Mycobacterium smegmatis during stationary phase of growth. By glycerol gradient ultracentrifugation, RNA binding assay, and RNA co-immunoprecipitation, we show that Ms1 interacts with the RNA polymerase (RNAP) core that is free of the primary sigma factor (σA) or any other σ factor. This contrasts with the situation in most other species where it is 6S RNA that interacts with RNAP and this interaction requires the presence of σA. The difference in the interaction of the two types of sRNAs (Ms1 or 6S RNA) with RNAP possibly reflects the difference in the composition of the transcriptional machinery between mycobacteria and other species. Unlike Escherichia coli, stationary phase M. smegmatis cells contain relatively few RNAP molecules in complex with σA. Thus, Ms1 represents a novel type of small RNAs interacting with RNAP.Small RNAs (sRNAs) are molecules essential for a number of regulatory processes in the bacterial cell. Here we characterize Ms1, a sRNA that is highly expressed in  Mycobacterium tuberculosis and Mycobacterium leprae-and nonpathogenic saprophytic species, such as Mycobacterium smegmatis. Approximately one third of the world's population is infected by M. tuberculosis and almost 9 million people developed the active disease in 2012. According to WHO, tuberculosis caused 1.3 million deaths in 2012. In addition, multi-drug resistant tuberculosis is present in most countries surveyed. An important feature of M. tuberculosis is that the bacteria can survive in the human body for decades as a latent infection with no symptoms  that is itself regulated by various auxiliary factors. Bacterial RNAP is a multisubunit enzyme composed of core subunits: α factors  while Esas seven . Typicalsubtilis . When coctivated .E. coli encodes hundreds of different sRNAs . sRNAs usually have a length of 50–300 nt and most of them base-pair with mRNA and regulate mRNA stability or the efficiency of mRNA translation . It is ent sRNAs . Only a nt sRNAs –14. In ent sRNAs . This nunt sRNAs ). MTS282in silico searches for 6S RNA homologs. 6S RNAs fold into a secondary structure that mimics an open promoter (σ factor (RNA polymerase holoenzyme). 6S RNA prevents the binding of the RNAP holoenzyme to promoter sequences and reduces its transcriptional activity . 6S RNAσ factor –28; mycoσ factor , it is sM. smegmatis as a model organism and search for the binding partner of Ms1. We show that Ms1 is a sRNA that directly interacts with the transcriptional machinery but in a different way than 6S RNA-Ms1 binds core RNA polymerase instead of RNAP holoenzyme. Thus, Ms1 represents a novel class of small RNAs. Finally, we discuss possible reasons why mycobacteria may differ from most other bacterial species in the interaction of RNAP with the sRNA.Here we use M. smegmatis mc2 155 and FLAG-tagged RpoB strain (600 ∼0.5) or 4–6 h after the entry into stationary phase (OD600 ∼2.5–3) unless stated otherwise. Transformations of M. smegmatis mc2 155 cells were performed by electroporation. When required for selection of transformants, media were supplemented with hygromycin (50 μg/ml) and/or kanamycin (20 μg/ml). Wild-type E. coli K12 KW72  or 3–4 h after entry into the stationary phase of growth unless stated otherwise. Growth phenotype experiments  and the cells were grown for additional 6 h . Plasmids were verified by sequencing and electroporated into the mc2 155 strain, yielding strains overexpressing either Ms1 (LK1323), or its mutant form Ms1nb (LK1337) or without any overexpression-a control strain with an empty plasmid (LK1302).Ms1, Ms1nb  and 80 bp promoter  DNA sequE. coli, M. smegmatis and B. subtilis RNA isolation. Briefly, 8–20 ml cells from the indicated growth phases were quickly pelleted and immediately frozen. The pellet was then suspended in 240 μl TE (pH 8.0) plus 60 μl lysis buffer  pH 8.0, 5% sodium dodecyl sulphate (SDS)) and 600 μl acidic phenol (pH∼3):chloroform (1:1). Lysates were sonicated in a fume hood, centrifuged, the aqueous phase extracted two more times with acidic phenol (pH∼3): chloroform and precipitated with ethanol. RNA was dissolved in water and DNase treated . Five μg of total RNA were mixed in a 1:1 ratio with the sample buffer , heated for 1 min at 90°C, then kept on ice and electrophoresed in 7 M urea 7% polyacrylamide gels.The same protocol (adapted from ) was useM. smegmatis total RNA consists of Ms1, we used RNase H treatment that degrades RNA in RNA/DNA duplexes. We set up 9 μl reactions with 3.5 μg total RNA, 2 μl 5xRT buffer  and 1 μl 100 μM DNA oligonucleotides . Reactions were incubated for 1 min at 95°C, 1 min at 60°C and cooled on ice. One μl (2 U) of RNase H  was then added and the reactions were incubated for 10 min at 37°C, mixed 1:1 with sample buffer , heated for 1 min at 90°C and electrophoresed in 7 M urea 7% polyacrylamide gels. The gels were stained for 20 min with GelRed (LabMark), 10 000× diluted in 1× TBE (Tris-borate-EDTA) gel running buffer and visualized using a UV transilluminator. RNA isolation after the glycerol gradient or immunoprecipitation is described in the relevant sections.To test whether the prominent 300 nt band in the M. smegmatis mc2 155 genome (GenBank # NC_008596.1). The transcription start sites for other species were retrieved from previous publications: MTS2823  (Mycobacterium bovis BCG Pasteur 1173P2 genome) (Mycobacterium avium MAH104)  for 1 h at 37°C. RNA was extracted with acidic phenol (pH∼3):chloroform (1:1), precipitated with ethanol and a 5′-adaptor DNA/RNA oligonucleotide  was ligated to the 5′ ends. RNA was then extracted and reverse transcribed into cDNA  with an Ms1-specific reverse primer (5′-CGTCCGCTTTTCGAAACTAC-3′). The same reverse primer and the 5′-ATCGTAGGCACCTGAAA-3′ forward primer were used for polymerase chain reaction (PCR) with Taq DNA polymerase (Biotools). The PCR products were sequenced and mapped to the  MAH104)  GenBank E. coli and M. smegmatis stationary phase cells were pelleted and resuspended in 20 mM Tris–HCl pH 7.9, 150 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and Calbiochem Protease Inhibitor Cocktail Set III protease inhibitors, sonicated 15 × 10 s with 1 min pauses on ice and centrifuged. Protein extracts (1.5 mg) were loaded on a linear 10–30% glycerol gradient prepared in gradient buffer  and fractionated by centrifugation at 32 000 rpm (130 000 × g) for 17 h using an SW-41 rotor (Beckman). The gradient was divided into 20 fractions, RNA from individual fractions was extracted with acidic phenol (pH∼3):chloroform, precipitated by ethanol and electrophoresed in 7 M urea 7% polyacrylamide gels. The gels were stained for 20 min with GelRed (LabMark) 10 000× diluted in 1× TBE gel running buffer and imaged using the UV transilluminator. Proteins were analyzed by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining  or detected by western blotting using mouse monoclonal antibodies to σ70/σA [clone name 2G10] or to the β subunit of RNA polymerase [clone name 8RB13] and secondary antibodies conjugated with a fluorophore dye and quantified with an Odyssey reader (LI-COR Biosciences). 6S RNA, Ms1, σA, σ70 and RNA polymerase (β subunit) were quantified from RNA gels/western blots using the ImageJ software.E. coli and M. smegmatis cell lysates were prepared in the same way as the lysates used for the glycerol gradient ultracentrifugation, and 300 μg (protein) of lysates were incubated for 2 h at 4°C with 20 μl of Dynabeads Protein A (Invitrogen) coated either with 4 μg mouse monoclonal antibody to σ70/σA [clone name 2G10], 2–8 μg mouse monoclonal anti-β subunit of RNAP antibody [clone name 8RB13] (both from Santa Cruz) or 10 μg mouse nonspecific IgG (Sigma-Aldrich) used as a negative control. The captured complexes were washed 4× with 20 mM Tris–HCl pH 7.9, 150 mM KCl, 1 mM MgCl2 and divided into two parts. A quarter of the beads were incubated in SDS sample buffer for 5 min at 95°C and eluted proteins were detected by western blotting. The remaining three quarters of the beads were resuspended in 200 μl 1% SDS, 150 mM KCl, 20 mM Tris–HCl pH 7.9, 1 mM MgCl2 and vortexed with 200 μl acidic phenol (pH∼3):chloroform (1:1) for 10 min. Eluted RNA was precipitated with ethanol, dissolved in water and DNase treated . RNA was visualized on a 7 M urea 7% polyacrylamide gel by staining with GelRed (LabMark). Alternatively, RNA was reverse transcribed into cDNA  using random hexamers and amplified by quantitative reverse transcription PCR (RT-qPCR) in a LightCycler 480 System (Roche Applied Science) in duplicate reactions containing LightCycler® 480 SYBR Green I Master and 0.5 μM primers (each). For the Ec_6SRNA primer pair, dimethyl sulfoxide (DMSO) was added to a 1% final concentration to optimize amplification efficiency. Primers were designed with Primer3 software and their sequences are in the Supplementary primer list. Negative controls (no template reactions and reactions with RNA as a template to control for contamination with genomic DNA) were run in each experiment, the quality of the PCR products was determined by dissociation curve analysis and the efficiency of the primers determined by standard curves. The proportions of coimmunoprecipitated RNAs were quantified on the basis of the threshold cycle (Ct) for each PCR product that was normalized to input values according to the formula 2(Ct(immunoprec)–Ct(input)).2 155 strain (negative control) were prepared in the same way as the lysates for glycerol gradient centrifugation, 300 μg of lysate incubated with 10 μl MS2 resin (Sigma-Aldrich) for 1 h at 4°C, captured complexes were washed 4× with 20 mM Tris–HCl pH 7.9, 150 mM KCl, 1 mM MgCl2 and proteins/RNA isolated and detected in the same way as the immunoprecipitations.Lysates from MR-sspB (FLAG tag on β) and the wt mc2, 10 mM Tris–HCl pH 7). RNAs were incubated in the same buffer for 1 h at 4°C with 100 μl magnetic streptavidin-coated beads (Sigma-Aldrich) while being gently agitated. The beads were then washed with lysis buffer  and incubated with ∼400 μg of cell extract (prepared in the same way as for glycerol gradient ultracentrifugation) for 1 h at 4°C, washed 4× with lysis buffer, resuspended in SDS sample buffer, heated for 5 min at 95°C and the eluted proteins were detected by SDS-PAGE and Coomassie staining .Ms1 and Ms1nb templates were amplified from Invitrogen DNA (GeneArt Strings DNA Fragments) with the primer carrying the T7 promoter at their 5′ end (see Supplementary Primer List). RNAs were prepared with a T7 RiboMAX Express Large Scale RNA Production System (Promega) and their 3′ends were biotinylated with an RNA 3′ End Biotinylation Kit (Thermo Scientific). After biotinylation, 2.5 μg of each RNA was denatured for 2 min at 90°C and refolded for 20 min at RT in 200 μl folding buffer  were plated on Middlebrook 7H10 agar. The plates were incubated at 37°C for 5 days before counting the colony forming units (CFU) and calculating CFU/ml. The obtained data were used in subsequent calculations of amounts of Ms1 per cell. The quantity of Ms1 in the total RNA sample (stained with GelRed) was determined by comparing the Ms1 band density to a standard curve derived from the RiboRuler Low Range RNA Ladder (Thermo Scientific). The quantitation was done with QuantityOne software (Biorad). The Ms1 quantity determined by northern blotting was done by comparing the signal intensity of Ms1 with a standard curve derived from serial dilutions of in vitro transcribed Ms1. Northern blotting was performed as described previously , and signals were captured in Fuji MS phosphor storage screens and scanned with a Molecular Imager_FX (BIO-RAD) and quantified with QuantityOne software (Biorad). For calculating the increase of Ms1 expression in stationary phase relative to exponential phase, the Ms1 northern blot signal was normalized to 5S rRNA.Cells from stationary phase were used to determine the number of viable cells and purify total RNA. Viable cell numbers were counted as follows: 100 μl aliquots of cells from serial dilutions (10eviously . BrieflyM. smegmatis and compare it to the amounts of known 6S RNAs in E. coli and B. subtilis (recently reviewed in (E. coli (184 nt) and the main 6S RNA in B. subtilis (represented by the 190 and 201 nt doublet) are highly expressed during stationary phase and both are clearly visible when total RNA is resolved on denaturing PAGEs . 6S ng PAGEs ,20. We il Figure . The gelA Figure .We then quantified the amount of Ms1 in mycobacterial cells using total RNA staining  and northern blot hybridization . In both cases we compared the Ms1 amount extracted from a known number of cells to serially diluted standards of known concentration. From the GelRed stained gels, we calculated the amounts of Ms1 and 5S rRNA (as a control) in stationary phase to be ∼400 and ∼2800 molecules per cell, respectively. From the northern blots  we obtained a comparable result of ∼600 Ms1 molecules per cell. Compared to exponential phase, the Ms1 amount increased ∼130-fold upon entry into stationary phase .sRNAs represented by these bands is currently unknown and they are the subjects of another study. Hence, we established that Ms1 is a highly abundant sRNA present in the cell in quantities comparable to the 6S RNA found in other species and we decided to further characterize Ms1.Besides Ms1, we also detected a <100 nt band in stationary phase Figure . AnotherM. smegmatis stationary phase cells and identified the first nucleotide of Ms1 to be an adenine transcribed from position 6 242 368 in the M. smegmatis genome , which belong to the group of actinobacteria. Based on sequence similarity searches . These sequences fold into a long double-stranded hairpin with a bubble in the middle and two short hairpins close to the ends. The internal bubble and the adjacent double stranded regions are the most conserved elements. Although the primary sequences of Ms1 homologs are not identical, their predicted secondary structures are similar, which suggests that these sRNAs could have the same function(s).We began characterizing Ms1 by performing 5′RACE (Rapid Amplification of the 5′ cDNA End) with total RNA isolated from the e Figure . Upstreas Figure . AlthougM. avium ,35 were nsTermHP  and is lsearches , Ms1 wassearches  of Ms1 hsearches  is partisearches . In siliM. smegmatis and E. coli (as a control) stationary phase cell lysates by glycerol gradient ultracentrifugation. After ultracentrifugation, large complexes such as ribosomes are sedimented at the bottom of the tube whereas e.g. 6S RNA is found in the middle of the gradient. Importantly, molecules that are in the same complex must be found in the same fractions of the gradient. This method was previously used to identify proteins that are in complex with E. coli 6S RNA , we fractionated i 6S RNA . The maji 6S RNA  indicatie Figure , since σwith Ms1 . Using min vitro transcription. In addition, we also prepared Ms1 with a deleted internal bubble-designated ‘Ms1nb’  might be below our detection limit.To identify which of the proteins from the Ms1 fractions truly interacted with Ms1, we pulled down Ms1 binding partners directly. First, we prepared Ms1 sRNA by ’ Figure . We then’ Figure . We subsA Figure . In addiM. smegmatis strain carrying the FLAG-tag on β (RpoB) . This experiment, however, did not enable us to distinguish the RNA polymerase core from the holoenzyme (β is present in both complexes).To verify that Ms1 associates with RNAP, we utilized a β (RpoB) , one of σA-containing) form of RNAP we performed immunoprecipitations with two specific antibodies. Both antibodies efficiently immunoprecipitated E. coli and M. smegmatis proteins . The anti-σA/σ70 antibody (2G10) recognized both free σA/σ70 as well as σA/σ70 in complex with RNAP (RNAP holoenzyme). The anti-RNAP antibody  was previously reported to mainly bind the RNAP core devoid of σ factors  and identified them by mass spectrometry (see Supplementary Table S4) as NusG (transcription elongation factor)  also did not interact with RNAP   and the  factor) . Importas Figure , lane 7 s Figure . We founs Figure , lane 7,P Figure . In agreP Figure  and glycP Figure , we did e Figure . This is complex   that is strongly active in exponential phase  interacted much less with core RNA polymerase compared to the intact Ms1   Figure . Moreovel Figure .σA, we asked the reverse question: Can σA affect the amount of Ms1 bound to the RNAP core? To test this in vivo, we overexpressed σA in stationary phase cells from the pJAM2-σA plasmid, which contains the σA (mysA) gene under an inducible acetamidase promoter (600 ∼1.6) to induce σA expression, cells were harvested after 6 h and the increase in the σA protein level was confirmed by western blotting  in exponential versus stationary phase both in M. smegmatis and in E. coli by analyzing the data from Figure E. coli, a similar amount of RNAP (monitored by the level of β) coimmunoprecipitated with σ70 both in exponential and stationary phases , the RNAP core had an even lower chance to form a holoenzyme with σA in M. smegmatis as we observed a decrease in the total amount of σA in the cells  in M. smegmatis is 2-fold: the binding of σA to RNAP is decreased and the total level of σA drops. Thus, M. smegmatis significantly differs from E. coli in the composition of the transcription machinery in stationary phase.Finally, we asked why mycobacterial Ms1 interacts with core RNAP whereas 6S RNA, which is present in the majority of other species, binds to the RNAP holoenzyme. A possible explanation could be that mycobacteria may differ in their amounts of the RNA polymerase holoenzyme in stationary phase. Therefore we compared the amounts of the RNAP core that coimmunoprecipitated with e Figure . This meM. smegmatis. We determined that Ms1 binds to the RNAP core devoid of σ factors. Until now, only 6S RNA was shown to bind to RNAP and the presence of the main σ factor is essential for this interaction. Thus, the Ms1–RNAP complex represents a novel type of sRNA–RNAP interaction. Moreover, we detected no other sRNA to interact either with the RNAP core or holoenzyme, suggesting that M. smegmatis likely does not contain a 6S RNA. This reflects the difference between mycobacteria and other bacterial species (see ‘The difference between mycobacteria and other bacteria in their interaction of RNAP with sRNAs’ section).In this study we characterized Ms1, a highly abundant sRNA from M. smegmatis RNA metabolism  locus in the genome of M. tuberculosis . In stationary phase, almost 40% of total Ms1 sRNA interacts with RNA polymerase, which suggests that ∼200 Ms1 molecules bind around 200 molecules of core RNA polymerase. As a control, we calculated that the number of ribosomes (based on 5S rRNA) is ∼2800 per cell. This is a lower number than what was reported for E. coli –56 but imegmatis ,58 as wemegmatis . The num present ,60–62. I0. In staare free , represeM. tuberculosis, where overexpression of the Ms1 homolog resulted in a moderate reduction of the growth rate (M. smegmatis grown in rich medium) the cells contain enough free RNA polymerase and thus are not affected by the presence of Ms1, or, there might be different levels of free core RNAP in these two species.We were able to express Ms1 sRNA in exponential phase in a comparable quantity to the amount of Ms1 present in stationary phase . In addition, no sRNA peaks were detected with the RNAP-σA holoenzyme in M. smegmatis stationary phase glycerol gradients  was present in M. smegmatis in either the stationary or exponential phase . Alte Figure , we do nmegmatis . Why, ths Figure  and the l Figure  68). In. InσA origure σA . RbpA warculosis , and untrculosis ,72, indiy Figure  and C. TSupplementary Data are available at NAR Online.


Early work utilized ‘naked’ RNA vaccines, whereas more recent efforts have attempted to encapsulate RNA thereby protecting it from degradation. However, feasibility has been limited by a lack of defined and safe targeting mechanisms for the in vivo delivery of stabilized RNA. As new cancer antigens come to the forefront with novel RNA encapsulation and targeting techniques, RNA vaccines may prove to be a vital, safe and robust method to initiate patient-specific anti-tumor efficacy.Tumor-specific immunotherapy holds the promise of eradicating malignant tumors with exquisite precision without additional toxicity to standard treatments. Cancer immunotherapy has conventionally relied on cell-mediated immunity while successful infectious disease vaccines have been shown to induce humoral immunity. Efficacious cancer immunotherapeutics likely require both cellular and humoral responses, and RNA based cancer vaccines are especially suited to stimulate both arms of the immune system. RNA is inherently immunogenic, inducing innate immune responses to initiate cellular and humoral adaptive immunity, but has limited utility based on its poor  Tumor-specific immunotherapy is a burgeoning field targeting tumor antigens in the form of peptides, nucleic acids, and cell lysates to induce host-immunity . The exqAlthough some infectious disease vaccines are capable of inducing cell-mediated immunity, humoral immune responses remain the hallmark of effective infectious disease vaccination strategy . These v+ T lymphocytes initiating Th2 responses that induce humoral immunity [Bacterial immunizations for diphtheria, tetanus, pertussis, (collectively known as DTaP- diphtheria-tetanus-acellular pertussis vaccine), all utilize aluminum  containing adjuvants, and demonstrated efficacious and prophylactic immune responses prompting their adoption into the primary vaccination series in children . Inactivimmunity -18. Addiimmunity . Since pimmunity . This haimmunity .+ T cell responses [+ T cell expansion, IFN production, and memory formation [Mycobacterium tuberculosis, is one of the only licensed vaccines thought to work primarily through Th1 T-cell responses [+ T-cell responses are highly variable rendering the efficacy of BCG vaccines unreliable in a clinical setting [Similar to inactivated bacterial vaccines, viral vaccines induce humoral immunity through a Th2 dependent response; however there is growing evidence that Th1 mediated immunity plays a prominent role in the induction of antigen specific CD8+ T cells after administration of live RNA viral vaccines -30. For esponses -24. Simiormation ,28,30. Lormation . Despiteesponses ,39. The  setting -42.Thus, while infectious vaccine strategies have had tremendous success, notwithstanding BCG, they have historically relied on humoral immune responses. RNA viruses administered as whole viral vaccines, evidenced by attenuated influenza and yellow fever conjugates, can induce combinatorial Th1 and Th2 responses suggesting that a synergistic approach between cellular and humoral immunity may mediate efficacy in malignancies and resistant infectious diseases.RNA is recognizable by pathogen-associated molecular patterns (PAMPs), which are potent stimulators of PRRs such as TLR receptors 7/8 thereby activating DCs to initiate anti-tumor responses -46. The ex vivo priming of autologous DCs or in vivo delivery to target APCs [Based on their propensity for inducing both antibody and T cell responses, nucleic acids encoding for cancer antigens are an attractive immunotherapeutic platform for the transfection of APCs . The magget APCs -53.in vivo gene expression [in vivo half-life of RNA from local and systemic administration, other studies have focused on direct injection of RNA into target organs [The initial experiments with RNA vaccines demonstrated that direct injection of messenger RNA (mRNA) into murine skeletal muscle induced pression . These ipression . One of pression . RNA trapression . Direct pression . Given tt organs -59. Diret organs . Additiot organs . Other mt organs .Much of this pre-clinical data has led to the translation of naked RNA vaccination into phase I trials for refractory malignancies ,62. In aAlternatively, strategies have focused on making ‘naked’ RNA vaccines self-replicating -66. In tOther methods have focused on condensing ‘naked’ RNA to enhance its stability and immunogenicity. RNA can be stabilized through incorporation of polypeptide cations such as protamines which function to condense nucleic acid . These min vivo [in vitro with mRNA coding for tumor associated antigens, elict cytotoxic T cell responses in murine models, and induce suppression of metastatic spread in a murine melanoma model [in vivo model [While the initial attempts using naked RNA have been promising, RNA degradation remains a concern limiting the amount of RNA available for cellular internalization . To protin vivo . Since lin vivo . These sma model -84. Lipoma model ,85. Theyma model ,86. Selfma model . Similarvo model ,86. In avo model . Fusion-vo model . Althougvo model .in vivo re-introduction [ex vivo generated cellular vaccines through multi-institutional clinical trials and eventual distribution for widespread clinical utility has proven to be fraught with developmental challenges prompting others to prioritize the advancement of RNA-loaded nanoparticle vaccines as an attractive, “off-the-shelf” targeted therapeutic platform.Future vaccine strategies will build on the success of previous advances while ameliorating the limitations of the immunogenic but highly degradable naked RNA vaccines, and the limitations of the protected but inefficient delivery of RNA-liposomal vaccines. The safety and stability profile of liposomes is attractive, and the immunogenicity of RNA is appealing for initiating prolific immune responses. Since RNA-liposomes cannot hone to targeted cells, engineering targeting ligands and moieties may be utilized to transfect desired cells. Pre-clinical studies have shown that IgG-coated liposomes are efficiently taken up and presented to T cells by dendritic cells via Ig FcR while mannosylated liposomes enhance uptake and activation of DCs ,89. Meanoduction ,92. Thesoduction ,93,94. Ioduction . Similaroduction ,96. In toduction . While tin vivo DC transfection and anti-tumor immunity against a murine melanoma model [Nanomaterials have been utilized to deliver drugs directly to malignant tissues, bypassing their systemic toxicity . Given tma model .in vivo system containing plasma proteins that may alter a nanomaterial’s surface property and affect its biocompatibility [Despite this active area of research, the arrival of approved nano-drugs to the market has been slow with only a handful of FDA-approved agents ,105. Beftibility . These ctibility -109. Bastibility .in vivo targeting technologies, particularly in the area of nanomaterials engineering, and our understanding of the requirements for successful induction of potent anti-tumor immunity promise to yield the development of effective and safe RNA vaccines for the treatment of refractory cancers.Cancer vaccines can meet the challenge of targeted cancer care, but require strong adjuvants to re-direct a ‘tolerant’ immune system. We can look to infectious disease vaccines as a model for successful immunization strategies. While most infectious disease vaccines function through induction of humoral immunity, RNA viral vaccines such as influenza and yellow fever have been shown to generate robust cellular and humoral immunity. To leverage successful cancer immunotherapeutic RNA vaccination strategies, traditional methods in conjunction with novel adaptations are requisite in developing safe and effective formulations that protect RNA from degradation, deliver payload to target, and remain amenable to central manufacturing and distribution. The rapid advancements being made in


RNA helicases are implicated in most cellular RNA-dependent events. In eukaryotes however, only few have been functionally characterized. Upf1 is a RNA helicase essential for nonsense-mediated mRNA decay (NMD). Here, using magnetic tweezers and bulk assays, we observe that human Upf1 is able to translocate slowly over long single-stranded nucleic acids with a processivity >10 kb. Upf1 efficiently translocates through double-stranded structures and protein-bound sequences, demonstrating that Upf1 is an efficient ribonucleoprotein complex remodeler. Our observation of processive unwinding by an eukaryotic RNA helicase reveals that Upf1, once recruited onto NMD mRNA targets, can scan the entire transcript to irreversibly remodel the mRNP, facilitating its degradation by the NMD machinery. Upf1 is a multifunctional helicase involved in various DNA- and RNA-related processes, including nonsense-mediated mRNA decay (NMD). Here the authors demonstrate that Upf1 is a highly processive ribonucleoprotein complex remodeler—a capability likely important for Upf1's NMD function. RNA helicases participate in all biological processes involving RNA and are often essential for life56889The ATPase activity of Upf1 is required for the correct progression of NMD in all eukaryotic organisms tested so far. Structurally, Upf1 is composed of a characteristic double RecA-like helicase domain (HD) flanked by two external and regulatory domains1314Here, to investigate the properties of Upf1, we study its behaviour on single RNA and DNA molecules by combining biochemical and biophysical experimental strategies. We find that the helicase domain of Upf1 is capable of both unwinding double-stranded nucleic acids and translocation on single-stranded nucleic acids over long distances. In addition, we compared the activity of Upf1 in the absence and in the presence of its partner Upf2. Finally, we investigated whether the translocation of Upf1 is hampered by the presence of proteins bound to DNA and RNA, revealing an unexpected potential of Upf1.F to the hairpin. Adjusting the distance between the magnets and the beads from 2 to 0.125 mm, results in forces ranging between 0.1 and 25 pN respectively. The extension of the molecule with time Z(t) was obtained by tracking the position of the bead using video-microscopy, which provides an accuracy of ∼3 nm (3–4 bases) between two video frames sampled at 30 Hz. For forces lower than ∼17 pN, the RNA hairpin of 156 bp is fully folded while at higher forces, the hairpin is mechanically unfolded  displayed long and characteristic saw-tooth events (τpu (τpt) ; the duration of the unwinding (translocation) events was also exponentially distributed (τu and τt). The values of these characteristic times reported in −1, respectively, at 26 °C (In the presence of Upf1 helicase domain (Upf1-HD) in saturating ATP conditions, we observed that h events . The burat 26 °C . Upf1-HDat 26 °C . To ensuat 26 °C .Together with the low enzyme concentration, several pieces of evidence support that a monomer of Upf1-HD unwinds the complete RNA hairpin. First of all, Upf1 has been previously shown to exist as monomer in solutionTogether, the results from this single-molecule approach showed that the helicase domain of Upf1 is able to both unwind a long dsRNA of 156 bp and translocate onto ssRNA.in vitro unwinding assays (21t to transiently unzip the hairpin entirely (z) measured when the force resumed its original value and the hairpin partially refolded , as shown by g assays , compellg assays . As for g assays . The abientirely . Upf1-HDrefolded .Next, we varied the force applied to the hairpin to determine whether its unwinding and translocation rates were force dependent. Typically, increasing the applied force enables a passive helicase, which waits for a spontaneous thermal opening of the base pairs, to unwind or translocate with higher ratesKM of 1.22±0.2 μM with a saturating speed Vmax of 1.36±0.1 bp s−1, showing that the enzyme works at its maximum rate in physiological ATP concentration  where u equals the number of unwinding events and b equals the number of binding events. For Upf1-CH-HD, Ac=49% , passed the apex and started translocating but it rapidly performed a strand-switching event. In this process the helicase direction was reversed and the helicase started unwinding the hairpin again .For 76±8% of the Upf1-CH-HD/Upf2 binding events, we noticed a characteristic burst shape with a normal rising edge but a falling edge interrupted by blocking  at 770 sobserved . Upf1F19r pauses . The meaTherefore, our single-molecule approach confirms that Upf2 activates Upf1-CH-HD, and reveals that the enzyme bound to its activator is processive, with a highly efficient activation mechanism (95%).F192E efficiently displaced the streptavidin with a comparable efficiency while Upf1-CH-HD showed a much weaker activity 34233624Escherichia coli RecBCD5Helicases can carry different biophysical properties including RNA binding, translocation onto ssNA or dsNA, dsNA separation and RNA–protein remodelling over more or less long distance ranging from few nucleotides to several kilobases30−1 refs . Upf1 alin vitro approach detected identical trends since single molecules of Upf1 unwind dsRNA molecules more slowly than they translocate  Rosetta cells. The Upf1 protein truncations were CBP-tagged at the N-terminal and His-tagged at the C-terminal, while Upf2 was His tagged. The cell cultures were grown in 1 l of LB medium and induced overnight at 16 °C. The cells were lysed in buffer A  NP-40, 20 mM imidazole and 10%(w/v) glycerol) supplemented with 100 μg ml−1 of egg white lysozyme (Sigma-Aldrich) and with 1 × protease inhibitor cocktail EDTA-Free (Sigma-Aldrich). Soluble lysate was applied to a prepacked nickel column  and fractioned on an Äkta Purifier  using a linear gradient from buffer A to B (buffer A added of 0.5 M imidazole) over 20 column volumes. The CBP-fused proteins were subsequently purified on a Calmodulin affinity column. Loading of the samples and column washes were performed with a buffer C  NP-40, 1 mM DTT, 4 mM calcium chloride and 10%(w/v) glycerol) and elution with the same buffer containing 20 mM EGTA instead of calcium chloride13142, 2% BSA, 1 mM DTT, 0,1 mM EDTA). The Gp32-B protein was cloned in petIMPACT plasmid and expressed into E.coli BL21(DE3) cells. The protein purification was performed using a chitin column and analysed for purity using SDS–polyacrylamide gel electrophoresisHuman Upf1-HD (295–914 amino acids), Upf1-CH-HD (115–914 amino acids) Upf1-CH-HD-F192E and Upf2  and the RNA product was purified on denaturing (8 M urea) polyacrylamide (5%) gel as previously described14A shorter palindromic sequence was produced from pLacD1 plasmidThe DNA 1.2 kbp substrate used in the single-molecule studies is a 1,239 bp hairpin with a 4-nt loop, a 76-nt 5′-biotinylated ssDNA tail and a 146 bp 3′-digoxigenin-labelled dsDNA tailwww.picotwist.com) to manipulate individual DNA and RNA hairpin molecules. The DNA hairpins were attached by the 5′ biotinylated extremity to streptavidin-coated magnetic beads  and by a 3′ digoxigenin modified extremity to a anti-Dig-coated glass surface. The RNA hairpins were attached to the magnetic bead by the 3′ biotinylated extremity and to a glass surface via a digoxigenin modified oligonucleotide handle. For both DNA and RNA substrates, the glass coverslip was treated with anti-digoxigenin antibody (Roche) and passivated with 1 × P Buffer  and 0.2% of BSA (Sigma-Aldrich). The beads were trapped in the magnetic field generated by a pair of magnets located above the reaction chamber. Forces in the pN range were calibrated using the horizontal Brownian motion21We used a PicoTwist magnetic tweezers instrument  annealed to three different complementary RNA to form 5′ ssRNA overhang of 5, 12 and 54 nt . Transcru/(u+b) where u equals the number of unwinding events and b equals the number of binding events.To measure activity we have used the following protocol: any traces shorter than 1,000 s or those presenting no binding or no unwinding were disregarded. From the remaining bead traces, we selected those where the enzyme was seen unwinding the hairpin as in  right frThe streptavidin displacement assay was performed using the helicase domain of Upf1 on a 3′-biotinylated 30-mer RNA substarte bound to a Streptavidin monomer (Promega). The mixture is prepared by mixing 1 nM of the substrate with 60 nm of Upf1-HD enzyme in a F-100 buffer . A preincubation of 5′ was performed before the enzymatic reaction was initiated by adding 2 mM ATP together with 4 μM of free biotin.At defined incubation times, 2 μl of reaction aliquots were mixed with 5 μl of quench buffer  SDS, 25% (w/v) Ficoll-400, 0.05% (w/v) xylene cyanole and 0.05% (w/v) bromophenol blue) in a preset tubes, and immediately loaded on the running gel. Samples were separated by 8% (w/v) polyacrylamide gel containing 0.3% of SDS and detected using a Typhoon 9400 phosphorimaging system and the ImageQuant software .How to cite this article: Fiorini, F. et al. Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities. Nat. Commun. 6:7581 doi: 10.1038/ncomms8581 (2015).Supplementary Figures 1-10 and Supplementary Tables 1-3


One characteristic of natural environments is that outcomes vary across time. Animals need to adapt to these environmental changes and adjust their choices accordingly. In this experiment, we investigated the sensitivity with which rats could detect, and adapt to, multiple changes in the environment. Rats chose between two spouts which delivered 5% sucrose rewards with distinct probabilities. Across three phases, reward probabilities changed in size  and direction (increase or decrease). A discrete trial-structure was used, which allowed the choice process to be decomposed into three distinct response latency measures . We found that a large decrease in reward probabilities rapidly produced the greatest change in choice proportions. The time taken to execute a choice reflected the differences in reward probabilities across the two spouts in some cases, but also reflected training history. By contrast, the amount of time rats spent responding at reward spouts in anticipation of reward consistently reflected the relative likelihood of reward across the two spouts and not the absolute probability of reward. The latency to initiate the subsequent trial reflected choice evaluation. These three response latencies thus indexed key behavioral correlates of the choice process as it unfolds in time. We discuss how this paradigm can be used to assess the corresponding neural correlates of decision-making. How do animals distribute their time and choices across options in order to maximize their procurement of food and other necessities? This is a key question in the context of foraging theory and adaptive decision-making. One fundamental feature of the environment within which animals make such choices is that the likelihood of resource availability changes over time. Hence, it is essential to study how choice behavior adjusts to changes in environmental conditions.Another feature of natural environments is that information about the likelihood of reward is often not directly indicated and, hence, the value associated with one choice over others can only be obtained through the animal's exploration of alternative options. Animals need to first detect the change in outcome contingencies and then identify the corresponding ideal behavior. Such adjustment in choice behavior can be quantified in terms of the degree of adjustment, its optimality and the speed with which it occurs. Here, we trained rats in a discrete trial choice paradigm, and examined how specific aspects of unsignaled changes in choice outcome  differentially impact on the speed and amount of changes in choice allocation. In addition we examined if behavioral latencies pre- and post-choice adjust to changes in the likelihood of the sucrose reward.The impact of changes in reward contingencies on behavior has been examined in studies of contrast effects. The typical observation when animals are presented with two options is that a change in reward availability from one option results in a change in behavior toward the other unchanged option. A classic example involves first presenting pigeons with a red and green light in succession, where each light is associated with equal reward rates. Reynolds  found thIn the current study, rats nose-poked to initiate a trial which consisted in two simultaneous options. These options were associated with reward contingencies that changed across three phases. Critically, the reward likelihood for one option remained unchanged, while the other option either increased or decreased by small or large amounts. We identified changes in choice allocation across the three phases, and further examined changes in pre- and post-choice response latencies. The discrete trial structure used here allowed us to decompose a single choice into three distinct components with which we could examine behavioral latencies as an index of choice behavior. Our previous work has shown that choice execution latencies (duration between leaving the nose-poke and first contact with a reward spout), spout sampling durations  and trial initiation latencies  are sensitive to reward contingencies  has a greater probability of reward compared to the other (Low); however, the absolute reward probabilities for High for one group would be identical to the reward probabilities for Low in another group. Contrast effects would be evident if behavior toward these two spouts is different . However, if behavior reflects absolute value, then behavior toward these two spouts will be identical . The present study additionally examines these contrast effects across more than one change in reward contingency.Subjects were 24 experimentally naïve, adult, male Wistar rats with initial weights of 159–191 g. Rats were housed in groups of four in opaque plastic boxes (22 cm high × 67 cm long × 40 cm wide) in a climate controlled colony room on a 12 h light-dark cycle, where lights were turned on at 7 a.m. All procedures were approved by the Animal Care and Ethics Committee at the University of New South Wales. Rats were given 1 h free access to food and water after each session and body weights were monitored to ensure they did not drop below 85% of their free-feeding weight.Rats were trained in a dark chamber (30 height × 20 width × 50 cm length) with a central nose-poke aperture (4 × 4 cm) located on the front wall of the chamber. Reward spouts were located at a distance of 5 cm on either side of the nose-poke aperture and delivered 5% sucrose solution via pumps located outside the testing chamber. Electrical sensors fitted to the reward spouts detected licking activity at a temporal precision of 1 ms. The experiment was controlled and data recorded using MATLAB . A schematic of the testing apparatus is shown in Figure In phase one, there was a 50% probability of reward on each spout, and an uncollected reward from a previous trial remained available, but did not accumulate. This “holding” of reward meant that in order to obtain the maximum amount of rewards, rats had to alternate between reward spouts because the probability of reward at one reward spout increased with a choice of the alternative spout Figure . This ren = 6) according to the change in reward probabilities scheduled in phase two  in an attempt to ensure that rats were at similar level of performance before moving on to the next phase. An additional constraint was that each phase had a maximum length of five sessions in the event that rats failed to reach criteria levels of performance. The choice of five sessions was based on our previous work where we found that rats made behavioral adjustments within 5 sessions following contingency reversal .Rats were presented with 50–50 Hold reward probabilities for a maximum of five sessions, or until they reached criterion of alternating between reward spouts on more than 75% of trials in a session. At this stage, the experimental contingencies were identical for all rats.Rats were presented with the reward probabilities that correspond to their experimental group identity . In addition to choice allocation, we focus on three response latency measures which our previous work has shown to be sensitive to reward contingencies . Supplementary figure The analysis of choice proportions revealed that changes could occur rapidly within one session. Therefore, to quantify the changes in behavioral latencies in greater detail across the three phases, we first divided individual sessions into bins of 50 trials. Latencies for each bin were calculated separately for High and Low. Each rat's High and Low bin latency was then expressed as a proportion of its own baseline . Hence, phase two latencies are expressed relative to phase one baseline, and phase three latencies are expressed relative to phase two baseline. Previous phase baseline was calculated using spout sampling durations for both High and Low as this represented the experience of the rats, where reward probabilities were equivalent across the two spouts at phase one and three. Group means for High and Low proportion latencies are shown in p-values. When comparing between trial initiation intervals after different trial outcomes at the same reward spout, randomized distributions were computed based on trial outcome (rewarded versus unrewarded). We also calculated the effect sizes of the differences between latencies using Cohen's d  which was used to determine significance using the randomization test. We plot group mean latencies separately for High and Low in Figures 5, 6 because this enables visualization of the source of the differences in means (increase in High from baseline versus decrease in Low from baseline), which is not captured by plotting the group mean difference in latencies.To determine whether the difference between High and Low proportion latencies were statistically significant within groups, we carried out a randomization test. High and Low proportion latencies were combined and shuffled. The mean of the first half of this shuffled sample was then subtracted from the mean of the second half of the shuffled sample. This was repeated 1000 times to generate a distribution of the difference in proportion latencies when spout identity (High versus Low) was disregarded. This test identifies the differences in proportion latencies which arise specifically from the differences in spout identity without making assumptions about the underlying distribution of proportion latencies. This randomization test was carried out separately for each group. Significance was determined by comparing the observed difference in group means with the 5th–95th percentile of the randomized distribution and calculating two-tailed d Cohen,  in ordern = 1; group +15: n = 1; group −15: n = 3; group −30: n = 5; Figure n = 5; group +15: n = 6; group −15: n = 3; group −30: n = 5; Figure Following the change in High/Low reward ratios at phase two, all groups increased choices to High. Group −30 showed the largest increase in both High choice Figure  and the n = 3; group +15: n = 1; group −15: n = 3; flat horizontal lines in Figure The pattern of changes in choice allocation between phases was further examined for each rat within phases two and three. Figure Since the changes in reward probabilities were reflected in changes in choice proportions, we sought to identify similar changes in other indices of choice behavior. A striking pattern was that spout sampling durations were longer for High than Low during phase two only, reflecting the change in relative reward probabilities between the two spouts across phases Figures . This wad = 0.42; group −15: d = 0.20; group −30: d = 0.25), while phase three effect sizes were greatest for group −15 .In order to quantify changes in behavioral latencies that result from contingency changes, we calculated the amount of change by each rat from its baseline . Figure p = 0.06). Effect sizes indicate that these differential spout sampling durations were greatest for groups −15 and −30 . The comparison of spout sampling durations with baseline also show that the differences in spout sampling durations were due to a decrease in the Low spout sampling durations from phase one rather than an increase in High spout sampling durations: Low proportion latencies were all less than 1, while High proportion latencies were close to 1 . These differences were due to an increase in spout sampling durations on Low, while High spout sampling durations remained close to baseline . These changes again reflected the current contingencies in that the increase in spout sampling time on the Low side mirrors the increase in reward probabilities for −15 and −30 groups and the increase in the relative value of the Low side for +15 and +30 groups.In phase three, consistent differential sampling times were again apparent Figure , right. d = 0.30; group +15: d = 0.33; group −15: d = 0.22; group −30: d = 0.45). There were no latency differences to initiate a new trial following unrewarded trials  for identical trial types at phase two showed that all groups were significantly slower to initiate a new trial subsequent to receiving a reward from Low compared to one from High . The corresponding comparison on the Low spout .Figure d = 0.3), while for the Low reward spout comparison between rewarded and unrewarded choices were significant for all groups except +30; effect size for group +15 was largest .In phase three Figure , right, d = 0.56; group −15: d = 0.24), groups +15 and −30 showed the opposite pattern . This may reflect individual differences between rats as to the temporal location of the decision point. For some rats, the decision as to which spout to respond next might have been formulated prior to nose-poke entry, while other rats might have come to such a decision upon seeing the go-signal.There were inconsistent patterns in the difference between High and Low choice execution latencies during phase three expressed as a proportion of phase one baseline. While some groups showed longer latencies for choice execution for High compared to Low  and for groups +15 and −30 for the comparison between unrewarded trials . The corresponding comparison of different trial outcomes for the same spout showed that all groups took longer to initiate a new trial after unrewarded trials. This was significant for groups +30 and −30 for the High spout comparison  and for groups +15 and −30 for the Low spout comparison .The comparison of trial initiation latencies for the same outcome but different spout showed that all groups took longer to initiate a trial after having visited the Low side, regardless of outcome  against which the reward probability manipulations in phase two could be assessed, where we inquired how quickly rats adopted the new optimal strategy. In phase two, different groups were exposed to a large or small increase or decrease in reward probability associated with one of the reward spouts, while probability of reward on the other spout remained unchanged at 50% for the entire experiment. In phase three, reward probabilities were returned to the initial phase one contingencies to identify how quickly rats reverted to the original optimal strategy of alternating. In addition to identifying the adjustment in choice allocation in response to phase contingencies, we examined adjustments in the latencies of pre- and post-choice behaviors. These latencies mirrored changes in choice allocation, but also reflected other aspects of the choice process not captured by choice allocation.The most direct measure of perceived changes in reward probability at phase two was choice allocation. In terms of criterion performance, group −30 appeared to be the most sensitive, with half of the group reaching criterion within the third session and all but one rat reaching criterion at the end of five sessions. The corresponding size of reward probability change experienced by group +30 did not result in comparable criterion performance. A change-point algorithm  led to longer trial initiation latencies. This window between trial outcome and initiation of a new choice might be a period of choice evaluation, when the immediate outcome is integrated with existing representations of environmental contingencies to guide upcoming choices. Hence, following surprising (unlikely) events, longer trial initiation latencies may reflect the updating of choice-outcome associations. Further, the differences in trial initiation latencies between the two outcomes were only evident for the spout which had been associated with new contingencies in phase two (High for + groups and Low for − groups). This is consistent with the suggestion that these latencies reflect choice evaluation, as outcomes from the unchanged spout were expected and did not require updating.It is interesting that we observed differences in behavior between the changed versus unchanged spout in this choice evaluation period, when spout sampling durations instead reflected the High-Low difference in spout status. It is unlikely that the differences in trial initiation latencies between the two different outcomes simply reflect the time taken for reward consumption for two reasons. Firstly, as noted, the differences in trial initiation latencies between rewarded and unrewarded trials were only evident for one of the spouts; reward consumption effects would be evident for both spouts. Secondly, trial initiation latencies after rewarded trials at Low were longer than trial initiation latencies after rewarded trials at High. Since the reward was identical in size and concentration between the two spouts, the differences in latencies cannot be due to differences in the times taken to consume the sucrose.The level of surprise associated with choice outcomes is a critical aspect of choice evaluation. Models of decision-making emphasize that learning to make adaptive choices is mediated by the ability to adjust our predictions of choice outcomes in line with experienced choice outcomes  and post-choice discrete periods  index the different stages of decision-making. This is an ideal way in which the role of brain regions which have been argued to be critical for certain aspects of decision-making can be assessed. For example, studies have argued that the ventral striatum is important for choice outcome evaluation (Cardinal et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila melanogaster strains, here we identify 545 editing QTLs (edQTLs) associated with differences in RNA editing. We demonstrate that many edQTLs can act through changes in the local secondary structure for edited dsRNAs. Furthermore, we find that edQTLs located outside of the edited dsRNA duplex are enriched in secondary structure, suggesting that distal dsRNA structure beyond the editing site duplex affects RNA editing efficiency. Our work will facilitate the understanding of the cis-regulatory code of RNA editing.Adenosine-to-inosine (A-to-I) RNA editing, catalysed by ADAR enzymes conserved in metazoans, plays an important role in neurological functions. Although the fine-tuning mechanism provided by A-to-I RNA editing is important, the underlying rules governing ADAR substrate recognition are not well understood. We apply a quantitative trait loci (QTL) mapping approach to identify genetic variants associated with variability in RNA editing. With very accurate measurement of RNA editing levels at 789 sites in 131  Adenosine-to-inosine (A-to-I) RNA editing plays an important role in neurological functions. Here, by a quantitative trait loci (QTL) mapping approach in 131 Drosophila melanogaster strains, the authors identify 545 QTLs associated with differences in RNA editing. Drosophila melanogaster (D. melanogaster), with over 5,000 RNA editing sites identified234Drosophila, knockout of the ADAR gene results in severe neurological phenotypes including impaired locomotion, defective flight and male mating difficultiesRNA editing is the modification of RNA nucleotides from their genome-encoded sequence. The most common type of RNA editing in metazoans is the deamination of adenosine into inosine catalysed by the adenosine deaminase acting on RNA (ADAR) family of enzymesAlu repeats are promiscuously editedcis-acting regulatory elements) surrounding the editing site guide the preference and selectivity of ADARs. ADAR has a preferred sequence motif, in particular the 5′ and 3′ nearest neighbouring positions to the editing site11121215A-to-I editing occurs cotranscriptionally in the nucleus when double-stranded RNA (dsRNA) is formed at the pre-mRNA level, which is subsequently bound and edited by ADARs1D. melanogaster and identify genetic variants that are associated with changes in editing levels. Using these RNA editing QTL (edQTL), we examine how changes in RNA secondary structure induced by genetic variants affects RNA-editing levels.Quantitative trait loci (QTL) mapping in natural populations is a strategy that has been used to successfully study the regulatory architecture of many molecular phenotypes such as gene expression (eQTLs)1719D. melanogaster, we quantified RNA editing levels in replicate from male whole bodies in 131 strains of the Drosophila Genetic Reference Panel (DGRP) using mmPCR-seq, an efficient method we recently developedTo study natural variation of RNA editing in de novo identification of RNA editing sites in each sample, we identified 1,202 novel A-to-I RNA editing sites with an estimated false discovery rate of <2%  for the top associated variant and found an abundance of very low P values  and found ECSs for 641 editing sites . We defiosophila . We obseosophila . This suWe characterized the properties of editing substrates . The proWe used the ECS predictions to characterize how edQTLs affect structures of RNA duplexes containing editing sites. Of the 545 total edQTLs identified, we were able to predict ECS locations for 276 of their associated editing sites. Of these 276 edQTLs, 45 lie within the edited dsRNA structure, either in the ECS or in the sequence surrounding the editing site that base pairs with the ECS. We also identified a set of 100 control variants that are not associated with editing level changes within the edited dsRNA structures (Methods section).We looked for structural features that differ between the edQTLs and control variants within the edited dsRNAs. We restricted our analysis to 27 out of the 45 edQTLs that had an effect size of 0.025 or greater (5% or greater difference in editing levels between the two homozygotes), because we did not expect to see major differences induced in RNA structure by edQTLs with very low effect sizes. We hypothesized that dsRNA stability may influence the editing efficiency of ADAR to its target substrates. To test this hypothesis, we looked at two RNA features, effect on base pairing and duplex free energy. We noticed that QTL variants were more likely than control variants to be affecting nucleotides that are base paired . Base paThe majority of edQTLs, 213 (77%), lie outside of the edited dsRNA substrate. Not surprisingly, these ‘distal' edQTLs have smaller effect sizes than edQTLs within the edited dsRNA . However15cis-regulatory architecture of RNA editing is largely unexplored. The mechanisms targeting ADAR proteins to specific adenosines in an imperfect dsRNA are not well characterized, especially in vivo. In this study, we quantified RNA editing in natural strains of D. melanogaster using mmPCR-seq and used these editing level measurements to identify genetic variants associated with the differences in editing levels between strains. These edQTLs allowed us to identify structural features within the edited dsRNA duplex important for ADAR efficiency. In addition, distal edQTLs located outside of the primary dsRNA duplex guided us to locate secondary dsRNA stems that modulate editing.The We utilized mmPCR-seq to overcome the inherent biases of RNA-seq towards highly expressed genes. Using mmPCR-seq, we can efficiently capture and sequence up to 605 different loci from 48 different samples on a single microfluidic chip. We ran all PCR reactions to saturation, which provides uniform capturing efficiencies for the majority of targeted lociRNA secondary structure is an important determinant of RNA editing specificity232425Previous reports have implicated secondary dsRNA elements that influence editing at a nearby dsRNA15We anticipate the application of edQTL mapping to human RNA editing. Human genome sequencing has identified many disease-associated variants, but their functional interpretation is challenging. Dysregulation of RNA editing has been implicated in a myriad of human diseases such as amyotrophic lateral sclerosis (ALS)Fly stocks were reared at 25 °C. RNA was extracted from whole bodies of 3–5-day-old adult males for 131 DGRPWe quantified RNA editing at 605 loci using a multiplex microfluidic PCR with deep sequencing method developed in our labD. melanogaster RNA editing sitesPaired-end reads were combined and mapped onto the genome dm3) using BWA samse allowing 9 mismatches per read using BWFor QTL mapping, we examined 789 RNA editing sites with editing level measurements in at least 35 strains. For each of the 789 RNA editing sites we normalized the editing level measurements. First, we centred and scaled each measurement by subtracting out the mean editing level value and dividing by the s.d. Then we quantile normalized the distribution to fit a standard normal distribution.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 PlinkP value (Pmin) across all variants tested for that particular editing site. (3) We repeat steps (1) and (2) for 10,000 permutations of the genotype sample labels and obtain 10,000 null P values . (4) We estimate an empirical P value for the most significant variant by determining where pmin lies within the null distribution . QTLs were called at FDRs of 10 and 5%, which were determined using the qvalue softwareThe following protocol was performed to map edQTLs in cisD. melanogaster yellow white (yw) strain nascent RNA-seq and ADAR null mutant nascent RNA-seq from NCBI SRA (GSE37232)We obtained I SRA GSE23237 to D. melanogaster flies. We used the same primers to amplify both cDNA and DNA: chr2L_2784071 5′- GAGGAATTTGCTTGCTGTGG -3′ and 5′- TACCCAAATGCCAACACAGA -3′, chr3L_4431719 5′- AGGATAACCCGGTCACACAC -3′ and 5′- GAACCGCTCGATTGTGGTAT -3′, chr3L_11546420 5′- TATTGACGACGACCTGCAAC -3′ and 5′- CCACTTTGCCGTGTTCTCTT -3′. For both RNA and DNA samples, we performed PCR using the KAPA SYBR Fast qPCR Kit (Kapa Biosystems) and the following protocol: initial enzyme activation at 95 °C for 3 min and 40 cycles of 95 °C for 3 s and 60 °C for 50 s. For RNA samples, we performed ribosomal RNA depletionWe validated three randomly chosen intronic RNA editing sites by Sanger sequencing of RNA and DNA from heads of adult yw To predict proximal ECSs, we predicted the secondary structure of the region within 200 bp of each editing site using the programs partition, MaxExpect, and ct2dot from the RNAStructure packageD. melanogaster, we first identified conserved intronic regions as candidates. We smoothed phastCons scores using a sliding window of 51 bp . For each single nucleotide variant (edQTLs and controls) we used the Fold and ct2dot programs from RNAstructureTo compare against edQTLs in edited dsRNAs, we identified a set of control variants in edited dsRNAs that do not affect editing levels. This set of 100 control variants consists of all variants that were not edQTLs and were not in linkage with an edQTL (R2≤0.05).To predict dsRNA stems around distal QTLs and matched controls, as with the ECS predictions, we predicted the secondary structure of the region within 200 bp of each variant using the programs partition, MaxExpect, and ct2dot from the RNAStructure packageAccession codes: The mmPCR-seq data was deposited to Gene Expression Omnibus at the National Center for Biotechnical Information under the accession number GSE67082.How to cite this article: Ramaswami, G. et al. Genetic mapping uncovers cis-regulatory landscape of RNA editing. Nat. Commun. 6:8194 doi: 10.1038/ncomms9194 (2015).Supplementary Figures 1-12, Supplementary Tables 1-2 and Supplementary ReferencesmmPCR-seq primer sequencesRNA editing QTLsnovel intronic RNA editing sitesediting complementary sequence predictions


Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3′ terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging. Satellite tobacco mosaic virus (STMV) was originally found in association with its helper virus (HV) Tobacco mosaic virus (TMV strain U5) in natural infections of tree tobacco (Nicotiana glauca)Tobacco mild green mosaic virus. Other strains of TMV such as U1 and U2 also have been shown to support the replication of STMV22. The 3D structure of STMV has been determined at 1.8 ÅA hallmark feature of STMV is that it is the only satellite virus, whose HV is a rod-shaped tobamovirus. Additionally, STMV stands out among other spherical viruses because of the high degree of order adopted by the RNA within the capsid7Agrobacterium-based transient expression system (agroinfiltration) we recently reported several interesting properties of STMV when expressed in the presence and absence of its HV-RdRpUsing an Agrobacterium-mediated transient expression system (agroinfiltration) developed recently for STMV in our lab162AUG164 → 162UAG164)  ; (ii) ag2UAG164)  and (iii2UAG164) .N. benthamiana leaves. Plants co-infiltrated with pSTMV and pRP served as positive controls. At five days post infiltration (dpi), total RNA and protein preparations were isolated and subjected respectively to duplicate Northern blot hybridization to detect progeny (+) and (−)-RNA and Western blot analysis. Duplicate Northern blots probed for assessing the affect of CP on the accumulation of progeny (+) and (−)-strands are shown in Each of the above-mentioned three agrotransformants was mixed with agrocultures of pRP to provide the HV-RdRp  and was N. benthamianaRecently, we demonstrated that production of a truncated form of STMV RNA is a hallmark feature associated with STMV replication in N. benthamiana leaves. Plants co-infiltrated with pSTMV and pRP served as positive controls while those infiltrated only with empty vector served as negative controls. At 4 dpi, virions were purified, negatively stained and examined by Transmission Electron Microscopy (TEM). Icosahedral virions of 18 nm characteristic of STMV were recovered from leaves infiltrated with pSTMV and pRP .Results of RT-PCR analysis evaluating the production of STMV Δ150 are shown in Next we wanted to evaluate the replication and packaging phenotypes of full-length STMV CP bearing each of the five N-terminal alanine substitution variants . ConsequNorthern blot hybridization results on the effect of the five-alanine mutations on STMV replication are shown in Results shown in N. benthamiana leaves. N. benthamianaN. benthamiana plants mechanically inoculated with virions of HV and each of the four CP variants  were competent to translate higher levels of CP than wt. Among the four variants, CP production was more prominent with variant 7A than others . By contN. benthamiana plants was infiltrated to co-express HV-RdRp . Plants co-infiltrated with P19 and either pSTMV or 13aac-FLAGserved as controls. At 4 dpi, total protein was extracted from infiltrated leaves and incubated with anti-126 kDa antibodies, followed by the precipitation of the complex with protein G-agarose beads and Western blotting with anti-126 kDa, CP or Anti-FLAG antibodies. Results are shown in Although replication of STMV depends on HV-RdRp, results shown in in vivo  and 32P-labeled STMV RNA as a binding probe. Interaction between STMV RNA and CPs of wt, variants 5A and 7A, but not with either CPKO or variant 3A, was detectable [Northern blot results , 3, 4 dein vivo , a NorthAlfalfa mosaic virus (AMV)1311171420Viral replication is a complex process involving numerous macromolecular interactionsOur results exemplify that STMV CP , specifiCo-expression of the wt 13aa  along wiAnother interesting outcome of our study is the intrinsic role played by a positively charged residue at position 3 of the N-terminal 13aa motif. For example, substitution of an alanine residue for an arginine at position 3  along with the full length RNA (9). Results of this study revealed that production of ∆150 is linked to HV-dependent replication since it was not detected in plants infiltrated with replication defective variants of STMV  and it is 0.3 electron/nm2 for the wt capsid. The solid black curve shows the free energy of RNA encapsulated by wt proteins while the dashed red curves correspond to that by the mutated coat proteins. Blue dots and orange squares respectively represent the encapsidation free energy of STMVΔ150 (908 nt) and wt RNA (1058 nt). As illustrated in the figure, in the case of the mutated capsid, the encapsidation free energy of STMVΔ150 RNA is significantly lower than that of wt RNA (14.28kBT) leading to the exclusive encapsulation of STMVΔ150 RNA. However, in the case of wt CP, the encapsulation free energy of STMVΔ150 RNA is lower than that of wt RNA by only1.47kBT, explaining why both STMVΔ150 and wt RNA can be encapsidated by wt CP. As a matter of fact, while the ratio of free STMVΔ150 to wt RNA is very low (24:76)Data shown in STMVΔ150 08 nt andAs noted above, electrostatics clearly explains the results corresponding to variants 5A, 7A, 10A and 11A presented in GCAGGTAAGGTTAAACC3′, 5′ GCTATGGGGAGAGGTGCAGTTAAACC3′,for 5K → A 5′ GGGGAGAGGTAAGGTTGCACCAAACC 3′; for7K → A 5′ GGTAAGGTTAAACCAAACGCAAAATCGACG 3′; FOR 10R → A 5′ GGTTAAACCAAAC CGTGCATCGACG 3′; 5′ for CPKO, 5′CTGTTTCCAGCTTAGGGGAGAGGTTAGGTTAAACC3′;for 13aa, 5′CGTAAATCGACGTAGGACAATTCGAATG 3′; for 13aa/A5′ GCTATGGGGGCAGGTAAGGTTAAACC 3′; 13aa/B 5′ CCAGCTATGGGGAGAGCAGCAGCAGCAGCAGCAGCAGCATCGACGTAGGACAATTCG 3′) and a commonly shared reverse primer . The resulting PCR products were gel purified and used as mega primers in a PCR reaction. A region spanning the sequence of pSTMV from nt768–614 was amplified using a forward primer  and each mega primer for the respective variant prepared above. The resulting PCR products were digested with HindIII and SalI and sub-cloned into a similarly treated pSTMV. The nature of all recombinant clones was verified by DNA sequencing. Transformation of wild type and variant pSTMV agroconstructs to Agrobacterium strain GV3101 followed by infiltration into the abaxial side of the fully expanded N. benthamiana leaves was as described previouslyThe construction and characteristic features of an agro-construct of STMV pSTMV;  have recN. bethamiana with STMV variant virions in the presence of TMV U5, isolation of total RNA from agroinfiltrated leaves, purification of STMV virions, and Northern blot analyses are as described previously933Procedures used for mechanical inoculation of E. coli poly A polymerase according to the manufacturer’s protocol (NEB). Following inactivation of poly A polymerase by heat (95 °C for 2 to 3 minutes), first strand cDNA was synthesized using a reverse primer  and M-MulV reverse transcriptase (New England Biolabs). The resulting product was subjected to PCR using a forward primer (5′ TACGTAAACTTACCAATCAAAAG 3′) and a reverse primer . PCR products were analyzed by agarose gel (1%) electrophoresis then visualized and photographed under UV light following ethidium bromide staining.Virion RNA from wild type and CP variants was subjected to Poly–A tailing using N. benthamiana leaves as described previouslyTotal protein extracts were prepared from healthy and agroinfiltrated N. benthamiana leaves were co-infiltrated with agrotransformants of STMV wt or CP variants 3A or 5A or 7A and TBSV P19, a suppressor of RNA silencingin vitro translation kit (Promega Corporation) and subjected to Western, Northwestern and Far-Northwestern blot analyses.32P-STMV RNA for 1 h at room temperature in re-naturing buffer containing 2 mg/ml of yeast tRNA. The membrane was washed twice with re-naturing buffer at room temperature to remove any unbound RNA, followed by autoradiography. For Far-Northwestern analysis32P-labelled STMV RNA transcripts and in vitro translated STMV CP.Western blot analysis was performed as described previouslyHow to cite this article: Sivanandam, V. et al. Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging. Sci. Rep.6, 26328; doi: 10.1038/srep26328 (2016).


Multi-subunit RNA polymerases are the enzymes that perform transcription in all living organisms and that have emerged before the divergence of domains of life. The structures of catalytic cores and their functions during elongation step of transcription cycle are very similar for all multi-subunit RNA polymerases. In contrast, the mechanisms for terminating the RNA synthesis have seemingly diverged in modern RNA polymerases. However, the recent finding that, much like during bacterial transcription, RNA secondary structure is involved in termination by eukaryotic RNA polymerase III (pol III), suggests that RNA-dependent termination may have emerged before the divergence of bacterial and archaeal/eukaryotic RNA polymerases. In the case of pol III, the terminating RNA secondary structures are not dedicated hairpins, but are formed by the bodies of highly structured transcripts, which are clearly the remnants from the RNA–protein world. Here I discuss the similarities and differences of RNA-dependent mechanisms of termination of transcription by bacterial RNA polymerase and pol III. Termination is an obligatory event that causes extremely stable transcription elongation complex to disassemble at the end of the gene with the release of RNA polymerase (RNAP) and the transcript from the template DNA. Termination is required for proper expression of neighboring genes, maturation and export of transcripts (in eukaryotes), recycling of RNAP, and clearing the template for subsequent transcription. Though mechanisms involved in transcription elongation are very similar for bacterial RNAP, archaeal RNAP and pol I, pol II, and pol III from eukaryotes (plant-specific RNAPs IV and V are not discussed here), the mechanisms of transcription termination seem to be strikingly different (see below). However, recently, we showed that, similarly to termination in bacteria, termination by eukaryotic pol III involves formation of RNA secondary structure,,In bacteria, destruction of the elongation complex during termination is facilitated by a dedicated > 7 base pairs-long G:C-rich RNA hairpin that folds behind RNAP.,,,Despite this apparent difference, the mechanisms of termination between bacterial RNAP and pol III appear to be very similar. As seen from ,,The ubiquitous hairpins that form during elongation cannot destruct the actively transcribing elongation complex.S. cerevisiae).Another unusual property of termination of pol III transcription is the insensitivity to the RNA–DNA hybrid sequence. Backtracking on the oligoT signal can shift pol III backward as far as ~12 bp, replacing the U:A-rich hybrid in the elongation complex with a sequence that precedes it in the gene.,Based on the structural similarities between the mechanisms of termination by bacterial RNAP and eukaryotic pol III, the requirement for the RNA stem for destruction of the elongation complex and the distance between this stem and the RNAP active center, it can be hypothesized that the RNA stem-dependent termination of transcription emerged before divergence of bacteria and archaea/eukaryotes. Pol II was shown to be able to terminate transcription in RNA hairpin-dependent manner in vitro,,Though the RNA stem-dependent termination may have been the ancestral mechanism, it apparently has been supplemented and/or displaced by protein-dependent mechanisms after the divergence of multi-subunit RNAPs. Bacteria, in addition to the hairpin-dependent intrinsic termination, acquired protein-mediated termination that uses an RNA helicase ρ or a DNA translocase Mfd to destruct the elongation complex. Pol I and Pol II have acquired mechanisms involving RNA exonucleases and/or an RNA helicase,


How could modern life have evolved? The answer to that question still remains unclear. However, evidence is growing that, since the origin of life, RNA could have played an important role throughout evolution, right up to the development of complex organisms and even highly sophisticated features such as human cognition. RNA mediated RNA-aminoacylation can be seen as a first landmark on the path from the RNA world to modern DNA- and protein-based life. Likewise, the generation of the RNA modifications that can be found in various RNA species today may already have started in the RNA world, where such modifications most likely entailed functional advantages. This association of modification patterns with functional features was apparently maintained throughout the further course of evolution, and particularly tRNAs can now be seen as paradigms for the developing interdependence between structure, modification and function. It is in this spirit that this review highlights important stepping stones of the development of (t)RNAs and their modifications (including aminoacylation) from the ancient RNA world up until their present role in the development and maintenance of human cognition. The latter can be seen as a high point of evolution at its present stage, and the susceptibility of cognitive features to even small alterations in the proper structure and functioning of tRNAs underscores the evolutionary relevance of this RNA species. In modern life forms, RNA is involved in a number of cellular activities. Three classes of RNA molecules, mRNA, tRNA and rRNA, have been known for a long time as players in the expression of genetic information: mRNA as the messenger that conveys information from the DNA in the nucleus to the site of protein synthesis in the cytoplasm, tRNA as the carrier of specific amino acids and rRNA as essential constituent of the ribosome, where proteins are made. Over the past three decades, the picture of RNA being just the minion of gene expression has dramatically changed. A large number of small and long non-coding RNAs have been discovered to act as catalysts (ribozymes), as modulators of molecular interactions in the cell, and most importantly, as regulators of gene expression. The functional diversity of RNA and the many RNAs involved in major cellular processes support the idea of the existence of an RNA world at the origin of life, where RNA was the primary living substance ,2. The hStill, at some point the world of RNA must have proceeded towards higher complexity, integrating other molecular entities for storage of genetic information and gene expression. Taking a closer look at what we know today as tRNA allows us to hypothesize that transfer of amino acids onto RNA as well as incorporation of modified nucleotides in RNA (or post-synthetic modification) may have been processes that marked the rise of higher structural, and consequently functional complexity. Indeed, several papers discuss the ability of RNA to self-aminoacylate as a key event at the transition from the RNA world to DNA/protein based life, and it has been suggested that modern protein synthesis may have evolved from a set of aminoacyl transfer reactions catalyzed by ribozymes ,18,19,20Focusing on tRNA development and modification, this review aims to highlight important stepping-stones in the fate of tRNAs from their origin in ancient RNA world organisms to their role in human brain development and function.in vitro selection, the first one being a small RNA that rapidly aminoacylates its own terminal 2′- or 3′-OH group with aminoacyl adenylate (aa-AMP) as activated donor [trans, thus behaving like a true enzyme, and moreover, to catalyze the formation of peptides up to a length of three amino acids [The RNA world hypothesis describes a period in the early history of life, where RNA  was the carrier of genetic information as well as the supporter of metabolic transformations ,2. As sued donor . Other red donor ,14,15. Ted donor , therebyed donor . This sced donor ,17,18,19no acids .An early translation system may have used ribozymes for aminoacylation of tRNAs or tRNA-like molecules. tRNA-like molecules as precursors of modern tRNA may have consisted for example of a short aminoacyl-acceptor stem; accordingly, a minihelix-loop RNA was suggested as primordial analog of tRNA . NumerouYet another model, the genomic tag hypothesis, suggests that minihelix RNAs containing the anticodon may have acted as tags to mark already existing ssRNA genomes for replication ,40. Thisin vitro evolved precursor tRNA, consisting of a catalytic 5′-leader sequence and an aminoacyl-acceptor tRNA, selectively self-charges an amino acid onto its 3′-terminus. Remarkably, this cis-active ribozyme was shown to be a substrate for RNaseP RNA to cleave the 5′-leader segment and thus to generate the mature tRNA [Nevertheless, there is also the possibility that aminoacylation catalyzed by ribozymes also occurred on more complex structures other than minihelix RNAs. In 2001, Suga and coworkers, for example, demonstrated that an ure tRNA . Moreoveure tRNA .N-biotinyl-N′-iodoacetyl-ethylendiamine (BIE) and to catalyze formation of a carbon-nitrogen bond . Interestingly, the ribozyme shows a tRNA-like structure, leaving space for the assumption that tRNAs might have modified themselves . Inspired by this RNA self-modification, Sharma and co-workers generated another self-alkylating ribozyme that is able to covalently link a fluorophore to RNA [Taking a closer look at tRNAs, one can observe that they contain a large spectrum of chemical modifications, which are necessary for correct tRNA folding and function. Modifications in particular of tRNAs and rRNAs are not unusual. According to the RNA modification database, there are 112 RNA modifications that are currently known . Thus, fe to RNA   [trans-acting ribozyme (96 nt) shows reaction rates of 0.16 min−1 under optimal conditions, which is in the typical range of ribozyme reaction rates. Moretti and Müller postulate that such a ribozyme could have been of significance for RNA world organisms, since RNA 5′-triphosphates could have been used for RNA polymerization [Still, RNA modifications did not necessarily occur in a single step. Looking at protein catalysts, many enzymes first activate a compound to let it react in the second step. Mostly, ATP is used as chemical energy for this activation step. Possibly, ribozymes have also used triphosphates as activating reagent. Moretti and Müller report on a ribozyme obtained by te (TMP) . The relrization ,48, RNA–rization ,51,52,53rization , RNA caprization ,56, and rization . Possibl4SUMP) [et al. isolated a purine synthase ribozyme that can synthesize tethered 6-thioguanosine-5′-monophosphates (6SGMP) [6SGMP as substrates [In modern metabolism, nearly all pathways use the activated ribose 5-phosphoribosyl-1-pyrophosphate (PRPP) as substrate for nucleotide synthesis. In 1998 Unrau and Bartel selected a PRPP-dependent pyrimidine nucleotide synthase ribozyme capable of synthesizing tethered 4-thiouridine-5′-monophosphate (4SUMP) , and six (6SGMP) . Interesbstrates  , backbone modified (phosphorothioate substitutions) or base modified (2-aminopurine or 4-thiouridine) segments into RNA. Another sophisticated RNA modifying strategy makes use of twin ribozymes [An interesting ribozyme-based way of incorporation of modified nucleotides into a RNA strand was reported by Mutschler and Holliger, who used the hairpin ribozyme as a non-canonical 3′-5′ nucleotidyl transferase . That wasplicing . The sitibozymes ,68,69. Tibozymes  leading i.e., the global analysis of the genome, transcriptome, proteome, etc.) it has also become possible to perform global mapping of methylation sites such as N6-methyladenosine   and e, e.g., ,74) and e.g., by ). This as  during et al. ), rangine, e.g., ). Moreove, e.g., ,85). Suce, e.g., ). Anotheactivity ,88.5C RNA methyltransferases known, which are comparatively well described, and which also play a role in brain development: DNMT2 and NSUN2 [Asp [Dnmt2 and Nsun2 in mice revealed complementary target-site specificities for the two as well as a complete loss of cytosine-C5 methylation of tRNAs, while the phenotype included impaired thickness and a reduced level of organization in the cerebral cortex [By the same token, correct RNA-methylation seems to be essential for proper functioning of the brain. In higher eukaryotes, there are presently at least two prominent mnd NSUN2 ,90. The nd NSUN2 . In miceUN2 [Asp . Loss ofUN2 [Asp  and doubl cortex .5C at cytosine 34 (Leu (CAA) [5C RNA modification in a variety of contexts, including tissue development, differentiation and cellular signaling  . It can eu (CAA) . Findingg -domain proteins  at position 26 (FTSJ1 [Escherichia coli (E. coli) heat shock protein FtsJ, which has been found to form a complex with S-adenosylmethionine (SAM or AdoMet) and to possess a methyltransferase fold [E. coli 23S rRNA at position U2552, with S-adenosylmethionine as methyl-group donor  [FTSJ1 mutations show an almost complete lack of Cm32 and Gm34 as well as reduced peroxywybutosine (o2yW37)  is a methyltransferase that dimethylates guanosines . U2552 r . Eukarytoplasm) ,118,119.(o2yW37)  in tRNAP37) (Phe .The evolution of aminoacyl–RNAs from the RNA world at the origin of life to what we know today as tRNA is reflected in two characteristic features: aminoacylation and modification. Once being just a prerequisite for gain of functionality and a selection advantage, aminoacylation of tRNA in modern biochemistry is highly specific, and involves specialized enzymes, aminoacyl tRNA synthetases. Correct aminoacylation ensures proper decoding of the charged tRNA in the ribosome and translation into protein according to the genetic code. To the same extent, modification of tRNAs is directly linked to accurate and efficient translation of the genetic code, as it affects the correct charging of the tRNA by the cognate aminoacyl tRNA synthetase, decoding, tRNA folding and stability. Subtle changes in the modification state of tRNAs may have drastic effects: inaccurate decoding, improper folding and loss of function or decay by various degradation pathways. Thus, RNA modification in general and tRNA modification in particular are highly complex and fine-tuned processes of tremendous significance for life. Not least with the findings from the Encode project , it is b