Using a temperature programmed desorption apparatus equipped with a quadrupole mass spectrometer showed the production of hydrogen molecules, methane, and ethane, suggesting that the protonation of n-octane takes place on graphite surface at near room temperature. It is known that acidic functional groups are terminated at edges on the air-cleaved HOPG surface and they increase their acidity via reactions with water. However, it is most unlikely that they protonate n-alkanes at near room temperature such as superacids. We anticipate that superacidic protons, which can protonate n-octanes, are produced on the graphite surface through a novel reaction mechanism.Graphite basal surface is inert, and decomposition of n-alkanes on the graphite surface has not been discovered. We here report the evidence of decomposition of n-octanes on highly oriented pyrolytic graphite (HOPG) surface, heat-treated up to 1200 °C under high vacuum (10 In the graphite planes, each carbon atoms are linked to the nearest three carbon atoms with sp2 covalent bond (the nearest distance between carbon atoms in the plane: 1.42 Å) to form honeycomb structure. The extended structure of graphite is a consequence of the 2 pz orbitals, which bond weakly between planes of the sp2 structures. Distance between the nearest two planes is 3.34 Å. However, we cannot obtain large single crystal graphite which enables us to determine the exact structure of graphite by using x-ray diffraction1μm. The step edges are formed on the cleaved HOPG surface. The grain boundaries are known to have somewhat lower chemical reactivity compared to the step edges4Graphite is a very popular material and widely used in science and engineering. The structure is simple, but its periodic flat-layer structure is serving as a source of variety material properties such as mechanical, chemical, electrical, and physico-chemical properties. Graphite is comprised of only carbon atoms, where each carbon atom is sp2)nH, with n = 5 to 60) was performed on the graphite surface which is held at ~120 K9105H12-C12H26) were also close to their melting points (143–263 K)16H34 adsorbed on the HOPG surface at room temperatureGraphite basal plane surface is chemically inert. Carbon atoms at edge sites on graphite surface are terminated by oxygen- and hydrogen-functional groups in air491011121314151617We here report a novel chemical reaction discovered between n-octane and graphite basal surface that is well heat-treated in high vacuum, by using the TPD method. The TPD measurements evidently showed that n-octane is decomposed on the graphite surface at near room temperatures and the decomposition of n-octane is accompanied by the evolution of hydrogen molecules, methane, and ethane, suggesting that protonation of n-octanes takes place on the basal graphite surface19−7 Pa) were measured without taking the sample out of the ultrahigh vacuum chamber. The TPD curves for m/z = 16, 18, 28 and 44 signals up to 1300 K are shown in TPD spectra of HOPG sample heated up to 1200 °C under ultrahigh vacuum  database of mass spectra on straight chain and branched alkanes, alkenes, and their isomers with 1–8 carbon atoms, we confirmed that a part of n-octane dropped onto the graphite surface decomposes into smaller alkanes or alkenes with 1–3 carbon atoms as follows; Assuming that all of the m/z 43 ion signal are attributable to n-octane, TPD curves of n-octane were calculated from the m/z 43 TPD curve using NIST mass spectrum data, and the results were displayed by red curves in 4H10, C5H12, C6H14, C7H16 and C8H18 and their isomers, the intensities of m/z 27, 28 and 29 signals are smaller than that of either m/z 43, 57, 71, or 85 signal. From these facts, the intensities of m/z 27, 28 and 29 signals cannot be explained by assuming the mass spectra of C4-C8 n-alkanes and their isomers.As can be seen from 3H6, the intensities of m/z 27, 28 and 29 signals are smaller than that of m/z 42 signal. In the mass spectra of 1-butene C4H8 and its isomers, the intensities of m/z 27, 28 and 29 signals are smaller than that of m/z 56 signal. In the mass spectra of 1-pentene C5H10 and its isomers, the intensities of m/z 27, 28 and 29 signals are less than or almost equal to that of m/z 70 signal. In the mass spectra of 3-hexene C6H12 and its isomers, the intensities of m/z 27, 28 and 29 signals are smaller than that of either m/z 55, 56, or 69 signal. In the mass spectra of 1-heptene C7H14 and its isomers, the intensities of m/z 27, 28 and 29 signals are also smaller than that of either m/z 55, 56, or 69 signal. In the mass spectra of 1-octene C8H16 and its isomers, the intensities of m/z 27, 28 and 29 signals are less than that of m/z 55 signal. The relative intensities of m/z = 27, 28, 29, 41, 42, 43, 55, 56, 69 and 70 signals obtained from the NIST mass spectrum database of propene, 1-butene, 1-pentene, 3-hexene, 1-heptene, and 1-octene are shown in Similarly, in the mass spectrum of propene C3-C8 alkenes.From these facts mentioned above, it is concluded that most of the m/z 27, 28 and 29 signals are not due to fragments from C1-C3 alkanes or C2 alkene.2O4 · H2O) which evolves only water at ~245 °C and the TPD spectrum of the m/z = 16 observed for the HOPG samples, respectively. According to the TPD spectra in +) at m/z 17 has a relative intensity of 26.5% in comparison to that of the base peak at m/z 18 and ion (O+) at m/z 16 has a relative intensity of 2.3%. TPD curves of m/z = 17 and m/z = 16 calculated from the TPD curve of H2O (m/z = 18) on the basis of the TPD spectra of CaC2O4 · H2O are plotted by red curves in 2O is considerably smaller than the measured intensity of m/z 16 signal. TPD curve of m/z = 16 (O+) originating in water is plotted by red curve (see TPD spectrum of m/z = 16 in 2) was detected at temperatures between 30 and 600 °C. Therefore, the observed m/z 16 ion signal does not arise from oxygen molecule. According to the NIST database, with the exception of methane, mass spectra of hydrocarbons do not show peak at m/z = 16, even if they show the peak at m/z = 16, it is extremely small compared to their base peaks. Assuming that in 2), respectively, the sum of m/z 16 ion signals that come from CO and CO2 molecules was obtained at temperatures between 30 and 600 °C from the observed TPD curves of m/z = 28 and m/z = 44 on the basis of NIST database. The sum of TPD curves of the m/z = 16 signals due to CO and CO2 is plotted by a blue curve in +) originating in water, which is calculated from the TPD curve of H2O (m/z = 18) on the basis of the TPD spectra of CaC2O4 · H2O, is also shown by red curve in 1-C3 hydrocarbon and dehydrogenation of alkanes to alkenes occur on the graphite surface at a temperature from about 70 °C. This is not due to thermal cracking, but possibly due to catalytic one. The TPD experiments showed that dihydrogen, methane, and ethane are produced in the cracking. This result indicated that this decomposition is not based on the classical (bimolecular) mechanism of catalytic cracking including β-scission and hydride transfer where the smallest alkane cracking product is propane19It is known that alkanes thermally decompose at temperatures in the region 500–800 °C2) or an alkane, for example, methane, ethane, and a carbenium ion.Here we propose that the protolytic cracking on the graphite surface19−7 Pa), to remove all contaminates such as oxygen- and hydrogen-containing functional groups on the air-cleaved HOPG surface. However, carbon atoms at the edge will be terminated by hydrogen and oxygen functional groups423a) are orders of magnitude weaker than those of superacids that can protonate n-alkanes at near room temperature. We therefore argue that superaidic protons, which serve to protonate n-octane, are generated not at the edge sites and that the protonation of n-octane occurs on graphite basal plane surface  by using TPD apparatus (ESCO. Ltd.) equipped with a quadrupole mass spectrometer (QMS)  in two operational modes, i.e., full scan and selected ion monitoring (SIM) modesThe chemical reaction between graphite and n-octane was studied under ultrahigh vacuum conditions . Four HOPG sheets (5 × 12 × 0.36 (average) mm) were prepared from the each purchased sheet and both sides of these four sheets were cleaved with scotch tape in air before experiment.−7 Pa). After that, these sheets were cooled to room temperature. As soon as the sheets were taken out from the ultrahigh-vacuum chamber, a 30 μl solution of n-octane was dropped onto the surface in air, using a micropipette in less than 15 s. These HOPG sheets were samples used in this experiment. After the completion of this procedure, the HOPG samples were inserted into the ultrahigh-vacuum chamber for TPD measurements. TPD measurements were carried out at temperatures from room temperature to 600 °C with a heating rate of 100 K min−1 under ultrahigh vacuum (~10−7 Pa). The mass spectrometer operated in the electron impact ionization mode at 70 eV. In the full scan mode (m/z 1–120), electron impact ionization (EI) mass spectra were recorded at 70 eV electron energy. Before each measurement, the background spectrum was recorded using as heat-treated HOPG sheets, from which no peaks were observed in the range of m/z 1–120 up to 800 °C.It has been known that oxygen- and hydrogen-containing functional groups exist on the air-cleaved HOPG surfaceHow to cite this article: Kawashima, Y. and Iwamoto, M. Protolytic decomposition of n-octane on graphite at near room temperature. Sci. Rep.6, 28493; doi: 10.1038/srep28493 (2016).


Muscleblind-like (MBNL) proteins are key regulators of precursor and mature mRNA metabolism in mammals. Based on published and novel data, we explore models of tissue-specific MBNL interaction with RNA. We portray MBNL domains critical for RNA binding and splicing regulation, and the structure of MBNL's normal and pathogenic RNA targets, particularly in the context of myotonic dystrophy (DM), in which expanded CUG or CCUG repeat transcripts sequester several nuclear proteins including MBNLs. We also review the properties of MBNL/RNA complex, including recent data obtained from UV cross-linking and immunoprecipitation (CLIP-Seq), and discuss how this interaction shapes normal MBNL-dependent alternative splicing regulation. Finally, we review how this acquired knowledge about the pathogenic RNA structure and nature of MBNL sequestration can be translated into the design of therapeutic strategies against DM. Muscleblind-like proteins (MBNL) belong to a family of tissue-specific RNA metabolism regulators, which in mammals are encoded by three genes MBNL1, MBNL2 and MBNL3 [Figure MBNL1 and MBNL2 genes rises during differentiation. This is especially evident in the brain, heart and skeletal muscle, in which there is a several fold increase in MBNL1 and 2 mRNA expression in adult tissues compared to the fetal  might be one of the transcription factors that drives the expression of MBNL1  that attract and sequester in the nuclei much of the available cellular pool of MBNLs. More severe type of DM, the DM1, results from a CTG repeat expansion in the 3′UTR of the dystrophia myotonica protein kinase (DMPK) gene (CNBP) gene  gene –28. The BP) gene . DespiteBP) gene –21,30.In vitro experiments indicate that once transcribed, expanded C/CUG repeats fold into very stable and long RNA hairpin structures, so-called toxic RNAs, that co-localize in the nucleus with MBNLs and other nuclear factors in the form of ribonuclear foci , insulin receptor (INSR), bridging integrator 1 (BIN1) and calcium channel voltage-dependent L type alpha 1S subunit (CACNA1S) results in myotonia, insulin resistance and muscle weakness, respectively, constituting key hallmarks of DM  and six  undergo alternative splicing , and ZnFs 3 and 4  pre-mRNA fragment with a conjugated fluorophore and a quencher at the 5′ and 3′ termini and led to separation of the molecule ends, as judged by the increased fluorescence level 12 molecule , INSR, MBNL1, nuclear factor I/X (NFIX) and very low density lipoprotein receptor (VLDLR). Only in a subset of targets, such as sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP2A1) and cTNT, ZnF3/4 sufficed for high MBNL1 activity Y sequence in intronic regions adjacent to exon 5 of chicken and human pre-mRNA of cTNT [Figure GCY, and particularly UGCU, as the most common MBNL1 binding motif, especially when repeated several times over a short sequence region [Figure GCU is one of the core elements of interaction not only for MBNL1 but also MBNL2 and 3 , CUGBP elav-like family member 1 (CELF1) and forkhead box 2 (FOX2), that favored fully single-stranded RNA motifs  pre-mRNA fragment and C/CUGexp are also within the same range , INSR and MBNL2 , comprised of roughly 300 000 small molecules, was employed to identify chemical compounds that disrupt the MBNL1/CUG the RNA . One of  the RNA . Howeverpeat RNA . This inNA decay . This wong cells . Howeverechanism . Intrigutability .exp hairpins. Modularly assembled ligands offer the advantage of targeting many RNA structure repeat units at a time, and hence offer more specificity toward expanded repeat RNA, higher potency and potentially, less off-target effects . For example, derivatives of Hoechst 33258 assembled modularly into a peptoid scaffold displaying multiple copies of the ligand separated by spacing modules were highly specific in binding multiple CUG-repeat motifs in vitro with low nanomolar affinities (in vitro (exp foci and rescued the cTNT pre-mRNA alternative splicing defect in a cellular DM1 model (exp complex in vitro at low micromolar concentrations (et al. developed cell- and nucleus-permeable dimeric ligand with oligoamine linkers, which very effectively dispersed ribonuclear foci in DM1 cell models (More recently, an alternative strategy was developed based on a rational design of modularly assembled ligands that target and neutralize CUGfinities ,111. TheM1 model . Furthertrations , Jahromil models . Noteworl models .exp-binding ligands. Two recent studies based on these approaches yielded a series of inhibitors of the toxic MBNL/RNA complex, which were much more potent than the query compounds Hoechst 33258 (9 molecular transporter, researchers were able to improve the ligand's cellular uptake and bioactivity and consequently improve DM1-associated defects in cell and animal models (Shape- and chemistry alignment-based virtual computational screens can also be successfully employed to improve the efficacy of CUGst 33258  and 4′,6st 33258 . Importast 33258 ,118. In st 33258 . Approprst 33258 . Howeverst 33258 , it was st 33258 . Interesl models .exp RNA. Some of the most recent examples include the use of gapmers to promote target cleavage via RNaseH (exp degradation via RNA interference pathway (et al. (HSALR mice decreased the number of CUGexp foci, redistributed MBNL1 protein, corrected abnormal alternative splicing of MBNL1-sensitive exons and reduced myotonia (HSALR transgene mRNA, which the authors attributed to enhanced natural decay of the transgene transcript due to its relocation to cytoplasm, as CAG25 was unable to support RNA cleavage by RNaseH. Importantly, the authors also postulated that the therapeutic effect of CAG25 likely results from displacement of the MBNL1 protein from the expanded CUG repeats (et al. utilized CAG25 with a 5′ primary amine modifications, linked to arginine-rich cell penetrating peptide, for a systemic administration into HSALR mice to demonstrate that it efficiently redistributes MBNL1 protein in myonuclei and corrects alternative splicing defects (The neutralization of RNA toxicity can be also achieved by antisense oligomers targeting CUG repeats. However, majority of available strategies that fall into this category are based on degradation of CUGa RNaseH , or the  pathway , as well pathway –126. The (et al. . This stmyotonia . It also repeats . Further defects .Summarizing, expanding knowledge of the structural basis for MBNL/RNA interaction opened up several options for researchers to prevent the pathogenic protein/RNA complex formation or revert its toxic effect. The future challenge lies in achieving effective cellular delivery and ensuring optimal bioactivity of the therapeutic compounds. Finally, in the case of small molecules targeting the RNA component of the DM complex, increasing their specificity toward expanded CUG repeat RNA and minimizing the off-target effect toward other CUG repeat-containing RNAs will be yet another demanding task in a therapeutic battle against DM.GCY motif in pre-mRNA and mRNA. However, other factors, such as proximity of additional YGCY motives, pyrimidine content of adjacent regions and RNA structure, might also play a role in MBNLs’ target recognition (In this review we highlighted the structural basis for MBNL1 target recognition and discussed how it impacts the regulation of alternative pre-mRNA splicing in differentiation and disease. MBNLs bind the common Yognition –56. It iognition . Of inteognition ,128–129.ognition . Anotherognition , but alsognition . Delinea


X-chromosome inactivation is the process that evolved in mammals to equalize levels of X-linked gene expression in XX females relative to XY males. Silencing of a single X chromosome in female cells is mediated by the non-coding RNA Xist. Although progress has been made toward identifying factors that function in the maintenance of X inactivation, the primary silencing factors are largely undefined. We developed an shRNA screening strategy to produce a ranked list of candidate primary silencing factors. Validation experiments performed on several of the top hits identified the SPOC domain RNA binding proteins Rbm15 and Spen and Wtap, a component of the m6A RNA methyltransferase complex, as playing an important role in the establishment of Xist-mediated silencing. Localization analysis using super-resolution 3D-SIM microscopy demonstrates that these factors co-localize with Xist RNA within the nuclear matrix subcompartment, consistent with a direct interaction.  •An shRNA screen identifies factors implicated in chromosome silencing by Xist RNA•Rbm15, Wtap, and Spen are required for Xist-mediated silencing•Rbm15 is important for efficient deposition of H3K27me3 on the inactive chromosome•Rbm15, Wtap, and Spen co-localize with Xist RNA in perichromatin spaces To identify primary silencing factors implicated in X-chromosome inactivation, Moindrot et al. set up a pooled shRNA genetic screen. The RNA-binding proteins Rbm15 and Spen, together with Wtap, a subunit of the RNA methylation complex, were identified as important factors required for Xist-mediated silencing. XCI is initiated by the X inactive specific transcript (Xist), a long non-coding RNA (lncRNA) that is expressed from the inactive X (Xi) elect. Xist RNA spreads in d in cis .Formation of the Barr body is a multistep process involving several pathways linked to formation of repressive heterochromatin. Notable examples are the acquisition or loss of specific histone tail modifications, enrichment or depletion of variant histones, DNA methylation at X-linked promoter CpG islands, and long-range topological reorganization of the chromatin fiber. How these different pathways are linked to Xist RNA accumulation and to one another remains poorly understood. A key challenge has been to identify the primary factors that initiate the silencing cascade during the early developmental window of opportunity. A priori, these factors are predicted to include one or more RNA binding proteins (RBPs). Polycomb repressive complex 2 (PRC2), which mediates the histone modification H3 lysine 27 methylation, has been suggested as a candidate for this role  and has Efforts to purify native Xist ribonucleoprotein complexes have been hampered by the association of Xist RNA with the insoluble nuclear matrix. Similarly, an attempt to purify key factors from nuclear extracts using defined regions of Xist identified only hnRNPC, a generic mRNA processing factor . Two indcis with the Xist transgene using homologous recombination to generate the MG-3E cell line , which are known to be Xist responsive . We madeell line A and S1.ell line . Consistell line B.We adopted a pooled lentiviral shRNA screening strategy  using a Following optimization and establishment of appropriate conditions, both libraries were screened to identify targets for which knockdown enhanced representation in GFP high cells following induction of Xist RNA C. BrieflA ranked list of the top targets from the nucleome (225) and ubiquitylome (34) libraries is shown in Selected high-ranked targets were validated by FACS analysis of Mylc2b-GFP following transduction of induced and non-induced MG-3E cells with individual shRNAs and scrambled control . A lucifThe aforementioned FACS validation experiments highlighted Mediator, Rbm15, Spen, and Wtap as potentially having a role in Xist-mediated silencing. A priori, the knockdown of these positive hits could affect Xist transgene expression, Xist RNA localization or interfere with the downstream silencing cascade. We therefore analyzed Xist RNA expression using RNA FISH C and RT-For Rbm15, Spen, and Wtap, Xist domains were apparently unaffected, although for Rbm15, levels of Xist RNA determined by RT-PCR were somewhat reduced C and S3Ecis with the Xist transgene. Initially we used RT-PCR to assess expression levels of four chromosome 17 genes, SatB1, Enpp5, Crb3, and Fbxl17, previously shown to be downregulated following Xist RNA induction in MG-3E cells . As shown in We went on to determine whether knockdown of Rbm15, Spen, and Wtap affect chromatin features of Xi, specifically the formation of H3K27me3 domains linked to Xist-mediated recruitment of the Polycomb complex PRC2. Initial analysis indicated Rbm15 knockdown leads to reduced intensity and size of H3K27me3 domains A. No obvTo further examine the function of Rbm15, Spen, and Wtap in Xist-mediated silencing, we used immunofluorescence to assess their nuclear localization in 3E mESCs expressing transgenic Xist RNA. The results, illustrated in Analysis of Xi features and Xist RNA by super-resolution 3DSIM has shown that Xist RNA localizes to the perichromatin or nuclear matrix compartment, spatially separated from chromatin . To analWe went on to determine the relative localization of BglG-mCherry and Rbm15 C, Wtap D, and SpThe genetic screen described here was designed to identify factors required for establishment of Xist-mediated silencing. Specifically, the reporter cell system functions within the critical developmental window of opportunity during which cells are Xist responsive. The use of an inducible Xist transgene system and of an unstable PEST-GFP reporter enabled us to focus the screen on the time period during which Xist-mediated silencing is initiated. The fact that we identified different subunits of defined complexes and also multiple factors linked to specific pathways indicates that the screen achieved a good degree of saturation. However, we cannot rule out that some factors evaded detection, for example, because of functional redundancy or incomplete coverage of the shRNA libraries.Pooled shRNA knockdown offered specific advantages in the context of this screen. First, loss of function occurs across a broad dynamic range (because of cell-to-cell variation in knockdown efficiency and between different hairpins designed to the same gene), creating a virtual allelic series that facilitates identification of essential factors for which significant loss of function affects cell viability. Second, using a number of shRNA hairpins to each target provided a critical parameter for the ranking of positive hits.Our screening procedure was developed after extensive optimization. Notably, we found that it was important to ensure that the number of cells transduced with a given shRNA was sufficient to reliably detect overrepresentation in the selected populations, particularly given that lentiviral transduction of ESCs is relatively inefficient. Linked to this, it was important to limit the coverage of the screen to factors in the nucleome and ubiquitylome in order to reduce false positive rates. Finally, inclusion of an HTS barcode in the shRNAs was essential to obviate the need to sequence across stem loops, which can introduce extreme bias.Possible sources of false positives in the screen include knockdown of factors that affect the TetOn-inducible promoter system used to drive Xist expression in the reporter cell line and factors that influence the levels of the GFP-PEST reporter other than at the level of transcriptional silencing. Using validation assays that discriminate these possibilities, we found that Mediator knockdown strongly reduces TetOn promoter-driven Xist RNA expression, most likely linked to its requirement for VP16 mediated transactivation, and that knockdown of core peroxisome proteins increased levels of GFP-PEST protein, possibly linked to defects in protein turnover. Although we discarded these hits as false positives, the fact that we identified multiple factors in the same complex/pathway in both cases further demonstrates that the screen achieved a high degree of saturation.cis but had no obvious effect on the formation of Xist RNA domains. In the case of Rbm15, we also observed a deficiency in the formation of Xist-mediated H3K27me3 domains. SR-3DSIM analysis revealed co-localization with Xist RNA within the nuclear matrix/perichromatin compartment. Given that Rbm15 and Spen are RBPs, our SR-3DSIM observations are consistent with a direct role in binding Xist RNA. This proposal is further supported by two very recently published studies that identified Spen, Wtap, and Rbm15 among several proteins that crosslink to Xist RNA following either formaldehyde  domain proteins, possibly indicating a common mode of action. The SPOC domain of Spen has been reported to interact with the co-repressor NCoR/SMRT, together with the histone deacetylase Hdac3 , and McHWtap and Virilizer are subunits of the m6A methylation complex, important for the regulation of mRNA stability . AdditioPolycomb repressor proteins and several other factors implicated in Xi chromatin structure show a strong enrichment within Xist silencing domains relative to other regions of the nucleus. Our findings indicate that this is not the case for Rbm15, Spen, and Wtap, even though they clearly co-localize with Xist RNA. A possible explanation is that these factors are constitutive components of a machinery that localizes to perichromatin spaces, which functions, for example, in mRNA surveillance following release from RNA PolII complexes, prior to translocation to the nuclear pores. Xist RNA may have evolved to interact with this machinery in a manner that triggers a checkpoint that involves both RNA entrapment by nuclear matrix proteins such as hnRNPU/SAFA and signaling back to chromatin to shut down transcription. In relation to the latter, it is interesting to note that Spen is an unusually large protein (450 kDa) and as such could bridge the distance between Xist RNA and underlying chromatin, observed using SR-3DSIM.In summary, we have used shRNA screening to identify novel factors that function in the establishment of Xist-mediated silencing. The screen achieved a high degree of saturation, indicating that the majority of key factors have been detected. Of particular interest, we validated that Rbm15, Wtap, and Spen are required for Xist-mediated silencing. Given that those three factors have very recently been identified as Xist RNA interacting proteins , we beliFor detailed experimental procedures, see cis with the Xist transgene with an unstable GFP  or 126 × 106 (Ubiquitylome library) MG-3E cells were seeded in 14-cm dishes in ESC medium containing 8 μg/ml Polybrene. Lentiviruses were then added to the medium and cells grown for 24 hr at 37°C 5% CO2. The following day, ESCs were trypsinized and seeded on feeders. On day 3, Puromycin selection (2 μg/ml) was initiated and maintained until day 7. From day 4, Xist expression was induced by supplementing the medium with doxycycline (1.5 μg/ml). At day 7, cells were trypsinized and processed for FACS analysis.ESCs were grown on feeders in DMEM supplemented with leukemia inhibitory factor (LIF)-conditioned medium. Before lentiviral infection, ESCs were trypsinized and pre-plated for 30 min to remove the feeders. For the pooled shRNA libraries 215 × 10Flow sorting (Beckman Coulter MoFlo XDP) was performed on 30–50 million MG-3E ESCs for each of three nucleome sublibraries and the ubiquitylome library. Cells with high GFP fluorescence (upper 5%) were collected. Candidates were identified and ranked based on the hairpin enrichment in the FACS-sorted compared with input populations see . A similPreparation of cells for RNA-FISH, IF, and 3DSIM was essentially as previously described . 3DSIM iA.C, T.B.N., and N.B. conceptualized the experiments. A.C. generated the reporter cell line, optimized and validated the initial screening strategy. B.M. performed and analyzed the genetic screen and validated the hits in the reporter cell line. T.B.N. performed the validations in differentiated ESCs. H.C. and L.S. performed the super-resolution imaging. A.G., G.P. and O.M. provided cell lines and reagents. N.B. wrote the manuscript. B.M., A.C., H.C., G.P., and T.B.N. reviewed and edited the manuscript. The manuscript has been approved by all authors. N.B. acquired and secured the funding.


Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains.Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone ‘foldases’ that are distinct from chaperone’ holdases’ that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein.We demonstrate using The ribosome can behave like a ‘holdase’ chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis. Saccharomyces cerevisiae and Trigger factor in Escherichia coli ensure that the nascent polypeptide chain is kept in a folding competent state until the whole sequence information is available E. coli ribosome Protein folding in biological cells is not yet well understood. Following ribosome mediated synthesis of the proteins the polypeptide chains are released into a highly crowded cellular environment where they require the assistance of a number of molecular chaperones to either fold or be rescued from misfolding and aggregation. The ribosome associated molecular chaperones like the complex of Hsp70 and J-type chaperones in the yeast E.coli ribosome and the domain V of its 23S rRNA to interact with a partially folded intermediate state of proteins and influence their aggregation and reactivation under refolding conditions. Our studies were performed on the proteins a) bovine carbonic anhydrase II (BCAII) which under mild denaturing conditions assumes an equilibrium molten globule state All earlier studies on ribosome assisted folding were performed on completely unfolded state of proteins Leishmenia donovaniE. coli ribosome synthesized by in vitro transcription also possess chaperoning ability. Studies on the mechanisms of domain V chaperoning activity showed that it is a two step process involving its two sub-domains RNA1 and RNA2 The protein folding ability of ribosome appears to be a universal one and have been demonstrated with ribosome isolated from wide range of sources including the eubacteria, archaebacteria, eukaryotes , rabbit reticulocyte, bovine mitochondria and mitochondria of the parasite The ability of the chaperones to interact with partially folded intermediates of proteins is well documented. The chaperonin GroEL primarily recognizes contiguous sequence elements or hydrophobic surfaces, such as those typically exposed in the molten globule intermediates that form in the early stages of folding due to the partial collapse of the hydrophobic residues Our studies show that the ribosome, more specifically domain V of 23S rRNA can interact with a range of different folded states of the BCAII and influence their reactivation. We also demonstrate that these chaperones can interact with and prevent aggregation of BCAII and lysozyme molten globule. Using variants of domain V RNA and its mutants, we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and that the reactivation of protein and suppression of aggregation might represent two distinct properties of the chaperone.Micrococcus lysodeikticus, cystine hydrochloride, fluorescein isothiocyanate (FITC), Guanidine hydrochloride (GuHCl), dithiothreitol (DTT), GTP, ATP and antibiotics that specifically bind to domainV of 23S rRNA (blasticidin and chloramphenicol) were purchased from Sigma. Nitrocellulose filter was purchased from Millipore, p-nitrophenylacetate (p-NPA) from SRL biochemical and reagents for molecular biology like T7 RNA Polymerase and RNase free DNase I were purchased from Fermentas, Ni+2-NTA was purchased from Qiagen. All other chemicals were local products of analytical grade. All data analysis was performed using OriginPro 8 software.Bovine carbonic anhydrase II (BCAII), hen egg white lysozyme, Ribonuclease A, 2, 100 mM NaCl; blasticidin binding buffer, 100 mM Tris-HCl (pH 7.2), 10 mM MgCl2, 100 mM NH4Cl (pH 7.2), 6 mM β-Mercaptoethanol; 2, 50 mM NH4Cl,100 mM KCl; 2, 1 mM DTT; redox buffer- non-redox buffer containing 1 mM cystine hydrochloride).The following buffers were used : BCAII refolding buffer, 50 mM Tris-HCl (pH 7.5), 10 mM MgClE. coli MRE 600 cells E.coli 70S ribosome, blasticidin bound ribosome, bDV RNA, mutants of bDV RNA, chloramphenicol bound bDV RNA, mDV RNA  was monitored by turbidity measurement in Hitachi spectrophotometer (U-1900). The effect of bDV RNA and mDV RNA on BCAII-m aggregation was monitored at 320 nm while the effect of ribosome was monitored at 450 nm since the ribosome itself interferes with turbidity measurements at 320 nm. All measurements were repeated three times and the data represents the average of all these experiments.2, final DTT concentration 1 mM) or redox (non-redox buffer containing 1 mM cystine hydrochloride) buffer systems to achieve final protein concentration 2 µM. During refolding lysozyme and the chaperones  were present at equimolar concentrations (2 µM). The effect on the reactivation yields of reduced-denatured lysozyme under redox conditions in presence of a) RNase treated bDV RNA and b) ribosome treated with Proteinase K, extracted using phenol, precipitated with ethanol and treated with RNase were also determined. The refolding mix was incubated at 30°C for a period of 16 hrs. Recovery of enzymatic activity was determined at 30°C by following the lysis of Micrococcus lysodeikticus−1 cell suspension was measured in a Hitachi UV-visible spectrophotometer. The activity of similar amount of native lysozyme was assumed as 100% for calculating the percent reactivations obtained in our experiments. Aggregation of lysozyme was monitored by turbidity measurement at 450 nm in a Hitachi spectrophotometer in a final concentration of 2 µM.Lysozyme (200 µM) was completely reduced and denatured by incubation at room temperature for 3 hours in presence of 6 M GuHCl and 100 mM DTT . The PTC of bovine mitoribosomal large rRNA (mDV RNA), that has major deletions in the RNA2 region shows delayed release and reactivation of the bound protein (60 min with mDV RNA vs 30 min with bDV RNA) E.coli domain V were cloned into the pTZ57R/T vector downstream to the T7 Polymerase promoter. The RNA corresponding to mDV RNA, bDV RNA, mutants of bDV RNA (see below), RNA1 and RNA2 were synthesized by run-off transcription and prepared as described earlier Earlier studies have shown that bDV RNA mediated folding is a two step process involving its two segments  [17],  UTP during in vitro transcription. Reduced-denatured lysozyme or BCAII-m was incubated with equimolar radiolabeled RNA at 4°C for different time intervals. After incubation the samples were cross-linked by UV irradiation at 254 nm for 90 seconds  and filtered through pre-soaked nitrocellulose filter paper (Millipore) with pore size of 0.22 µM. The filter papers were dried and 32P counts were taken in a liquid scintillation counter (Perkin Elmer). The RNA bound to denatured protein was retained on the filter while the free RNA passed through it. The percentage of radioactivity retained on the filter paper was calculated and plotted against incubated time. Comparing the radioactive count incorporated in the total RNA to that on the filter, the percentage of radioactivity retained was calculated.Filter binding studies were performed as described in earlier studies BCAII and lysozyme were fluorescent labeled with FITC. BCAII-m or reduced-denatured lysozyme was diluted in refolding buffer in presence of the ribosome. Aliquots were withdrawn at 30 seonds after initiation of refolding, UV- cross - linked and analyzed by SEC using a Sephacryl S-300 column (length×diameter = 8 inch×0.6 inch) with refolding buffer as mobile phase (flow rate of 0.250 ml/min). Proteins in the eluted fractions were detected by FITC fluorescence at an excitation wavelength of 494 nm and an emission wavelength of 518 nm and a band pass of 5 nm using a Hitachi 2700 fluorescence detector. Ribosome in the eluted fractions was detected by absorbance at 260 nm in a spectrophotometer. The elution profiles of the protein and the ribosome were plotted together to compare between the ribosome bound and unbound proteins.Earlier studies have demonstrated the ability of the ribosome to assist in folding of BCAII from its completely unfolded state. To assess the ability of the chaperone to fold the protein from its partially denatured states, the following experiments were performed.,BCAII, denatured in presence of GuHCl was refolded upon rapid dilution of the denaturant in presence of equimolar concentration of the chaperones 70S ribosome, bDV RNA or mDV RNA. The reactivation yields after 30 minutes of refolding were compared to that attained in absence of the chaperones. The final BCAII concentrations during refolding were 0.3 µM or 0.9 µM. As shown in All subsequent studies on the chaperoning ability of ribosome, bDV RNA or mDV RNA were performed with BCAII-m. As shown in Aggregation of BCAII-m under refolding conditions was followed by turbidity measurements at 320 nm. As shown in The suppression of aggregation of BCAII-m observed in presence of mDV RNA was confirmed by electron microscopy. BCAII-m (0.9 µM) was incubated in presence of equimolar concentration of mDV RNA under refolding conditions as stated above. A large number of small BCAII aggregates were observed in absence of the chaperones .i. In pr. Multiple ribosome associated chaperones like the Trigger factor and DnaK primarily ensure improper association of polypeptides during or immediately following their synthesis. A comparison of the ability of these chaperones to increase reactivation or suppress aggregation during refolding of BCAII-m to that of the ribosome is shown in The ribosome is viewed as a ‘platform’ for nascent polypeptide folding Lysozyme, that has been reduced and denatured, folds via formation of a transient molten globule intermediate E. coli ribosome, its bDV RNA and mDV RNA. These results are akin to that observed with refolding of reduced-denatured lysozyme in presence of GroEL:GroES (in absence of ATP) where the reactivation of the protein is fully suppressed due to formation of a complex between GroEL-GroES and lysozyme folding intermediate Lysozyme was reduced and denatured in presence of 100 mM DTT and 6 M GuHCl and refolded by 100 fold dilution in presence of ribosome and its domain V RNA. The protein: chaperone was present in 1∶1 stoichiometric ratio and the final DTT concentration is 1 mM (non-redox buffer). Spontaneous refolding of reduced and denatured lysozyme under these condition shows only marginal (2% reactivation yield) that remain unaffected in presence of bDV RNA, mDV RNA or ribosome (data not shown). The bar diagram in ). Evidence of complex formation between refolding lysozyme and domainV RNA or 70S ribosome under these conditions are shown below.Turbidity measurements performed at 450 nm  shows thSuppression of lysozyme aggregation by bDV RNA was confirmed by electron micrograph images of reduced-denatured lysozyme under refolding condition  in absence and in presence of bDV RNA . LysozymThe ability of the ribosome associated chaperones like the Trigger factor and DnaK chaperones to affect reactivation or suppress aggregation during refolding lysozyme was assessed in absence of any added co-chaperones or co-factors. The chaperone: protein ratio was 1∶1.As shown in Earlier studies on bDV RNA mediated refolding of unfolded BCAII had shown that the RNA1 sub-domain of bDV RNA interacts with the refolding protein and the RNA2 subdomain is responsible for the releasing the bound protein in a folding competent state within 180 seconds from the initiation of interaction. To follow the time course of interaction between the chaperone and protein , the following experiments were performed.To study the effect of binding and release of the bDV RNA associated protein on refolding and aggregation of BCAII-m, mutations U2585C (in the RNA1 region) and delG2252 (in the RNA2 region) were introduced in DNA corresponding to domain V RNA .32P labeled domain V RNA under refolding conditions. Aliquots of refolding mix were withdrawn at different time intervals after initiation of folding, crosslinked and passed through nitrocellulose filter. The radioactivity retained on the filter represents the RNA-protein complex present at each time point. The time course of interaction of BCAII-m or reduced-denatured lysozyme to bDV RNA mutants and mDV RNA were monitored by filter binding studies. Briefly, BCAII-m or reduced-denatured lysozyme was incubated with Filter binding studies with reduced-denatured lysozyme showed that neither bDV RNA nor mDV RNA could release the bound protein even after 3000 seconds after initiation of refolding under non-redox conditions . The stuWe wanted to study the time course of interaction of the refolding proteins with the ribosome. We incubated FITC labeled BCAII-m or reduced-denatured lysozyme with 70S ribosome in the respective refolding buffer . Aliquots of the refolding mixture were withdrawn 30 seconds after initiation of refolding in presence of the ribosome, UV- crosslinked and analyzed by gel filtration. The elution of the ribosome and proteins were followed by absorbance at 260 nm and fluorescence at 518 nm respectively. The elution profile of the ribosome and the native BCAII and lysozyme were also separately determined. As shown in Taken together, these studies show that the early refolding species formed during BCAII-m and lysozyme refolding interact with the ribosome and the bDV RNA. The efficient and complete binding of these BCAII intermediates to the ribosome is achieved within the first 30 seconds of return to refolding conditions compared to ∼60 seconds required with the bDV RNA. This might explain why the ribosome was more effective in suppressing aggregation and therefore increasing reactivation and highlights the importance of the time of binding to the chaperones in determining its chaperoning ability.The relative extents of aggregation suppression of BCAII by the bDV RNA mutants were determined. The bar diagram  shows thThe effect of bDV RNA mutant on BCAII-m reactivation studies are shown in When early release of BCAII-m (0.9 µM) from either delG2252 bDV RNA or mDV RNA was induced with wild type RNA2, marginal reactivation yield was observed. A delay of 10 minutes in addition of wild type RNA2 to BCAII-m refolding mix  leads toThe present study is the first in which the chaperoning activities of ribosome and its domain V RNA on partially folded intermediates of proteins have been characterized and it has been demonstrated that the chaperones can suppress aggregation of the proteins that competes with refolding process. Earlier studies have shown that the ribosome can enhance solubility of aggregation prone proteins that are coupled to its surface. The intrinsic charge of the ribosomal RNA or the steric effect of association with the ribosome has been proposed to be responsible for this property Recent experimental evidences suggest that the discontinuities in the rates of translation which are determined by the presence of rare codons in the mRNA might have significant effect on cellular protein folding Figure S1Structure of 23S ribosomal RNA and domain V RNA. A) The 23S rRNA of E. coli large ribosomal subunit (PDB: 2I2V) has been displayed (orange). The domain V rRNA is highlighted in ribbon (grey). B) Secondary structures of domainV of large ribosomal subunit RNA of E.coli (bDV RNA) with RNA1 and RNA2 regions marked. The black square in RNA1 and RNA2 represents the nucleotide U2585 and G2252 respectively. (C) Secondary structures of domainV of large ribosomal subunit RNA of Bovine Mitochondria (mDV RNA).(TIF)Click here for additional data file.Figure S2Delay in addition of bDV RNA reduced BCAII reactivation yield. Refolding of BCA II-m was initiated in refolding buffer lacking domain V RNA. The chaperone was then added at the indicate times. After 30 min the samples were assayed for BCAII activity. The reactivation yield was determined with reference to equal concentration of native BCAII. The effect of time interval between initiation of refolding and addition of Domain V on reactivation yield is indicated.(TIF)Click here for additional data file.Figure S3Effect of ribosome associated chaperones on refolding of BCAII and lysozyme. A) Comparison of the reactivation yield of BCAII-m (0.9 µM) after 30 minutes of refolding in absence of chaperone (1) and in presence of 70S ribosome (2), 70S+DnaK+Trigger factor (3). B) Time course of change in turbidity at 450 nm of BCAII-m (0.9 µM) upon dilution of denaturant and in absence of chaperone (-▪-) or in presence 70S ribosome (-•-), DnaK (-▾-), and Trigger factor (-▴-) are shown. C) Comparison of the reactivation yield of reduced- denatured lysozyme (2 µM) after 16 hrs of refolding (redox buffer) in absence of chaperone (1) and in presence of 70S ribosome (2), 70S ribosome+DnaK+Trigger factor (3), 70S ribosome+DnaK+Trigger factor+ATP (4). D) Time course of change in turbidity at 450 nm of reduced-denatured lysozyme upon dilution of denaturant (non-redox buffer) in absence of chaperone (-▪-), in presence of DnaK (-▾-), Trigger factor (-▴-) and 70S ribosome (-•-) are shown.(TIF)Click here for additional data file.Figure S4Binding and release of BCAII-m in the presence of wild type and mutant RNA. The time course of binding of BCAII-m with wild type bacterial RNA1 (-▴-) and bDV RNA1 mutant U2585C (-▪-) and wild type bacterial RNA2 mediated release of the protein from wild type RNA1 (.Δ.), bDV RNA1 mutant U2585C (.□.) are shown here. The binding and release experiments were repeated thrice and their average values were taken for final data plotting.(TIF)Click here for additional data file.


Background: Optimized RNA extraction from tissues and cell lines consists of four main stages regardless of the method of extraction: 1) homogenizing, 2) effective denaturation of proteins from RNA, 3) inactivation of ribonuclease, and 4) removal of any DNA, protein, and carbohydrate contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of RNase. Various technical difficulties occur during extraction of RNA from pancreatic tissue due to spontaneous autolysis. Since standard routine protocols yield unacceptable results in pancrease, we have designed a simple method for RNA extraction by comparing different protocols.Methods: We obtained 20-30 mg pancreatic tissues in less than 2 min from 30 rats. Several methods were performed to extract RNA from pancreatic tissue and evaluate its integrity. All methods were performed three times to obtain reproducible results.Results: Immersing pancreatic tissue in RNA-later for 24 h at -80ºC yielded high quality RNA by using the TriPure reagent which was comparable to the commercial RNeasy Micro Kit. The quality of RNA was evaluated by spectrophotometer, electrophoresis and RT-PCR. We separated intact 28S and 18S ribosomal RNA (rRNA) when our procedure was compared with the RNeasy Micro Kit. Finally, full length of the actin gene was amplified by RT-PCR.Conclusion: We designed a simple, fast, cost-effective method for complete RNA extraction from the least amount of quantitatively intact pancreatic tissue. Recent studies have focused on RNA analysis as a gene expression tool in cells to detect differential gene expression between two conditions. Different methods have been presented for extracting nucleic acids such as guanidinium thiocyanate followed by phenol-chloroform extraction, chromatography by cellulose, extraction using silica matrices, magnetic bead based nucleic acid purification, and anion-exchange.3,4Accurate detection of gene expression is influenced by status of the RNA that is isolated from tissues. The quality of isolated RNA should be checked prior to its use in subsequent tests and studies. The purity and quality of the isolated RNA is a vital step in RNA dependent assays. Performing complementary molecular tests with low-quality RNA may compromise the results of downstream applications which are often labor-intensive, time consuming, and highly expensive. Researchers need high quality RNA for molecular biological tests that have various diagnostic applications such as quantitative RT-PCR, micro-arrays, ribonuclease protection assay, northern blot analysis, RNA mapping, and cDNA library construction.5,6The quality of purified RNA from tissues and cells is variable. Often, after extraction, RNA is rather unstable over a long storage time. Long mRNA fragments up to 10 kb are especially sensitive to degradation.7,8Researchers must consider various factors that affect the quality of purified RNA. Purified RNA must not be contaminated with RNases, proteins, genomic DNA, and enzymatic inhibitors. Additionally, the UV absorption ratio (260/280) of total RNA should be between 1.8-2.0 and RNA should have a minimal degree of fragmentation during electrophoresis. Recently developed laboratory techniques allow scientists to more adequately control the quality of samples used for molecular analyses.9-15Although RNA is easily and successfully isolated from most cells and tissues, intact RNA extraction from the pancreas is difficult due to the high level of its ribonucleases (RNases). Despite the improvement in several approaches, including rapid removal of pancreatic tissue from the abdominal cavity and homogenization at cold temperatures to inhibit RNases, the isolation of intact, high-quality RNA from this tissue remains challenging because of the complexity and indefinite reproducibility of the above mentioned techniques.We aimed to design a simple, fast, and cost-effective method for complete RNA extraction that utilized the least amount of pancreatic tissue. We compared different protocols of RNA extraction and optimized the most feasible extraction method by which the highest quality RNA could be qualitatively obtained. In the current study, pancreatic tissues were taken from 30 rats and divided into several pieces (20-30 mg) for use in the following methods.In the first method, these small pieces of pancreatic tissue from 30 rats were placed into two microtubes. The first tube contained 1 ml RNX-plus solution  and the second tube contained 1 ml TriPure isolation reagent . Both solutions contained guanidinium thiocyanate which inhibits RNase. Subsequently, both tubes were snap-frozen in liquid-nitrogen for inhibition of RNase activity after which the integrity of RNA was evaluated with denaturing agarose gel electrophoresis  and 2.In the second method, pancreatic tissues were perfused with 1 ml RNA-later as the RNA stabilization reagent  by an insulin syringe. Tissues were subsequently cut into small pieces with sterile scissors. The tubes that contained pancreatic tissue and RNA-later were processed for extraction by using the RNX-plus solution, TriPure, and RNeasy Micro Kits  according to the manufacturers’ instructions after either 30 min, overnight in 4ºC, or following storage at -80ºC for one, three or seven days in order to compare the effect of preservation time on RNA integrity. In all conditions, the livers were removed from 30 rats and used as control tissue in a comparison of RNA quality between pancreatic and liver RNAs. 16The isolated RNAs were separately diluted at a ratio of 1/100 in water treated with diethylpyrocarbonate (DEPC) and the 260/280 UV absorption ratio was calculated. The integrity of total RNAs was evaluated by denaturing agarose gel (MOPS gel) electrophoresis. MOPS buffer was used as running buffer to separate several ribosomal RNA (rRNA) bands  during electrophoresis.We did not obtain acceptable bands when RNA was extracted with the RNX-plus reagent or RNA-later. However, we observed the best results when TriPure reagent was used. These results were dependent upon the tissue preservation time, temperature and perfusion method. Immersion of pancreatic tissue in RNA-later for 24 h at -80ºC yielded high quality RNA with sharp, distinct 28S/18S bands.Evaluating RNA Integrity with the RNX-Plus SolutionNo specific band was seen when we used the RNX-plus solution. According to electrophoresis results, the RNA was completely degraded .RNA Integrity with TriPure ReagentIn comparison to the liver tissue control, we noted that RNA separation was not successful when the TriPure reagent was used .RNA Integrity of Samples Immersed in RNA-Later and Extracted with RNX-Plus or TriPure ReagentThere was no band visualized when we used RNA-later along with the RNX-plus reagent . DependiRNA Integrity with RNA-Later and the Qiagen KitIn terms of purity and integrity, high-quality RNA was extracted by using RNA-later along with the Qiagen reagent .RT-PCR for β-Actin Gene Expression in Pancreatic Tissues with RNAs Extracted by Using the TriPure ReagentHigh quality RNA extracted with the TriPure procedure amplified a specific β-actin locus. The full length of actin was amplified by RT-PCR in order to evaluate the synthesized cDNA by intact RNA. The presence of 138 bp bands confirmed the cDNA quality .14,17,18Obtaining high-quality RNA is the first and most critical step in many molecular techniques such as reverse transcription real-time PCR (RT-qPCR), transcriptome analysis that uses next-generation sequencing, array analysis, northern analysis, and cDNA library construction. RNA extraction is complicated because of the presence of ribonuclease enzymes in cells and tissues that can rapidly degrade RNA.19RNases act without cofactors and are stable enzymes. The inactivation of RNases is difficult. Small amounts of RNase are sufficient to destroy RNA. In order to inactivate any RNases prior to surgery and in cases of removal of rat pancreatic tissue from the abdominal cavity, all surgical instruments should be cleaned with strong detergents, thoroughly rinsed, and placed in an oven for at least 4 h at 240ºC. The place of surgery should be sterilized with NaOH and mild bleach to inactive the RNases.18,19The pancreas has an extremely high level of RNase. RNA degradation occurs while the pancreas tissue is removed during dissection. In order to increase efficiency dissection should be performed as quickly as possible.20The pancreas is one of the important tissues that functions in the body’s homeostatic mechanisms. Therefore, improvement of RNA extraction procedures from the pancreas increases understanding of active pathways such as glucose regulation and redifferentiated β islets of the pancreas before and after treatment with different drugs. Knowledge of gene expression assays may potentially lead to the development of therapeutic drugs to restore β-cells and prevent apoptosis in diabetics.21-23 Therefore we have attempted to design an efficient, simple optimized method to extract RNA from the pancreas based on our laboratory facilities.To identify novel metabolic genes and pathways that may play a role in the pathogenesis or treatment of diabetes, differential expression of metabolic genes is necessary. However, RNA extraction from pancreatic tissue is difficult because of the abundance of RNase.In order to enhance and correct this method, we assessed different common RNA extraction methods from pancreatic tissue with particular focus on the effect of frozen storage and RNase inhibition strategies, both of which affect RNA quality.10,15,18 Therefore, in this study we obtained only 20-30 mg of pancreatic tissue in less than 2 min from the anesthetized rats.The duration of surgery and amount of collected pancreatic tissue are the most crucial steps for obtaining intact RNA. Recent studies have shown a positive correlation between RNA degradation and the amount of pancreatic tissue.24 RNA extraction performed with guanidinium thiocyanate followed by phenol-chloroform on different tissues has yielded good results, with the exception of pancreatic tissue (data not shown). Several studies determined that the RNA isolation procedure must include a number of important steps before, during, and after the actual RNA purification.25 Therefore we changed the RNA extraction process by using RNA-later.Several methods are used in molecular biology to isolate RNA from different samples. The most common isolation method is guanidinium thiocyanate followed by phenol-chloroform extraction that uses liquid nitrogen along with a specialized motorized grinding device which prevent RNA activity.Our results showed that RNA later rapidly permeated the pancreatic tissues, protected cellular RNA and minimized the need to immediately process the tissue samples. The best results were obtained after the samples that contained RNA-later were stored for 24 h at -80ºC. Our data confirmed that it was necessary to stabilize RNA within the pancreatic samples by using an effective reagent to delay RNA degradation, even the extraction was performed by using a Qiagen RNA extraction kit. Several kits such as TriPure and Qiagen (foreign kits) and RNX-Plus  are commercially available for RNA extraction in Iran. These kits offer the dual advantages of ease of use and effectiveness. These kits often work well and are widely used. Foreign kits are also more expensive per sample than homemade kits. 15 however we were unable to obtain good quality RNA. The only difference between these two protocols was the use of RNX-plus in our study which did not seem to be an appropriate solution for RNA extraction from pancreatic tissues compared to TRIzol. Although RNX-plus works well for extracting RNA from other tissues, we did not use RNX-plus for RNA extraction from pancreatic tissue. In this case, the possibility of the presence of active RNase during surgery possibly led to RNA degradation and could not be ruled out.We were unable to obtain high-quality RNA with Iranian reagents and snap-frozen tissues. Possibly, the low yield of RNA from the immediately frozen pancreatic samples was attributed to rapid degradation initiated by RNases in the pancreas. Although our entire experimental process was similar to a previous study that used TRIzol reagent where the researchers obtained high-quality intact RNA from the rat pancreas,In order to evaluate the quality of RNX-plus, the second RNA extraction procedure was performed using the TriPure reagent under snap-frozen conditions which led to decreased RNA degradation. However, the problem with using TriPure solution was the lack of reproducibility. In the second step, we decided to decrease autolysis during dissection and RNA extraction by using RNA-later as a pancreas RNase inhibitor. The use of RNA-later before starting the isolation method with RNX-plus did not change the quality of RNA. The RNA was degraded and 28S/18S rRNA smear bands were observed. However, when TriPure was used, RNA quality was incredibly high. Additionally, this process was reproducible. The quality of RNX-plus was questioned. We also detected DNA contamination with the use of RNX-plus that had to be reduced.°C or for one, three and seven days at -80°C. As shown in In the third step we have focused on how to perfuse RNA-later into the pancreatic tissues. Complete tissue perfusion with RNA-later after total pancreatic tissue dissection is not cost-effective. Therefore, we researched three perfusion conditions for the later use of RNA as follows. We dissected a small section of the pancreas (20-30 mg) during surgery from anesthetized rats and immersed these tissues in 1 ml RNA-later for 30 min at 4The above mentioned methods enabled a smaller amount of RNA-later to penetrate into the organ. The degradation process was halted faster because small pieces were dissected. Modifications to the basic procedures introduced by Li and Griffin et al enabled us to obtain high-quality reproducible RNA from rat pancreatic RNA which was suitable for RT-PCR of the actin gene as shown in Although isolation of intact RNA from the rat pancreas is compromised by autolysis and by the presence of endogenous RNases, our pancreas perfusion method yields excellent, high quality and integrity RNA for molecular biology studies which is comparable with Qiagen kits. In summary, the presented method is a simple, reproducible and economical procedure which does not require the use of higher amounts of RNA-later total perfusion. Using TriPure solution after RNA-later perfusion can be a good substitute for expensive and column-based RNA extraction kits. Furthermore, use of the RNX-plus kit for RNA extraction from pancreatic tissue is not recommended.


Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway. The innate immune system is the first line of defense against viral infection. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that recognizes viral double-stranded (ds) RNA and trigger antiviral innate immune responses . The RIGRIG-I recognizes relatively short dsRNA (<1 kbp) with a 5′ tri- or di-phosphate group –13, wherRecent studies have revealed the post-translational modification of RLRs Figure . Gack anMDA5 assembles along viral long dsRNA and forms a nucleoprotein filament structure required for MAVS activation . MDA5 acThe phosphorylation of RIG-I and ubiquitination of MDA5 have also been reported and are required for the regulation of RLR signaling .Several RNA helicases lacking CARDs are involved in RLR-mediated signaling . Bowie aLGP2 is a member of RLRs but lacks N-terminal CARDs , whereasDDX60 KO mice and reported that DDX60 KO moderately reduced RIG-I-mediated type I IFN production from peritoneal macrophages and MEFs but not from bone-marrow-derived cells, suggesting the cell-type specificity (DDX60 KO first) and a DDX60 KO mouse   , whose construct , 40. Thenditions –40.Recently, we identified another role of DDX60 in the antiviral response. The DDX60 protein exhibits the similarity to SKI2 RNA helicase, a component of the RNA exosome, which degrades host and viral RNA , 40. We RIG-I and MDA5 forms a nucleoprotein filament . It is wThe LGP2 protein increases the initial rate of MDA5–RNA interaction, resulting in the formation of numerous shorter MDA5 filaments . These nAll authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The terminal RNA (positions 19–21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15–18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5′ proximal nucleotides, which include seed sequence (positions 2–8 from the 5′ end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the  Drosophila, mammalian RISC is formed through dynamic changes in protein–protein and protein–RNA interactions in RISC-loading complexes (RLCs)  is a phenomenon whereby double-stranded RNA (dsRNA) leads to posttranscriptional gene silencing through base-pairing interaction . RNAi ans (RLCs) , but thetrans-activation-responsive RNA binding protein (TRBP) as major components , Piwi/Argonaute/Zwille (PAZ), Middle (MID), P-element-induced wimpy testis (PIWI)  highly similar in amino acid sequence to one another . Only Ags (PIWI) . An RNass (PIWI) . N domais (PIWI) . The 5′ s (PIWI) , while ts (PIWI) . The curs (PIWI) .Drosophila, photo-crosslinking studies indicated that the Dcr-2/R2D2 heterodimer determines which siRNA strand associates with Ago and serves as a guide strand  are responsible for RNA binding. The remaining C-terminal domain (dsRBD3) is considered to be in charge of binding to Merlin, Dcr and PACT, but not dsRNA . In Drose strand . Dcr-2 ie strand .In the present study, we systematically introduced 2–3 bp DNA substitutions in the non-seed-duplex region of siRNA with a dsDNA-substituted seed duplex, and identified two subdomains that cannot undergo DNA substitutions without a profound loss of RNAi activity. We found the RNA subdomain capable of enhancing the binding of the Ago-PAZ domain to the terminal non-seed region of siRNA. We also showed that both of these dsRNA cores are essential for effective binding of Dcr-free TRBP to siRNA. RNAi based on siRNA with a dsDNA-substituted seed duplex was found to be completely dispensable for Dcr. These results enable therapeutic approaches for harnessing the untapped potential of RNAi based on RNA–protein interaction.Drosophila S2 cells were cultured in Schneider’s Drosophila medium (Gibco BRL) at 25°C.Chinese hamster ovary CHO-K1 (RIKEN Cell Bank) and human HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium  at 37°C. Media for both cell lines were supplemented with 10% heat-inactivated fetal bovine serum (Mitsubishi Kagaku) and antibiotics (10 units/ml penicillin (Meiji) and 50 µg/ml streptomycin (Meiji). Mouse embryonic stem (ES) cells E14TG2a were cultured in DMEM supplemented with 20% heat-inactivated fetal bovine serum (Hyclone), 0.1 mM 2-mercaptoethanol (Wako), 8 µg/ml adenosine, 8.5 µg/ml guanosine, 7.3 µg/ml cytidine, 7.3 µg/ml uridine, 2.4 µg/ml thymidine, 0.1 mM each nonessential amino acid and 1000 units/ml leukemia inhibitor factor . Passenger- and guide-strand RNA or DNA-substituted RNA oligonucleotides were chemically synthesized (Sigma), mixed in a 1:1 fashion in 10 mM NaCl and 20 mM Tris–HCl (pH 7.5), and annealed by incubation at 95°C for 15 min, 37°C for 30 min and 25°C for 30 min. Annealed products were examined using 3% agarose gel electrophoresis in Tris-borate-EDTA buffer, which can separate 21-bp long double-stranded siRNA from 21-nt long single-stranded RNA. Almost all RNA was recovered as dsRNA.5 cells/ml), HeLa (1 × 105 cells/ml) or E14TG2a (2 × 105 cells/ml), S2 (1 × 106 cells/ml) cell suspension was inoculated in a 1.5-cm well 24 h before transfection. Cells were transfected with pGL3-Control DNA  or pGL2-Control DNA  encoding the firefly luciferase (luc) gene and pRL-SV40 DNA  encoding the Renilla luc gene with or without siRNA or DNA-substituted siRNA against the firefly luc gene. Knockdown of each protein was performed by transfection of specific siRNA for each protein designed by siDirect2.0 (luc-encoding vectors. Lipofectamine 2000 reagent (Invitrogen) was used for transfection for CHO-K1, HeLa and E14TG2a cells, and calcium phosphate precipitation method was used for transfection for S2 cells. Cells were harvested 24 h after transfection, and luc activity was measured using the dual-luciferase reporter assay system (Promega). In this system, two luc (firefly and Renilla) genes were expressed simultaneously in cells. The reduction of firefly luc activity by the addition of siRNA against the firefly luc gene was normalized to the Renilla luc activity .One milliliter of a CHO-K1 (3 × 10irect2.0  24 h befEscherichia coli BL21 codon plus and Rosetta (DE3) pLysS, as amino-terminal hexa-histidine fusion proteins, and purified with NTA agarose (32P-labeled guide strand (0.5 nM) for 30 min, and samples were electrophoresed on a 9% polyacrylamide gel and analyzed quantitatively using a FLA-2000 image analyzer (Fujifilm).Amplification of the coding sequence of TRBP by polymerase chain reaction (PCR) has been described . TRBP pr agarose . ElectroNot I site of pGEX-6 P-2 . For construction of pGST-PAZ-His, a DNA fragment corresponding to human Ago1 PAZ domain (amino acid residues 222–376) was amplified by PCR with the following primers: 5′-CCGGATCCTTTTATAAGGCACAGCCAGTGATTGAGTTC-3′ and 5′-TCATCAGGGCGGCCGCAGGCAGCAGCCACCACCACCACCACCACTGACTCCTCCTGTCTGTCTGGAGCGGATCTAGC-3′. The amplified fragment was digested with BamHI and NotI, and inserted into pGST-His. The PAZ domain was expressed in E. coli BL21 codon plus (Stratagene) as a glutathione S-transferase (GST) fusion protein, and purified with a glutathione sepharose column (Amersham) according to the manufacturers’ instructions. PAZ protein (1 pmol/µl) was incubated with siRNA or DNA-substituted siRNA containing 32P-labeled guide strands (5 fmol/µl each) in 20 mM Tris–HCl (pH 7.5) with 100 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 50 ng/µl poly(dA-dT) and 2 U/µl RNasein (Promega) for 30 min at room temperature. The mobility shift was analyzed by 5% polyacrylamide gel electrophoresis and quantified using a FLA-2000 image analyzer (Fujifilm).For EMSA analysis of the PAZ domain of human AGO, the control pGST-His vector expressing 6xHis and pGST-PAZ-His expressing human Ago1 PAZ domain were constructed. pGST-His was constructed by insertion of annealed oligonucleotides (5′-GGCCGCAGGCAGCAGCCACCACCACCACCACCACTGA-3′ and 5′-GGCCTCAGTGGTGGTGGTGGTGGTGGCTGCTGCCTGC-3′) into the At first, we would like to briefly describe the results of our previous experiment , as thisWe hereafter referred to the DNA-modified siRNA schematically shown in the lower half of siRNA with a dsDNA seed duplex was found to be associated with little passenger-strand–dependent gene-silencing activity or seed-dependent off-target effects . Thus, iIn contrast to siRNA with a dsDNA seed duplex, the authentic siRNA is rotationally symmetric with respect to RNA structure. Because of this, Ago binding may occur in two orientations, resulting in two forms of functional RISCs, one including the guide strand and the other the passenger strand C. BalancSupplementary Figure S1A. One hundred percent in relative luc activity in Supplementary Figure S1 corresponds to the absence of RNAi activity. We used three mammalian cell lines  and Drosophila S2 cells for transfection. siRNA sequences used were of highly functional ones against luc . These siRNAs were same siRNAs used in our previous study . Subdomain B corresponds to two nucleotide pairs at position 13 and position 14. Subdomain B is replaceable with DNA.DNA substitutions at positions 13 and 14 and of the 3′ overhang of the guide strand changed little, if any, RNAi activity . DNA replacement at nucleotide position 19 gave some RNAi activity reduction in a cell-type–dependent manner (lanes 13): gene silencing activity was considerably reduced in HeLa and E14TG2a cells but that in CHO-K1 and S2 cells was not significant. If not otherwise mentioned, the 3′ overhang of the guide strand and the nucleotide pair at position 19 are collectively called the subdomain T. Based on these observations, we considered that the non-seed region of siRNA are divided into four subdomains and that two of them, A (from nucleotide position 9 to 12) and C (from position 15 to18) subdomains, should be RNA for active RNAi . Note that all nucleotides of the passenger and guide strands, respectively, in Supplementary Figure 1B and C) are ribonucleotides. Although these results may indicate that RNA requirement in the non-seed region is due primarily to interactions with RLC/RISC protein moieties, it should be noted that 2–3-bp DNA substitutions have never completely abolished relative luc activity , possibly suggesting that either 2–3-bp-long DNA segments are too short to completely suppress RNAi or there is some ambiguity in positioning of putative RNA-binding proteins in RLC/RISC.Drosophila than mammalian cells . However, this is not due to the effect of introduction of an additional 2–3-bp DNA substitution in the non-seed region, as virtually no difference in gene silencing activity between parental and additionally DNA-modified siRNAs .The contribution of the passenger strand to gene silencing is much greater in Supplementary Figure S2), various DNA substitution variants were constructed and their RNAi-inducing activity was examined . Lane 1 and lane 2 are positive controls, indicating RNA in the entire non-seed-duplex region to give a highly active RNAi activity. Lane 11 is a negative control and the relative luc activity induced was virtually 100%, indicating no or little functional RNAi to be induced by siRNA in which all ribonucleotides are replaced with cognate deoxyribonucleotides (siDNA). If necessary, DNA substitution variants are hereafter simply referred to as ‘an RNA domain combination’. Thus, for example, TC-RNA means a DNA-substitution variant of siRNA in which only subdomains T and C are RNA.To make certain that the siRNA non-seed-duplex region consists of four subdomains  resulted in only a slight, if any, reduction of RNAi activity (lane 3), indicating that RNA in subdomain B is not essential for functional RNAi in all cell types examined. DNA substitutions in both subdomains B and T (CA-RNA) (lane 4) induced a considerable reduction of RNAi in human and Supplementary Figure S2). But RNA in subdomain T could not solely recover the abolished RNAi activity at all (compare lane 8 and lane 11). Thus, we consider that synergistic interactions among three RNA subdomains A, C and T may be important for maintaining siRNA-based RNAi activity.RNA only in subdomain A  or only in subdomain C  could not trigger effective RNAi, whereas the introduction of RNA into subdomain T to the C-RNA derivative slightly improve RNAi activity in particular in the cases of siLuc-36 derivatives , while in the other, the guide strand domains (lane10). Again, we noted the guide strand preference. In fact, a partial and cell-type–dependent RNAi activity was found to be associated with the presence of RNA in the non-seed-duplex guide strand but not the non-seed-duplex passenger strand.luc siRNA, which consist of authentic siRNA, siRNA with a dsDNA seed duplex (TCBA-RNA) and siRNA with simultaneously dsDNA-replaced seed-duplex and subdomain B (TCA-RNA) . In each case, the concentration of 50% inhibition (IC50) was normalized by that for siGY441 treatment and the resultant relative IC50 s were shown for comparison. Although relative IC50 values considerably varied depending on target luc sequences, it is virtually evident that Ago2 and TRBP are profoundly involved in RNAi due to all three types of siRNAs used and that Ago2 knockdown results in changes much larger than TRBP knockdown .TRBP knockdown experiments may suggest that TRBP contribution to RNAi due to siRNA with a dsDNA seed duplex is similar, if not identical, to that due to nonmodified siRNA see B and C. Supplementary Figure S6C). As shown previously , 15∼26 μM . Note that the latter is ∼500 times as high as the former concentration. As described in ‘Discussion’ section, we interpret these results as suggesting that TRBP binding to siRNA with a dsDNA seed duplex within cells is not weak, and that only TRBP associated with an unidentified RNA-binding protein ‘X’, which is capable of strong binding to siRNA with a dsDNA seed duplex, can effectively bind to siRNA with a DNA-modified guide sequence.At a first glance, TRBP binding to siRNA with a dsDNA seed duplex strikingly differed from that to the authentic siRNA , and it appeared slightly increased by an additional DNA replacement in subdomain T (lane 4), suggesting low levels of contribution of RNA in subdomains B and T for TRBP binding. In contrast, as expected, siDNA exhibited virtually no siRNA binding activity (lane 5).Next, we examined TRBP capability for binding to four subdomains consisting of the non-seed region of siRNA. As shown in Supplementary Figure S6D). siRNAs with RNA in (T+C) subdomains (TC-RNA) also gave low values . Taken together, these results may indicate that the physical association between TRBP protein and the non-seed duplex of siRNA requires a simultaneous presence of nonterminal subdomains, A and C.DNA-modified siRNA containing RNA only in one of the non-seed-duplex subdomains other than subdomain B gave much reduced values of the TRBP concentration giving 50% binding of siRNA .Finally, we examined the effect of guide- and passenger-strand-specific DNA-replacement in the non-seed region on the activity of TRBP binding to siRNA. Consistent with a previous experiment , we coulDrosophila S2 cells as well  for general value and obtained an identical conclusion.Our results see  showed tIt is known that the PAZ domain specifically recognizes an RNA with a 2-nt 3′ overhang. Then, if the subdomain T is RNA, this substrate would not be expected to perform a binding defect with Ago-PAZ protein domain. Thus, the mechanistic implications of a weak Ago binding to the DNA-substituted siRNA variant with RNA only in subdomain T (T-RNA) , lane 7 Drosophila RLC consisting of Dcr-2, Ago2 and R2D2 promotes siRNA loading . The TRBP concentration giving 50% binding of siRNA with a dsDNA seed duplex was estimated at ∼50 μM , which roughly corresponds to one molecule in a sphere of 400 Å diameter, leading us to the notion that a functional intracellular TRBP should be associated with an unidentified RNA-binding RLC/RISC protein. One of the possible candidates may be Ago protein. However, as interaction between TRBP and Ago protein is conflicting, the other unidentified protein(s) ‘X’, which is a possible substitute for Dcr and stimulates the access of TRBP protein to siRNA as in the case of Drosophila R2D2/Dcr-2 complexes, might not be excluded  and Core Research Project for Private University matching fund subsidy (to K.U.-T.) and a Research Fellowship for Young Scientists of the Japan Society for the Promotion of Science (to T.T.). Funding for open access charge: Research grant form Ministry of Education,Culture, Sports, Science and Technology of Japan [21115004 to K.U.-T.] and JSPS (to T.T.).This work was supported by the Conflict of interest statement. None declared.


Elusimicrobia and Verrucomicrobia phyla; Desulfovibrionaceae, Elusimicrobiaceae, and Sphaerochaetaceae families; and Methanobrevibacter woesei were only detected in the RNA-Seq and RNA Amplicon-seq datasets, whereas Streptococcaceae was only detected in the DNA Amplicon-seq dataset. In addition, the relative abundances of four bacterial phyla, eight bacterial families and one archaeal taxon were different among the three datasets. This is the first study to compare the outcomes of rumen microbiota profiling between RNA-seq and RNA/DNA Amplicon-seq datasets. Our results illustrate the differences between these methods in characterizing microbiota both qualitatively and quantitatively for the same sample, and so caution must be exercised when comparing data.Taxonomic characterization of active gastrointestinal microbiota is essential to detect shifts in microbial communities and functions under various conditions. This study aimed to identify and quantify potentially active rumen microbiota using total RNA sequencing and to compare the outcomes of this approach with the widely used targeted RNA/DNA amplicon sequencing technique. Total RNA isolated from rumen digesta samples from five beef steers was subjected to Illumina paired-end sequencing (RNA-seq), and bacterial and archaeal amplicons of partial 16S rRNA/rDNA were subjected to 454 pyrosequencing (RNA/DNA Amplicon-seq). Taxonomic assessments of the RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq datasets were performed using a pipeline developed in house. The detected major microbial phylotypes were common among the three datasets, with seven bacterial phyla, fifteen bacterial families, and five archaeal taxa commonly identified across all datasets. There were also unique microbial taxa detected in each dataset.  Microbiota play essential roles in many ecosystems, including the animal gastrointestinal tract, and have attracted much attention in the past decade due to the understanding of their functions in host productivity and health . Although Amplicon-seq has been widely used, it can be biased due to primer selection  at −20°C for further analysis.Rumen digesta samples were collected from five 10-month-old crossbred beef steers, which were raised under feedlot conditions on a high-energy finishing diet, as previously described  from each sample was used to construct an RNA library following the TruSeq RNA sample Prep v2 LS protocol , without the mRNA enrichment  step. The quality and concentration of cDNA fragments (~260 bp) containing artificial sequences  and inserted cDNA sequences (~140 bp) were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and a Qubit 2.0 fluorometer (Invitrogen), respectively, before sequencing. RNA libraries were paired-end sequenced (2 × 100 bp) using an Illumina HiSeq2000 platform .For the DNA Amplicon-seq, partial bacterial and archaeal 16S rRNA genes (the V1-V3 region for bacteria and the V6-V8 region for archaea) were amplified as previously described by Kittelmann et al.  and sequhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The program Trimmomatic  described by Kozich et al. . Finally, chimera-depleted sequences were subject to taxonomic assignment to different phylotypes using a naive Bayesian algorithm , Prevotella sp. , and Tolumonas auensis . The copy numbers for each standard curve were calculated as described previously  and followed the same procedures mentioned above to verify DNA Amplicon-seq results.Quantitative reverse transcription PCR (qRT-PCR) and quantitative PCR (qPCR) were further performed to validate the relative abundance data obtained from RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq. Three primer pairs  of the detected taxa, and Pearson's correlation analysis for qRT-PCR/qPCR validation were all performed using R 3.1.2  under the accession number This study assessed active rumen microbial communities using RNA-seq and is the first study to compare RNA-seq outcomes with the well-accepted Amplicon-seq methods to evaluate rumen microbiota. It has been reported that rRNA levels directly relate to the protein synthesis potential of microorganisms  and 31,031  bacterial reads, respectively, as well as 8303 (1661 ± 20 for RNA Amplicon-Seq) and 6663 (1333 ± 95 for DNA Amplicon-Seq) archaeal reads, respectively, (Supplementary Table de novo (database-independent) mode to determine chimeras rather than reference-based chimera detection. This is because the existing chimera reference databases only contain sequences from cultured organisms  have well-studied 16S rRNA genes for use as references  were detected only in the RNA-seq dataset and not in the RNA/DNA Amplicon-seq datasets. Henderson et al.  of Sphaerochaetaceae in the rumen digesta when using DNA Amplicon-seq with the same PCR primers, suggesting that the amplicon-based approach and/or the primers used may have masked the detection of these taxa in the rumen. In addition, lower sequencing depth of RNA/DNA Amplicon-seq could also lead to the missing detection of these taxa. As shown in Table Elusimicrobia and Verrucomicrobia) and one bacterial family (Elusimicrobiaceae) were detected only in the RNA-based datasets (RNA-seq and RNA Amplicon-seq) and not in the DNA Amplicon-seq dataset. Our results suggest that these two phyla may be more active in the rumen, and they may be underestimated based on the DNA Amplicon-seq dataset. Moreover, the absence of Elusimicrobia and Verrucomicrobia in the DNA Amplicon-seq dataset may be due to the unsuccessful isolation of their DNA, and it has been reported that various DNA extraction methods could impact the taxonomic outcomes of rumen microbiota assessments  of rumen and foregut digesta samples and DNA-based Amplicon-seq, Streptococcaceae also showed low prevalence in all animals , eight bacterial families , and one archaeal taxon  had significantly different relative abundances among the three datasets  of the relative abundances of commonly detected bacterial phyla, bacterial families, and archaeal taxa revealed dissimilarities in microbial profiles between RNA- and DNA-based approaches Figure . For eacs Figure . Among tBacteroidetes , Firmicutes (29.4 ± 6.2%), and Proteobacteria (14.3 ± 8.5%), which is consistent with previous studies using DNA-based methods. For example, Bacteroidetes  and Firmicutes  were reported as the most abundant phyla, and Proteobacteria was commonly detected but less abundant  in the rumen , followed by Bacteroidetes (23.3 ± 7.9 and 22.7 ± 8.1%), and Firmicutes (16.2 ± 4.5 and 19.2 ± 6.0%). The higher proportion of Proteobacteria in the RNA-based datasets confirmed similar findings by Kang et al. (Succinivibrionaceae (belonging to the phylum Proteobacteria) in the RNA-seq (45.6 ± 14.0%) and RNA Amplicon-seq (45.1 ± 14.4%) datasets, while it was Prevotellaceae (belonging to the Bacteroidetes) in the DNA Amplicon-seq dataset (38.7 ± 8.6%). Succinivibrionaceae was an abundant family at the DNA level when ruminants were fed high-energy diets  than in the DNA Amplicon-seq dataset . These two families probably had higher cellular abundances but relatively lower activities in the bovine rumen.The dominant bacterial phyla detected by DNA Amplicon-seq were g et al.  and Kangg et al. , who appMethanobrevibacter gottschalkii (28.2 ± 9.7 /37.1 ± 10.6%), Methanobrevibacter ruminantium (37.1 ± 12.4/36.4 ± 13.1%), and Methanomassiliicoccales (30.9 ± 10.0/15.7 ± 2.0%) were dominant but with different rankings. The DNA Amplicon-seq outcomes are generally consistent with previous studies that used the same approaches , and Methanobrevibacter gottschalkii (22.4 ± 7.4%). The high proportion of Methanomassiliicoccales from the RNA-based datasets supports the hypothesis that they are more active in the rumen, as many studies have suggested  among all three datasets. Gammaproteobacteria was selected to represent Proteobacteria because 95.8, 98.0, and 96.5% of the Proteobacteria reads from the three datasets belonged to the class Gammaproteobacteria. The qRT-PCR results for Bacteroidetes were in agreement with the RNA-seq  and the RNA Amplicon-seq  results, and the qPCR results for Bacteroidetes were also consistent with the DNA Amplicon-seq results  and the RNA Amplicon-seq dataset , and there was also a high degree of correlation between qPCR and DNA Amplicon-seq  for Gammaproteobacteria. The overall consistent trends between the RNA-based approaches and qRT-PCR (as well as between DNA Amplicon-seq and qPCR) confirm the different relative abundance detected in the three datasets.qRT-PCR and qPCR were performed to estimate the relative abundances of two predominant phyla  among the three datasets . The values of Good's coverage were all above 99% for the bacterial and archaeal data from the three datasets, indicating that the numbers of reads after normalization were sufficient to represent the microbial communities. The Shannon index and the inverse Simpson index were not significantly different  in rumen using DNA Amplicon-seq , and all of them were negatively correlated with Succinivibrionaceae and Methanomassiliicoccales (P < 0.1) in the RNA-seq dataset  as suggested by Henderson et al. . No other taxa exhibited strong consistency between the RNA-seq and DNA Amplicon-seq datasets, indicating that cellular abundance did not correspond to the activities of most rumen taxa.The relationships that exist among microbial taxa could be a determining factor for microbial community composition (Prosser et al., t Figure . Howevern et al. . MeanwhiProteobacteria and Bacteroidetes are Gram-negative, while most Firmicutes members in the rumen are Gram-positive, which may explain the higher abundances of Proteobacteria and Bacteroidetes, and the lower abundance of Firmicutes in the RNA-seq dataset. Moreover, significant differences in microbial community structures were also demonstrated to correspond to different DNA extraction methods in a report by Henderson et al. (This study has limitations that should be taken into account. Firstly, the sampling timing may have more of an effect on RNA-based than DNA-based approaches of profiling microbial communities. If assessing the activities of rumen microbiota is the main study objective, the sampling timing should be carefully considered. In this study, rumen digesta samples were collected before the feeding, which probably resulted in different RNA profiles than in digesta samples collected after feeding. However, the same rumen digesta sample was used for RNA and DNA extraction in our study, so the detected differences between RNA- and DNA-based analyses are valid and not biased due to different sampling times. Second, during RNA isolation processes, RNA yield may differ according to extraction method (such as between physical, mechanic, enzymatic, and chemical methods; Stark et al., n et al. . Third, n et al.  for co-en et al. , such inn et al. . Howevern et al. . Howevern et al. , but difA comparison of the microbial profiles generated from RNA-seq and RNA/DNA Amplicon-seq revealed the generation of different taxonomic profiles of the same rumen microbiota between these methods, and thus, their results could not be simply combined. The RNA-based methods could more robustly detect microbial phylotypes with potentially metabolic activities in the rumen and also detect more interactions among these phylotypes than DNA Amplicon-seq. In addition, compared to RNA/DNA Amplicon-seq, the RNA-seq approach showed more diversity and could detect more bacterial and archaeal phylotypes in the rumen. Although the RNA-seq approach has the advantage of simultaneously identifying and quantifying active microorganisms within a microbiota, the data are not conclusive on which method is the best for analyzing animal gastrointestinal microbiota due to the different technologies and constraints of DNA vs. RNA and due to differences in nucleotide extraction, sequencing, and analysis protocols. Nevertheless, this is the first study to compare RNA-seq and RNA/DNA Amplicon-seq for the taxonomic assessment of rumen microbiota, and differences among these methods should be carefully considered to accurately assess gastrointestinal microbiota in future studies.FL: experimental design, data generation, data analysis and interpretation, manuscript writing; GH: DNA Amplicon-seq, data interpretation and manuscript writing; XS: protocol optimization of RNA-seq library construction; FC: DNA isolation and DNA Amplicon-seq; PHJ: data interpretation and manuscript writing; LLG: experimental design, data analysis and interpretation, manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.