time point sufficient to yield 5 mg of SOX per 50 ml of cells. Protein expression was confirmed by western blot with an anti-GST antibody .KSHV SOX was codon optimized for Sf9 expression and synthesized from GENEWIZ. SOX was then subcloned using restriction sites BamHI and SalI (New England BioLabs) into pFastBac HTD. This vector was modified to carry a GST affinity tag and PreScission protease cut site as described . All SOXSf9 cell pellets were suspended in lysis buffer containing 600 mM NaCl, 5% glycerol, 0.5% Triton X-100, 3 mM DTT, 20 mM HEPES pH 7.0 with a cOmplete, EDTA-Free protease inhibitor Cocktail tablet (Roche). Cells were sonicated on ice using a macro trip for 30 s bursts with 1 min rests for 6 min at 50 A. Cell lysate was cleared using a pre-chilled (4°C) Sorvall LYNX 6000 Superspeed Centrifuge spun at 18 000 rpm for 45 min. The cleared lysate was incubated for 4 h at 4°C with rotation with 4 ml of a GST bead slurry  that had been pre-washed 3× with wash buffer (WB) containing 600 mM NaCl, 5% glycerol, 3 mM DTT, 20 mM HEPES pH 7.0. The bead-protein mixture was washed 3× with 15 ml of WB, then transferred to a 10 ml disposable column (Qiagen) and washed with an additional 50 ml of WB followed by 100 ml of low salt buffer (LSB) containing 250 mM NaCl, 5% glycerol, 3 mM DTT, 20 mM HEPES pH 7.0 with periodic resuspension to prevent compaction. SOX was then cleaved on column with PreScission protease  overnight at 4°C, and protein eluate was collected for a final volume of 10 ml in LSB.2 using nuclease-free 0.5 ml microfuge tubes (Ambion Life Technologies) and stored at –80°C.Cleaved protein was concentrated to ∼1 ml using Amicon filter concentrator membrane cut off 30 kDa (EMD Millipore), then loaded onto a HiLoad Superdex S200 pg gel filtration column . Protein elutions were concentrated using an Amicon concentrator described above to 5 mg/ml and 25 μl aliquots were snap frozen in liquid N32P]-ATP-6000 Ci/mmol 150 mCi/ml (Perkin Elmer) using T4 PNK (New England BioLabs). RNAs were 3′ end labeled with 5′-[32P]-pCp 3000 Ci/mMol 10 mCi/ml using T4 RNA ligase 1 (New England BioLabs). Labeled RNA substrates were purified using 20% urea–PAGE and were isolated from gel slices by incubating overnight at 8°C in a buffer containing 10 mM Tris–HCl, 1 mM EDTA pH 8.0. Eluted RNAs were ethanol precipitated and resuspended in RNase-free ddH2O.All RNA substrates . Samples were resolved by 15% urea–PAGE, imaged using a Typhoon variable mode imager , and quantified using ImageQuant and GelQuant software packages (Molecular Devices). The data were plotted and fit to exponential curves using Prism 7 software package (GraphPad) to determine observed rate constants.escribed . BrieflyLIMD1 54 Flo) was purchased from Dharmacon  in the presence or absence of 2 μM SOX for 10 min at room temperature. RNA was then ethanol precipitated, resuspended in 95% formamide solution containing 10 mM EDTA, and resolved on a 12% urea–PAGE analytical grade sequencing gel together with a ssRNA Decade ladder (Ambion Life Technologies) for 1.5 h at 22 W before imaging as described above.For assays designed to detect endonucleolytic cleavage intermediates, 1 μl of labeled RNA substrate was combined with 9 μl of reaction solution  backbone using the BamHI and XbaI restriction sites. Mutations were introduced by the Quickchange site directed mutagenesis protocol (Agilent). The 100 nt sequence surrounding the GFP cut site was inserted using the BamHI and XhoI restriction site.in vitro transcription with the HiScribe T7 High Yield RNA synthesis Kit (NEB) and afterwards subjected to Turbo DNase (Ambion by Life Technologies) treatment. RNA was resolved by 8% Urea PAGE, and full length transcripts were excised from the SYBR Gold stained gel (Thermo Fisher Scientific), eluted overnight in G50 buffer , phenol/chloroform extracted, and ethanol precipitated. The RNA (∼40 pmol) was dephosphorylated using shrimp alkaline phosphatase , labeled with 1 μl [γ32P] ATP (150 mCi/ml) using USB Optikinase (Affymetrix), then gel purified as described above and dissolved in 20 μl of nuclease free water. For the in-line probing reaction, 1 μl RNA (≥20 000 cpm) was incubated in 2× reaction buffer  at room temperature for 24 or 48 h. The reaction was quenched with 2× loading buffer . To generate ladders, 1 μl of the purified RNA was separately subjected to hydrolysis using the Next Magnesium RNA Fragmentation module (–OH) or RNase T1 digestion (T1) (NEB). Reactions were resolved by 8% urea–PAGE, exposed on a phoshorimager screen, and scanned using the Storm 820 imaging system . Deduced RNA structures were drawn using the RNA secondary structure visualization tool forna (Vienna RNA Web Services).In-line probing was performed as described previously . Briefly2, 0.01% Tween-20, 0.5 TCEP, 0.2 mg/ml BSA (Sigma-Aldrich), 40 μg/ml of yeast tRNA (Ambion Thermo Fisher), and the indicated amount of purified SOX protein. Calcium Chloride was used in these binding assays to prevent substrate processing and stabilize RNA–protein interactions. Reactions volumes were kept at 10 μl and stopped with 3 μl 7× EMSA loading dye . Reactions were resolved by 8% native PAGE, and gels were imaged on a Typhoon multivariable imager  and quantified using GelQuant software package (Molecular Dynamics).RNA probes used in EMSA experiments were radiolabeled using the protocol described for ribonuclease activity assays. Reactions were incubated at RT for 30 min in buffer containing 20 mM HEPES pH 8.0, 30 mM KCl, 5 mM CaClLIMD1-54 RNA was 5′ end labeled with γ-[32P]-ATP-6000 Ci/mmol 150mCi/ml (PerkinElmer) using T4 PNK (New England BioLabs). RNA was then gel purified as stated previously. EMSA gel shifts were first used to determine optimal binding conditions . Binding buffer contained 0.01% Tween 20 (Sigma-Aldrich), 5 mM CaCl2, 5 mM KCl, 50 mM NaCl, 0.5 mM TCEP, 20 mM HEPES pH 8.0, 0.04 mg/ml yeast tRNA (Ambion), 0.2 mg/ml nuclease free Bovine Serum Albumin (BSA) (Ambion). A dilution series of SOX (8–0.5 μM) was incubated with 1 μl of radiolabeled LIMD1-54 in the presence of 0.1 unit of RNase T1 (Epicentre Illumina). Reactions were incubated at RT for a total of 10 min before being ethanol precipitated. RNA pellets were then resuspended in 5 μl of 95% formamide solution containing 10 mM EDTA and boiled for 5 min. Samples were then loaded onto a 10% analytical grade urea–PAGE gel and run at 22 W for 1.5 h. Gels were imaged and analyzed as stated above. In order to produce an RNase T1 ladder, 1 μL of LIMD1-54 was incubated with 0.1 units of RNase T1. Reactions were incubated at RT for 5 min before being quenched and prepared as stated previously. The LIMD1-54 hydrolysis ladder was generated as stated in the in-line probing methods.Kd) were determined from kon and kdis values derived from the fitted curves. A complete table of all values is provided in RNA probes (3′ end labeled with biotin) were synthesized from Dharmacon  and HPLC and PAGE purified See . The Octin vitro, RNA fragments are rapidly degraded by 5′-3′ exonucleolytic activity intrinsic to purified SOX , and we therefore chose this as our model substrate to study SOX targeting in vitro  as a function of SOX concentration, yielding a Hill coefficient of n = 1.11 . We confirmed this RNA was resistant to degradation by the 5′-phosphate dependent exonuclease terminator . Incubation of this mutant with cleavage . Thus, rin vitro assay, we first compared SOX degradation of LIMD1-54 to a 51-nucleotide sequence of the mRNA encoding GFP (GFP-51). We have previously shown that GFP mRNA is cleaved by SOX in cells, and that GFP-51 is the minimal sequence required to elicit cleavage  SOX (see LIMD1-54 are in the single digit nanomolar range (Kd = 7 nM), while P176S and F179A display >2 log defects  . ,21. 8,21 SOX see . However SOX see . Further) Figure . Thus, tIn silico RNA folding predictions of SOX targeting motifs, coupled with RNA mutagenesis experiments, have indicated that an RNA stem loop structure is an important determinant in SOX targeting both in vitro and in vivo . In this structure, the only observed contacts between SOX and K2-31 occurred between the four active site residues of SOX  and the UGAAG motif surrounding the cleavage site of the RNA  from RNase T1 digestion in a dose dependent manner . Second, we largely abolished the loop structure by providing complementary base pairing (LIMD1-54 Zipper). Third, we mutated the residue located at the predicted SOX cut site that was also protected in the footprinting assay (LIMD1-54 A-G). This mutant has been previously identified to block SOX cleavage in vivo . All RNA probes were 3′ biotinylated and immobilized to a streptavidin-coated BLI probe, whereupon the binding and dissociation of SOX was measured. To prevent degradation of the probe, excess calcium ion was used in place of magnesium (LIMD1 A-G (Kd = 25 nM) as to WT LIMD1-54 (Kd = 16.3 nM)  and to the LIMD1-54 zipper mutant lacking the loop region (Kd = 904 nM)  that did not disrupt the polyadenosine sequence. In agreement with the loop structure playing a critical role in target recognition, this LIMD1-54 zipper 2 mutant also displayed a substantial defect in binding  , suggesting that despite having an UGAAG motif upstream of a predicted bulge, this is unlikely to be a SOX target  Figure . In cont) Figure . To rule; K2-31) . Notablyt Figure .LIMD1-54 A-G, there was a 7-fold defect in its ability to degrade this mutant  or maintaining the bulge but mutating the polyadenosine stretch (LIMD1-54 3xA-G) resulted in a ∼100-fold reduction in binding affinity, correlating with a dramatic decrease in cleavage efficiency. Collectively, these data demonstrate that variability in the efficiency of SOX targeting observed in cells is likely due to differences in RNA sequences that mediate SOX binding.A key open question related to SOX function is how it can target the majority of mRNAs in cells, yet with significant site specificity. Our observations suggest that there must be specific mRNA features that influence targeting. Indeed, PARE-seq analyses of cleavage intermediates in SOX expressing cells revealed that cleavage sites were associated with a degenerate sequence motif . Sequencin cells . Becausein cells , we reasin cells . MutatinK2-31 pre-miRNA captured the importance of the exposed loop region for SOX cleavage  compared to its DNA substrates (Kd = 1 μm) (2 interaction surface), when compared to the K2-31 RNA bound structure (∼240 Å2 interaction surface). It is therefore possible that more interaction along the length of SOX protein might occur with optimal substrates such as LIMD1 that are more tightly bound.A recent crystal structure of SOX bound to the cleavage . Howeverm region , rather  = 1 μm) ,10,21. HLIMD1-54 at the precise site observed in SOX-expressing cells demonstrates that cleavage site selection on an mRNA is not mediated by a cellular cofactor. Instead, targeting at particular RNA motifs is strongly influenced by the strength of SOX binding. Our observation that the P176S and F179A SOX mutants display significant RNA binding defects indicates that their failure to cleave mRNAs in cells is due to an inability to efficiently bind the targeting motif.The fact that purified SOX endonucleolytically cleaved in vitro. We hypothesize that cellular co-factors, perhaps though interactions with SOX, enable this distinction. More broadly, endonucleases are instrumental in RNA processing and degradation. Nuclease processing defects lead to several human pathologies ranging from cancer to neurodegeneration (The mechanism by which SOX initially distinguishes RNA polymerase II transcribed mRNAs from other types of RNA in cells remains an important open question, as this feature of SOX selectivity is not preserved neration , and ourSupplementary DataClick here for additional data file.