Sulfolobus solfataricus, the transposon-derived paralogous RNAs, RNA-2571–4, show extended complementarity to the 3′ UTR of the 1183 mRNA, encoding a putative phosphate transporter. Phosphate limitation results in decreased RNA-2571 and increased 1183 mRNA levels. Correspondingly, the 1183 mRNA is faster degraded in vitro upon duplex formation with RNA-2571. Insertion of the 1183 3′ UTR downstream of the lacS gene results in strongly reduced lacS mRNA levels in transformed cells, indicating that antisense regulation can function in trans.We report the first example of antisense RNA regulation in a hyperthermophilic archaeon. In  In prokaryotes, small non-coding RNAs are involved in various biological processes, including transcriptional and translational regulation, RNA processing, RNA-guided modification of RNA and chromosome replication . In eukacis- and trans-acting RNAs. The prototypic trans-acting sRNAs of Enterobacteriaceae have a typical size between 50 and 200 nucleotides, are usually not genetically linked to the loci of their target genes and are often expressed under specific growth or stress conditions -5′-end labelled oligonucleotide 5′-GGCAGACCCGTTCATAC-3′ specific for RNA-2571, the oligonucleotide 5′-GGTGGTGCGTCATCAGATTAT-3′ specific for RNA-2573 or the oligonucleotide 5′-GATTGTCTTACCAACCTTTC-3′ specific for RNA-2572 and RNA-2574. The 5S rRNA was probed with oligonucleotide 5′-CACTAACGTGAGCGGCTTAAC-3′ and served as loading control.RT–PCR and qPCR. Total RNA from S. solfataricus strains P2, PH1-16(pMJ05), PH1-16(pMJ05-1183), PH1-16(pMJ05-1183R26), PH1-16(pMJ05-Δ), PH1-16(pMJ05-6454-1183), grown in either full medium (phosphate present) or under phosphate-limiting conditions was isolated and complementary DNA was synthesized using random hexamer oligos (Fermentas) and SuperScriptIII Reverse Transcriptase (Invitrogen). See In vitro RNA degradation assay. S30 extracts of Sulfolobus solfataricus P2 were either pre-incubated for 20 min at 75 °C or heat-inactivated for 20 min at 100 °C. To achieve duplex formation, full-length 1183 mRNA (0.16 μM) was incubated with RNA-2571 (0.48 μM) followed by addition of either the ‘active’ or the heat-inactivated extracts (0.3 μg protein/μl) and incubated at 75 °C. Samples were withdrawn at 0, 2, 4 and 6 min, the reaction was terminated by addition of 1 mM EDTA and an equal amount of 2 × RNA loading dye, and then transferred to a nitrocellulose membrane. After crosslinking either the [32P] 5′-end labelled oligonucleotide 5′-GAAATCCCATGAAGCCCAAAC-3′ (complementary to nucleotides 979–999 of 1183 mRNA) or the [32P] 5′-end labelled oligonucleotide 5′-CTGTGTTCAGCCATTATCG-3′  of 1183 mRNA) was added to detect 1183 mRNA. See reports online (http://www.emboreports.org).


RNAi is widely appreciated as a powerful regulator of mRNA translation in the cytoplasm of mammalian cells. However, the presence and activity of RNAi factors in the mammalian nucleus has been the subject of considerable debate. Here we show that Argonaute-2 (Ago2) and RNAi factors Dicer, TRBP and TRNC6A/GW182 are in the human nucleus and associate together in multi-protein complexes. Small RNAs can silence nuclear RNA and guide site-specific cleavage of the targeted RNA, demonstrating that RNAi can function in the human nucleus. Nuclear Dicer is active and miRNAs are bound to nuclear Ago2, consistent with the existence of nuclear miRNA pathways. Notably, we do not detect loading of duplex small RNAs in nuclear extracts and known loading factors are absent. These results extend RNAi into the mammalian nucleus and suggest that regulation of RNAi via small RNA loading of Ago2 differs between the cytoplasm and the nucleus. Since the discovery of mammalian RNA interference (RNAi) , over 50The assumption that mammalian RNAi is confined to the cytoplasm has been supported by reports that siRNAs cannot silence introns . In addiMicroRNAs (miRNAs) enter the RNAi pathway by binding Ago proteins . In the During cytoplasmic RNAi, small RNA loads into the RNA induced silencing complex (RISC), the complex recognizes a complementary RNA target, and target cleavage can occur at a specific site . SeveralAgo2 binds small duplex RNA and forms the core of RISC . Other kTo resolve the controversy over mammalian nuclear RNAi, we investigated the localization, interaction, and activity of known RNAi factors in human cell nuclei. Here we show that Ago2 and other RNAi factors are present in the nucleus and can associate in multi-protein complexes. Small RNAs in complex with Ago2 can silence nuclear RNA and induce site-specific cleavage. Nuclear Dicer is catalytically active and miRNAs are bound to nuclear Ago2. In contrast, we did not detect loading of duplex RNA in nuclei and most RISC loading factors are absent. These results place the protein machinery necessary for RNAi recognition inside the mammalian cell nucleus but suggest key differences between nuclear and cytoplasmic RNAi.We began our study by using HeLa cells to examine the localization of Ago2, the catalytic core of RNAi . We usedOur microscopy revealed a substantial amount of Ago2 in the nucleus in addition to the expected distribution within the cytoplasm. Images of slices through several micrometer thick sections along the z-axis, combined with 3-D rendering of composite focal sections, revealed Ago2 within the nuclear compartment . The appAs a second method for testing nuclear localization, we used cellular fractionation and Western blot analysis to evaluate the levels of Ago2 and other RNAi factors in the nucleus. We developed a step-wise protocol for isolating cytoplasmic, whole nuclear, nucleoplasmic (soluble nuclear), and chromatin-associated fractions from the same cell population. Fractions were used for various assays, including protein and RNA detection, chromatographic or biochemical fractionation, and enzymatic assays .RNAi factors can localize to the endoplasmic reticulum (ER) . This poWestern blot analysis of purified nuclei revealed endogenous Ago2, Dicer, TRBP, and TRNC6A in the nuclei of multiple human cell lines . ProteinRNAi factors interact to form RISC and execute RNAi in the cytoplasm . To deteTo further support the observed association of RNAi factors with Ago2 in nuclei, we generated T47D cells stably expressing FLAG-HA-tagged Ago2 (FHA-Ago2). Immunoprecipitation of FHA-Ago2 with anti-FLAG antibody confirmed co-purification of Dicer, TNRC6A and TRBP . Co-immuTo visualize the interaction of TNRC6A and Ago2 inside of cell nuclei, we performed immuno-fluorescence microscopy. These experiments revealed co-localization of Ago2 and TNRC6A staining within HeLa nuclei . Co-locaTo further characterize nuclear complexes containing RNAi factors, we separated nuclear extracts by size and charge. Fractionation by size-exclusion chromatography revealed high molecular weight complexes containing all four RNAi factors . To testTo compare nuclear and cytoplasmic RNAi protein complexes, we performed similar size exclusion chromatography with cytoplasmic extract . The retAfter demonstrating that RNAi factors were present in human cell nuclei, we investigated whether they could also direct silencing of nuclear RNA substrates. We examined the silencing within cytoplasmic, nucleoplasmic, and chromatin-associated cell fractions. Our target RNAs were Malat1 and Neat1, long noncoding RNAs (lncRNAs) primarily associated with chromatin  Dodd et. For comWe treated cells with siRNAs targeting each RNA transcript, fractionated the cells, and then used qPCR to measure RNA levels. As typically observed with siRNAs, levels of all four RNA transcripts were reduced in the cytoplasmic fraction . We obseCleavage at a predicted location is diagnostic for substrate processing by Ago2 and we used 5′ rapid amplification of cDNA ends (5′ RACE) to further test whether silencing of RNA in the nucleoplasm and on chromatin was due to Ago2-mediated cleavage. We isolated RACE products and sequencing revealed that site-specific cleavage had occurred at the predicted phosphodiester bond in all cellular fractions for Malat1 . Identifin vitro cleavage assay using Ago2 from either cytoplasmic or nuclear fractions and a radiolabeled RNA substrate derived from luciferase (Luc) mRNA  mRNA . Cells wuc) mRNA . Ago2 waAgo2 immunoprecipitated from nuclear extracts of cells treated with siLuc caused sequence-specific cleavage of the Luc substrate . These rin situ hybridization (FISH). FISH prior to siMalat1 treatment revealed distinct nuclear speckles. Upon treatment with siMalat1, the Malat1 speckles disappeared. In contrast to the disappearance of speckles after treatment with siMalat1, a siRNA with no cellular target (siLuc) had no effect  and performed fluorescence o effect . Taken tWe evaluated the localization of miRNAs within cell nuclei and their association with Ago2. Sequencing of small RNAs revealed that out of 456 miRNA species identified in the whole cell, 346 of them also exist in cell nuclei, suggesting that roughly 75% of miRNAs in the cytoplasm are shuttled into the cell nucleus . The ideWe then examined the association of miRNAs with Ago2 in cell nuclei. Ago2 was immunoprecipitated from nuclear extract using a non-specific mouse IgG as a negative control. Bound small RNAs were isolated and sequenced on a Helicos single molecule sequencer using direct RNA sequencing mode. In this mode, the steps of making complementary DNA and PCR amplification are avoided so that potential sequencing biases are eliminated. The number of sequencing reads better represents the original miRNA expression level. We prepared two biological replicates and averaged the sequencing read number for each small RNA. Single molecule sequencing revealed substantial binding of numerous miRNAs to nuclear Ago2 .in vitro processing assay in which we immunoprecipitated Dicer from cytoplasmic or nuclear extracts, mixed it with radiolabeled pre-miR-19a, and analyzed cleavage products by denaturing polyacrylamide gel electrophoresis. We observed the expected 23-nucleotide product for both nuclear- and cytoplasmic-derived Dicer , we considered the possibility that nuclear extracts could harbor a nuclease activity that might interfere with loading. When duplex RNA was incubated with either nuclear or cytoplasmic extracts, we observed similar levels of degradation over time , suggestin vitro loading assay. We added duplex siLuc with a radiolabeled guide strand to either nuclear or cytoplasmic extracts. When Ago2 was immunoprecipitated with antibodies against either endogenous or FLAG-tagged Ago2, we observed co-purification of radiolabeled siLuc guide strand with cytoplasmic, but not nuclear, Ago2  buffer or with a low salt (0.15 M NaCl) buffer to potentially preserve formation of unstable Ago2-RNA complexes. When RNA isolated from Ago2 immunoprecipitations was resolved by native gel electrophoresis, both duplex and single-stranded RNAs were bound to cytoplasmic Ago2, indicating formation of both complexes during loading  Ago2, the catalytic engine of RNAi, and the RNAi factors Dicer, TRBP, and TNRC6A are all present in human cell nuclei; 2) these nuclear-localized RNAi factors can associate in stable multi-protein complexes; 3) small RNAs can reduce levels of nuclear-localized RNA targets through site-specific cleavage; 4) Ago2 and Dicer isolated from nuclei exhibit catalytic activity; 5) RNAi activity can be visualized inside of human cell nuclei by FISH; 6) endogenous miRNAs are bound to nuclear Ago2 and miRNA pathway components are in the nucleus; 7) programming of RNAi through Ago2 loading with duplex small RNAs is not observed in nuclear extracts; and 8) necessary RISC loading and maturation factors are absent from the nucleus.A previous study from Meister and co-workers reported on the presence and complex formation of EGFP-tagged Ago2 in human cell nuclei using fluorescence correlation and cross-correlation spectroscopy . ConsistThe activity and regulation of RNAi factors in mammalian cell nuclei might have multiple impacts on gene regulation. Small RNAs could potentially guide RNAi factors like Argonaute to nuclear RNA species, such as noncoding RNAs at gene promoters to affect transcription or intronic RNA to alter splicing. Nuclear RNAi-like pathways exist in various eukaryotic organisms like plants, flies, worms, fungi, and ciliated protozoa . These pWe have recently reported a role for Ago2 and TNRC6A in endogenous control of the inflammatory pathway genes cyclooxygenase 2 (COX-2) and phospholipase 2G 4A (PLA2G4A) by miR-589 miRNA . COX-2 aMammalian RNAi has typically been assumed to localize to the cytoplasm, limiting the vision of researchers investigating the regulation of gene expression. Clarity about RNAi factors and their activity in the nucleus widens investigation of RNAi beyond traditional targets in the cytoplasm to targets in the nucleus that may regulate processes like transcription or splicing. While much remains to be learned about nuclear RNAi, such control in the nucleus would add a new layer of gene regulation and offer new options for RNAi-based therapeutics.2. Lipofectamine RNAiMAX (Invitrogen) was used to deliver siRNAs into cells following the manufacturer's recommended protocol. Sequences of siRNAs used are listed in HeLa, T47D, fibroblast, and A549 cells were cultured in their standard media at 37°C in 5% CO2, 0.3% NP-40) supplemented with 1% Protease Inhibitor (PI), 1 mM NaF, and 1 mM Na3VO4. Cells were spun and supernatant kept as cytoplasmic extract after addition of NaCl to 0.15 M and glycerol to 10%. Pelleted nuclei were washed 3× with HLB. To make nuclear extract, nuclei were resuspended in nuclear lysis buffer (same as HLB but containing 0.15 M NaCl and 10% glycerol) also supplemented with PI, NaF and Na3VO4. Nuclei were sonicated and supernatant kept as nuclear extract.Cells were lysed in hypotonic lysis buffer (HLB)  and co-purified proteins eluted with SDS-PAGE loading buffer. Specific proteins were detected by Western blot.Protein G Plus/Protein A resin , antibody and pre-cleared nuclear extract were mixed at 4°C for 2-3 h. When indicated, 20 μg RNas e A was added before incubation. Resin was washed with IP wash buffer , and separated by FPLC. Eluted fractions were assayed by Western blot. For subsequent fractionation by anion exchange, size-exclusion fractions were concentrated and injected onto a Mono-Q FPLC column (Amersham Pharmacia) equilibrated with FPLC buffer at 0.1 M NaCl. Elution was performed by linear gradient from 0.1 to 1 M NaCl. For ammonium sulfate precipitation, saturated ammonium sulfate solution was added to cell extracts at the indicated final percentages, incubated on ice for 15 min, spun down at 18,000 × g at room temperature, and pelleted precipitate resuspended in SDS loading buffer. Supernatant was kept and additional ammonium sulfate added for the next cut. Fractions were analyzed by Western blot.For size-exclusion chromatography, extracts were either treated with RNase A or SUPERase-In (Ambion), filtered, injected onto a Superdex 200 HiLoad 16/60 column (Amersham Pharmacia) pre-equilibrated with FPLC buffer   . Superna2, 0.5 M NaCl, 0.05% NP-40) then mixed with 5′ radiolabeled synthetic target RNA substrate in 1× RNAi buffer  supplemented with 1 mg/mL yeast tRNA, 20 units SUPERase-In (Ambion), and 0.5 mM ATP. Reactions were incubated at 30°C for 1.5 hr with periodic mixing then target RNA and cleavage products phenol extracted. Extracted RNA was resolved on a 15% denaturing polyacrylamide sequencing gel. The gel was dried and exposed to a phosphorimager screen overnight to visualize radioactive bands.HeLa cells were either untreated or transfected with 25 nM siLuc or siLuc_mm see  then harImmuno-fluorescence was performed similarly to that previously described  with modCells were grown on 35mm MatTek dishes and transfected with 25 nM siLuc or siMalat1 as described above. Cells were fixed in ice-cold 4% PFA and permeabilized in 70%. From this point forward, the protocol recommended by the manufacturer of the FISH probes for Malat-1  was followed. Cells were set with mounting medium with DAPI and imaged as above for IF.Small RNA-seq libraries were constructed from either whole cell RNAs or nuclear RNAs isolated from T47D cells and sequenced on Illumina Hiseq 2000. The reads were aligned to human genome hg19, UCSC miRNA database, and/or miRBase (mature miRNA). Ago2-associated miRNA in cell nucleus was identified by first carrying out a RNA immunoprecipitation using a specific anti-Ago2 antibody. Small RNA (<40nt) including miRNA was further gel-selected. The small RNA was then subjected to poly-A tailing and sequenced on a single-molecule Helicos sequencer with a Direct RNA Sequencing (DRS) module.2, 0.5 M NaCl, 0.05% NP-40) then phenol-chloroform extract to isolate co-purified RNA. Extracted RNA was resolved on a 15% denaturing polyacrylamide sequencing gel or a 15% native TBE-buffered polyacrylamide gel. The gel was dried and exposed to a phosphorimager screen overnight to visualize radioactive bands.Duplex siRNA or single-strand guide RNA radiolabeled at the 5′ end was incubated with extract supplemented with 1 mM ATP for 1 h at room temperature with rotation. Ago2 was immunoprecipitated using anti-Ago2 antibody  and Protein G Plus/Protein A agarose . Resin was washed with IP wash buffer (IPWB) (20 mM Tris-HCl, pH 7.5, 4 mM MgCl010203


Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF- In recent years, siRNAs have generated tremendous interest in therapeutics . NeverthIn addition to these modifications, siRNA architecture is also crucial in the design of effective and specific siRNA. The architecture itself can be altered by chemical synthesis. In addition to the canonical siRNA architecture of 21-nt antiparallel, double-strand RNA with 2-nt 3′-overhangs , severalSeveral strategies can be used to prepare branched RNA structures. Although the synthesis of these compounds is complex and tedious, commercially available synthons have improved the complexity and yields of these structures. The assembly of branched nucleic acids on a solid support can be achieved by convergent or divergent strategies. In the former, synthesis of branched oligonucleotides containing two or more identical strands can be achieved by branching derivatives 1,3-diaminopropanol, pentaerythritol, the commercially available symmetric doubler –20, or bSynthetic branched oligonucleotides have been applied for several purposes. Initially, most of the interest in this area was focused on the study of branched oligoribonucleotides as splicing intermediates of eukaryotic mRNAs –26. Moreα) protein, which is involved in the apoptosis, inflammation, and immunity processes [Here, we synthesized branched RNA structures  and evalrocesses . We reasSigma-Proligo, Dharmacon): sense or passenger scrambled 5′-CAGUCGCGUUUGCGACUGG-dT-dT-3′, antisense or guide scrambled 5′-CCAGUCGCAAACGCGACUG-dT-dT-3′, antisense or guide anti-TNF-α: 5′-GAGGCUGAGACAUAGGCAC-dT-dT-3′, and sense or passenger anti-TNF-α: 5′-GUGCCUAUGUCUCAGCCUC-dT-dT-3′. RNA monomers in capital letters, dT represents thymidine. The anti-TNFα siRNA was previously described to efficiently downregulate murine TNFα mRNA [The following RNA sequences were obtained from commercial sources (NFα mRNA .Glen Research). Guanosine was protected with the dimethylaminomethylidene group, cytidine with the acetyl group, and adenosine with the benzoyl group. t-Butyldimethylsilyl (TBDMS) group was used for the protection of the 2′-OH function of the RNA monomers. The phosphoramidites were dissolved in dry acetonitrile (0.1 M), and a modified cycle was used with increased coupling time to 10 min. Oligoribonucleotide 1 was synthesized on a CPG solid support with a symmetric branching unit of two arms containing two DMT-protected hydroxyl groups, as described in [2 and 3 were synthesized using standard low-volume polystyrene thymidine columns. After the solid-phase synthesis, the supports were treated with concentrated aqueous ammonia-ethanol (3 : 1) for 1 h at 55°C. After filtration of the supports, the solutions were evaporated to dryness. The residue was dissolved in 85 μL of 1 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) for 12 h. Then, 85 μL of 1 M of triethylammonium acetate was added and the oligoribonucleotides were desalted on a NAP-10 column using water as eluent. The compounds were purified by HPLC under the following conditions. Column: Nucleosil 120–10 C18 (250  ×  4 mm); 20-min linear gradient from 15% to 100% B (DMT ON conditions); flow rate 3 mL/min; solution A was 5% acetonitrile in 0.1 M aqueous triethylammonium acetate (TEAA) buffer and B 70% acetonitrile in 0.1 M aqueous TEAA. The purified products were analyzed by MALDI-TOF mass spectrometry. Yields  were between 5–10 OD units at 260 nm.ribed in . Oligori1–3 and their unmodified RNA complementary strands (guide strand) were performed following the absorption change at 260 nm. Samples were heated from 20°C to 80°C, with a linear temperature ramp of 0.5°/min in a JASCO V-650 spectrophotometer equipped with a Peltier temperature control. Sample concentration of the samples was around 2 μM. All the measurements were repeated three times and conducted in 15 mM HEPES 1 mM Mg(OAc)2  and 50 mM KOAc pH 7.4.The thermal melting curves for duplexes of the oligoribonucleotides 2, Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, 2 mM L-glutamine, supplemented with penicillin (100 U/mL) and streptomycin (100 mg/mL)). All in vitro experiments were performed at 40–60% confluence. HeLa cells were transfected with 250 ng of a plasmid expressing murine TNF-α using lipofectin (Invitrogen), following the manufacturer's instructions. One hour after transfection cells were transfected with 100 nM double strand concentration of siRNA against TNF-α, using oligofectamine (Invitrogen). Previously, siRNA duplex annealing was performed by mixing modified  and unmodified passenger strands (unm) with the appropriate amount of the corresponding unmodified guide strand. HeLa cells were cultured under standard conditions  following the manufacturer's instructions. The inhibitory capacity of the siRNA duplexes is expressed as double strand concentration for comparative purposes. A 100 mM double strand concentration is equivalent to a 50 nM concentration of two-branched siRNA (1 or 2) and to 25 nM of four-branched siRNA (3).TNF-1–3 of the passenger strand of a siRNA directed against TNF-α (1 was synthesized using a controlled pore glass (CPG) solid support containing a symmetric doubler [2 and 3 with two or four strands, respectively, were synthesized on a low-volume polystyrene support (LV200) functionalized with dimethoxytrityl- (DMT-) thymidine. The commercially available symmetric doubler phosphoramidite was used to introduce two and four branches on the 3′-position of the starting thymidine (t-butyldimethylsilyl (TBDMS) group. Coupling yields, determined by the absorbance of the DMT cation released in each synthesis step, were more efficient (98%) on low-volume (LV200) polystyrene supports than on CPG support (95%). After assembly of the sequences, the DMT-containing oligonucleotides were released from the supports with ammonia, and the resulting compounds were treated with fluoride to remove the TBDMS groups. HPLC analysis of the resulting products is shown in Figures 1 and 2; In order to prepare branched RNA for RNA interference, the potential steric hindrance of the branching unit with RISC must be considered. As the passenger strand is removed from the siRNA duplex upon binding to RISC, we introduced the branching modification at the protruding 3′-end of the sense strand. This position has been demonstrated to allow the introduction of a large number of modifications without affecting the inhibitory capacity of siRNA , 7. We tst TNF-α . Sequenc doubler , as showhymidine . Sequencsupport 9%. After 3). In this case, three peaks in the DMT-containing area were observed. Although resolution of these peaks was not as good as in the previous case, the last eluting peak corresponded to the desired tetra-DMT compound (3). The purified compound had the expected molecular weight, was homogeneous by analytical HPLC  was selected as a target for RNA interference studies. This protein is a major mediator of apoptosis as well as inflammation and immunity, and it has been implicated in the pathogenesis of a wide spectrum of human diseases. Consequently, inhibition of this protein is of particular relevance. Modified oligoribonucleotides (1–3) were annealed with equimolar amounts of the unmodified guide, and the resulting duplexes were used to inhibit the expression of TNF-α gene. HeLa cells were transfected first with the murine TNF-α plasmid using lipofectin, and 1 h later they were cotransfected with the siRNA duplex using oligofectamine. After 24 h, cellular TNF-α production was analyzed by enzyme-linked immunosorbent assay (ELISA). The inhibitory capacity of the siRNA duplexes is shown in α, we normalized the data taking in account the number of strands of each siRNA. Thus, a 100 nM double strand concentration is equivalent to 100 nM of unmodified siRNA duplex, 50 nM of siRNA 1, and 2, and 25 nM of siRNA 3. 1 and 2) were more efficient than four-stranded ones (3). In addition, siRNA 1 showed slightly greater efficiency at inhibiting TNF-α than siRNA duplex 2. This small difference may be related to the lower melting temperature of the former (Tumor necrosis factor (TNF-e former .For several years, research has focused on chemical modifications and delivery technologies to improve the pharmacokinetic properties of siRNA. Many of the chemically modified siRNA with interesting inhibitory capacity contain one or multiple modifications in the sugar, nucleobases, and phosphate linkages or at the 3′- or 5′-ends. In addition to these modifications, duplex architecture of siRNA itself is also relevant, and several modifications have been reported to show satisfactory inhibitory capacity. Here we demonstrate that branched siRNA is compatible with RNAi and that, when transfected with cationic lipids, siRNA has similar inhibitory capacity than unmodified duplex siRNA. Although the potency of branched siRNA containing two or four strands was not increased, we consider it a suitable starting point for further development. Given that the method described here is compatible with most of the RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives. In addition, they offer an internal mid position that could be suitable for attachment to delivery systems. In this regard, optimization of the branching approach for the synthesis of asymmetric branched siRNAs may lead to the development of siRNA for the combined inhibition of multiple targets. These asymmetric siRNA duplexes carrying two RNA sequences attached or bound to an appropriate delivery system will insure the 1 : 1 ratio of two RNA sequences for the combined inhibition of two genes that may improve the treatment of a particular disease.


Termination of transcription by RNA polymerase II requires two distinct processes: The formation of a defined 3′ end of the transcribed RNA, as well as the disengagement of RNA polymerase from its DNA template. Both processes are intimately connected and equally pivotal in the process of functional messenger RNA production. However, research in recent years has elaborated how both processes can additionally be employed to control gene expression in qualitative and quantitative ways. This review embraces these new findings and attempts to paint a broader picture of how this final step in the transcription cycle is of critical importance to many aspects of gene regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation. ► Pol II transcription termination and 3′ end processing are different processes. ► Termination and 3′ end processing are intimately connected and influence each other. ► Pol II transcription termination employs two non-mutually exclusive pathways. ► Many cues transmitted to Pol II influence its choice of termination pathway. ► Termination choice is affected by external stimuli, which controls gene expression. Initial scrutiny of protein-coding genes transcribed by RNA Polymerase II (Pol II), suggested that this was a fairly discrete process; upon recognition of a Pol II termination signal, the transcribed nascent RNA is processed and Pol II released from the template S. cerevisiae, providing H. sapiens nomenclature, where applicable, in brackets. We refer the reader to very detailed reviews covering additional mammalian specificities In this review we will focus on the connected mechanisms of transcription termination and RNA 3′ end rocessing. We will also provide selected examples of how transcription termination and RNA 3′ end processing feed into the larger context of gene expression. Several recent excellent reviews have covered the mechanism of transcription termination and RNA 3′ end processing in mutually complementary ways 2The advancement of genome wide techniques has not only allowed the generalisation of findings from well-studied, isolated examples, but has also helped uncover hitherto unnoticed biological phenomena. Whereas classically the three eukaryotic RNA polymerases  had clearly separate responsibilities in the gene expression repertoire, deep sequencing techniques have uncovered many new transcription units for both Pol II and III that often give rise to short lived or regulatory non-coding RNA In addition to allowing for unscheduled transcriptional events, perturbation of nucleosome positioning will severely affect the ability of Pol II to progress into productive elongation or even to terminate transcription In our subsequent discussion of the mechanism of transcription termination we will therefore distinguish between controlled or scheduled transcription termination and unscheduled transcription termination. The latter describes a large hub of partially ill-defined events ranging from termination of abortive transcripts to premature termination of polymerases that encounter obstacles during transcription along a gene. In making this distinction, we would like to emphasize that as for the newly identified “short” (or longer) non-coding transcripts, even in a regular chromosomal context transcription processivity is often low. As a consequence, prematurely terminated transcripts can outnumber correctly terminated ones 3in vitro studies suggest that this cleavage process is only sufficient to lead to Pol I and Pol III release at designated sites All three eukaryotic RNA polymerases have the ability to cleave RNA within their active sites. However 3.1Where a PAS serves as a TTS, the transcript is processed by endonucleolytic cleavage and subsequently protected from 3′ end degradation by addition of a poly(A) tail of about 70 nt  In contrast to polyadenylation, termination of transcription occurs where Pol II is released from the DNA template over a range of about 200 (yeast) to 1500 (humans) nt downstream of the PAS S. cerevisiae PAS are more degenerate so that their consensus is sometimes difficult to recognize  or thymine (T)), an A-rich positioning element , typically located 30 nt upstream of the cleavage position, as well as an uridine-rich element spanning the cleavage position and site of poly(A) addition (1SPTSPS) repeats that are dynamically phosphorylated during the transcription cycle The PAS of humans consists of a highly conserved hexanucleotide sequence, AAUAAA, in addition to other less conserved sequence recognition elements, whereas ecognize A, left [addition B, left [addition C, left [However, even if yeast cells are momentarily induced to express Pol II devoid of its essential CTD, some 3′ end processing can still be observed, arguing that interaction with CTD is not an absolute prerequisite for 3′ end processing .In yeast, the RNA cleavage site is defined by binding of CF IA component Rna15 to the PE and a loosely associated protein Hrp1 (CF IB) to the EE The enzymatic activities for cleavage and polyadenylation are concentrated in the 15 subunit CPF complex that binds further downstream over the site of 3′ end cleavage (associated with Pta1) contains several proteins that have additional functions at early transcriptional stages, such as Ssu72, the CTD Ser5P phosphatase activity and Swd2, also a component of the Set1 methyl transferase complex COMPASS  signals in yeast has two consequences. First, a slight perturbation in mRNA packaging or altered CTD binding of the 3′ end processing factors, may result in changes of 3′ end selection through usage of more diverse poly(A) signals 3.2in vivo even though in vitro the surrounding sequences may stimulate some residual cleavage activity Transcription termination occurs up to several hundred bases downstream of the 3′ end processing site even though both processes are intimately linked and mutation of many 3′ end processing factors results in transcription termination defects in vivo and in vitro and these examples have now been generalised by genome wide studies showing that polymerase speed decreases after many poly(A) signals After many years of debate on exactly how Pol II is disengaged from the template at a poly(A) signal, it seems plausible that two key aspects influence Pol II destabilisation at poly(A) signals; the speed of the Pol II elongation complex (EC), as well as the stability of the RNA:DNA hybrid within the active centre of the polymerase body. Whereas the RNA:DNA hybrid is probably the main determinant of Pol II EC stability, modulating the EC speed will affect how much time other proteins have to act on it. In a plethora of examples Pol II was observed to pause downstream of poly(A) signals E. coli employs both these mechanisms as manifested by the hexameric ring-shaped transcription termination factor Rho. This factor associates with transcribing polymerases and appears to thread the RNA through its body, until the polymerase undergoes a conformational change that disengages RNA polymerase from the DNA template in vitro reaction, whereas it has no effect on termination of paused Pol I or III in vivo, even though the isolated enzyme fails to dismantle Pol II from DNA templates in in vitro reactions in vitroin vivo Rat1 acts in combination with Rai1 to degrade rRNAs that are polyadenylated as part of a quality control mechanism involving the nuclear exosome component Rrp6 Several models for how the RNA:DNA hybrid within the polymerase body could be destabilized have been suggested: Firstly, a processive protein was suggested to engage with the nascent RNA, follow and destabilize a paused Pol II by reducing the RNA:DNA hybrid length in its active site. Secondly if a protein is associated with Pol II close to the RNA exit channel, this could invade the polymerase active site and unwind the hybrid without directly possessing processivity itself being processive in vitro 3′ end processing itself is not affected Because Rat1 prefers to engage with 5′ monophosphorylated RNA ends, a possibly heightened role for Rat1 in termination came from the observation that human CF IIm component ClpI (hClpI) has a 5′OH RNA kinase activity 45–106 copies per cell) snRNAs of the spliceosome, and snoRNAs, involved in rRNA biogenesis. In contrast, CUTs are transcribed at varying levels, presumably from accessible chromatin acting as “adventitious” and bi-directional promoters In addition to polyadenylated transcripts, Pol II transcribes several classes of non-coding RNA, such as small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), meiotic unannotated transcripts (MUTs) and Xrn1 stabilized transcripts (XUTs); the short RNAs found at mammalian TSS or promoter upstream transcripts (PROMPTs) 4.1Mammalian promoters of either polyadenylated or non-polyadenylated RNA genes are clearly distinguishable S. cerevisiae snRNA and snoRNA are transcribed from independent TATA box containing promoters (51 of 76 snoRNA genes encoded in S. cerevisiae). The 5′ NFR of many of these promoters is bound and demarcated by Tbf1, a protein known to prevent heterochromatin spreading at telomeres During transcription of snRNA and snoRNA, these transcripts associate with specific proteins to form snoRNA particles  or snRNPs (formed by the Sm-class proteins) The termination mechanism of snoRNA genes, although employing some common factors, differs markedly in several key aspects from PAS mediated termination. First, the main RNA processing step involves exonucleolytic transcript trimming from both ends; 5′ to 3′ by the exonucleases Rat1 (Xrn2) or Xrn1 and 3′ to 5′ by the nuclear exosome, a protein complex with two 3′ to 5′ exonuclease activities in vitro the requirement of Ysh1 for transcript cleavage and polyadenylation can be suppressed by deletion of Syc1 . It has therefore been proposed that Syc1 competes with Ysh1 for either inhibitory or stimulatory interactions Second, snoRNA termination and processing relies on a different use of the 3′ end processing machinery. In addition to CTD Ser2P (and the responsible kinase Ctk1), they require all components of CF IA , but only those of the APT sub-complex of CPF  In contrast, if adventitious polyadenylation of a snoRNA occurs, this interrupts snoRNP interactions and so may direct the RNA for degradation 4.2G and (U/A)GUA(A/G)  nuclear pre-mRNA down regulation 1, or the human SCAF8 (SR-like CTD-associated factor 8)) possesses a central RNA recognition motif (RRM), and an N-terminal CTD interacting domain (CID), which establishes interaction with Ser5P and Ser2P CTD nuclear poly(A) RNA binding 3), similarly an RRM and a potential C-terminal arginine/glutamine multimerisation domain containing protein in press). In contrast, Nrd1 and Nab3 binding site sequences are more prevalent in the AT-rich intergenic regions of short or long genes, and are also found in some promoter proximal regions, ORF antisense orientations, as well as tRNA elements. This suggests their involvement in gene expression regulation of a wide spectrum of transcripts snoRNA termination sites are defined by arrays of two oligo-nucleotide motifs UCUUMutation of the third essential gene within the NRD complex, Sen1 (Senataxin), causes the most severe and pleiotropic phenotype, affecting termination of both Pol I and Pol II transcription Alternatively, Sen1 has been suggested to prevent hybridisation of the nascent RNA to underwound transcribed DNA which results in stable RNA–DNA structures 4.3As with snoRNA and mRNA, transcription termination of the plethora of other RNA Pol II transcripts appears to be tightly linked to their processing. Although the growing list of such transcripts renders this discussion inevitably incomplete, we will attempt to summarize current emerging principles. Transcription termination of CUTs also depends on the NRD complex. However, lack of stable interactions of these RNA with proteins, ensues their immediate degradation through the NRD associated nuclear exosome 5in vivo that initiating S. cerevisiae Pol II receives any pre-determined cues in favour of one or another transcription termination pathway. In contrast it seems that Pol II behaviour is dynamically adapted in an opportunistic response to the factors influencing it. For example, nucleosome positioning determines chromatin accessibility to Pol II albeit not its directionality. An initial block to transcription can only be overcome if various positive transcription signals in combination exceed a threshold. By overcoming this threshold, transcription is selectively favoured in one direction. Transcription only proceeds throughout the body of the gene if obstacles do not hamper processivity. Equally, nucleosomes, removed by the passage of elongating Pol II, have to be rapidly re-assembled behind the transcribing polymerase. Pol II release only results in the production of a functional mRNP if a battalion of processing events are correctly coordinated in a timely manner.In the above discussion we have reviewed the two major termination pathways, PAS and NRD dependent, as well as the different coupled transcript processing strategies available to Pol II. Even though this distinction may be useful for the dissection of the molecular mechanisms involved, there is little evidence NRD1, HRP1, PCF11, RPB10, MUD1, URA2, URA8, IMD2, IMD3, HPT1 , GUA1, ADE17, SER3, SRG1, CLN3, SMK1) this termination mechanism is exploited for active transcript control in response to changing growth conditions IMD2 and IMD3, key regulators in the purine nucleotide biosynthesis pathway, whereas low nutrient concentrations will increase transcription of SER3, the 3-phosphoglycerate dehydrogenase HRP1), Pcf11 and the U1 snRNA binding protein A1 (MUD1), thereby establishing a negative feedback control on termination When polymerase engages with the DNA template, transcription can be directed in both directions by PICs NRD1 or NRD-counteracting elongation factor genes such as PAF1, are only now being dissected, adding a fascinating new layer to gene regulation et al. in this issue) How external signals are conveyed to activate Finally, when reaching a PAS, some Pol II fails to terminate transcription, especially at highly transcribed genes, where up to 60% Pol II complexes may read through the normal transcript end 6et al. 2012 in press) With all these obstacles to productive transcription it appears likely that organisms have evolved additional means to ensure sufficient transcriptional activity on the one hand and to allow for rapid response to external stimuli on the other. As mentioned above, NRD activity is modulated in response to external stimuli On the other hand, PAS choice also feeds back to transcriptional levels; a strong PAS correlates with higher levels of PIC formation and for many genes with the physical association of TSS and TTS, forming a structure referred to as a gene loop Fast growing cells exploit this connection by over-expressing polyadenylation factors and selectively express RNAs with short 3′UTRs In the light of the research summarized here, we argue that transcription termination constitutes an important layer in the control of gene expression. We anticipate that revelations from future research will uncover even more intricate interweaving between transcription termination and chromatin associated gene expression.


A total of 124 transporter genes were detected. Genes for fatty acid, peptide, amino acid and glucose and multidrug resistance/multidrug resistance-associated protein (MDR/MRP) transport were expressed at relatively higher levels than the other transporter types. The dogs exhibited abundant mRNA expression of the fatty acid transporters  FABP1 and FABP2, the ATP-binding cassettes (ABCs) ABCB1A and ABCC2, the amino acid/peptide transporters SLC3A1 and SLC15A1, the glucose transporters SLC5A1, SLC2A2 and SLC2A5, the organic anion transporter SLC22A9 and the phosphate transporters SLC20A1 and SLC37A4. In mice, a similar profile was observed with high expression of the glucose transporters SLC5A1 and SLC2As, the fatty acid transporters FABP1 and FABP2, the MDR/MRP transporters ABCB1A and ABCC2 and the phosphate transporter SLC37A4. However, the overall data reveal diverse transcriptomic profiles of the intestinal transporters of dogs and mice. Therefore, the current database may be useful for comparing the intestinal transport systems of dogs with those of mice to better evaluate xenobiotics.This study was performed to produce a transcriptional database of the intestinal transporters of beagle dogs. Total RNA was isolated from the duodenum and the expression of various mRNAs was measured using GeneChip Intestinal drug transporters have great potential for drug absorption ,2. They Dogs have emerged as a primary species for the study of biology and human diseases . The org® reagent and SuperScript Choice System cDNA synthesis kit were purchased from Invitrogen . The BioArray high-yield RNA transcript labeling kit was obtained from Enzo Biochem . The RNeasy kit was supplied by Qiagen . The Canine 2.0 and Mouse 430A 2.0 GeneChips were provided by Affymetrix . GeneChip hybridization and scanning were performed at the Seoulin Molecular Biology Technique Center .The TRIzolad libitum. All procedures were approved by the Konkuk University Institutional Animal Care and Use Committee.Beagle dogs  were housed in a controlled semi-barrier system room at Chemon Co. . The animals were considered to be healthy based on clinical examination . ICR mice (30–40 g) were obtained from Orient Bio Co.  and housed in a controlled animal room at Konkuk University . The animals were fed solid pellets and provided with water 2. Tissue (∼100 mg) was added to 1 ml TRIzol reagent and homogenized with a razor on ice. The homogenate was transferred to a new tube and then 200 μl chloroform was added to the TRIzol mixture. Following centrifugation at 12,500 rpm for 15 min at 4°C, the aqueous phase was transferred to a new tube. The RNA was then precipitated with 500 μl isopropanol and washed with 80% ethanol. The RNA was further purified with an RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. The concentration of the purified RNA was measured at 260 nm and 5 μg purified RNA was mixed with RNA loading buffer and heated at 75°C for 15 min. After cooling on ice for 5 min, the RNA was loaded onto 1% agarose/formaldehyde gel in 1X MOPS buffer. The gel was run at 80–100 V for 50 min and the presence of two sharp 18S and 28S bands was confirmed under UV light.Mucosal tissues obtained from dog and mouse duodenums were immediately scraped with a clean glass slide, transferred to a new frozen vial and dipped into liquid Nin vitro transcription (IVT). cDNA was transcribed using the GeneChip IVT Labeling kit  in the presence of biotin-labeled CTP and UTP. The resulting biotin-labeled IVT-RNA was again purified with a Sample Cleanup Module  and subsequently fragmented. Fragmented cRNA was hybridized at 45°C for 16 h according to the manufacturer’s instructions. Following hybridization, the arrays were washed in a GeneChip Fluidics Station 450 with a non-stringent wash buffer at 25°C and then by a stringent wash buffer at 50°C. The arrays were then stained with a streptavidin-phycoerythrin complex. After staining, the intensities were determined with a GeneChip scanner. The duodenal mRNA expression profile obtained from microarray data analyses for SLC15A1 was validated using semiquantitative RT-PCR. The RT-PCR assay was performed as described previously . The cDNA was synthesized using the One-Cycle cDNA Synthesis kit. Single-stranded cDNA was synthesized using Superscript II reverse transcriptase and T7-oligo(dT) primers at 42°C for 1 h. Double-stranded (ds) cDNA was obtained through a reaction using DNA ligase, DNA polymerase I and RNase H at 16°C for 2 h, followed by T4 DNA polymerase at 16°C for 5 min. After clean up with a Sample Cleanup Module , ds-cDNA was used for http://www.genenames.org). Data analysis was performed using GeneSpring 7.2 software . The numeric data were extracted from DAT images and normalized using Microarray Suite software. Gene function analysis was performed using the gene ontology-mining tool of NetAffx, which is based on the Gene Ontology database (http://www.geneontology.org). GeneSpring also uses data from public genomics databases to build gene ontologies based on annotation information. For the present GeneChip probe array study, the data for each gene represented data from 11–20 probe pairs, each ∼25 bp in length. The overall target-specific intensity was measured as the difference between the intensity of the perfectly matched and mismatched probes. For normalization, data from each expression array were scaled so that the overall fluorescence intensity across each chip was equivalent (average target intensity set at 500). The One-Sided Wilcoxon Signed Rank test was employed to generate the detection P-value. If the overall intensity of the perfect match was significantly larger than that of the mismatch, the detection P-value was small. The probed gene set was considered to be present if the P-value was <0.04. If the P-value was >0.06, the probe set was considered to be absent. The change algorithm generated a change P-value and an associated fold-change value. The second algorithm gave a quantitative estimate of the change in gene expression in the form of a signal log ratio. The level of gene expression was considered to be increased if its change P-value was <0.0025 and the gene expression was considered to be decreased if its change P-value was >0.9975.Official symbols and gene names were used in accordance with the symbol and name lists approved by the Human Genome Organization (HUGO) Gene Nomenclature Committee , SLC20 (phosphate transporter), SLC34 (sodium phosphate transporter) and SLC37 (glycerol-3-phosphate transporter) family genes were detected in the dogs . Only 4 The completion of the DNA sequencing of the human, mouse, dog and rat genomes and knowledge of cross-species gene homologies enables the study of differential gene expression in animal models . The chaMembrane transport is a critical process for the intestinal absorption of glucose, one of the primary energy sources for various physiological and pharmacological functions . All gluet al(The classification of amino acid transporters is well defined according to their substrate specificity and tissue distribution . The amiet al also repFatty acids are absorbed by passive diffusion. However, numerous studies have demonstrated that various transporters are involved in fatty acid absorption . IntestiAside from the crucial roles of purines and pyrimidines in DNA and RNA synthesis, they are also significant components of a number of important biomolecules . TherefoTypical efflux transporters involve MRPs and MDRs, which are members of the ABC superfamily. These proteins translocate a wide variety of substrates, including sugars, amino acids, nucleosides, lipids, bile salts, metal ions, peptides, proteins and a large number of hydrophobic compounds and metabolites across extra- and intracellular membranes ,30. In tet al(Organic anion transporters serve as the efflux system for a number of endogenous compounds, anionic drugs, environmental substances and their metabolic products, which are usually harmful to the body. In particular, these families are known to affect the pharmacokinetics and drug-drug interactions of various drugs . In the et al renamed In the analysis of phosphate transporters, the data reveal that members of the SLC17, SLC20, SLC34 and SLC37 families were expressed in the dogs, while only SLC37 family genes, including SLC37A1, SLC37A2, SLC37A3 and SLC37A4, were expressed in the mice. The most highly expressed genes in the dogs and mice were SLC20A1 and SLC37A4, respectively. SLC20A1 is a ubiquitously expressed sodium-phosphate symporter that plays a fundamental housekeeping role in the maintenance of phosphate homeostasis. SLC20A1 may also function as a retroviral receptor as it confers susceptibility to certain viral infections in human cells.Although similar profiles were observed for several transporters in both species, including the glucose transporters SLC5A1 and SLC2As, the fatty acid transporters FABP1 and FABP2, the efflux transporters ABCB1A and ABCC2 and the phosphate transporter SLC37A4, overall, the data of the present study reveal markedly different transcriptomic profiles for the intestinal transporters of dogs and mice. The dog is an extremely important animal species, not only as a laboratory animal, but also as a major animal in veterinary medicine. However, genetic information has not been explored extensively for the dog in comparison with other model animals. Therefore, the database generated in the present study may be useful for comparing the intestinal xenobiotic transport systems of dogs and other animals.


Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays.We developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzolHere we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays. Over the last decade small RNAs (sRNAs) have arisen as key regulators of diverse biological processes in eukaryotic organisms, including for instance development or stress responses, among others (reviewed in: -4). sRNAThe presence of endogenous sRNAs is now reported for many model plants and various non-model species e.g. ,6), and , and 6])® Reagent ) Reagent ,16 follo Reagent ,8. These® Reagent or commercial columns and omits the total RNA isolation step and, therefore, becomes a simpler and cheaper sRNA isolation method for plants.This motivated us to investigate whether it would be possible to find a generic protocol to isolate sRNAs, which would also work for plant tissues with a high polysaccharide content. In this report, a sRNA isolation method is presented that works efficiently for different plant species like cactus, banana and tomato fruits, and agave leaves, but also for Arabidopsis and tobacco with less polysaccharide content. The method presented here is not based on the use of the TRIzolOpuntia robusta) cactus pads and floral buds were collected at INIFAP  Campo Experimental Norte de Guanajuato, in San Luis de la Paz, Gto., Mexico. Agave leaves were collected at the campus of CINVESTAV, Irapuato, Gto., Mexico. Arabidopsis thaliana (ecotype Ws-3) and Nicotiana tabacum plants were grown under conventional long day growth conditions . Banana and tomato fruits were purchased at the local market. The samples were sliced, ground to a fine powder in a mortar with liquid nitrogen and stored at -80°C until further use.Prickly pear 0.5× TBE buffer, pH 8.07 M Urea5 ml of polyacrylamide stock25 μl of 20% APS (Ammonium persulfate)5 μl TEMED 0.001% SYBR Gold (Invitrogen)0.5× TBE buffer, pH 8.0• 3 M sodium acetate, pH 5.2• absolute ethanol• phenol, pH 8.0• chloroform-isoamyl alcohol • phenol-chloroform-isoamyl alcohol • 5 M NaCl solution• 40% polyethylene glycol 8000 solution (PEG 8000)• DEPC treated (0.05%) water245 μl of 12.5 M methylimidazole, pH 8.00.5 g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)10 g dextran sulphate5 ml of 20% SDS20 ml of 5 M NaCl5 ml of 1 M Tris-HCl, pH 7.52× SSC0.1% SDS1. Place 0.1 g of pulverized frozen tissue in a 1.5 ml microcentrifuge tube and add 500 μl of LiCl extraction buffer and 500 μl of phenol pH 8.0.2. Shake or mix well using a vortex for 1 min. Place each sample on ice until all samples are ready.3. Incubate tubes for 5 min at 60°C.4. Centrifuge for 10 min in a microcentrifuge at max speed at 4°C.5. Transfer the upper phase to a new microcentrifuge tube and add 600 μl of chloroform-isoamyl alcohol .6. Centrifuge 10 min at max speed at 4°C.7. Transfer the upper phase to a new microcentrifuge tube and incubate for 15 min at 65°C.8. Add 50 μl of 5 M NaCl and 63 μl of 40% polyethylene glycol 8000 (w/v) and mix using a vortex for 1 min, followed by incubation on ice for at least 30 min.9. Centrifuge for 10 min at max speed at 4°C (Note: the supernatant contains LMW RNA and the pellet consists of HMW RNA and DNA).10. Transfer supernatant to a new microcentrifuge tube and add 500 μl of phenol-chloroform-isoamyl alcohol .11. Centrifuge for 10 min at max speed at 4°C.12. Transfer supernatant to a new microcentrifuge tube and precipitate LMW RNA by adding 50 μl of 3 M sodium acetate pH 5.2 and 1200 μl of absolute ethanol.13. Incubate overnight at -20°C.14. Centrifuge for 10 min at max speed at 4°C.15. Discard supernatant and dry pellet. When dry, resuspend in 20 μl RNAse-free water.16. Determine RNA purity and concentration by measuring their absorbance at 230, 260 and 280 nm, and calculate the A260/A280 and A260/A230 ratios.1. Prepare a denaturing 12.5% polyacrylamide gel by mixing all components . Let the polyacrylamide polymerize for at least 30 min and then remove combs, gels may be stored at 4°C. Note: Polymerization time affects the quality of the run, and we have noted that gels prepared two days previous to their use showed improved band definition.2. Pre-run the gel(s) in 0.5× TBE buffer (to remove ammonium persulfate residues) for 2 h at 90 V.3. Prepare samples. For 2 μg LMW RNA, add 0.3 (v/v) loading buffer . Incubate samples for 5 min at 65°C to denature RNA and immediately place on ice for at least for 1 min.4. Before loading each sample in the gel, wash each gel slot with 0.5× TBE using a syringe.5. Load the samples in the gel (fill empty slots with loading buffer) and run for around 2 h at 90 V in 0.5× TBE buffer (until bromophenol blue of the loading buffer reaches the end of the gel).6. When the electrophoresis run is ready, take the gel out the chamber and stain for 30 min in 15 ml 0.5× TBE buffer with 0.001% SYBR Gold. Afterwards, rinse for 5 min with RNAse-free water.7. Visualize the gel under UV light.1. The gel may be used for northern blot analysis. In this report, the northern blot analysis was performed following the protocol by Pall and Hamilton (2008)  with mod2. Pre-hybridize with 15 ml hybridization solution  for 1.5 h at 60°C, followed by replacing the hybridization solution and adding the labelled probe of interest, and incubate for 24 h at 60°C. In this report, two probes were used (5'-AGGGGCCATGCTAATCTTCTC-3' and 5'-AAGAGCTCCCTTCAATCCAAA-3'), each labelled with [γ-32P]ATP to detect the small nucleolar RNA U6 and miRNA159a, respectively.3. Wash the membrane twice with wash solution  at room temperature, followed by exposure to a storage phosphor screen for ~48 h at room temperature.® , CTAB [Opuntia robusta), because there are already many examples of successful total RNA isolation for model plants like Arabidopsis.The isolation of total RNA is a common step prior to LMW RNA isolation. Three different total RNA isolation protocols were compared: TRIzolA), CTAB , and LiCA), CTAB . We perf® Reagent, while total RNA from floral buds was best isolated using the LiCl protocol. The 260/280 ratios were similar among the different methods (1.76-2.19), as presented in Table ® Reagent was used. Possibly, guanidine thyocianate or other contaminants could be producing this effect. Electrophoretic analysis showed that the LiCl method produced the best RNA quality  and enriched for LMW RNA . A positive relationship between the quality and yield of LMW RNA and total RNA obtained by the different methods was observed . Besides comparing the different protocols of total RNA extraction for the more difficult cactus plant Figure , it was d Figure . Both thAs sRNAs were obtained both from the supernatant and the total RNA pellet Figure , it was Small RNA extraction protocol; data not shown).The extractions were performed in triplicate and the LMW RNA extracts were electrophoresed using denaturing polyacrylamide gels Figure . The preAs described above, positive results were obtained for the isolation of LMW RNAs using the fourth modification and well defined sRNA bands could be visualized in a gel for 6 different plant species Figure . To testHere we provide a simple and effective method suitable for sRNA extraction from polysaccharide-rich material such as cactus, agave, banana, and tomato tissues, which also works well for less complex plant tissues form e.g., Arabidopsis and tobacco. This modified extraction method gives good yield and quality of LMW RNA species. Moreover, the LMW RNA obtained from the different plant species was successfully used for northern blot assays. Well defined bands were detected when using the miRNA159a and the small nucleolar RNA U6 probes. Therefore, the sRNA molecules that can be obtained with this low-cost short method are suitable for downstream assays like northern blot hybridization, and most likely also for cloning and sequencing of sRNAs.The authors declare that they have no competing interests.FFRC did the major experimental work. NDF and JPVC contributed to the northern blot assays and with technical advice. FFRC, ACH, NMM, and SDF conceived the project and designed the experiments. FFRC, NMM, and SDF drafted the manuscript. All authors read and approved the final manuscript.


Thunnus alalunga) and yellowfin tuna  frequently consume reef prey with higher probability of consumption closer to land and in the western part of the Pacific Ocean. For surface-caught-predators consuming reef prey, this prey type represents about one third of the diet of predators smaller than 50 cm. The proportion decreases with increasing fish size. For predators caught at depth and consuming reef prey, the proportion varies with predator species but generally represents less than 10%. The annual consumption of reef prey by the yellowfin tuna population was estimated at 0.8±0.40CV million tonnes or 2.17×1012±0.40CV individuals. This represents 6.1%±0.17CV in weight of their diet. Our analyses identify some of the patterns of coastal-oceanic ecosystem interactions at a large scale and provides an estimate of annual consumption of reef prey by oceanic predators.The Western and Central Pacific Ocean sustains the highest tuna production in the world. This province is also characterized by many islands and a complex bathymetry that induces specific current circulation patterns with the potential to create a high degree of interaction between coastal and oceanic ecosystems. Based on a large dataset of oceanic predator stomach contents, our study used generalized linear models to explore the coastal-oceanic system interaction by analyzing predator-prey relationship. We show that reef organisms are a frequent prey of oceanic predators. Predator species such as albacore ( Compared to these oceans and to the Eastern Pacific, the WCPO is uniquely scattered with many atolls, high islands and island groups The tropical area of the Western and Central Pacific Ocean (WCPO) represents a vast area of about 35 million kmThe WCPO region is also characterized by tuna fisheries that generate the highest tuna catches in the world  with skipjack, yellowfin, bigeye and albacore tuna annual catches estimated at nearly 2.5 million tonnes in recent years The complex structure of the WCPO coastal system and its spread over such a large area where important oceanic fisheries operate create the opportunity for a high degree of interaction between coastal and oceanic ecosystems. Organisms with an obligate coastal, reef or lagoon life-history phase, named reef prey in our study, drifting in the oceanic domain before coming back to the reef, have a role in transferring energy between the coastal and the oceanic realm and vice versa.Interactions between coastal and oceanic ecosystems have been explored through predator-prey relationship studies. Reef organisms have been identified as prey of the oceanic predators in a number of diet analyses of large pelagic fish However, most of these studies were based on limited sample size (fewer than 400) collected in restricted areas, in the open ocean less than 100 km from land, close to land or around near-shore fish aggregating devices (FAD) anchored in deep waters. These studies do not offer the possibility to systematically analyze the influence of multiple factors such as distance to land or reef, predator species, or predator length, latitude and longitude so as to properly characterize reef prey consumption by oceanic predators at a large scale. Moreover, to our knowledge, the total amount of reef prey consumed by predator’s populations has never been estimated.Trophic studies conducted by the Secretariat of the Pacific Community (SPC) in the whole WCPO provide a unique opportunity to explore the potential importance of reef prey for the offshore pelagic ecosystem at an ocean basin scale. We examine patterns of interaction between coastal ecosystems and oceanic ecosystems in the WCPO by applying generalized linear models (GLM) to this large dataset of oceanic predator stomach contents data, and present an example of estimating total annual consumption of reef prey by oceanic predators in the WCPO.The most important reef prey found in the stomach contents were Acanthuridae (surgeonfish), Balistidae (triggerfish), Chaetodontidae (butterflyfish), Holocentridae (squirrelfish), Monacanthidae (filefish), Pomacanthidae (angelfish), Siganidae (rabbitfish), Synodontidae (lizardfish) larval and juvenile fishes, and Stomatopoda (mantis shrimp), Brachyuran (crabs) and Palinuroidea (lobsters) crustacean larvae . AverageThe chosen model for explaining the reef prey occurrence in stomach contents included predator species, distance-to-land and longitude . At the The preferred model for explaining the weight proportion of reef prey in the stomach of the predator consuming reef prey only included fishing gear . Predato12±0.40CV individuals (2.02×1012±0.41CV crustaceans and 0.15×1012±0.41CV fishes).The yellowfin tuna population, estimated at 1.47 million tonnes in 2009 in the equatorial Western and Central Pacific Ocean between 20°N and 10°S 12 specimens) is estimated to be consumed by the yellowfin tuna population in the region, representing 6.1% in weight of their diet.Our analyses suggest that reef organisms are a frequent prey for small oceanic predators in the WCPO. They also suggest that the degree of interaction is affected by the spatial distribution of reef prey: the majority of reef prey is consumed in the western part of the region and at distance-to-land less than 100 km. The predator’s behavior also influences the reef prey consumption as small specimens and species foraging at the surface eat more reef prey. A total of 0.8 million tonnes of reef prey . The decreasing trend observed from west to east can be explained by the topography and its related oceanography. The western region of the WCPO has more islands, more coast line and higher reef surface area than the eastern region of the studied area . In geneThe school type (FAD vs. non-FAD school) did not appear as a significant explanatory variable in our chosen models. However it came forward in models ranked within the 5 best models with lowest BIC . In the In the water column reef prey are commonly distributed in the upper 100 to 200 m, with maximum abundance observed between 10 and 100 m for fish and crustaceans larvae The size ratios between predator and prey also influence the consumption of reef prey. Our analyses suggested that the diet contribution of reef prey, smaller than non-reef prey on average (35 vs. 78 mm SL for fish prey), was higher for predators smaller than 40–50 cm caught at the surface than for larger predators. Graham 2) is in the same order than previous estimates for yellowfin tuna in the same region 2), in the Eastern Pacific Ocean 2), for Thunnus tonggol in Australian waters 2) and for Euthynnus affinis in eastern Australia 2). To our knowledge, our study provided for the first time an estimate of reef prey consumed by oceanic fish predation  which indicate their importance in the diet of top predators and highlight the role of pelagic predators on mortality of coastal organisms during their pelagic offshore phase. However it is difficult to estimate if the impact of this type of predation on the recruitment rate of reef larvae to the reef. Moreover it likely does not have influence on abundance of juvenile and adult reef organisms at the reef as it was suggested that post-recruitment mortality had much greater effect on abundance than recruitment rate The estimated annual amount of prey (reef and oceanic) consumed by the yellowfin population in the WCPO , different species , different school types (FAD and non-FAD versus non-FAD) and different locations , with limited overlap between these factors. Location, FAD schools and distance-to-land are also related parameters as FAD are preferentially anchored in the western part of the region (Papua New Guinea and Solomon Islands). Despite the number of stomachs examined, the large number of co-variates makes it difficult to explore the influence of each covariate independently as the degrees of freedom in the analyses were insufficient to explore all relationships. Close relationships between some of the parameters are apparent when testing all the possible models, as the 4 or 5 best models usually highlighted the importance of related factors . We chosA total of 7633 stomachs of pelagic predators were collected between January 2001 and April 2011 in the Western and Central Pacific Ocean (9°N - 27°S and 127°E - 132°W) . All samStomachs were considered empty when containing only digestive fluids. For non-empty stomachs, prey were identified to the lowest taxonomic level possible. Identification keys used for prey identification were, for fish: Smith & Heemstra Quantitative analysis of stomach contents can be achieved in different ways http://shoreline.noaa.gov/data/datasheets/wvs.html). Distance between the sampled predator and the closest reef was calculated based on predator’s position and reef data from the Millennium Coral Reef Mapping Project (MCRMP) www.unep-wcmc.org) for locations where MCRMP was not available .The explanatory variables included in the model were predator’s species (pred_code), predator’s length (pred_L), latitude (lat_dec) and longitude (lon_dec) of catch, gear code (gr_code), school code (sch_code) and distance to closest land (dist_land) or closest reef (dist_reef). Surface of reef or lagoon in a 100-nautical miles-diameter-circle around the predator were also included in preliminary models, however considering that these data were not available for about 20% of the samples, these variables were not included in the model. Considering the opportunistic nature of the sampling, the number of samples per year or month did not allow taking into account temporal variability. Fishing gears were grouped into longline (catching fish from the surface down to approximately 400 meters depth) and surface gears . School associations were grouped into FAD  and non-FAD schools . Distance between the sampled predator and the closest land was calculated based on predator’s position and land information established by the National Geospatial Intelligence (NGA) World Vector Shoreline (WVS) . To account for this data structure we analyzed the data in two parts using a delta generalized linear models (GLM) by modelling occurrence separately from the quantity observed Model1: response variable ∼ (1|set).Model2: response variable ∼ pred_code+ pred_L+ sch_code+ ns+ ns+ gr_code+ log(dist_land+1) or (dist_reef+1) +(1|set).where (1|set) represents the random effect for fishing set.Due to their high degree of correlation, co-variates distance-to-land and distance-to-reef were evaluated in separate models. Log transformation and splines with various degrees of freedom were explored for continuous co-variates longitude, latitude, distance-to-land and distance-to-reef. Model fits were compared using the BIC Bayesian Information Criterion p by a predator of species i, caught at longitude j and distance-to-land k is defined as follow:For the model of occurrence, the chosen model was preferred to the model with the smallest BIC since the BICs were almost equal and the chosen model was simpler with 3 explanatory variables instead of 5 . The expf is a cubic spline estimated with two degrees of freedom and C1 is a constant calculated in the model.−16); but including both variables rather than only one significantly improved the model. Despite the correlation these 2 variables affect p independently and should be conserved into the model.Correlation between distance-to-land and longitude was statistically significant  was estimated by combining probability of reef prey occurrence, proportion of reef prey given reef prey was consumed, number of predators and predator’s daily ration. The area was divided into 1/10 degree cells and the longitude and the distance to land of the center of the cell were determined. Probability and proportion were determined by the statistical models as shown above. Numbers of yellowfin tuna at age and per quarter were extracted from the 2011 yellowfin stock assessment for the year 2009 b represents the number of cells of 1/10 degree square with n = 277436, i,j,kp is the probability of consuming reef prey as defined above by species i which is yellowfin (YFT) in this case and for each cell b defined by its longitude j and the distance-to-land k of its center, a represents the predator age class (1 to 28 quarters), g,mq is the proportion of reef prey consumed given that reef prey was consumed as defined above with gear g equals to purse seine (ps) and m length of the fish, t represents the quarter of the year, N is the number of yellowfin tuna of age a in cell b at quarter t, R is the predator daily ration at age a expressed in proportion of predator’s weight, W is the average weight of the predator at age a.According to model results above and considering yellowfin tuna catches by longline represent only about 12% of the catches in the equatorial area Uncertainty around the annual consumption estimate was calculated using a randomization method (n = 1000) to combine the uncertainty estimates for each of the main input values. Uncertainties around the probability and proportion estimates were determined from the statistical models detailed in results. For the daily ration no uncertainty was provided by Maldeniya Figure S1Quantification of island and reef coverage per longitudinal band in the western and central Pacific. Within the 15°N to the 25°S latitudinal band and excluding Australia.(TIF)Click here for additional data file.Table S1Species composition, raw value and percentages of the cumulated weight (W), number (N) and frequency (F) of the reef preys in the diet of the main pelagic predators.(DOCX)Click here for additional data file.Table S2Results of the five second-best models of reef prey occurrence in stomach content.(DOCX)Click here for additional data file.Table S3Results of the five best models of reef prey proportion in stomach containing reef prey.(DOCX)Click here for additional data file.Table S4Results of the five best models of reef prey proportion in stomach containing reef prey for predators caught with surface fishing gears.(DOCX)Click here for additional data file.Table S5Results of the five best models of reef prey proportion in stomach containing reef prey for predators caught with longline fishing gear.(DOCX)Click here for additional data file.Table S6Number of non-empty stomachs examined for the 58 species collected.(DOCX)Click here for additional data file.


However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis) and gram negative (E. coli) bacteria, an archaeon (S. solfataricus) and a eukaryote (S. cerevisiae), followed by massively parallel sequencing. We were able to recover most previously documented m5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m5C was absent were also discovered. Intriguingly, we detected m5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.The presence of 5-methylcytidine (m 6A), and 5-methylcytidine (m5C) can change the coding sequence, alter splicing patterns, or change RNA stability. However, no mRNA modifications in bacteria or archaea have been documented to date. We have used an approach that enables mapping of the m5C modifications across all expressed genes in a given organism. Applying this approach on model bacterial, archaeal, and fungal microorganisms enabled us to reveal the modified RNA bases in these organisms, and to provide an accurate and sensitive map of these modifications. In archaea, we documented multiple genes whose mRNAs are subject to RNA modification, suggesting that similar to eukaryotes, these organisms may utilize mRNA modifications as a mechanism for gene regulation.Ribonucleic acids are universally used to express genetic information in the form of gene transcripts. Although we envision RNA as a mere copy of the DNA four-base code, modification of specific RNA bases can expand the information code. Such modifications are abundant in transfer RNA (tRNA) and ribosomal RNA (rRNA), where they contribute to translation fidelity and ribosome assembly. Recent studies in eukaryotes have shown that mRNA modifications such as RNA-editing (conversion of an adenosine base to inosine), N6-adenine methylation (m In eukaryotes, this DNA modification has been extensively studied over the past years, and was found to play a crucial role in genomic imprinting, X-chromosome inactivation, and suppression of repetitive elements 5C sites in RNA. In bacteria, m5C positions were described only in rRNA, whereas in archaea and eukaryotes m5C was mapped to both tRNA and rRNA. In tRNA molecules, m5C sites are typically present at the variable region and the anticodon loop, where they have been shown to stabilize the secondary structure of the tRNA and affect codon identification and tRNA aminoacylation 5C at position 40 of the yeast tRNA-Phe enables conformational transition of the entire anticodon loop, inducing Mg2+ binding at a distant site and resulting in structural stabilization 5C modification in the anticodon wobble-base (position 34) of tRNA-Leu in yeast was associated with decreased functionality, observed when nonsense suppressor function was estimated in vivo5C, are dynamically modulated during cellular responses to several stress conditions, suggesting that these modifications may play a role in cellular response to stress, potentially by mediating translation rates 5C is thought to play a role in translational fidelity 5-methylcytidine  and in which the methylation levels exceeded 50%. Analysis of the data yielded a total of 10 sites in rRNAs and 85 sites in tRNAs. Below we provide a summary of the identified methylated sites in the different RNA classes in each of the organisms.We began by examining methylation sites in tRNAs and rRNAs that passed a statistical metric . Not only were the positions conserved, but also the methylation stoichiometry appeared to be conserved between E. coli and B. subtilis, with methylation levels of 98.7% at position 977 compared to 97.5% in E. coli, and 63.7% at position 1411 compared to 58% in E. coli.We identified two modified residues at positions 977 and 1411 of the 16S rRNA of subtilis . While nS. solfataricus we identified one m5C site at position 1369 of the 16S rRNA, and another at position 2643 of the 23S rRNA. An additional site with a methylation level of 43% was observed at position 2121 of the 23S rRNA , is consistent with the prediction of an additional m5C site in the 26S unit of yeast rRNA, which was not mapped at the time In yeast we identified two modified positions within the 26S rRNA subunit. Position 2278, which was found to have a methylation level of 72%, was previously reported to contain a methylated site 5C modifications at specific positions. Consistent with this finding, we identified 46 modified sites in 34 tRNAs in S. solfataricus, and 39 sites in 30 tRNAs in S. cerevisiae. To allow comparative analysis of methylation levels in different tRNAs, we generated a multiple alignment of the archaeal and yeast tRNAs, and color-coded methylated positions along them based on their methylation levels (p<0.01) methylated positions even when methylation levels were below 50%, to allow full comparative inspection of the data.Eukaryotic and archaeal tRNA molecules, but not bacterial tRNAs, are known to undergo mn levels . NotablyE. coli tRNA positions, as in 5C modification in yeast was different: usually, either position 48 or position 49 was methylated, with only two tRNAs showing modifications at both positions. Two other tRNAs also contained modifications at position 50 in the variable region.Consistent with previous findings, the methylated positions in both archaea and yeast were clustered at positions 48–49 along the tRNA molecule . This may imply that the typical single, strongly methylated site in yeast may compensate for the presence of two more weakly methylated sites in S. solfataricus. This interpretation, however, needs to be taken with caution, as other factors may account for the observed differences in methylation levels . In general, levels of methylation in yeast were significantly higher than those in archaea, with mean values of 96% per position in vels see .5C modifications in the variable region, despite the presence of a cytosine in one of the positions 48–50. These include tRNA-AlaAGC,TGC and tRNA-ArgCCT in yeast and tRNA-GluTCC,CTC in S. solfataricus. This selective absence of the modification in a subset of tRNAs might provide a potential anchor towards elucidating the role of this modification on tRNAs. Alternatively, other types of RNA modifications, not detectible through bisulfite-conversion, may replace m5C in these tRNAs.Surprisingly, a small fraction of the tRNAs were found to be devoid of m5C sites were observed in S. solfataricus tRNA-TrpCCA and tRNA-LeuTAA, but given their relatively low methylation levels, these sites require further verification. Finally, we failed to detect the known methylation at position 40 of tRNA-Phe Several additional methylated positions were observed in our data, including the previously identified methylation in the anticodon (position 34) of yeast tRNA-Leu S. cerevisiae we made use of a dataset of short sequences previously obtained using bisulfite-treatment of polyA-selected RNA We were next interested in searching for methylated sites within mRNA molecules. Since our initial experiment lacked sufficient sequence coverage to identify such sites within the vast majority of mRNAs due to the high coverage of rRNA and tRNAs, we performed a second bisulfite-seq experiment on the bacterial and the archaeal RNA using size-selected total RNA (>200 bp) to deplete tRNAs, combined with rRNA depletion protocols to enrich for mRNAs . For S. B. subtilis, 1,110 genes in S. solfataricus, and 4,489 genes in yeast. In E. coli, due to very high levels of ribosomal RNA reads (>99%), only 287 protein-coding genes had sufficient coverage  in 1,394 protein coding genes in coverage .5C on an mRNA in that organism, occurring on a protein of unknown function. No m5C events were found in the expressed bacterial mRNAs that were covered in our experiment, although the lack of coverage for most E. coli genes prevents us from drawing a strong conclusion as to the possibility of methylated sites in mRNAs for that organism.Despite significant coverage of most yeast genes, we were only able to detect a single event of mS. solfataricus, the mRNAs of two protein coding genes contained a nucleotide methylated to a level of >50%, and 12 additional genes contained nucleotides with methylation levels of 20–50% (5C modification were enzymes involved in energy and lipid metabolism (notably oxidoreductases and dehydrogenases), which might imply a possible role for this modification in regulating specific metabolic processes. Interestingly, all 14 m5C positions in S. solfataricus mRNAs were located within a strong sequence motif of AUCGANGU . Of these, 31 (41%) were methylated at levels >10%, and 13 (17%) were methylated at levels higher than 20%. These levels are far higher than expected by chance alone: examining all sites  covered by at least 5 reads, but significantly differing from the consensus sequence, only 5.9% had methylation levels of >10%, and 0.9% of the sites had methylation levels exceeding 20%. These results suggest that the presence of this motif is to a large extent sufficient for recognition and methylation by the putative methyltransferase. However, the varying levels of methylation suggest that additional factors other than the immediate sequence environment – such as RNA availability or more distant motifs – may also play a role in determining methylation levels.To determine if the presence of this consensus sequence is sufficient to drive methylation, we searched for the motif within all P = 0.07). The apparently non-uniform distribution of modified sites may provide an angle for future elucidation of the function of this RNA modification.We next examined the relative position of the modified sites within the genes they reside. Methylated bases tended to be localized towards the beginning of the gene  but not m5C. The hm5C modification is similar to m5C but carries an additional hydroxyl group on top of the added methyl group. Total RNA of S. solfataricus was sheared into ∼100 nt-long fragments (the “input”) and immunoprecipitated using the anti-m5C or anti-hm5C antibodies. Libraries were prepared from immunoprecipitated as well as non-immunoprecipitated input control RNA fragments, and subjected to massively parallel sequencing on the Illumina platform.Several modifications other than mDNA e.g. , but sinS. solfataricus genome resulted in a highly non-uniform coverage (5C modified (positions 1369 in the 16S and 2121 and 2643 in the 23S). These peaks represented enrichment of 18–30 fold over the rest of the rRNA molecule, but no enrichment was observed in these positions in the non-immunoprecipitated (input) sample or the sample immunoprecipitated by the hm5C antibody. Most  of the sites we identified in the mRNAs of S. solfataricus , for example, could contribute noise to this measurement. In addition, bulky modifications  may hinder reverse transcriptase processivity, and hence, completely modified tRNA molecules may be under-represented in the sequenced data. Therefore, the stoichiometric measurements we recorded might be biased. Methodologies that are not based on reverse transcription should be applied to decisively determine methylation stoichiometry.Our approach not only detects methylated positions in RNAs, but also provides a quantitative measure of the fraction of transcripts harboring this methylation (to which we refer as “methylation level”). The close similarities in rRNA methylation levels of orthologous positions between the gram positive bacterium p values, but with much lower methylation levels. About 95% of these sites had methylation levels lower than 10%, including many putative positions in rRNAs and tRNAs. As most of these positions have not been previously reported, despite the fact that tRNAs and rRNAs have been extensively characterized, it is highly probable that the majority of these sites represent artifacts from various sources. This notwithstanding, this group contains a single site in the E. coli 16S rRNA (position 1407), which is, indeed, known to be modified. This suggests that there may be additional real methylated nucleotides within this group. However, our approach is currently unable to differentiate between rarely methylated positions and experimental artifacts.An additional limitation of our approach is that it will fail to detect positions methylated at low levels, given the conservative cutoff we set of filtering out rarely methylated positions. Indeed, apart from the positions we described with high methylation levels, there were thousands of additional positions with significant 5C 5C modifications are functionally relevant, and whether other RNA modifications also exist on prokaryotic mRNAs, remains to be determined.The emergence of ultra-high throughput sequence interrogation technologies is revolutionizing research on RNA modifications. Application of such technologies, including RNA-seq and RIP-seq, to study such modifications in eukaryotes has recently revealed that mRNAs carry a plethora of conserved modifications, such as A-to-I Escherichia coli (MG1655) cells were grown in LB medium to log phase at 37°C. Bacillus subtilis str. 168 cells were grown in LB medium to mid log phase at 37°C. S. solfataricus P2 (DSMZ 1617) cells were grown in defined modified Brock's mineral medium with final pH 3.5, and S. cerevisiae (BY4741) cells were grown in YPD medium to log phase at 30°C. All cells pellets were suspended in RNAlater  for 30 min at room temperature. Pre-treatments for RNA isolation included Lysozyme digestion for E. coli cells, glass beads vortex for B. subtilis and Lyticase digestion for S. cerevisiae. Total RNA was then extracted using Tri-Reagent (Molecular Research Center Inc.) according to manufacturer's instructions. RNA samples were treated with Turbo DNA-free kit (Ambion) and rRNA was removed from the E.coli and B.subtilis samples using MICROBExpress mRNA enrichment kit (Ambion).Bisulfite conversion was performed based on the protocol by Schaefer et al. For all 4 organisms, untreated RNA and bisulfite-treated RNA were used as templates for cDNA synthesis using SuperScript II RT (Invitrogen) and random hexamers according to manufacturer's protocol. cDNA was amplified by PCR (ABgene) using regular primers (for untreated RNAs) and primers specific to cytosine-deaminated sequences (for bisulfite treated RNAs). In order to improve sequencing results, generic M13 sequences  were combined with the 5′ end of all the deaminated primer sequences. PCR products were separated on agarose gel and extracted using gel extraction kit (Qiagen). Sanger sequencing of the PCR product was conducted from the M13 primer sequence.Equal amounts (50 ng) of each of the four organism's bisulfite-treated total RNA were mixed together. The mixed bisulfite-treated RNA sample was used as a template for cDNA library preparation according to the mRNA-seq Illumina protocol, omitting the polyA-based mRNA purification step. In brief, RNA was first fragmented by divalent cations at 94°C for 5 min. Double stranded cDNA was generated using SuperScriptII and random primers. cDNA was then end-repaired, adenylated and end-ligated to adapters. Following gel separation, a ∼200 bp fragment was gel-purified. The cDNA library was further amplified and sequenced using 40 single-read cycles on a Genome Analyser II (Illumina).http://www.novocraft.com) to bisulfite-converted genomes of Bacillus subtilis (NC_000964), Escherichia coli (NC_000913), Sulfolobus solfataricus (NC_002754) and Saccharomyces cerevisiae (NC_001133-NC001148) downloaded from the NCBI website. Reads that did not align to the reference sequence at their original length, were iteratively trimmed by two base-pairs from the end of the read and then realigned, as long as their length exceeded 35 nt. To allow identification of methylated positions in tRNAs and rRNAs occurring in multiple copies in the genome, reads mapping to multiple regions in the genome were randomly assigned to one such region. Parameters used for the indexing step were “–b”, and for alignment were “-t 60 -h 120 -b 4 -l 35 -s 2 -F STDFQ -r Random -u 6”. For the transcriptome-based study of rRNAs and tRNAs, reads were aligned against a database consisting of unique copies of all fully processed tRNAs obtained from the tRNAdb 40-nt long reads were aligned using Novoalign , all positions sharing an identical 31-nt sequence surrounding the identified position were collapsed together into a single sequence. Curation of the data confirmed that this efficiently collapsed together sequences from identical genes. The joint methylation level for each collapsed position was recalculated based on the cumulative number of converted and non-converted reads in its ‘parents’.For each cytosine in the genome in both orientations, the number of reads in which the cytosine underwent conversion to uridine (suggesting that it was not methylated), and those in which it was not converted (suggesting methylation) were counted. To eliminate artifacts of various sources, the following filters were applied: (1) identical reads were considered a single read, to eliminate PCR amplification artifacts, (2) reads with ≥3 unconverted cytosines were eliminated, as they might reflect transcripts that for did not obtain sufficient exposure to bisulfite, (3) only positions with a sequencing quality >20 were counted, to eliminate low-quality positions. In the transcriptome based analysis we did not apply the first filter, since for the highly expressed rRNA and tRNA molecules identical reads generally do not reflect PCR artifacts, but merely reflect the high coverage of these genes. Each position was then assigned a methylation level, equivalent to the proportion of reads in which it was not converted, and a For the alignment of tRNAs from tRNAdb AUCG-A/U-G/UG-U/G was searched in all protein coding genes in which the potentially methylated ‘C’ in the center of this motif was covered by at least 3 reads, and methylation ratios were recorded. As a negative control the analysis was repeated taking only motifs with at least three mismatches compared to the consensus . For the analysis presented in P value<0.05. Notably, to gain more statistical power for the downstream analysis, here we did not set a threshold on the minimal methylation level. For each site, its relative position within the gene was calculated, from a scale of 0 to 1 . We compared the distributions of these methylation sites to 22,665 controls lacking a consensus site, and obtained marginally significant results .The motif E.coli genome, containing the consensus sequence found in Sulfolobus solfataricus, were amplified via PCR from the genome of E. coli MG1655 . These amplified sequences were used as template for an in-vitro transcription reaction using MaxiScript kit  to produce RNA transcripts. These RNA transcripts were spiked into Sulfolobus solfataricus total RNA prior to the bisulfite conversion treatment. Reads obtained from this library were aligned separately against the S. solfataricus genomes, and against a synthetic genome comprising the two E. coli templates. Non-converted cytosines were quantified using the pipeline and filters described above.Two ∼200 bp sequences from the Sulfolobus solfataricus total RNA was chemically fragmented  to an average size of ∼100 bp and was subjected to immunoprecipitation with an anti m5C monoclonal antibody  or anti-hm5C polyclonal antibody  based on the protocol described in ref Figure S1Position of the modified sites respective to the gene in which they reside. Distribution of modified sites carrying the consensus (n = 38) appears in red; negative set of sites lacking the consensus  is in blue. The relative position of each methylated site within the gene was normalized to a scale between 0 and 1 , indicated by the X axis. Y-axis, distribution density.(PDF)Click here for additional data file.