While high‐density DNA microarrays have been available for over three decades, the synthesis of equivalent RNA microarrays has proven intractable until now. Herein we describe the first in situ synthesis of mixed‐based, high‐density RNA microarrays using photolithography and light‐sensitive RNA phosphoramidites. With coupling efficiencies comparable to those of DNA monomers, RNA oligonucleotides at least 30 nucleotides long can now efficiently be prepared using modified phosphoramidite chemistry. A two‐step deprotection route unmasks the phosphodiester, the exocyclic amines and the 2′ hydroxyl. Hybridization and enzymatic assays validate the quality and the identity of the surface‐bound RNA. We show that high‐density is feasible by synthesizing a complex RNA permutation library with 262144 unique sequences. We also introduce DNA/RNA chimeric microarrays and explore their applications by mapping the sequence specificity of RNase HII. High‐density DNA microarrays refer to extensive libraries of oligonucleotide sequences immobilized onto a surface.The development of RNA microarrays was limited for a long time to the spotting of pre‐synthesized RNA strands.O‐silyl protected nucleosides,O‐ALE ribonucleosides, which were then transformed into their corresponding phosphoramidites . The arrays were scanned immediately after synthesis. The stepwise coupling yields were found to range between 99 %  and 99.9 % , similar to the best results typically obtained in DNA synthesis  step. Next, the oligonucleotides were hybridized to their Cy3‐labelled DNA complement and showed similar hybridization intensities . After synthesis, the microarrays are deprotected in a stepwise manner. First, the cyanoethyl protecting groups of the phosphodiester bonds are removed in EtTo confirm the identity of the 25mer DNA and RNA oligonucleotides in Figure Finally, not only can DNA and RNA synthesis be carried out in parallel, RNA phosphoramidites can also be incorporated within a DNA oligonucleotide. For instance, the above‐mentioned DNA 25mer substituted with rU units at every dT position (six incorporations) correctly hybridized . The slightly weaker fluorescence signals for the rU‐modified 25mer relative to the pure DNA sequence may be attributed to multiple A‐ and B‐form helical junctions within the DNA/RNA duplex.9 (262 144 sequences) and fit comfortably on a single array, along with replicates (Table S1). We envisaged the fabrication of a high‐density RNA microarray hosting the entire permutation library of a 9‐nt sequence  relies on a digital micromirror device (DMD) in an imaging system to pattern and deliver 365 nm light onto spatially defined, micrometer‐sized features on the glass substrate.5=1024 sequences) in the variable region were synthesized in multiple replicates, deprotected and subjected to RNase HII‐mediated cleavage. A final treatment with water at 40 °C ensured complete removal of the cleaved portion of the hairpin. RNase HII‐mediated cleavage results in loss of fluorescence, as treating the array in buffer alone led to no significant difference in fluorescence relative to the DNA‐only hairpin. Interestingly, we found that the hairpin sequences were not all cleaved to the same extent, but with noticeable disparities spanning a 40 % range. The least‐cleaved nucleotide combination, TTrGCT, only lost 30 % of its initial fluorescence relative to the DNA hairpin, while the most‐cleaved sequence, GCrCCC, decreased by 70 %. Notably, the DNA hairpin containing a single rU insert within the TCCT loop was also found to be cleaved by up to 20 %, indicating some level of structural recognition by RNase HII even on unpaired ribonucleotides, which is not unheard of.To highlight the potential applications of complex arrays of RNA and mixed DNA and RNA chemistries in molecular biology and chemical biology research, we chose RNase HII, an enzyme whose preferred substrates are double‐stranded DNA carrying a single RNA baseThe vast majority of sequences (846 out of 1024) showed cleavage rates between 40 and 60 %, and the remaining combinations displayed lower (30–40 %) or higher (60–70 %) cleavage rates. We then searched for sequence motifs within the low and high cleavage subgroups. The large majority of highly‐cleaved hairpins (100 sequences) contains rC as the RNA base, and rU in a few select cases. In fact, rC becomes the only RNA base found in the 20 most‐cleaved hairpins  should be addressed to the authors.SupplementaryClick here for additional data file.


RNA-protein interactions permeate biology. Transcription, translation, processing, and mRNA decay all hinge on widespread use of regulatory information decoded by RNA-binding proteins. The final committed step of protein synthesis, translation, is intimately linked to nociceptor excitability. Understanding the factors that control translation is essential as nociceptor plasticity is a hallmark of persistent pain. Here, we review the growing body of evidence for widespread involvement of RNA-binding proteins in pain. Many of the relevant factors have been implicated in post-transcriptional and translational mechanisms of mRNA control. We propose that recent advances in the development of RNA-based therapeutics provide a potential means to exploit our current understanding of liaisons between RNAs and proteins for therapeutic purposes. Transcription results in the production of ribonucleic acid (RNA) and RNA can be translated into protein. This framework provides a simplistic view into how instructions present in the genomic blueprint manifest in complex and dynamic patterns of protein expression. The final committed step in this process, translation, is intimately linked to nociceptor plasticity and the development of pain hypersensitivity . UnderstAs the essential intermediate in protein synthesis, messenger RNA (mRNA) is subject to regulation at virtually every level. Precise control of mRNA processing, stability, translation, and localization dictates the efficiency, timing, and site of protein production. For instance, mRNA stability spans nearly four orders of magnitude ranging from minutes to months . Similareukaryotic translation initiation factors (eIF)4A, eIF4G, and eIF4E (collectively referred to as eIF4F) (untranslated region (UTR). The UTR serves as a repository for regulatory information including secondary structural elements, internal ribosomal entry sequences (IRES), upstream open reading frames (uORFs) and RNA-binding protein motifs. IRES elements bypass the requirement for specific translation factors and thus mediate cap-independent translational control  . eIF4E is eIF4F) . Adjacen control . 5′ UTRs control . The 5′  control . Like th control . After t control . Loss of control .Fig. 1RNIn this review, we summarize recent progress into conserved mechanisms of mRNA control implicated in pain. We focus first on the trans-acting factors involved in RNA recognition. We describe new potential players based on analysis of cis-acting elements found in key transcripts. Finally, we discuss the potential use of RNA-based inhibitors for understanding underlying mechanisms of chronic pain and as a new source of pain therapeutics.Several RNA-binding proteins have been identified to play a role in pain through various animal models of chronic pain conditions see . The funnerve growth factor (NGF) and interleukin-6 (IL-6) promote translation through convergent effects on eIF4F association with the m7G cap  neurons, the pro-inflammatory mediators  m7G cap . In both m7G cap . mTOR bi m7G cap . At leas m7G cap . Second, m7G cap . Third a m7G cap , althoug m7G cap .Adenylate-uridylate-rich elements  are abundant in the 3′ UTR of mRNAs encoding cytokines and immune-responsive genes . The preCPEBs are a well-established paradigm in activity-dependent translational regulation . CPEBs rThe underlying cause of Fragile X syndrome is loss of function of the RNA-binding protein, Fragile X mental retardation protein (FMRP). FMRP regulates synaptic plasticity and binds to a subset of mRNA and directly to the L5 protein on the 80S ribosome . Fmr1-knin vitro, block mechanical hyperalgesia and priming induced by pro-inflammatory cytokines, capsaicin, or incision injury  binding protein with selectivity in vitro attenuates translation initiation and functions as a robust anti-hyperalgesic in vivo. Because this approach targets the RNA-binding region of a given target, there is no inherent reliance on processing factors from the host. An additional advantage of the SPOT-ON approach is the ability to inhibit entire protein families that bind similar, if not identical sequences, to enact similar functions. By way of comparison, genetic dissection of paralogues gene families is extremely laborious and may result in negative results due to functional compensation. The simplicity of design suggests that this method will be broadly applicable for mechanistic studies of RNA-binding proteins in vivo. SPOT-ONs complement existing methods for protein reduction that are suitable for in vivo studies including siRNAs and ASOs. These experiments will be critical to elucidate new mechanisms of action with the coveted goal of improved therapies for chronic pain sufferers.While the specificity of many RNA-binding proteins is known, rational design of competitive inhibitors is challenging given the unstable nature of RNAs . Tremendbackbone . We recebackbone . As a prThe authors declare that they have no conflicts of interest.


The effects of coral cover, exposure, fishing pressure, lagoon size and geographical context, on observed patterns of fish assemblage structure were modelled using Multivariate Regression Trees. Reef fish assemblages were clearly separated in space with geographical location explaining ~53 % of the observed variation. Lagoon size, within each isolated reef system was an equally effective proxy for explaining fish assemblage structure. Among local‐scale variables, ‘distance from port’, a proxy for the influence of fishing, explained 5.2% of total variation and separated the four most isolated reefs from Cocos (Keeling) Island, from reefs with closer boating access. Other factors were not significant. Major divisions in assemblage structure were driven by sister taxa that displayed little geographical overlap between reef systems and low abundances of several species on Christmas Island corresponding to small lagoon habitats. Exclusion of geographical context from the analysis resulted in local processes explaining 47.3% of the variation, highlighting the importance of controlling for spatial correlation to understand the drivers of fish assemblage structure. Our results suggest reef fish assemblage structure on remote coral reef systems in the tropical eastern Indian Ocean reflects a biogeographical legacy of isolation between Indian and Pacific fish faunas and geomorphological variation within the region, more than local fishing pressure or reef condition. Our findings re‐emphasise the importance that historical processes play in structuring contemporary biotic communities.We quantify the relative importance of multi‐scale drivers of reef fish assemblage structure on isolated coral reefs at the intersection of the Indian and Indo‐Pacific biogeographical provinces. Large (>30 cm), functionally‐important and commonly targeted species of fish, were surveyed on the outer reef crest/front at 38 coral reef sites spread across three oceanic coral reef systems  Islands and the Rowley Shoals), in the tropical Indian Ocean ( The objectives were as follows: (i) to determine the relative importance of multiscale environmental drivers on the structure of reef fish assemblages across the Indo‐Pacific biogeographical boundary. Specifically, we compared the role of geography, reef geomorphology, wave exposure, coral cover, and fishing pressure on the structure of large and functionally important coral reef fishes. (ii) We identify the key species driving taxonomic patterns in reef fish assemblage structure among and within reef systems and (iii) evaluate how taxonomic patterns affect the abundance of key functional groups of fish among reef systems. We discuss the extent to which patterns in reef fish assemblage structure are likely to reflect legacies of contemporary and evolutionary processes, based on phylogenetic and ecological characteristics of species observed across this dynamic biogeographical region.22.16 km2 of ocean environment. Cocos (Keeling) Islands (CKI), Christmas Island (XI), and the Rowley Shoals (RS) have very different geological histories and geographical locations in the Indian Ocean, albeit with at least 300 km separating each of them from continental land masses  between 2004 and 2010. Counts were made at CKI in February 2004, RS (Clerke & Imperiuse Reefs) in October 2007 and XI in March 2010. A total of 38 sites were analyzed consisting of 12 sites at CKI, 16 sites at XI, and 10 sites at the RS Table . For RS,2.3http://www.bom.gov.au/climate/data/stations/ : date of access March 2015).Hard coral cover was estimated in a semiquantitative way with four categories representing the range of abundances found on the reefs. Categories were as follows: 0%‐0%, 1‐0 to 15%, 2%‐15% to 30%, 3%‐30% to 50%, and 4%–>50%. Surveys were conducted in XI by A.M. Ayling, CKI by J.P. Hobbs, and the RS survey data were provided by K. Fabricius . As no quantitative catch data were available for these reef systems, we derived two separate variables to represent estimates of fishing effort. The first used qualitative expert opinion  to generate a categorical scale of fishing effort at each site. Categories for perceived fishing effort were as follows: 1 – negligible fishing effort, 2 – low, 3 – medium, 4 – high. The categorical scale of fishing effort was informed by a range of factors including: proximity to populated areas; exposure of the site to prevailing ocean swells; local fisher knowledge from interviews; charter vessel catch data (if applicable); commercial vessel catch data (if applicable); number of fishable days; observations of fishing activity; evidence of fishing activity ; and level of compliance activities (if applicable). The second proxy fishing variable was calculated as the distance of each survey site from the nearest port or vessel launching area (DFP) and represents the potential effect of weather on reducing fishing effort at those sites furthest from safe harbor. An exposure index was also calculated for each site by determining the 22.5° sectors  from which the site was fully exposed to an unobstructed wave fetch of 3 km or more, and summing the product of the mean annual wind speed at each reef complex (km/hr) and duration (proportion) in each of these sectors  analysis De'ath,  was usedAs well as the environmental variables, latitude and longitude were explicitly incorporated into the model to account for spatial autocorrelation, reduce the risk of Type I error, and prevent the spurious inflation of other variables significance—a result we demonstrate using a permutational linear model  were run  among reef systems. Data were Log+1 transformed to improve normality and homogeneity of variance following visual inspection of scatter plots and the distribution of residuals.The density of key trophic and taxonomic groups of reef fishes was compared among reef systems  using analysis of variance (ANOVA). Species densities were averaged from adjacent sites in CKI and XI, to establish equal sample sizes  that were replaced by sister species  to the east  and mesopredators  , and the large iconic species Cheilinus undulatus and Bolbometapon muricatum at XI. The final split identified the species that distinguish between the three main locations and the exposed versus leeward side of CKI. Four species displaying relatively high abundances on isolated CKI reefs were primarily responsible for this division, namely, Lutjanus fulvus, Naso elegans, N. lituratus, and Scarus prasiognathus.The indicator species analysis identified the species that were significantly associated with each of the groups identified through the MRT analysis Table . The outt Figure . CKI rees) Table . XI asseus Table . The othU‐shaped pattern in fish density , while epinephelid piscivores were altogether absent from XI  followed by XI (41). However, the assemblage structure of the faunas differed substantially among the reef systems. The differences were driven mainly through absence or rarity of certain taxonomic and trophic groups from different reef systems, particularly at XI  and mesopredators from the family Epinephelidae  displayed higher densities in XI than CKI or RS , Variola louti , Gracila albomarginata , and Cephalopholis miniata  were most abundant at XI. Similarly, the large lutjanid piscivore, Lutjanus bohar, displayed densities of 9.8 ± 1.2 individuals/Ha, compensating for the rarity of sharks  and epinepheline groupers  at XI.The absence of certain groups from XI was compensated for by higher abundances of other taxa. Browsing herbivores from the family Kyphosidae  and Hipposcarus harid  at CKI and Scarus rubroviolaceus  at XI. An opposite trend, with numbers increasing from east to west, was seen in the omnivorous acanthurids, resulting from a larger number of shared species  in RS compared with the other regions . When examined hierarchically, local‐scale factors such as fishing pressure, coral cover, and wave exposure explained very little variation (~6%) in the fish assemblage structure. Interestingly, if geographical location was not accounted for, then local‐scale factors such as fishing and coral cover explained 47.2% of the variation in assemblage patterns—a result inconsistent with the taxonomic and functional characteristics of the species responsible for differences in the assemblage patterns among reef systems. Major divisions in assemblage structure were driven by sister taxa that displayed little geographical overlap between reef systems and low abundances of several species on Christmas Island that are reliant on the small area of proxy lagoon habitat available within the sole cove of the island. These findings suggest that the geomorphology and geographical legacies of these reef systems are acting as the primary driver of contemporary assemblage structure ahead of fishing pressure or reef condition. Our findings emphasize the importance of adopting a multiscale perspective to understand the underlying mechanisms structuring biotic communities.The combined influence of geographical isolation and island geomorphology on reef fish assemblage structure around the Indo‐Pacific Barrier builds on previous research that examined fish assemblages through the prism of island biogeography theory  from the more central (XI) and eastern locations (RS). Geographic location and the effects of lagoon size were responsible for this division and were indistinguishable as proxies for regional‐scale differences in the assemblage structure. Indicator species analysis revealed that the division between CKI and RS/XI was differentiated by a number of abundant sister taxa such as 2 and 92 km2, respectively, with a variety of habitat types ranging from sheltered, algal dominated sites to patch reefs supporting high densities of living coral interspersed with extensive areas of sand and seagrass beds. In contrast, XI comprises a continuous but narrow fringing reef with the only area equivalent to a lagoon habitat being a ~1.2 km2 area of sheltered bay (cove) that is dominated by coral, has minimal sand (1%), and lacks seagrass. Some lagoon‐associated fish species occur at low densities in this bay and nowhere else around XI (Hobbs et al., Cheilinus undulatus and Bolbometopon muricatum, groupers, and reef sharks, especially Carcharhinus melanopterus and some epinephelids that are present in much larger numbers at CKI and RS. The continued presence of a number of large rare species at Christmas Island is likely to be dependent on the continued health and protection of this small area of lagoon habitat. These results provide a cautionary note about the influence of local habitat condition on fish assemblage structure. While local coral condition had a relatively small influence on fish assemblage structure in the current study, structural and functional loss of this habitat due to repeated mass coral bleaching across the region (Hughes et al., In parallel to geographical isolation, differences in island geomorphology had an important influence on the fish assemblage structure throughout the region. While all surveys were conducted in comparable coral reef habitats, the size of neighboring lagoon habitats appears to be an important driver of fish assemblage structure on reefs. Lagoon habitats influence both the trophic biology and recruitment patterns of numerous reef fishes (Wilson et al., Lutjanus fulvus, Naso elegans, N. lituratus, and Scarus prasiognathus. In turn, our study also suggests that the scarcity or absence of commonly targeted species, such as B. muricatum, C. undulatas, and C. melanopterus at XI (Hobbs et al., B. muricatum, C. undulatas, and C. melanopterus could be incorrectly interpreted as having been overfished (c.f. Ruppert, Travers, Smith, Fortin, & Meekan, The low abundances of several charismatic, fishery targeted, and functionally important species from XI are interesting in the context of this study given the relatively low importance of local‐scale processes in driving the differences in assemblage structure among reef systems. Overfishing is a major driver of depauperate fish assemblages in many locations around the world (Jackson et al., Reef fish assemblage structure at remote coral reef systems in the tropical eastern Indian Ocean appears to primarily reflect a biogeographical legacy of isolation between Indian and Pacific fish faunas and geomorphological variation among reef systems, rather than local fishing pressure or reef condition. Studies that do not include these evolutionary and large‐scale processes risk attributing too much variation in assemblage structure to local processes and risk overemphasizing the need to manage local factors. Explicit recognition of the relative importance of multiscale processes on assemblage structure, including the evolutionary context of an area, is crucial for the effective management of remote and isolated reef systems.None Declared.SJN, SB, ARH JHC, and ESH conceived the ideas. JHC, AMH, and J‐PAH conducted the fieldwork and collected the data. SB, ARH, and JSG analyzed the data. SB led the writing with assistance from all authors. Click here for additional data file.


Studies of mRNA and miRNA expression profiling increasingly use stabilized whole blood. Commercial RNA extraction kits do not provide information about the simultaneous recovery of both mRNA and miRNA. This study evaluated yield, quality, integrity and representation of mRNA and miRNA from whole blood stabilized in Tempus tubes using three RNA extraction kits; two filter-based (Tempus and Norgen) and one bead-based .All RNA extraction kits and methods resulted in similar yields of mRNA  whereas there were differences in yields of miRNA. MagMax, either manual or semi-automated, with or without DNase treatment, yielded 1.6–2.2-fold more miRNA than Tempus and Norgen kits. In addition, MagMax and Norgen methods gave greater than 12-fold more and 3.3-fold less enrichment of specific miRNA targets, respectively, in comparison to Tempus extraction reagents. This study identified MagMax kit for simultaneous recovery of both mRNA and miRNA from whole blood collected in Tempus tubes. Gene expression profiling is only meaningful when the starting RNA is representative of the starting material. Representation and yield of RNA are impacted by a number of pre-analytic factors including RNA extraction . While iPhase one included 12, and phase two included four anonymous healthy volunteers recruited from Centers for Disease Control and Prevention’s voluntary blood donation program. The clinic collected peripheral blood through antecubital venipuncture directly into 3 ml Tempus Blood RNA tubes (Thermo Fisher), followed by immediate vigorous shaking of each tube for 10 s. Three tubes were collected from each participant for phase one, and four tubes for phase two. Samples were stored at room temperature for 2 h, then stored at − 20 °C until RNA extraction.In phase one, we manually extracted total RNA to compare three different kits: two filter-based kits [Tempus Spin RNA Isolation kit (Thermo Fisher) and Norgen Preserved Blood RNA purification kit I (Norgen Biotek Corp)], and one magnetic bead-based kit [MagMax for stabilized blood tubes RNA isolation kit (Thermo Fisher)]. Frozen blood tubes were thawed prior to RNA extraction following the manufacturer’s protocol for each kit. Tempus Spin and Norgen protocols did not include a DNase treatment whereas the MagMax protocol included a DNase treatment. For the second phase, the manual MagMax protocol (with and without DNase treatment) was compared to semi-automated MagMax extraction (with and without DNase treatment) per the instructions of the MagMax Express 96 instrument (Thermo Fisher).We determined RNA yield and purity based on 260/280 and 260/230 ratios with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher). We determined RNA integrity number  and the fraction of small RNAs  with the Agilent 2100 Bioanalyzer  using the Eukaryote RNA 6000 Nano and the small RNA kits (Agilent). The small RNA analysis indicates the region enriched for miRNA.Two RT reactions were prepared for each sample, one for mRNA and one for miRNA. For mRNA, 0.5 µg total RNA in a 20 µl reaction was transcribed using the PrimeScript™ RT reagents Kit with gDNA eraser  following manufacturer’s recommendations. After gDNA eraser treatment for 2 min at 42 °C, a 1 µl aliquot was removed for use as the no-RT control. The remainder of the reaction volume underwent the RT reaction at 37 °C for 15 min. For miRNA, 0.5 µg total RNA in a 25 µl reaction was transcribed using the All-in-One™ miRNA qRT-PCR Reagent Kit  following manufacturer’s recommendations (37 °C for 1 h). The GeneCopoeia kit includes reagents for polyadenylation of mature miRNA followed by reverse transcription using modified oligo (dT) with a universal adaptor.We selected nine mRNA and nine miRNA targets based on their reported abundance in blood . mRNA ta−ΔΔCq [−((A−B)−(C−D)) where A = Cq of miR-16-5p (MagMax), B = geomean Cq of PPIB and PGK1 (MagMax), C = Cq of miR-16-5p (Tempus) and D = geomean Cq of PPIB and PGK1 (Tempus). The resulting relative value assumes primers for both targets and reference genes amplify with nearly equal efficiencies. All samples were restricted to the same set of PCR conditions for targets and reference genes.Total RNA yield, RNA purity (absorbance ratios), RNA integrity and small RNA yield were expressed as mean ± standard error of mean (SEM) for all samples processed with each kit, and comparisons used One-way ANOVA. Fold differences in the representation of mRNA or miRNA targets were calculated by 2−ΔΔCq  where Cq−ΔΔCq , and theRNA yields from all three protocols Table  were witRIN values for individual RNA extracts with the Tempus and Norgen kits ranged from 8.4 to 9.0 whereas those with MagMax kit ranged from 5.9 to 7.5 (data not shown); the mean RIN for all samples with each kit reflect these differences Table . While tWhile there was only small differences among kits in their total RNA yields, perhaps reflecting their design for mRNA, they did differ in mean yields for miRNA as well as proportion of miRNA/small RNA Table . The mea−ΔΔCq analysis using raw Cq values for the selected target, mRNA representation differed by less than twofold between kits  performed on different days throughout the study gave highly reproducible Cq values for each mRNA and miRNA target. Based on 2its Fig. a. Howeveits Fig. b. SampleWhile MagMax RNA extraction has the advantage for miRNA retention, automation is desirable because the manual method is tedious and potentially prone to errors. We compared results achieved with an automated protocol to those with manual. Both methods, with or without DNase treatment, gave similar results on all parameters [total RNA yield, 260/280 ratio, 260/230 ratio, RNA integrity, small RNA and miRNA yield, mRNA representation (Fig. A key finding from this study is that RNA extraction kits have little impact on representation of mRNA but show a marked impact on miRNA representation. For simultaneous recovery of both mRNA and miRNA from stabilized blood in Tempus tubes, the MagMax protocol is favored. Hantzsch et al.  reportedComparison of three kits recommended for the extraction of total RNA from whole blood stabilized in Tempus blood tubes found the magnetic bead-based MagMax kit superior to the filter-based Norgen and Tempus extraction kits in terms of simultaneous recovery of both mRNA and miRNA with significant enrichment of the miRNA fraction. While our results favor the MagMax kit, the determination is based on only a snapshot of the complexity and representation of RNA in whole blood that a limited number of mRNA and miRNA targets provide. However we feel the guidance provided is useful because of judicial selection of targets that represent the spectrum of abundance and sizes of total RNA species for most applications.


Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used.We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a ‘gold standard’ control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit.RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts.The online version of this article (10.1186/s12864-018-5066-2) contains supplementary material, which is available to authorized users. Considerable scientific effort has been dedicated to understanding cell type-specific expression profiles from complex tissues, such as brain, liver, pancreas, testes, eye or ear –7. To ovWhile ribosome-attached RNA sequencing for expression analysis has been validated from a biological standpoint , 19, theGfi1-Cre knock-in mice generated by Dr. Lin Gan (University of Rochester) were kindly provided by Dr. Jian Zuo of the Developmental Neurobiology Department at St. Jude Children’s Research Hospital. RiboTag mice generated by Dr. Paul S Amieux (University of Washington) were kindly provided by Dr. Mary-Kay Lobo of the Department of Neurobiology and Anatomy at University of Maryland Baltimore. B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice  were purchased from the Jackson Laboratory . Experimental animals for RNA-seq, Gfi1Cre/+;RiboTagHA/HA, were obtained by crossing RiboTag mice with Gfi1-Cre mice. Animals for immunostaining, Gfi1Cre/+;Ai14, were obtained by crossing Gfi1-Cre mice with Ai14 mice . Dots above or below the red line represent the enriched or depleted transcripts. (JPG 587 kb)Additional file 6:Figure S5. Venn diagram of enriched/depleted transcripts . The mean ratio IP/input is ≥2 or input/IP is ≥2. (PDF 1063 kb)Additional file 7:Figure S6. Histogram of length distribution for enriched or depleted transcripts. (PDF 353 kb)


DIR2s adopts a compact, tuning fork-like architecture comprised of a helix and two short stem-loops oriented in parallel to create the ligand binding site through tertiary interactions. The OTB-SO3 fluorophore binds in a planar conformation to a claw-like structure formed by a purine base-triple, which provides a stacking platform for OTB-SO3, and an unpaired nucleotide, which partially caps the binding site from the top. The absence of a G-quartet or base tetrad makes the DIR2s aptamer unique among fluorogenic RNAs with known 3D structure.The DIR2s RNA aptamer, a second-generation, in-vitro selected binder to dimethylindole red (DIR), activates the fluorescence of cyanine dyes, DIR and oxazole thiazole blue (OTB), allowing detection of two well-resolved emission colors. Using Fab BL3-6 and its cognate hairpin as a crystallization module, we solved the crystal structures of both the apo and OTB-SO The DIR2s RNA aptamer activates the fluorescence of cyanine dyes allowing detection of two well-resolved emission colors. Here authors solve the crystal structures of the apo and OTB-SO3 fluorophore-bound DIR2s and show how the fluorophore ligand is bound. They consist of an RNA aptamer that can fold into a three-dimensional structure, recognize a profluorescent small molecule, and activate its fluorescence3. Although RNA-based fluoromodule development remains less advanced compared with protein-based fluoromodules4 due to the lack of inherently fluorescent RNAs, recently discovered fluorescent RNA aptamers have facilitated cellular imaging, localization, and tracking of RNAs in real time5. In vitro selection from random RNA populations has given rise to numerous RNA aptamers that bind and activate the fluorescence of small-molecule chromophores such as flavin mononucleotide7, sulforhodamine B10, malachite green (MG)2, derivatives of Hoechst 3325811, black hole quencher (BHQ)-conjugated fluorophores13, dimethylindole red (DIR)3, dinitroaniline-conjugated dyes14, and small-molecule analogs based on the chromophore in fluorescent proteins16. RNA aptamers such as Spinach15, Spinach217, Broccoli18, and Corn16, which activate fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone and 3,5-difluoro-4-hydroxybenzylidene imidazolinone-2-oxime (DFHO),  and RNA Mango20, which activates fluorescence of thiazole orange derivatives, have been used for RNA imaging studies both in vitro and in cellulo22. These aptamers can be encoded genetically into RNAs of interest and upon incubation with their cognate fluorophores allowed to study RNAs in a manner analogous to the use of green fluorescent proteins23 to study proteins4. For example, Spinach has been used as an RNA visualization probe in bacterial and mammalian cells21, as biosensors for detection of metal ions24, cellular metabolites27, and proteins28, and for construction of molecular beacons29. Nevertheless, existing fluoromodules remain constrained by limitations, including inadequate fluorophore-binding affinity, misfolding tendencies, and loss of fluorescence through photobleaching31. Advanced generations of these fluoromodules partly overcome these limitations but their use for in vivo applications remains problematic. Another potential limitation concerns the presence of an essential G-quartet motif33 in the Spinach35, Corn36, and Mango37 RNA fluoromodules as eukaryotic cells have machinery that specifically inhibits the formation of G-quartet folded structures38. It is therefore important to continue investigation of other RNA aptamers that bind and activate small-molecule fluorescence both to develop a framework for relating their structure to their functional properties and to identify motifs that can function without a quadruplex motif. For example, a recently developed RNA imaging platform for tracking mRNA and small noncoding RNAs uses the cobalamin riboswitch as an RNA tag and fluorescent dye fused to cobalamin as fluorescence quencher that exhibits fluorescence turn-on upon binding to the riboswitch39.RNA-based fluoromodules have become a valuable tool in biomedical research for RNA sensing and imaging applications40. First, to reduce the background fluorescence of the dye in an intracellular environment, a prerequisite for fluoromodule-based imaging applications, they precluded nonspecific intercalation by the dye through the incorporation of a geminal dimethyl group on the indole ring and a negatively charged anionic sulfonate substituent on the quinoline ring, which introduces repulsive electrostatics to the polyanionic backbone of RNA  RNA aptamer obtained represents the second generation of DIR binding RNA aptamers that bind to DIR dye and activate its red fluorescence. The aptamer exhibits promiscuity with respect to dye recognition, binding with nanomolar affinity to red fluorescence emitting DIR, as well as blue fluorescence emitting oxazole thiazole blue (OTB)5 dyes and their derivatives. Tan et al. used this property to monitor the cell surface expression and internalization of the epidermal growth factor receptor (EGFR) by fusing a known EGFR-binding aptamer to the DIR2s aptamer and imaging at different time points using either red (DIR) or blue (OTB) dyes40. Also, both dyes have shown excellent photostability when complexed with the DIR2s RNA aptamer compared with the MG aptamer40.Armitage and colleagues generated an RNA fluoromodule against the fluorogenic cyanine dye, DIRRNA Fig. . Next, t43. Originally selected against the class I ligase ribozyme45, Fab BL3-6 binds to a stem-loop structure containing an AAACA pentaloop closed by a G-C base pair, which can be grafted into target RNA constructs to create a binding site for the Fab34. We used this strategy to crystallize and solve the crystal structures of DIR2s RNA aptamer in the absence of ligand and co-crystallized with OTB-SO3 dye at 2.0 Å and 1.8 Å resolution, respectively. Our structures reveal that the DIR2s aptamer adopts a tuning fork-like fold consisting of two short stem-loops and a proximal helix. Tertiary interactions between the two loops create a stacking platform for the fluorophore ligand formed by three purine nucleobases, including a guanine nucleobase that forms hydrogen bonds to the ligand’s –SO3 moiety. From above, only a single adenosine residue at the apex of the aptamer sandwiches the fluorophore, likely accounting for the relaxed specificity.To understand the promiscuity of the DIR2s aptamer and its ability to bind DIR and OTB dyes, we sought to obtain the crystal structure of the aptamer in complex with fluorogens. We used the Fab BL3-6 antibody as an RNA crystallization chaperoneKd = 67 ± 20 nM; error reports SD from three independent measurements) similar to that of binding affinity reported for either Spinach RNA grafted with GAAACAC motif34 or isolated hairpin from class I ligase RNA43. The fluorescence activation of DIR and OTB-SO3 dyes complexed with the DIR2s aptamer remained unaffected by the hairpin graft or binding of the Fab compared with the parent aptamer , indicating that the BL3-6 binding loop had no adverse effects on its overall folding  from the constant stem-loop domain of the wild-type (WT) DIR2s aptamer with a 7-nucleotide GAAACAC motif to create a binding site for the chaperone Fab BL3-6 , including a U-A-U base triple and stacking interactions. Two of these base pairs, A14-U38 and C18-G43, form canonical Watson–Crick (WC) pairings, whereas the other two (A14•U42 and A15•A41) form noncanonicalons Fig. . Among t 47 Fig. . Includiely Fig. . A14 of ely Fig.  and formely Fig. . Below tely Fig. . In addiine Fig. . G12, whine Fig. . In the ine Fig. .3 fluorophore sits in a planar conformation predominantly upon the G39 and A41 nucleobases, which form stacking interactions with the benzothiazolium and benzoxazole rings, respectively .In the crystal lattice, two RNA aptamers from neighboring symmetric units pack against each other in a head-to-head fashion to bury 420 Årea Fig. . In thisrea Fig. . The nucKd < 1 nM) in which the interface between monomers binds and activates fluorescence of DFHO36. Our crystal structure raised the possibility that DIR2s could function analogously. To address this possibility, we investigated whether the DIR2s aptamer forms a homodimer in solution using several experimental approaches. First, we titrated a fixed concentration of fluorophore (10 µM) with increasing concentrations of the aptamer and observed a linear increase in fluorescence (R2 = 0.996) over the 0–2.5 µM concentration range  measurements at various DIR2s–OTB–SO3–Fab ternary complex concentrations. Fitting the observed scattering profiles against calculated scattering from the crystal structure indicates that, at increasing concentrations, the RNA exists as a monomer:dimer mixture (80:20% at 4 mg/mL). However, at the lower concentration , which is >10- to 15-fold higher than the RNA concentrations used in fluorescence studies performed in vitro and in vivo, the RNA populates predominantly ( > 90%) the monomeric form.  RNA standards. As expected for the monomeric form of the aptamer (MW ~ 18 kDa), we observed a single peak that eluted slightly later than the 21 kDa standard  residue (which replaces the entire nucleobase with a hydrogen atom) by enzymatic ligation of synthetic RNA oligonucleotides. Both G39ab and A40ab variants exhibited diminished fluorescence signal compared with the WT aptamer. From the structure, we hypothesized that insertion of an additional purine nucleotide between A40 and A41 might be able to provide stacking interaction to the benzoxazole ring of the OTB from the top; however, this insertion had an adverse effect on OTB-SO3 fluorescence. Together these results support the functional significance of these nucleotides and suggest that that the crystal structure captures the aptamer–OTB–SO3 complex in a functionally relevant conformation. To test the functional significance of the P1 stem, we constructed truncates of the DIR2s aptamer lacking 5 base pairs (nucleotides 1–5 and 56–60) and 10 base pairs from the end (nucleotides 1–10 and 51–60). For the latter construct, we replaced U11 and U20 with G and C, respectively. The construct with the five base pair P1 stem retained fluorescence similar to that of WT aptamer, and the construct lacking P1 entirely showed slightly lower fluorescence (ca. 80%), indicating that the P1 stem has little functional importance  as a search model. The apo form of the DIR2s aptamer adopts a highly similar architecture to its ligand-bound form , whereas RNA contributed only 13% through RNA–RNA interactions. In addition, the Fab facilitated structure determination by allowing the use of molecular replacement to solve the phasing problem.Using Fab BL3-6 and its cognate RNA hairpin motif as a crystallization module36, which binds its DFHO ligand with 2:1 aptamer:fluorophore stoichiometry. However, Corn functions as a dimer in solution, whereas DIR2s, which buries only half as much surface area at the dimer interface (880 Å2 vs. 420 Å2 for DIR2s with OTB dye), remains monomeric at micromolar concentrations.The co-crystal structure of the DIR2s aptamer and supporting biochemical studies reveal a compact, tuning fork-like architecture comprised of a helix and two stem-loops oriented parallel to each other. Interactions between the two loops create the fluorophore-binding site, which consists of an unpaired nucleotide (A40) that caps the benzothiazolium ring from the top and a base triple formed by three purines  that provide a stacking platform for the two rings of OTB to sit in a planar configuration. Together the platform and the unpaired nucleotide form a claw-like structure that clamps down on the ligand. OTB’s propylsulfonate projects downward from the open end of the claw to position the sulfonate moiety for chelation by the G39′s amidinium group. In the crystal lattice, two complexes homodimerize via head-to-head stacking interactions involving the ligand-binding sites, resulting in nearly complete surface burial of the fluorophore. Dimerization governed by stacking interactions resembles a feature of the recently reported Corn RNA aptamer3, DIR-SO3, and TO1-Biotin. If the aptamer functioned as a dimer, the snug fit for OTB-SO3 at the dimer interface in the crystal would be expected to preclude binding and fluorescence activation of DIR, the actual ligand targeted in the selection. Indeed, crystallization trials in the presence of DIR-SO3 did not yield crystals of the ligand-bound form. DIR-SO3 and OTB-SO3 bind the DIR2s aptamer competitively and exhibit similar responses to mutation  compared with when bound to certain scFv proteins selected from a yeast-displayed library for binding to this particular fluorogen (ϕf = 0.84–1.00)49.According to the structure, the monomeric form leaves the top face of the benzoxazole ring uncapped and exposed to solvent. This open feature likely relaxes the ligand specificity and accounts for the ability of the aptamer to bind both OTB-SOion Fig. , consistion Fig.  that cou35 and Corn36, respectively, as well as RNA Mango37, all contain a G-quadruplex in their fluorophore-binding site. The MG aptamer lacks a G-quadruplex motif, but the fluorophore-binding site contains a non-G-quartet base quadruple51. In contrast, the fluorophore-binding site of DIR2s aptamer uses a base triple as a stacking platform and contains no tetrad. All fluoromodule aptamers identified thus far use nucleobase stacking and shape complementarity to bind their profluorescent ligands and restrict motion, which decreases nonradiative decay and thereby increases fluorescence quantum yield. Similar to previous observations for the Spinach aptamer34, the DIR2s aptamer retains its global architecture in the absence of ligand and undergoes only a local structure perturbation to accommodate the fluorophore. Ligand binding to this largely preorganized aptamer requires a simple rotation about the glycosidic bond of A40, and thus, likely imparts only a modest entropic penalty.The absence of a G-quadruplex in the DIR2s fluorophore-binding site is unique among fluoromodules as RNA mimics of green and red fluorescent protein SpinachIn summary, the DIR2s aptamer recognizes planar, aromatic chromophores without the use of a G-quadruplex or nucleobase tetrad, thereby expanding the scope of fluorogenic RNA activation. Our structures streamline RNA construct and ligand design for future applications of this fluoromodule and provide a physical framework to guide efforts to engineer DIR2s aptamers and ligands with improved photophysical and recognition properties.52 , DNA template 30 pmol/mL, 40 μg/mL homemade T7 RNA polymerase. Transcription reactions were quenched by adding 10 U/mL RNase free DNase I  and incubating at 37 °C for 30 min. After the phenol/chloroform/isopropanol, pH 4.3 extraction, the RNA was purified by denaturing polyacrylamide gel electrophoresis (dPAGE). Corresponding RNA band was visualized UV shadowing and excised from the gel. RNA was eluted overnight at 4 °C in 10 mM Tris, pH 8.0, 2 mM EDTA, 300 mM NaCl buffer. The buffer in eluted RNA was exchanged three times by pure water using 10 kDa cutoff size exclusion column (Amicon). RNA was collected, aliquoted into small fractions, and stored at −80 °C until further use.The double-stranded DNA templates for transcription reactions were prepared by PCR amplification of the single-stranded DNA oligomer purchased from Integrated DNA Technologies (IDT). The first two nucleotides of the reverse primer contained 2′-OMe modifications to reduce transcriptional heterogeneity at the 3′ end4OH/EtOH at 55 °C for 8 h, desilylated with 300 μL 6:3:4 N-methylpyrrolidinone/triethylamine/triethylamine-3HF at 65 °C for 2 h and precipitated by n-BuOH. The oligomers were further purified by dPAGE, collected in pure water and stored at −80 °C until further use.DIR2s aptamers containing abasic site were prepared by chemical ligation of two short oligonucleotides “Donor” and “Acceptor” synthesized in house by solid-phase synthesis on a 1-μmol scale using an Expedite Nucleic Acid Synthesis System (8900) by following standard RNA synthesis protocols. The oligonucleotides were released from solid support with 3:1 NH2, 1 mM DTT, 400 μM ATP, pH 7.5) and T4 RNA ligase 2 (NEB) were added, and the resulting reaction mixture was incubated at room temperature for 12 h. Ligation reaction mixtures were purified by 15% dPAGE as described above and concentrations for pure oligos were determined using Nanodrop (Thermo Scientific).The Donor oligonucleotide was 5′-end phosphorylated with T4 polynucleotide kinase  in the presence of excess ATP at 37 °C for about 1 h followed by heat inactivation of the enzyme at 60 °C for 20 min. For the ligation reaction, the DNA splint oligonucleotide was mixed with donor and acceptor RNA oligonucleotides at a 1:1.5:1.1 ratio of donor/acceptor/splint and annealed in the presence of monovalent ions by heating to 90 °C and gradual cooling to 20 °C over a period of 1 h. After annealing, T4 RNA ligase buffer  were measured at 20 °C, using a Fluorolog-3 spectrofluorometer equipped with a thermo controller (Horiba Inc.) and reported results were the average of three measurements. The samples for fluorescence measurements were prepared by following a standard refolding protocol: the RNA was denatured by heat treatment for 1 min at 90 °C in deionized water followed by refolding in the buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2 (supplied as 10 × ) for 15 min at 50 °C, 10 min at 20 °C and incubated in the presence of dye for 30 min at 20 °C.The fluorescence emission of DIR2s or mutant aptamers in complex with either DIR or OTB–SO2 (supplied as 10 × ). For refolding, RNA was heated at 90 °C for 1 min in deionized water and then incubated at 50 °C for 15 min in folding buffer followed by incubation at room temperature for 10 min. The refolded RNA was then incubated in the presence of dye for 30 min at 20 °C followed by 1.1 equivalents of the BL3-6 Fab , respectively43) at room temperature for 30 min and concentrated to 6 mg/mL using 10 kDa cutoff, Amicon Ultra-15 column. The formation of Fab–RNA complex was confirmed by native polyacrylamide gel electrophoresis. To decrease the number of nucleation events, RNA was then passed through the 0.2 μm cutoff, Millipore centrifugal filter units. A Mosquito liquid handling robot (TTP Labtech) was used to set up high-throughput hanging drop vapor diffusion crystallization screens at room temperature using commercially available screening kits from Hampton Research, Sigma, and Jena Bioscience. The best-diffracting crystals of the OTB-SO3–DIR2s–Fab complex were obtained in a condition from the Natrix screen: 50 mM MES, pH 5.6, 10 mM MgCl2, 1.8 M Li2SO4. The complex without the OTB-SO3 fluorophore, the optimal condition was from the Jena Bioscience screen: 15 % v/v 2-Propanol, 50 mM MES pH 6.0 20 mM MgCl2. Crystals appeared and grew to full size within 2 weeks in 1 μl + 1 μl hanging drops on siliconized glass slides. The crystals were socked in buffer containing 30% glycerol and saturated OTB dye for crystal with OTB dye before being flash-frozen in liquid nitrogen for data collection at Argonne National Laboratory.An aliquot of RNA sample was refolded in a buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgClhttps://rapd.nec.aps.anl.gov/rapd/). The structures were solved by molecular replacement. Initial electron density maps were obtained from Phaser53 after searching for N-terminal variable domain and C-terminal constant domain of Fab BL3-6 (PDB: 3IVK) sequentially. DIR2s RNA and OTB-SO3 ligand were built into the initial electron density maps using COOT54. The structures were refined using PHENIX55 package. The restraint for OTB ligand was generated by using PRODRG56. Solvent-accessible surface area and area of interaction were calculated using PDBePISA57. All figures were made in PyMOL (Schrodinger).The X-ray diffraction datasets were collected at 100 K at the Advanced Photon Source NE-CAT section beamline 24-ID-C. All the datasets were then integrated and scaled using its on-site RAPD automated programs (32P-radiolabeled DIR2s RNA was refolded in a buffer (900 μL) containing 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2 at 50 °C for 10 min, 10 min at RT and added 0.1 U/μL RNase inhibitor (Amersham). The refolded RNA was incubated for 30 min with Fab BL3-6 ranging from 2 µM to 2 nM in a final volume of 40 µL. A nitrocellulose membrane (Whatman) at top and HyBond filter (Amersham) at bottom were placed in a 96-well Dot-Blot apparatus (Bio-Rad), and the wells were pre-equilibrated with 100 μL refolding buffer. The Fab–RNA mixture was applied, and the filter was washed with another 100 μL of refolding buffer. The membrane and filter were air-dried, exposed to Phosphorimager screens and scanned with a Typhoon Trio imager . The amount of radiolabeled RNA bound to each filter was quantified with ImageQuant software (Molecular Dynamics). Binding constants were obtained by fitting the fraction of nitrocellulose-bound RNA to the following Hill equation: Kd is the dissociation constant; F0 and Fmax are the minimum and maximum fractions of RNA bound; and n is the Hill coefficient.Approximately 50,000 c.p.m. of 5′-59. RNA and Fab–RNA complex samples bound to the OTB dye were prepared and purified as described above. Sample preparation and measurements were performed in a buffer consisting of 50 mM Tris pH 7.4, 150 mM NaCl, and 5 mM MgCl2. For each complex, data on three concentration samples, 1.0 mg/mL, 2.0 mg/mL, and 4.0 mg/mL were collected, along with buffer blanks before and after each sample. Data were collected as 32 0.3-s exposures, in the range q = 0.01087–0.565291. For buffer subtraction, averages of the 32 exposures of pre- and post-sample buffer were subtracted from each exposure of the sample. Each buffer-subtracted exposure was checked for radiation damage by examining the Gunier region using AUTORG60, outlier exposures were removed, and the remaining exposures averaged using DATMERGE61 to produce a final curve for each concentration/sample. Fitting of the Gunier region to calculate Rg for each sample was performed using AUTORG60. For calculation of scattering from the crystal structure atomic coordinates and subsequent fitting to experimental data, ALMERGE58 was used to merge the low- and high-q segments of the 1 mg/mL and 4 mg/mL samples, respectively, and this was used along with atomic coordinates as input to FOXS62. To improve the fit to the data, OLIGOMER63 was used to fit a mixture of crystallographic monomer and dimer to each concentration, using the fits from FOXS62 to monomer and dimer alone as the input form factors. A summary of data collection parameters, programs used for data analysis, and fits to the data is presented in Supplementary Table SAXS experiments were conducted on the SIBYLS beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory, essentially as described previouslySupplementary InformationPeer Review File


One central hypothesis of molecular biology is that a protein sequence can be deduced from the DNA sequence. However, diverse processes at the DNA, mRNA, and even protein level can lead to protein sequences that differ from the deduced sequence. One such process is co- or post-transcriptional RNA editing. RNA editing is found in all domains of life and in diverse RNA species from bacteria and archaea as well as plastids, mitochondria, and nuclei of eukaryotes ,2,3,4. TmRNA editing is the occurrence of base substitutions and short insertions or deletions (indels) in an mRNA that could alternatively be directly encoded by the genomic DNA . TypicalMany land plant lineages show extensive C-to-U editing and sometimes U-to-C editing in mitochondrial and plastid mRNA, a process that seems to be independent of transcription ,6,7. SimGanoderma lucidum and Pleurotus ostreatus as well as for the filamentous ascomycetes F. graminearum, F. verticillioides, Neurospora crassa, N. tetrasperma, Pyronema confluens, and Sordaria macrospora [G. lucidum, editing shows neither a base change nor a tissue preference [F. graminearum and N. crassa, editing was detected only in datasets from sexually developing samples, not from asexual spores or vegetative mycelia [Schizosaccharomyces pombe does not show evidence of mRNA editing during meiosis [The occurrence of mRNA editing in fungi was revealed only recently for the basidiomycetes crospora ,13,14,15eference ,12,13. I mycelia ,13. InteMetazoan A-to-I RNA editing of nuclear transcripts is catalyzed by adenosine deaminases acting on RNA (ADARs) . These eF. graminearum ADAT-encoding gene FgTAD1 had no effect on editing, and deletion of FgTAD2 and FgTAD3 may be lethal [Fungi, like plants, do not encode ADAR homologs, and it remains unknown how fungi catalyze A-to-I RNA editing ,17. Funge lethal . Thus, tAs mentioned above, A-to-I editing of nuclear transcripts leads to proteome diversification. A-to-I editing in humans occurs in a tissue-specific fashion and targets mostly noncoding regions. The few targeted protein-coding transcripts are related to neurological functions .F. graminearum PUK1 kinase [A-to-I editing in filamentous ascomycetes is similarly restricted to a specific stage in the fungal life cycle see . However1 kinase .N. crassa [F. graminearum, N. crassa, and N. tetrasperma—only 454 are conserved between all three species [The question whether editing is adaptive or nonadaptive is still under debate. RNA editing allows the generation of divers proteins from one gene, and it has thus been hypothesized that it may facilitate adaptive evolution . In ceph. crassa . However. crassa . A neutr. crassa , might a species .T. brucei and T. cruzi, causing sleeping sickness and Chagas disease in humans, respectively [coxII gene does contain frame-shifts, implying a faulty gene sequence that needs to be corrected for proper biological function of the encoded protein [T. brucei displays an adaptation to the parasite lifestyle; the procyclic form in the tsetse fly has a standard mitochondrion, whereas the slender bloodstream form (BF) has a tubular mitochondrion with a nonfunctional respiratory chain [T. brucei [A correlation of RNA editing and pathogenesis has long been known from trypanosomes, e.g., vertebrate parasites like ectively . The fir protein . The sinry chain . Editing. brucei . However. brucei ,28,29.Escherichia coli. There, editing targets evolutionary conserved toxin—antitoxin pairs. Editing of the hokB toxin transcript increased toxicity and was conserved in the pathogenic bacteria Klebsiella pneumoniae and Yersinia enterocolitica [Recently, mRNA editing by ADAT activity was shown to occur in colitica , revealiF. graminearum, Sclerotinia sclerotiorum, causing stem rot, or Blumeria graminis f. sp. tritici, causing wheat powdery mildew [Fungal nuclear genes whose transcripts are affected by editing tend to have a role in late sexual development, i.e., meiotic spore (ascospore) formation and/or ascospore discharge ; Fig 1. y mildew ; Fig 1.


NELF-E (RD), SKIV2L (SKI2W), DXO (DOM3Z) and STK19 (RP1 or G11) and dubbed as NSDK. NELF-E is the subunit E of negative elongation factor responsible for promoter proximal pause of transcription. SKIV2L is the RNA helicase for cytoplasmic exosomes responsible for degradation of de-polyadenylated mRNA and viral RNA. DXO is a powerful enzyme with pyro-phosphohydrolase activity towards 5′ triphosphorylated RNA, decapping and exoribonuclease activities of faulty nuclear RNA molecules. STK19 is a nuclear kinase that phosphorylates RNA-binding proteins during transcription. STK19 is also involved in DNA repair during active transcription and in nuclear signal transduction. The genetic, biochemical and functional properties for NSDK in the MHC largely stay as a secret for many immunologists. Here we briefly review the roles of (a) NELF-E on transcriptional pausing; (b) SKIV2L on turnover of deadenylated or expired RNA 3′→5′ through the Ski-exosome complex, and modulation of inflammatory response initiated by retinoic acid-inducible gene 1-like receptor (RLR) sensing of viral infections; (c) DXO on quality control of RNA integrity through recognition of 5′ caps and destruction of faulty adducts in 5′→3′ fashion; and (d) STK19 on nuclear protein phosphorylations. There is compelling evidence that a dysregulation or a deficiency of a NSDK gene would cause a malignant, immunologic or digestive disease.At the central region of the mammalian major histocompatibility complex (MHC) is a complement gene cluster that codes for constituents of complement C3 convertases . Complement activation drives the humoral effector functions for immune response. Sandwiched between the genes for serine proteinase factor B and anchor protein C4 are four less known but critically important genes coding for essential functions related to metabolism and surveillance of RNA during the transcriptional and translational processes of gene expression. These four genes are  In 1984, Professor Rodney Porter’s team in Oxford reported the cloning and confirmed the physical linkage of the genes for human complement C4A and C4B, factor B and C2 through overlapping cosmids ,4,5,6,7.NELF-E, SKIV2L, DXO and STK19 (NSDK), respectively. Unlike the Class I and Class II and complement that have distinct immunologic functions involving proteins, NSDK seems to play fundamental life functions in eukaryotes and actively engaged in the surveillance of RNA integrity including the 5′ cap and 3′ tail. In the process of cell metabolism, these four genes play critical roles on transcriptional control, RNA turnover and signaling. They may also fine-tune the innate immune response or type I interferon-mediated inflammation elicited by infections of RNA viruses. Here we give a brief account on the molecular genetics and biology of this RNA metabolism and surveillance “quartet” in the MHC  and the SKIV2L gene. This gene was discovered by Meo and colleagues in 1988 . RD is oThe RD gene is 6.7 kb in size and consists of 11 exons. It encodes 380 amino acids. The calculated molecular weight of the RD protein is 46 kDa ,18. The negative elongation factor (NELF) and DRB  sensitivity inducing factor (DSIF). NELF acts cooperatively with DSIF to strongly represses transcriptional elongation by RNA polymerase II (RNAPII), about 25–50 nucleotides downstream of transcriptional initiation, particularly in the absence of P-TEFb (positive transcription elongation factor b) [7G caps, proper transcript processing and elongation checkpoint control. In an active transcription process, the cyclin dependent kinase 9 (Cdk9) in P-TEFb may phosphorylate the carboxyl domain of the RNAPII, DSIF and NFLF-E at sites next to its RNA recognition motif, and release the paused transcripts for processive elongation [One feature for the regulation of gene expression in metazoans is the promoter-proximal transcriptional pausing of RNA polymerase II. This phenomenon is carried out by two complexes known as the actor b) . Promoteongation   and participates in the 3′ end processing of mRNA. Both cap-binding proteins CBP-80 and CBP-20 of CBC bind to NELF-E. The region from amino acids 244 to 380 of NELF-E containing an RNA-recognition motif is necessary and sufficient for the interaction between NELF and CBC-RNA. Knockdowns of NELF-E and CBP80 in HeLa cells rendered the free NELF subunits and CBP20 unstable, which are subjected to degradation rapidly, and abolished functions of the NELF holocomplex and the CBC .On the transcription and processing of small nuclear RNA (snRNAs) that are essential for the assembly and functions of spliceosomes, and small nucleolar RNAs (snoRNAs) that are integral to the biosynthesis of ribosomes by RNAPII, NELF-E and ARS2 (arsenite resistance protein 2) play their respective roles progressively through binding to the cap-binding complex CBC. The binding of NELF-E allows promoter proximal pausing, and the subsequent replacement of NELF-E by ARS2 to the same binding site on CBC plus concurrent binding by PHAX (phosphorylated adaptor for RNA export) enable a continuous process to produce these distinct groups of RNA and prepare them for further processing in the cytoplasm or the nucleus .The mRNAs for histones are special as they may not contain polyadenylated 3′ ends. The RRM of NELF-E may bind to the stem-loop structure in the 3′ end of the “replication-dependent” histone mRNAs and enhance their stability. Notably, NELF appears to physically associate with histone gene loci and forms the so-called “NELF-bodies”, which are probably engaged in the processing of histone mRNA. Knockdowns of NELF-E and CBP-80 in HeLa cells led to increased accumulation of the aberrant, polyadenylated form of histone mRNAs .NELF-E was studied on the regulation of gene expression for human immunodeficiency virus HIV. Recombinant protein with the RNA-recognition motif (RRM) of NELF-E was shown to bind the stem of viral RNA with TAR (transactivation response element) . Such bi+ onto substrate proteins, consequently modulates diverse biological processes of ribosylated proteins. In a human cell line, Gibson et al. reported that PARP1 interacted with NELF and mediated ADP ribosylation of NELF-E [Poly-(ADP-ribose) polymerase 1 (PARP1) can covalently transfer of ADP-ribose from NADf NELF-E ,30. In af NELF-E ,30.In a study of double-stranded (ds) DNA break and DNA repair, NELF-E is found preferentially recruited to nearby transcriptionally active genes at sites of DNA ds-breaks to underpin transcriptional repression in a PARP-1 dependent manner. The transcriptional pause allows the machinery for DNA repair to carry out its function and avoid a potential collision of the two systems (DNA repair and transcription) at the damage site. A slight decrease in ribosylation of NELF-E (by PARP-1) at the site of DNA breaks also ensures the pause of transcription imposed by NELF .In a different study of UV-light induced DNA damage (but not ds-breaks) and changes of RNA metabolism, it was found that phosphorylation of NELF-E at serine 115 by MK2 (MAPK activated protein kinase II) promotes the recruitment of 14-3-3 dimers and rapid dissociation of the NELF complex from chromatin. This process is accompanied by elongation of transcription through RNA polymerase II .A cryo-electron microscopy structure with paused transcription complex consisting of RNAPII purified from porcine, human DSIF expressed in bacteria, and human NELF complex expressed in insect cells has been presented with 3.2 Å resolution A. This pPol II associated factor PAF. Homolog of suppressor for transposon Ty insertion 6 (SPT6) binds to RNAPII and then set off the transcriptional elongation process , especially in HCC with portal vein invasion (PVI) . NELF-E While overexpression of NELF-E may be associated with carcinogenesis, a genetic element embedded in the NELF-E gene serves as a tumor suppressor. Between nucleotides 2104 and 2205 of the human NELF-E gene is the precursor for a micro-RNA, miR-1236. This is a splice-dependent mirtronic miRNA that co-expresses with NELF-E ,38. miR-metastasis-associated protein MTA2 [miR-1236 can inhibit tumors migration, proliferation and invasion activity by binding to target mRNAs and down-regulating their protein synthesis. It can also induce target gene expression by binding to gene promoter in the nucleus. Binding of miR-1236 to target mRNAs can lead to abolition of hypoxia-induced epithelial–mesenchymal transition (EMT) and inhibition of migration and invasion of tumor cells. miR-1236-3p can suppress ovarian cancer metastasis by binding to the 3’ untranslated region of zinc-finger E-box binding homeobox 1 (ZEB1) mRNA, and downregulate ZEB1 gene expression . ZEB1 isein MTA2 . miR-123ein MTA2 .The Ski complex is a heterotetrameric complex consisting of Ski2, Ski3 and Ski8 in 1:1:2 stoichiometry that is associated with the cytoplasmic RNA exosome for 3′→5′ mRNA decay . Early iExosomes are highly conserved complex structures responsible for mRNA turnover and therefore fundamental for regulation of gene expression and cell differentiation in eukaryotes. Each exosome consists of 9 or 10 structural proteins that forms a channel structure by which mRNA molecules thread through and get degraded 3′→5′ by an exoribonuclease . The SkiThe functional human SKI2W gene in the HLA was discovered in 1995 . It alsoThe Skiv2l protein has a molecular weight ~140 kDa that consists of 1,246 amino acids ,17,52. T32P-labeled ATP, which would be an energy source for helicase activities [Fusion proteins with human Ski2w immobilized on polyvinylidene fluoride (PVDF) membrane were able to hydrolyze γ-tivities . Immunoctivities . Co-seditivities .7G cap for host mRNA, or double-stranded RNA that seldom exists in a eukaryotic cell, are achieved by RNA helicases such as retinoic acid activating gene 1 (RIG1) or melanoma differentiation-associated protein 5 (MDA5) [knockdown of Skiv2l, but not Xrn1 or Ski3, led to substantial increase of transcription for interferon-β. Thus, Skiv2l specifically suppressed or regulated the production of type I interferon from macrophages in response to ligands that mimic viral infections. This is because Xrn1 knockdown or Ski3 (TTC37) knockdown in similar experiments had no effects on IFN-β expression [The roles of human Skiv2l on the fine regulation of antiviral defense and RNA turnover was examined recently . Discrim5 (MDA5) ,57. Stimpression . Skiv2l unfolded protein response (UPR) through inositol-requiring enzyme 1 (IRE-1) for endonuclease cleavage of specific cellular mRNAs. One of them is a premature mRNA, which retains an unspliced intron, for the X-box binding protein 1 (XBP-1). Splicing out the intronic sequences from the premature mRNA of XBP-1 in the cytoplasm generates ligands for endogenous retinoic acid-inducible gene-like receptors (RLRs). In the absence of Skiv2l, such UPR would trigger elevated production of type I interferon (IFN) production. Therefore, SKIV2L is an important negative regulator of the RLR response to exogenous RNA ligands including RNA viruses [During a viral infection or an inflammation, the host cells are under stress and endoplasmic reticula in cells are subjected to excessive protein synthesis that overwhelms the posttranslational machinery to properly fold and modify protein molecules. Such stress triggers the  viruses . Under sIt was mind-boggling to find that a deficiency in Skiv2l or another Ski complex protein Ski3 or TTC37 causes the syndromic intractable diarrhea known as tricho-hepato-enteric syndrome or THES ,59. ThisFurther analyses revealed that most THES patients had defects in the immune system with low levels of switched immunoglobulins, highly variable responses to vaccination including unresponsiveness of specific antigens, impaired T cell and NK cell functions such as low production of γ-interferon and abnormal T cell proliferation, and severe Epstein Barr Virus (EBV) infection . It appeIt is not clear for the mechanism between the loss of function for SKIV2L or TTC37 and the detrimental phenotypes of THES, which include the failure to thrive, inability in food digestion, poor quality in hair texture, and impairments of liver and immune functions.Intriguingly, elevated expression levels of type I interferon stimulated genes in the absence of apparent viral infections is a marked phenomenon of systemic autoimmune diseases including systemic lupus erythematosus and juvenile dermatomyositis ,83,84. TThe discovery of human DOM3Z (DXO) was reported by Yang and colleagues in 1998 . This geCaenorhabditis elegans (DOM-3), baker’s yeast, fission yeast and possibly flowering plant Arabidopsis [The DOM3Z gene has CpG-rich sequences at its 5′ region, a feature conserved in housekeeping genes in simple and complex eukaryotes. Human DOM3Z is ubiquitously expressed, but the highest expression levels are found in the adrenals and the testes. The first exon (123 bp) of DOM3Z is noncoding because there is an in-frame TGA stop codon at nucleotides 19–21. The full length DOM3Z cDNA is 1386 bp and its genomic DNA is 2.2 kb in size, which consists of 7 exons encoding 396 amino acids. Between residues 103 to 124 of the Dom3z protein is a leucine zipper motif for protein-protein interactions . Yang anbidopsis ,15,17. HRai1. Through genetic deletion and complementation analyses, Xue and coworkers showed in year 2,000 that Rai1 interacts Rat1 . The yeast Rat1 is a nuclear 5′→3′ exoribonuclease that is important in RNA processing and RNA degradation [Most progress in studying the biologic functions of DOM3Z homologs occurred in yeasts, which is named radation . Rai1 stradation , suggestS. pombe Rat1 in complex with Rai1 in 2.2 Å resolution [+2 or Mn+2. They demonstrated that Rai1 has pyrophosphohydrolase activity towards 5′ triphosphorylated RNA [Kluyveromyces lactis (named as DXO1) was solved and its biochemical activity investigated [Xiang, Jiao, Tong, Kiledjian and colleagues in 2009 reported crystal structures of solution , and Raiated RNA . This teated RNA . Thus, istigated . Ydr370Cstigated . In mammstigated ,90. Collstigated .m7GpppNmpRNA) or the second nucleotide (m7GpppNmpNmpRNA) of the mRNA were methylated at the ribose 2′-O position, it would be protected from Dxo-mediated decapping and degradation [If the first (radation .7G cap helps the processes for (a) RNA splicing to remove intronic sequences, (b) the nucleus-cytoplasm transport of mature mRNA for translation, and (c) protection of mature mRNA from degradation. It has been presumed that such capping process in eukaryotes was constitutive and efficient with little errors until yeast Rai1 is shown to play an important role on the quality control of mRNA capping and stability. Moreover, it has been found recently that some bacteria RNA can be capped at the 5′ end with nicotinamide adenine dinucleotide (NAD+) to increase their stability. Unexpectedly, such NAD+ caps are also present in 5–10% eukaryotic mRNA and some small nucleolar RNA (snoRNA). Notably, NAD+-capped mRNAs are not translatable. They are also less stable and subject to Dxo-mediated decapping and 5′ →3′ decay, a process known as deNADding  was found in the promoter region of each C4 gene, suggesting the presence of a novel gene. Thus, cDNA clones were isolated and genomic DNA sequences for this novel gene were determined ,11,94. TRP, C4, CYP21 and TNX . The RP1 protein contains a bipartite nuclear localization signal and was shown to be a nuclear protein [serine/threonine kinase [RP1 is expressed in all tissues and its highest expression is in the adrenals and the testes of human and rodents. Homologs of RP1 are predominantly present among vertebrates but an exception is found in a sea anemone . The 7-exon version of STK19 is 9.1 kb in size that has a 267 bp intergenic distance from DOM3Z. It codes for 254 amino acids with a calculated molecular weight of 30 kDa. The first 110 amino acid residues encoded by the first two exons are absent in the shorter RP1 transcript. Murine STK19 has the small version with 254 amino acids.short interspersed nuclear elements named Alu. One of them is an integral component of a mobile retroelement SVA that consists of a distinct SINE, 21 copies of GC-rich variable number tandem repeats (VNTRs), and an Alu element [Scattered in the introns of the human RP1 gene are 8 copies of  element ,15,17. T element . The hig element ,101,102.https://www.ncbi.nlm.nih.gov/gene/8859, [preferentially expressed antigen in melanoma PRAME that acts as a repressor of retinoic acid receptor and may promote growth of cancer cells, and tripartite motif protein TRIM23 that may be a ADP ribosylation factor for guanine nucleotide binding protein and plays a role in the formation of intracellular transport vesicles and for their movement from one compartment to another in the cell. These interacting proteins may be substrates or cofactors of STK19 kinase, implicating that STK19 could be engaged in DNA replication, RNA transcription, RNA splicing, nucleoside/nucleotide synthesis and transport, and control of gene expression for development and differential cellular functions.In terms of gene function, STK19 encodes a nuclear protein  that is ne/8859, ). These While STK19 knockdown in cells revealed little effects on transcription, it is required for recovery of transcription after DNA damage and therefore important for the transcription-related DNA damage response . STK19 iBy analysis of large-scale exome data from melanoma and uninvolved tissues, Eran Hodis and colleagues showed STK19 is a novel melanoma gene for its somatic hotspot of point mutations. While the D89N mutation of SKT19 has a 5% frequency in a melanoma , no SKT1In addition, by next-generation sequencing and bioinformatics analysis, STK19 was found to have novel T/C missense single-nucleotide variants in Chr6:31947203 in induced pluripotent stem cell (iPSC) lines, which can be used to trace the genotype of original cells .Through meta-analyses of published GWAS data, STK19, SKIV2L and NELF-E appeared to be potential pleiotropic genes for metabolic syndrome and inflammation . Moreovehttps://www.ncbi.nlm.nih.gov/home/ or https://www.ncbi.nlm.nih.gov/pubmed/).We ask when did the genes for NELF-E, SKIV2L, DXO and STK19 start to link together physically as a group in the animal kingdom, and their relationships with the class I and class II genes in the emergence of the MHC in the vertebrates. We attempted to obtain answers to these questions through analyzing data deposited in the National Center for Biotechnology Information . The pr70084.1, ). Relate elegans . STK19 hFor the data deposited in the NCBI, most chromosomal segments are relatively short, and their linkage status is not yet known. The neighboring gene linkages of NELF-E and SKIV2L in one chromosomal segment, and DXO and STK19 in another chromosomal segment are observable in shark, zebrafish, and coelacanth. Genes in each of these pairs are organized in “head-to-head” configuration and therefore they share the same 5′ regulatory sequences in opposite orientations B. The phIn egg-laying mammal platypus, MHC class I and class II genes are present on chromosome X3, but most  of the class III genes form a linkage unit on chromosome X5. The assembly of the class III genes (including NSDK with complement convertases), class I genes and class II genes as the MHC locus appears intact in placental mammals.While the NSDK quartet genes express ubiquitously, it is not known whether there are concerted controls of gene expression (such as genes in operons in prokaryotes), or differential responses to regulation during growth and development.NELF-E-SKIV2L-DXO-STK19 is a complex genetic unit located between complement factor B and component C4 in the class III region of the major histocompatibility complex (MHC) ,117. NEL7G) capped, or NAD+ capped mRNAs from the 5′ end [Genetic studies revealed that homozygous deficiency of human SKIV2L contributes to the pathogenesis of syndromic diarrhea or trichohepatoenteric syndrome (THES). Regulation of gene expression is critical in determining cell identity, development and responses to the environment. DXO or Dom3z possesses pyrophosphohydrolase, decapping and 5′-3′ exoribonuclease activities that can preferentially degrade incompletely 7-methylguanosine (me 5′ end . STK19 ie 5′ end ,109. STKThus, NSDK seems to engage in the surveillance of host RNA integrity at the “head” and at the “tail” , in the


By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and 6N-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m Modificsine (I) . Howeverin vivo transcription errors and errors arising from reverse transcription or library preparation , pseudouridine (Ψ), 5-methylcytidine (m5C), 5-methyluridine  and 5-hydroxymethyluridine (hm5U) triphosphates. After synthesis of modified RNA, first and second strand reverse transcriptase error rates were measured by SMRT sequencing. SMRT sequencing can be used to generate very accurate sequencing data with a low background rate of substitution errors  sequencing to measure the fidelity of transcription and reverse transcription of modified and unmodified RNA. T7 RNA polymerase synthesized RNA from nucleotide pools containing n errors . True reAll reagents are from New England Biolabs, unless otherwise stated. All oligonucleotides were synthesized by Integrated DNA Technologies.+ and 1× ThermoPol Buffer were added to the HpaI digest for total volume of 1.1 ml, and incubated for 30 min at 37°C). Transcription templates were cleaned using a Zymo DNA Clean & Concentrator-25 kit (Zymo Research). 5 μg linearized and repaired plasmids were transcribed using the HiScribe T7 High Yield RNA Synthesis kit (#E2040) and the Standard RNA Synthesis protocol in a total volume of 100 μl for 2 h at 37°C. For unmodified RNA, 10 mM each ATP, UTP, GTP and CTP were used. For modified RNA synthesis, the equivalent unmodified nucleoside triphosphate was replaced with 10 mM N6-methyladenosine-5′-triphosphate , pseudouridine-5′-triphosphate (#N-1019), 5-methylcytidine-5′-triphosphate (#N-1014), 5-methyluridine-5′-triphosphate (#N-1024) or 5-hydroxymethyluridine-5′-triphosphate (#N-1086). After transcription, 4 U DNase I was added and incubated for 30 min at 37°C and RNA was purified using a MEGAclear Transcription Clean-Up Kit (ThermoFisher Scientific). RNA quantity and quality were assessed by running transcription products on a Bioanalyzer using a RNA 6000 Nano kit (Agilent Genomics) and a Novex 6% TBE–urea gel stained with 1× SYBR Gold Nucleic Acid Gel Stain (both from ThermoFisher Scientific). Incorporation efficiency of modified nucleoside triphosphates were determined by LC–MS or LC–MS/MS and found to be: 102% (m6A), 99% (Ψ), 95% (m5C), 104% (m5U) and 92% (hm5U) relative to guanosine , and DNuanosine .cDNA synthesis and the second strand synthesis were performed using the ProtoScript II First Strand cDNA Synthesis Kit (#E6560) with 10 μg RNA, 0.5 μM reverse primer, 1× ProtoScript II Reaction Mix and 1× ProtoScript II Enzyme Mix in 100 μl total reaction volume. Reactions were incubated for 1 h at 42°C. After cDNA synthesis, 20 U RNase H was added to the reaction and incubated for 1 h at 37°C. Single-stranded cDNA was purified using a Zymo DNA Clean & Concentrator-25 kit (Zymo Research) according to the manufacturer's protocol for purifying single-stranded DNA and with an elution volume of 44 μl. Second strand synthesis reactions (100 μl) contained: 40 μl purified cDNA, 5 μM forward primer, 1× ProtoScript II Reaction Mix and 1× ProtoScript II Enzyme Mix and incubated at 42°C for 1 h. Alternatively, cDNA and second strand synthesis were performed with M-MuLV Reverse Transcriptase (2.5 μg or 50 U) and 1× M-MuLV Reverse Transcriptase Reaction Buffer, or AMV Reverse Transcriptase (50 U) and 1× AMV Reverse Transcriptase Buffer. Synthesis products were purified with another Zymo column.Escherichia coli) and 5 U Exonuclease VII were added to the reaction and incubated at 37°C for 1 h. SMRTbell libraries were cleaned using a Zymo column, then additionally size selected with AMPure PB beads (Pacific Biosciences) using 1× volume (500 bp libraries) or 0.6× volume (1 kb libraries). Pacific Biosciences Binding Calculator was used to generate a protocol for annealing sequencing primers and polymerase binding using the DNA/Polymerase Binding Kit P6 v2 (Pacific Biosciences) and default settings. DNA-1 and DNA-2 samples sequenced using the MagBead OCPW protocol (with 6 h movies) and DNA-3 and DNA-4 samples using the Standard protocol (diffusion loading with 5 h movies).Double-stranded DNA products were digested with 30 U BssSαI and 20 U DpnI in 1× CutSmart Buffer  for 1 h at 37°C and cleaned using a Zymo column. Digest products were ligated to 300 pmol SMRTbell adaptors with compatible BssSαI overhangs (/5Phos/TCGTATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3′) with 800 U T4 DNA Ligase in 1× T4 DNA Ligase Reaction Buffer  at room temperature for 30 min, followed by heat inactivation for 10 min at 65°C. Then, 50 U Exonuclease III (High-accuracy consensus sequences were determined for the first and second strand for each sequenced double-stranded DNA as described in Potapov and Ong . The resError rates were derived as follows: first strand errors were determined by comparing cDNA to the reference sequence (RNA strand), and mutations were required to be present in both cDNA and the second-strand DNA Figure . For secFor comparing polymerase error rates and to estimate the statistical significance of observed differences, multiple measurements obtained on different templates  were compared using t-test at a significance value α = 0.05. When multiple comparisons were done, Bonferroni correction was applied to adjust the significance level according to the number of comparisons.E. coli) to degrade excess primers, cleaned using an Oligo DNA Clean & Concentrator (Zymo Research), and transcribed using the HiScribe T7 High Yield RNA Synthesis kit . After transcription, DNA was degraded by adding 4 U DNase I, 10 μl 10× DNase I Reaction Buffer in a 100 μl total volume and incubating for 30 min at 37°C. Transcription products were purified using a Zymo RNA Clean & Concentrator kit. Reverse transcriptase assays were performed by mixing 10 μl of enzyme mix (preheated for 1 min at 42°C) containing 0.026–0.26 pmol of reverse transcriptase, 1 mM each dNTP, 1× Reaction Buffer) and 10 μl of preheated substrate mix  and incubating at 42°C for 2 min. Reactions were quenched with 20 μl 20 mM EDTA, and analyzed using a 3730xl Genetic Analyzer (Applied Biosystems) and PeakScanner software as described in Greenough et al. (Transcription templates were prepared by amplifying a DNA oligo containing a T7 promoter (5′-AATTAATACGACTCACTATAGAAGTATTTCTCCTCGCTGACTGAGATCGGAAGAGCACACGTCT-3′) with forward (5′-AATTAATACGACTCACTATAG-3′) and back (5′-AGACGTGTGCTCTTCCGATCT-3′) primers using Q5 High-Fidelity 2X Master Mix. PCR products were treated with Exonuclease III . Base composition analysis by LC–MS determined that the modified nucleotides were efficiently incorporated (Bst 2.0 or 3.0) to produce the first strand (cDNA). A second strand was synthesized by the same reverse transcriptase or DNA polymerase to produce double-stranded DNA, which was then sequenced. For analyzing cDNA errors, sequenced bases were compared to the reference to identify first strand errors, and only errors that were also confirmed in the second strand were counted. An error in the first strand can either be generated during transcription or reverse transcription, and first strand synthesis errors represent the cumulative errors of both T7 RNA polymerase and the reverse transcriptase. For example, if the expected RNA base was an A, but a C was observed in the cDNA, these substitutions would be classified as rA→rG/dT→dC, where the first pair represents the equivalent RNA polymerase error and the second pair represents the equivalent reverse transcriptase error that could have generated this substitution event  indicating that N6-methyladenosine was more mutagenic than adenosine, by either increasing the mutation rate of reverse transcription or causing RNA polymerase misincorporation across dT. The increase in substitution events at adenosine positions was statistically significant for ProtoScript II and AMV reverse transcriptases. However, m6ATP was not misincorporated across other bases by T7 RNA polymerase, as there was no significant difference in substitution errors involving the incorporation of m6ATP .The presence of modified nucleotides had differing effects on first strand errors. Pseudouridine (Ψ), hms Figure . To furt5U displayed the largest effect on first strand synthesis fidelity, by statistically significantly increasing substitutions at hm5U RNA reference positions by up to 21-fold , or the RNA polymerase inserting the wrong nucleotide in place of hm5UTP . In contrast, there was no statistically significant difference in errors involving RNA polymerase misincorporation of hm5UTP across non-complementary template bases , indicating that when hm5UTP was incorporated into RNA, it was paired with template adenosine as frequently as UTP. Compared to hm5U, a smaller modification at the C5 position (m5U) also did not alter the fidelity of T7 RNA polymerase or reverse transcriptase, as almost all substitutions for m5U-substituted RNA showed no statistically significant difference between modified and unmodified uracil (the exceptions were rA→rU for M-MuLV and AMV reverse transcriptases). Base composition analysis of the RNA templates (5U- (4%) and m5U- (11%) containing RNA samples. While this had little effect on the error rate for m5U-substituted RNA, it likely led to an underestimation of the overall misincorporation levels measured across hm5U.Of the uracil modifications tested, hmemplates  showed tPseudouridine (Ψ) is an isomer of uridine where the base is attached by a carbon-carbon bond at the C5 position. Pseudouridine was misincorporated across thymidine by T7 RNA polymerase during transcription: rA→rU substitutions were increased 7- to 12-fold compared to unmodified RNA, while other substitutions (rG→rU or rC→rU) were unaffected Figure . There wTo determine if replication errors were enriched in particular sequence contexts, the identity and frequency of the bases surrounding all first strand errors were analyzed. The artificial sequences used in the present study contained all possible 4-base combinations, ensuring an equimolar distribution of bases. However, for modified RNA bases, certain sequence contexts were enriched for substitution errors. For example, for pseudouridine, which mispairs with dT during transcription by T7 RNA polymerase, rA→rΨ substitutions were preferentially preceded by incorporation of CTP, a bias that is not seen with unmodified rA→rU substitutions Figure .Bst 2.0 and 3.0 DNA polymerases, were measured  of all errors were at either adenosine or thymidine template bases, with the largest error class resulting from dA→dG substitutions. AMV reverse transcriptase had a statistically significant increase in dA→dG substitutions compared to M-MuLV and ProtoScript II reverse transcriptases. For isolated reverse transcriptase second strand errors, analysis of the bases surrounding substitution events indicated no discernible bias (Second strand errors reflect the isolated error rate of the reverse transcriptase on DNA templates and were identified by a mispair between the template base (first strand) and the incorporated base (second strand) Figure . The toted Table . The difble bias .In addition to substitutions, insertion and deletion errors were also identified. Compared to the rate of base substitution, indels were relatively rare, representing <20% of the total errors by a particular polymerase Table . ExaminiMulti-base indels were considered a single slippage event in our study and as such were counted as a single insertion or deletion event, regardless of the number of bases inserted or deleted. Larger indels (four or more bases) occurred statistically more frequently in first strand synthesis than second strand synthesis, and large deletion events were more common than large insertions in the first strand. We additionally observed large-scale rearrangements in sequencing reads, where each rearrangement was a complex combination of multiple insertions, deletions and substitutions. These large-scale rearrangements were likely generated by template switching during transcription or cDNA synthesis and were excluded from error rate analysis by filtering for sequencing reads of the expected length.6N-methyladenosine was generally replicated less efficiently by ProtoScript II, M-MuLV and AMV reverse transcriptases. Template pseudouridine had no effect on the turnover rate of all three reverse transcriptases, nor did the methylated bases  for ProtoScript II or m5C for AMV reverse transcriptases. Template hm5U was least efficiently replicated by ProtoScript II, reducing turnover by more than half that of unmodified RNA. Generally, with ProtoScript II, modifications that were accurately replicated did not affect reverse transcriptase specific activity, and modifications that decreased reverse transcriptase activity also decreased replication fidelity.In addition to studying the accuracy of modified RNA, we measured the replication efficiency of substituted RNA by reverse transcriptases. By following replication of a 23-mer RNA template using a fluorescently labeled primer, the nucleotide incorporation turnover rate for each reverse transcriptase was determined Table . SubstitPacific Biosciences SMRT sequencing was used to measure the fidelity of incorporation and replication of modified and unmodified ribonucleotide bases. In this study, the effect of base modification was determined by normalizing the error rate for modified bases to unmodified RNA for each specific type of substitution. Seemingly small modifications, such as methyl-, hydroxymethyl- and isomeric bases, were found to increase combined RNA polymerase and reverse transcriptase (first strand) error rates.Studies from DNA polymerases have shown how synthetic modified nucleotides have been used to probe the mechanism of polymerase fidelity . DNA−5 errors/base in this study, compared to 5.9 × 10−5 errors/base in Roberts et al. (−5 errors/base in this study, compared to 3 × 10−5 errors/base previously determined using a forward mutation assay with DNA templates (Reverse transcriptase-specific error rates on DNA templates were also determined by analyzing mismatches between the first and second strands. Results from this study can be compared to previously published error rates of reverse transcriptase acting on DNA templates using forward mutation assays. For AMV reverse transcriptase, we determined an error rate of 5.2 × 10s et al. . Howeveremplates ,17,39. Iemplates ,19.Polymerase slippage events, resulting in small insertions and deletions, were also characterized, and were shown to be a small percentage of the overall error rate for non-homopolymer sequence runs. The templates used in this study were artificial sequences designed to avoid homopolymers, which likely reduces observed error rates. In spite of the effort to reduce repetitive sequences, the artificial sequences still contained a particular trinucleotide repeat that was enriched for insertions of the triplet codon after cDNA synthesis. In contrast, this same repeat element did not increase triplet insertion for second strand synthesis, indicating that either T7 RNA polymerase was more prone to triplet insertions at this site during RNA synthesis or reverse transcriptases are more susceptible to insertions at this site when replicating RNA, but not DNA, templates. Both mechanisms are possible and would produce the same result in the current study.Bst DNA polymerase variants with innate reverse transcriptase activity (Bst DNA polymerase and viral reverse transcriptases, similar first strand error rates and mutational spectra were observed between the disparate polymerases. However, it should be noted that the relative contributions of RNA synthesis and RNA-templated reverse transcription to the overall first strand error rate cannot be determined in the current study and should be taken into account when interpreting results. Specifically, it is unclear whether T7 RNA polymerase and reverse transcriptases have similar or vastly different error rates, making it challenging to compare reverse transcriptase fidelity even on comparable RNA templates. Further characterization of polymerase fidelity by methods that can distinguish between RNA polymerase and reverse transcriptase errors is needed (Although reverse transcriptases are frequently utilized in RNA workflows, some DNA-dependent DNA polymerases have intrinsic activity on RNA templates . Utiliziactivity ,41 in ors needed .In conclusion, we developed a method to assess the relative accuracy of incorporation and replication of modified ribonucleotides and studied the effect of naturally-occurring RNA modifications on frequently used enzymes in biotechnology. Single-molecule next-generation sequencing assays provide comprehensive information on the frequency, type, and sequence context of replication errors and contribute to a better understanding of polymerase accuracy.https://github.com/potapovneb/rt-fidelity.Sequencing data pertaining to this study has been deposited into the Sequencing Read Archive under accession number SRP122531. Custom software tools are available in the GitHub repository at: Supplementary DataClick here for additional data file.