Pantoea stewartii contains 41 copies of 15-bp repeats and has a replicon that is homologous to that of ColE1. Although deleting the repeats (pSW207) does not change the copy number and stability of the plasmid. The plasmid becomes unstable and is rapidly lost from the host when a homoplasmid with the repeats (pSW201) is present. Deleting the repeats is found to reduce the transcriptional activity of RNAIp and RNAIIp by about 30%, indicating that the repeats promote the transcription of RNAI and RNAII, and how the RNAI that is synthesized by pSW201 inhibits the replication of pSW207. The immunoblot analysis herein demonstrates that RNA polymerase β subunit and σ70 in the lysate from Escherichia coli MG1655 bind to a biotin-labeled DNA probe that contains the entire sequence of the repeat region. Electrophoretic mobility shift assay also reveals that purified RNA polymerase shifts a DNA probe that contains four copies of the repeats. These results thus obtained reveal that RNA polymerase holoenzyme binds to the repeats. The repeats also exchange RNA polymerase with RNAIp and RNAIIp in vitro, revealing the mechanism by which the transcription is promoted. This investigation elucidates a mechanism by which a plasmid prevents the invasion of an incompatible plasmid and maintains its stability in the host cell during evolution.Plasmid pSW200 from  Stability is the most important determinant of the survival of a plasmid during evolution cis element, cmp, which functions at a distance and optimizes the utilization of oriV by a replication initiation protein, RepC et al.cmp from pT181 does not affect a plasmid’s replication, stability, or copy number, a homoplasmid without a cmp is rapidly lost when pT181 is present in the same cell Plasmid competition is a mechanism by which a plasmid is lost in the presence of an incompatible plasmid in the same cell. One of the best-documented instances of this phenomenon concerns pT181. This plasmid contains a oriV, allowing cleavage of the RNA by RNase H to produce RNAII cis and cannot initiate the replication of another ColE1 molecule. Also, ColE1 synthesizes RNAI, which forms a duplex with the 5′ region of preprimer RNA oriV and, thereby, the initiation of plasmid replication trans-acting factor that acts as an inc determinant that controls the copy number and causes plasmid incompatibility Plasmids of the ColE1 family replicate by a different mechanism from that used by pT181 and pSC101. Rather than using replication initiation proteins to initiate plasmid replication, ColE1 synthesizes preprimer RNA Pantoea stewartii SW2 is a corn pathogen that induces necrosis and systemic wilting, called Stewart’s wilt P. stewartii is a member of Enterobacteriaceae, these plasmids can replicate and be stably maintained in Escherichia colisps (sequence for plasmid stability) for the efficient synthesis of RNAII and maintenance of the stability of the plasmid RNAI and preprimer RNA genes. The elevated RNAI expression from pSW201 prevents preprimer RNA from coupling to the oriV in pSW207, inhibiting replication and resulting in the loss of pSW207.E. coli HB101  E. coli MG1655  DraI site in pSW201 to produce pSW201D1, pSW201D2, pSW201D3 and pSW201D4, respectively, to study the effects of the distance between the 15-bp repeats and the RNAI and RNAII promoters on competition. Plasmids pSW230, pSW231 and pSW232 are derivatives of pSW201 that lack the NheI-PstI (nt 1189 to nt 1752), PstI-AflIII (nt 1752 to nt 2178), and AflIII-SpeI fragments (nt 2178 to nt 3212), respectively, of pSW200. Plasmid pSW252 contains the BglII-NheI fragment (nt 380 to nt 1189) and the SspI-BglII fragment (nt 3356 to nt 3981) in pSW200, and a kanamycin-resistance gene. A BspHI-AccI fragment, a BglII-AccI fragment, a BstI-AccI fragment and a BglII-AccI fragment, which contained the minimal replicons of pBR322 BglII-AccI fragment in pSW200 to form pBR322-210, pACYC-210, pSW100-210 and pRK-210, respectively. Plasmid pKK175-6-lux was constructed by inserting a HindIII-BamHI fragment of pUCD1752, which contains the luxAB genes, into the HindIII-BamHI sites of pKK175-6 RNAIIp, with primers A1 (5′-CCCTGCAGATGACGGAGCTGGAAAAAC) and B1 (5′-GGAAGCTTCAGTTAATAAGATTACGGCGGG) using pSW210 as a template. The PCR product was cut using PvuII and HindIII and inserted into the SmaI-HindIII sites in pKK175-6 NcoI and BglII to form pSW262. The region from nt 532 to nt 957 that contained RNAIp in pSW200 was amplified by PCR using primers A2 (5′-GGAGGATCCTTCCAGTGTAGCCGCAG) and B2 (5′-CCGTCGACGCTGCGATGCCGTTTTTCC) and inserted into the BamHI-SalI sites in pSW261 to yield pSW2611. A DNA fragment that contained a 5′ portion of the tet gene was amplified by PCR using pKK175-6 as a template and primers A3 (5′-CCAAGCTTCCTAATGAGGAGTCGCATAA) and B3 (5′-CCCGGATCCATAAGTGCGCGACGATAGT). The fragment was inserted at the BamHI-HindIII sites in pSW2611 to form pSW2612. NcoI and BglII were used to delete RNAIIp from pSW2612 to form pSW263. The NcoI-HindIII fragment, which contained the DR region and RNAIIp in pSW2612, was then deleted to generate pSW264. AatII and SpeI were used to delete the DR region in pSW201, which was then treated with ExoIII and mungbean nuclease using an Exo-size deletion kit (New England Biolabs). The DNA fragments were then ligated to generate plasmids with various numbers of repeats. DNA sequencing was performed to count the repeats in the plasmids.Plasmids that were used in this study are summarized in E. coli HB101 was cotransformed with pSW207 (Tcr) and a deletion derivative of pSW201 (Kmr). Transformants were cultured overnight in LB broth that contained Km and Tc, and subcultured to the mid-log phase in the same medium without antibiotics. Plasmids in the cell were analyzed using the alkaline-lysis method of Kado and Liu 9 cfu). The cell pellet was dissolved in 20 µl TAE and 80 µl extraction buffer 5 cfu) were subcultured in 5 ml of antibiotic-free LB broth for 7 h with shaking at 37°C, plated on LB agar, and then replica-plated onto plates that contained Km or Tc. About 500 to 1000 colonies were examined to determine the fraction of the colonies that were resistant to kanamycin and tetracycline. Cells that had been transformed using a single plasmid were used as a control. The ratio of copy number of two plasmids in the same cell was estimated following agarose gel electrophoresis by measuring the intensity of the plasmid bands using the Gel-Pro software program (Media Cybernetics). Plasmid pSW201, which has ten copies per cell E. coli MG1655 was cultured in 50 ml LB overnight. After centrifugation, cell pellet was suspended in ice-cold homogenization buffer . Cells were homogenized using a Mini BeadBeater (Biospec Products Inc.). The cell lysate was centrifuged at 17,000×g for 30 min at 4°C. A biotinylated DNA probe, DR-I, which contains the entire sequence of the DR region (nt 3314 to nt 3975) in pSW200, was amplified by PCR using biotin-labeled primers 200-Bio-F5 (5′- GTTAGTCCCTTCCACATTAA) and 200-R5 (5′-ACGATGGGGTTATCAATCTG). The lysate (1 mg protein) was mixed with 2.5 µg of DR-I probe in a binding buffer that contained 60 mM KCl, 12 mM HEPES, pH 7.9, 4 mM Tris-HCl, 5% glycerol, 0.5 mM EDTA, and 1 mM dithiothreitol. The reaction mixture was incubated on ice for 45 minutes and mixed with 30 mg M280 streptavidin beads . The beads were then captured using a magnet and washed five times in binding buffer while they were attached to the magnet. A 2× electrophoresis sample buffer was used to extract proteins that were bound to the probes, and the proteins were then boiled for 10 min 70 antibody  fen operon 2, 1 µg poly(dI-dC), and 0.05% NP-40. The mixture was incubated at room temperature for 20 min. EMSA was performed using the method of Lin et al. 32P]ATP according to a method that has been described elsewhere 32P-labeled DNA was then purified using an Easy Pure PCR/Gel Extraction Kit . RNA polymerase (5 nM) was incubated with 32P-labeled DR probe (0.1 µM) in 20 µl EMSA reaction mixture. After 20 min of incubation, unlabeled 167, RNAIp and RNAIIp probes (0.1 µM) were added to the mixture to detect the exchange of RNA polymerase from the DR probe to 167, RNAIp and RNAIIp probes. In another set of exchange experiments, RNA polymerase (5 nM) was incubated with biotin-labeled DR probe (1 nM) for 15 min. The DNA-protein complex was captured using the method that was used for DAPA. 32P-labeled 167, RNAIp and RNAIIp probes (0.1 µM) were then added to the DNA-protein complex. Exchange of RNA polymerase from the DR probes to RNAIp and RNAIIp was analyzed by gel electrophoresis and autoradiography.Biotin was used to label double-stranded DNA probes using a 3′-end DNA labeling kit . Each probe (5 nM) was then mixed with RNA polymerase holoenzyme  in 20 µl of a reaction mixture that contained 1 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM dithiothreitol, 2.5% glycerol, 5 mM MgClE. coli cells that had been cultured to the log phase. Purified RNA was suspended in water that had been treated with diethyl pyrocarbonate and reverse-transcribed to cDNA using both random hexamers and Superscript II reverse transcriptase (Life Technologies). An ABI PRISM 7700 sequence detection system (Applied Biosystems) was used to quantify and analyze the cDNA. 5′-TCCTAATGCAGGAGTCGCATAA and 5′-CATAAGTGCGGCGACGATAGT were used in PCR to amplify a 115-bp region in the tetracycline-resistance gene. The 16S rRNA, which was used as an internal control to normalize the amount of tested transcript, was reverse-transcribed and amplified using primers F1 (5′-CCATGAAGTCGGAATCGCTAG) and R1 (5′-ACTCCCATGGTGTGACGG) RNAIp and RNAIIp were normalized by calculating the difference between the threshold number of cycles of 16S rRNA and that of the tetracycline-resistance gene (ΔCt). The difference between the promoter activity in the presence of DR and that in the absence of DR was determined from the difference between ΔCt values (ΔΔCt). The amount of tet mRNA that was obtained from pSW261 and pSW263 was set to 100%. The difference between the amounts of mRNA was calculated as 2ΔΔCtAn RNA/DNA Mini Kit  was used to purify RNA from E. coli HB101 was cotransformed with pSW252 and pSW207, 94% of the colonies were found to be resistant to kanamycin (pSW252), and 6% were resistant to tetracycline (pSW207)  (mob genes are not associated with competition.Our earlier study demonstrated that the repeats in pSW201 are responsible for the instability of pSW207  as a pSW(pSW207) , verifyi(pSW207) , caused (pSW207) . Further of 99∶1 . In this pSW232) . These p pSW232) , suggestSince the 15-bp repeats in pSW200 importantly affected plasmid competition, whether altering the number of the repeats could influence the capacity of pSW201 to compete with pSW207 was studied. The competition assay revealed that only 3% of the population contained pSW207 when the cells were cotransformed with pSW201. The fraction of the cells that contained pSW207 increased to 10% when the number of repeats in pSW201 was reduced from 41 to 19; it increased to 31%, 45% and 42% when the number of repeats in pSW201 was reduced to three, one and zero, respectively . The resinc determinant that inhibits the coupling of preprimer RNA to the oriV and thereby prevents the replication of ColE1 plasmids. This study posits that deleting the DR region from pSW200 reduces the transcription of the RNAI and preprimer RNA genes. Restated, in a cell that contains both pSW201 and pSW207, pSW201 expresses more RNAI than does pSW207, overwhelming the preprimer RNA from pSW207 to inhibit the replication of pSW207, which is therefore excluded from the cell. Therefore, in this study, transcriptional fusions were generated to test whether the DR region affected the activities of RNAIp and RNAIIp, which were determined by measuring the amount of RNA that was transcribed from the promoters by RT-qPCR. Additionally, instead of amplifying the region between RNAIp and RNAIIp, which has a complex secondary structure and is difficult to amplify by PCR, the regions downstream of the two promoters were replaced with a sequence from a Tc-resistance gene  yielded only the background level of luciferase activity  using purified enzyme. PAGE revealed that 5–25 nM RNA polymerase shifted 5 nM DR probe  was inseRNAIp and RNAIIp, enhancing the transcription of RNAI and preprimer RNA genes. Hence, 5 nM RNA polymerase was mixed with 0.1 µM 32P-labeled DR probe to enable the binding of the enzyme to the probe. Cold RNAIp, RNAIIp, and 167 probes (0.1 µM) were then added to the reaction mixture to compete for binding. Although adding probe 167 slightly reduced the amount of RNA polymerase that was bound to the DR probe  that had been captured with streptavidin beads to reaction mixtures that contained 0.1 µM 32P-labeled RNAIp, RNAIIp and 167 probes. The results revealed that RNA polymerase-DR complex shifted the RNAIp and RNAIIp probes  in pSW210 (E. coli HB101 (The n pSW210  that conli HB101 , lane 1,li HB101 , lane 3,li HB101 , lane 2,E. coli HB101 (RNAI and preprimer RNA genes. RT-qPCR results show that deleting the DR region reduces the activities of both RNAIp and RNAIIp by about 30%. The results of the RT-qPCR study also suggest that, despite the reduction in transcription, the ratio of RNAI to RNAII that are synthesized by the pSW207 is equal to that of pSW201. This finding may explain why the number of copies of pSW207 is similar to that of pSW201. This study also shows that DR functions in a manner similar to eukaryotic enhancers Our earlier study revealed that DR is essential to the destabilization of pSW207 by pSW201 in li HB101  [35]. ThRNAIp and RNAIIp, promoting the transcription of the RNAI and preprimer RNA genes. The first piece of evidence for this claim is that the 15-bp repeats in the DR region contain sequences that resemble that of a -35 box (70 to probes that contain the 15-bp repeat sequences (This study also supports the claim that RNA polymerase binds to the repeats in the DR region and is then exchanged with  -35 box . Althougequences . Finallyequences , revealiRNAIp and RNAIIp since the EMSA results show that adding cold RNAIp and RNAIIp probes to the reaction mixture considerably reduced the amount of RNA polymerase that was bound to the DR probe (luxAB reporter genes that are inserted downstream of the DR region are not transcribed from DR (32P-labeled probes that contain the sequences of RNAIp and RNAIIp are added to the reaction mixture (RNAIp and RNAIIp to enhance transcription. Finally, pUC18, pACYC184, and pSW100 exhibit the competition phenotype when a DR fragment is present (RNAI and preprimer RNA genes of pSW201 by exchanging RNA polymerase that is bound to DR with RNAIp and RNAIIp. Although pSW207 synthesizes less preprimer RNA than does pSW201, the ratio of RNAI to preprimer RNA remains unchanged, so the copy number of pSW207 is similar to that of pSW201. Meanwhile, the RNAI that is generated by pSW201 efficiently inhibits the coupling of preprimer RNA to oriV to decrease the copy number of pSW207, while maintaining the copy number of pSW201 at ten per cell. Therefore, pSW200 uses DR to inhibit the replication of an incoming incompatible plasmid without the DR sequence to prevent curing from the cell by incompatibility. This study elucidates a mechanism by which a plasmid causes the loss of an invading incompatible plasmid to maintain its stability during evolution.This study establishes that RNA polymerase that is tethered to DR is exchanged with DR probe . Despite from DR . Additio mixture , suggest present , suggest present . Based o


Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5′ triphosphate (5′ppp)-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3), a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5′ppp. We show here that a 5′ppp-containing cloverleaf (CL) RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand. Pathogen recognition is a key step in the initiation of the host antiviral innate immune response. Specialized pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) detect evolutionarily conserved structures known as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral response. TLRs detect various PAMPs commonly found in both bacteria and viruses, such as lipopolysaccharides (LPS), and non-methylated CpG-containing DNAs , which together establish an antiviral state in cells Recently, major efforts have been directed to the use of PRR agonists as antiviral agents. A TLR9 agonist, CpG oligodeoxynucleotides, was shown to prevent coxsackievirus B3 (CVB3)-induced myocarditis in vitro studies, it is known that RIG-I activation requires short double-stranded RNAs containing an intact 5′ppp group in vitro transcripts of the Epstein–Barr virus (EBV)-encoded small RNAs (EBERs) From in vitro transcription, would activate RIG-I signaling and the antiviral response. We demonstrate that a coxsackievirus B3 (CVB3) cloverleaf containing a 5′ppp moiety stimulated the RIG-I pathway, but other RNA stem-loop structures of similar lengths only poorly activated RIG-I, demonstrating the complexity and specificity of ligand recognition by this receptor. These results contribute to the identification and characterization of RIG-I ligands as potential antiviral agents.Picornaviruses do not produce 5′ppp-containing RNA species during infection, and are known to activate MDA5, but not RIG-I, through its double-stranded replication intermediate cis-acting RNA element (CRE) and additional stem-loop structures within the 3′ UTR (in vitro transcribed RNAs (ivtRNAs) corresponding to 2 kb fragments of the CVB3 genomic sequence with 1 kb overlaps, and investigated their individual abilities to activate RIG-I by an IFN reporter assay. Quality and concentrations of these transcripts were confirmed by gel electrophoresis prior to transfection (data not shown). Upon transfection in wt mouse embryonic fibroblasts (MEFs), ivtRNA corresponding to nucleotide (nt) 1–2000 of CVB3 genome (CVB3 1–2000) induced significantly higher levels of IFN-β promoter activation than all other segments of the genomic RNA  at the extreme 5′ terminus, the stem-loop structures within the viral internal ribosomal entry site (IRES), the e 3′ UTR . To inveomic RNA . When RNtivation , demonst−/−, MDA5−/− and IPS-1−/− MEFs. All wt MEFs showed similar patterns of IFN-β induction, and the first 1 kb (CVB3 1–1000) was the most potent IFN-β inducer amongst all fragments tested . Under these conditions, nt 1–742 induced IFN-β promoter activation to the same extent as nt 1–1000 , which digests the 5′ppp moiety that is required for ssRNAs to activate RIG-I, and studied the resulting RNA to activation IFN-β response in RIG-I+/− and RIG-I−/− MEFs. As shown in +/−, but not RIG-I−/− MEFs, and CIP treatment completely abolished the observed IFN-β response. To assess whether CL-induced RIG-I activation is due to copy-back species, which have been described as byproducts of T7 polymerase-produced RNAs Having established that the CL is a potent IFN-β inducer, we set out to confirm that this response is truly dependent on RIG-I. To this end we transfected RIG-IWe next sought to compare the antiviral activity of the CVB3 CL to that of the recently described short hairpin RNA derived from VSV UTR sequences  that wasBy employing the BioMark large-scale qRT-PCR analysis platform, we also analyzed the cytokine and chemokine induction profile of CVB3 CL as compared to that of VSV hairpin. As shown in To further confirm the potency of CVB3 CL in triggering antiviral immunity, we performed infections with dengue virus (DENV) and VSV, in cells pre-treated with varying concentrations of CVB3 CL or VSV hairpin. As shown in Not all 5′ppp stem-loop structures induced a RIG-I-mediated type I IFN response , and we PRR ligands are currently being investigated as potential antiviral agents. In this study, we examined several small, structured RNAs derived from CVB3 sequence in their RIG-I-stimulatory activities. Although none of these RNA ligands are natural PAMPs during CVB3 infection, the CL structure, when produced in vitro as a 5′ppp-carrying RNA, proved a highly potent RIG-I ligand. Upon transfection, CVB3 CL induces expression of type I IFNs and type III IFNs, as well as many proinflammatory cytokines and chemokines. This ligand protected cells against various viral infections including EV71, DENV and VSV – completely unrelated viruses that exploit different host factors and pathways for replication.in vivo. Altogether, our data demonstrate the reliability of utilizing RIG-I pathway activation as a means of immune stimulation.Another RIG-I agonist, a short hairpin derived from VSV genome, was previously characterized as a potent immune stimulator, capable of inducing ISG expression, similar to recombinant type I IFNs, but also pro-inflammatory cytokines such as TNF-α, IL-1α/β, IL-6, and CXCL10 as a result of NF-kB activation. We compared the potency of this ligand and our CVB3 CL in both immune stimulation as well as functional antiviral efficacy. Both ligands induced ISG and cytokine responses to similar extents at the RNA and protein levels. Consistent with this observation, both ligands fully protected cells against DENV and VSV infections at the same concentration. Moreover, the VSV hairpin was shown to protect mice against lethal influenza virus infection To date, most established RIG-I ligands, such as the panhandle structure of negative-strand RNA viruses and 5′ppp RNAs derived from Sendai virus and VSV, all adopt a hairpin structure. In comparison, our CVB3 CL adopts a rather unique structure, forming in total 4 short stems, one of which contains a 3-nt bulge, and 3 small loops. Interestingly, three other 5′ppp-containing RNA molecules tested in this study - Domains III, VI and VII - completely failed to activate RIG-I. These RNAs are all predicted to form hairpin structures, and contain 5′ppp on blunt ends, making them appear typical RIG-I ligands. Although Domains III, VI and VII are smaller  than CVB3 CL (90 nt), they are still larger than some of the established synthetic RIG-I ligands , chemical synthesis of the 5′ppp moiety is currently challenging. In this regard, dsRNA ligands may be favorable since it has been suggested 5′ppp is not essential for RIG-I activation by dsRNAs longer than 200 bp in vivo safety profile, delivery efficiency and potency of single-stranded or double-stranded RIG-I ligands. Having a large repertoire of various RIG-I-activating RNA molecules (as well as those that fail to do so) may be very important to make better-informed decisions in the future.Known RIG-I ligands not only include 5′ppp-containing ssRNAs such as CVB3 CL and VSV hairpin, but also short 5′ppp-containing dsRNAs Recombinant pegylated IFNs has been used as an antiviral therapy against hepatitis B and C virus infections with tremendous success, but with the price of prolonged severe side effects. Therefore, efforts have been focused on the use of ligands of TLRs and RLRs as inducers of antiviral immunity, because these agonists likely mimic the earliest immune recognition events of a natural antiviral response and induce a more extensive and balanced immune activation. Because of the ubiquitous expression profile of RIG-I, RNA agonists would be able to stimulate virtually any cell type, including those at the site of infection. The identification of a novel RIG-I agonist comprising the CVB3 cloverleaf will contribute to the development of RIG-I-based antiviral agents. The identification of RNA ligands that possess all the characteristics required for RIG-I induction - but fail to do so – further demonstrates that there is much to learn about the stimulation of the innate immune response by natural RNA ligands.in vitro transcribed RNA was prepared as previously described (Goulet 2013 PLoS Pathogens). CVB3 ivtRNAs were produced by T7-driven in vitro transcription using PCR products amplified the CVB3 infectious clone p53CB3/T7 The sequence of 5′pppVSV hairpin RNA was derived from the 5′ and 3′ UTRs of the VSV genome as previously described (Schlee 2009 Immunity) and 2. A549 MEFs Enterovirus 71 (strain BrCr) was produced on HeLa R19 cells and titers determined by cytopathic effect-based end-point titration. DENV (serotype 2 strain New Guinea C) was produced on C6/36 cells and purified by ultracentrifugation through a 20% sucrose cushion. DENV titers were determined by FACS upon E protein staining on Vero cells (ATCC). VSV-GFP bearing the methionine 51 deletion in the matrix protein was kindly provided by J. Bell . Virus stocks were grown on Vero cells, concentrated from cell-free supernatants by centrifugation at 15,000 rpm for 90 minutes at 4°C. Virus concentration was determined by plague assay.SQ PVDF membranes  and then subjected to immunoblotting using indicated antibodies. Anti-pIRF3 Ser 396 and anti-RIG-I antibodies were purchased from EMD Millipore, anti-IRF3 from IBL, Japan, anti-IFIT1 from Thermo Fisher Scientific, anti pSTAT1 Tyr701 and anti-STAT1 from Cell Signaling, and anti-β-actin from Odyssey.5′ppp-treated cells were lysed in RIPA buffer  and cleared by centrifugation at 17,000×g for 15 min at 4°C. Cleared lysates were subjected to SDS-PAGE analysis on 4–20% acrylamide Mini-Protean TGX precast gels . Proteins were electrophoretically transferred to Immobilon-PIFN-β luciferase reporter assay was performed as previously described Real-time quantitative PCR (RT-qPCR) was conducted as previously described Cell surface expression of phosphatidylserine was measured using an APC-conjugated annexin V antibody, as recommended by the manufacturer (Biolegend). Briefly, specific annexin V binding was achieved by incubating A549 cells in Annexin-V binding buffer (Becton Dickinson) containing a saturating concentration of APC-annexin V antibody and 7-amino-actinomycin D (7-AAD) (Becton Dickinson) for 15 min in the dark. APC-annexin V and 7-AAD binding to the cells was analyzed by flow cytometry, as described previously using an LSRII flow cytometer and FACS Diva software.www.universalprobelibrary.com) and obtained from the Integrated DNA Technology company (Table S1). cDNA along with the entire pool of primers were pre-amplified for 14 cycles using TaqMan PreAmp Master Mix as recommended by manufacturer's protocol (Applied Biosystems). cDNA was treated with Exonuclease I (New England Biolabs). cDNA samples were prepared with 2× FastStart TaqMan Probe Master (Roche), GE sample loading buffer (Fluidigm) and Taq Polymerase (Invitrogen). Assays were prepared with 2× assay loading reagent , primers (IDT) and probes (Roche). Samples and assays were loaded in their appropriate inlets on a 48.48 BioMark chip. The chip was run on the Biomark HD System , which enabled quantitative measurement of up to 48 different mRNAs in 48 samples under identical reaction conditions. Runs contained 40 cycles. Raw Ct values were calculated by the real time PCR analysis software (Fluidigm) and software-designated failed reactions were discarded from analysis. All data are presented as a relative quantification with efficiency correction based on the relative expression of target gene versus the geomean of (GAPDH+Actin+β2 microglobulin) as the invariant control. Fold of induction as compared to mock-treated sample was plotted on a log10 scale and used to generate the heat map. Statistical analyses were performed as described above.Total RNA and cDNA were prepared as described above. Intron-spanning PCR primers were designed using Roche's Universal Probe Library Assay Design Center Click here for additional data file.Table S1List of primers and probes used for high throughput RT-q-PCR.(DOCX)Click here for additional data file.


Although visual fixations are commonly used to index stimulus-driven or internally-determined preference, recent evidence suggests that visual fixations can also be a source of decisional bias that moves selection toward the fixated object. These contrasting results raise the question of whether visual fixations always index comparative processes during choice-based tasks, or whether they might better reflect internal preferences when the decision does not carry any economic or corporeal consequences. In two experiments, participants chose which of two objects were more aesthetically pleasing (Exp.1) or appeared more organic (Exp.2), and provided independent aesthetic ratings of the stimuli. Our results demonstrated that fixation parameters were a better index of choice in both decisional domains than of aesthetic preference. The data support models in which visual fixations are specifically related to the evolution of decision processes even when the decision has no tangible consequences. Aesthetic experiences are highly subjective: what one person finds beautiful, another may find dreadful. These idiosyncratic differences occur because aesthetic preferences are shaped not only by visual features such as symmetry The finding that visual fixations played an active role in biasing decisions, potentially in opposition to personal preferences, is particularly interesting within the context of aesthetic choices because looking times have long been used as a measure of preference  . One reaAesthetic choices therefore exist as an interesting boundary condition. If visual fixations actively contribute to decision-making, irrespective of the nature of the choice, then we would expect longer fixations on subsequently chosen objects even when it was rated as being less preferred than alternatives. However, if aesthetic decisions are arrived at through processes dominated by preference, then visual fixations should index preference over choice. Our results show that visual fixations reflect comparative processes during choice decisions, irrespective of the nature of the decision and even in opposition to aesthetic preference. The results suggest that the use of visual fixations must consider the context of the paradigm in order to avoid confounding mechanisms of choice with preference.The goal of this experiment was to determine if visual fixations more closely reflected the aesthetic preference of objects viewed in isolation or comparative processes during a two alternative-forced-choice decision.Twenty volunteers consented and participated . In this and all subsequent experiments, participants had normal or corrected-to-normal vision.Written informed consent was obtained, and the study complied with the guidelines and was approved by the Internal Review Board of the University of California, Davis.Stimuli were novel black-and-white patterns spanning 4.3° in diameter . To ensuunbiased preference for a stimulus and we refer to these the “baseline” ratings During “single-item” trials, only one pattern appeared , but in duplicate bilaterally to be visually identical to paired trials In order to test whether fixations were indicative of aesthetic choice or preference, it was first necessary to demonstrate that the two measures were imperfectly correlated. In the same fashion as the analysis by Krajbich et al. (2010), we calculated the probability of the left item being chosen as a function of the difference in aesthetic ratings between the left and the right items shown during the paired trials. The difference scores were calculated by subtracting the rating for the right-sided stimulus from the left-sided stimulus, and binning values into five categories: −2 and less, −1, 0, 1, 2 and greater. Difference scores were calculated for both the ratings from the baseline and paired trials. Note that in paired trials, the ratings followed the explicit choice whereas in baseline trials, there were two identical objects and no explicit choice; the baseline ratings could not have been influenced by any immediately preceding choice behaviors.To quantify the relationship between aesthetic choices and aesthetic ratings, the proportion of left choices (bounded by 0 and 1) were modeled using a logistic function given by, y = exp(A*(x−B))/(1+exp(A*(x−B))), where A is the slope and B is the intercept of the function . We fit 2 = .50). However, on paired trials, the data were well fit by a logistic function with a large slope , which reflected categorically higher ratings for the chosen item. The A parameters were significantly different from each other using a paired t-test, t(18) = 5.7, p<.0001. The results indicated that paired ratings were highly influenced by the preceding choice, which occurred on the same trial ×3 relative baseline rating  repeated measures ANOVA. An object’s relative baseline rating was classified as higher, lower or the same if the object’s baseline rating was greater than, less than, or equal to its pair counterpart, respectively.We next entered the proportion of time spent looking at each object in a paired trial into a 2  2 = .87). There was no main effect of the relative baseline rating, F = 2.69, p = .08, η2 = .12, nor an interaction, F = .59, p = .56, η 2 = .03. Thus, proportional fixation durations were clearly longer for the subsequently chosen object  was fixated for longer compared to the unchosen object , F = 128.75, p<.001, ηn object . FixatioTo identify the point at which fixation durations became significantly longer for the subsequently chosen object, we calculated the cumulative proportional fixation duration over 500 ms bins, time-locked to the choice response . We did before manual response). If the difference at a time bin was significant, a t-test of the next time bin  was conducted. This analysis demonstrated that the proportional looking duration of the chosen item was significantly longer than the unchosen item beginning at 3500 ms prior to the actual manual response when two measures were congruent, t(19) >3.99, p<.001 with Bonferroni correction for seven comparisons, at which point the difference was no longer statistically significant. On incongruent trials where the chosen object was given a lower baseline rating  the cumulative fixation duration on the chosen object was significantly longer than the unchosen object beginning from 3000 ms before the manual response, t(19) = 2.99, p<.05, with Bonferroni correction for six comparisons. On same-rating trials, the difference in the cumulative looking time also began as early as 3000 ms before the response, t(19) = 3.58, p<.05, with Bonferroni correction for six comparisons.In order to test for significant differences between the cumulative fixation durations on the chosen vs. unchosen object, we conducted sequential t-tests, beginning from the data bin nearest to response  was significant, F = 14.83, p<.001, η2 = .44 . Post-hoc paired t-tests revealed that amongst the chosen objects, those with lower ratings were less likely to be looked at last compared to those with higher or same ratings, both t(19) = 3.78, p<.001, d = 1.01 There was no difference between those rated higher and the same, t(19) = 1.51, p>.14.In addition to total fixation durations, we also examined whether the last fixation was a better indicator of the subsequently chosen item, or the more preferred item. This was motivated by literature showing that final fixations vary with choice and aesthetic preference The goal of Experiment 1a was to examine if eye movement parameters  would serve as indices of the choice process and/or aesthetic preference. We observed that fixation durations and final fixation corresponded to the subsequent explicit choice: when choice and aesthetic ratings differed, these parameters predicted choice and not ratings. Moreover, we observed that the cumulative fixation time on the subsequently chosen object was reliably longer on the chosen object approximately 3000 ms before the manual button press that indicated that choice. These results suggested that aesthetic choices engage similar mechanisms as decisions in other cognitive or corporeal domains In Experiment 1a, ratings and choice were based on an aesthetic judgment. It was possible that effects of choice simply masked those of ratings because they were in the same dimension. To address this question, we conducted an identical experiment, but now removed the choice component. If the influence of preference on looking durations were masked in Experiment 1a by the choice decision, we should see a difference in the cumulative looking times as a function of aesthetic ratings in this experiment when no choice was required.Twenty volunteers participated .The materials and procedure were identical to Experiment 1a except that the explicit choice portion of each trial was removed. On each trial, the stimuli were terminated when participants pressed a button to indicate their readiness to provide paired aesthetic ratings.Similar to the Experiment 1a, we first compared the total proportion of looking time on each of the paired objects as a function of their baseline rating. Because the decision component was removed, the looking time was analyzed as a function of the relative baseline ratings. The cumulative looking times for the higher-rated item  was not statistically different from the lower-rated item , t(19) = 1.0, p>.05.Given that the baseline and paired ratings were imperfectly correlated , a second analysis was conducted using the aesthetic ratings from the paired trials. Note that this was not possible in Experiment 1a because the paired ratings were contaminated by the choice . Still, the total proportional looking durations were not significantly different for the higher- and lower-rated items, t(19) = .24, p>.5 . Looking times did not reflect aesthetic ratings when these ratings were made in the absence of choice.These results suggest that longer fixation observed in Experiment 1a was driven more by the decision process rather than by aesthetic preference. Furthermore, the mean RT per trial was not statistically different from Experiment 1a, t(38) = .68, p>.4; M = 3553.35 ms, SE = 360.77, suggesting that the two tasks required comparable processing times.The decision and rating processes in Experiment 1a were both based on aesthetic judgments. Due to this overlap, it was difficult to rule out contributions from aesthetic ratings on eye movement parameters. Thus, in Experiment 1b we dissociated the choice and rating processes by asking participants to only rate items based on aesthetic value. Critically, the choice component was removed. Nevertheless, the eye movement parameters still did not correlate with measurements of preference rating. This provides further evidence that the eye parameters did not reflect preference.To further test the relationship between fixations, choice and aesthetic preference, we next dissociated the dimensions of the choice decision and preference by asking subjects to choose which of the two objects was more “organic” looking, but to rate each based on aesthetic preference. To confirm the results from Experiment 1a which showed stronger correlations between the choice process and eye parameters and not between aesthetic preference and eye parameters, it would be necessary to show in Experiment 2 that the correlation holds even for non-aesthetic based choice. Thus, in this experiment, the participants were asked to choose on the ‘organic’ dimension. We predicted that looking time would be longer for the chosen item, and that the chosen item would be fixated last. We did not expect parameters to vary with aesthetic ratings.Twenty new volunteers consented and participated  in the study.The materials and procedure were identical to Experiment 1 with the exception that participants were now asked to choose which of the two objects looked more “organic.” Procedures for acquiring baseline and paired aesthetic ratings were identical to Experiment 1a.2 = .4; paired: mean A = 1.88; rmse = .17, R2 = .56), suggesting a weak relationship between the item chosen as being more organic and their respective aesthetic ratings. The difference in A parameters was marginally significant, t(19) = 1.77, p = .092). Interestingly in contrast to Experiment 1, the paired ratings were less influenced by the same-trial choice now that the choice was made on a different stimulus dimension .As in Experiment 1a, we first modeled the proportion of left choices as a function of baseline and paired trial ratings using a two parameter logistic function . The slo2 = .58. There was no significant effect of rating, F(2.26) = 1.12, p = .34, η2 = .06, nor an interaction between the two, F = 1.16, p = .32, η2 = .06. This result was consistent with Experiment 1a and suggested that looking durations reflected comparative processes involved in choice decisions and not aesthetic preference.The total proportion of looking time on paired trials was entered into a 2 choice ×3 relative baseline rating  repeated-measures ANOVA . There wFurthermore, as in Experiment 1a, we analyzed the time series analysis data separately for trials in which the chosen item was congruent, incongruent, or the same as the one with the higher baseline aesthetic rating. Note that in this experiment, congruency between the organic choice and ratings were on different dimensions, but still referred to the convergence (or divergence) of choice with the baseline aesthetic rating. For example, the two dimensions would be congruent if the item chosen as being more organic was also given a higher baseline aesthetic rating.The data were analyzed separately for trials in which the choice and ratings were congruent , incongruent , or the same . The difference in proportional looking time for both the congruent and incongruent trials was statistically significant beginning at 2500 ms prior to the response, both t(19) >3.28, p<.05, with Bonferoni correction for five comparisons. The difference in proportional looking time for trials with the same baseline ratings was statistically significant starting at 3000 prior to response, t(19) = 3.31, p<.05, with Bonferoni correction for six comparisons. As with Experiment 1, these data suggest that the cumulative looking times reflected decision processes and not aesthetic ratings. The overall RT data from Experiment 1a  was not statistically different from Experiment 2 , t(38) = 1.53, p>.1.2 = .40. However, unlike Experiment 1a, this effect was not modulated by the aesthetic ratings of the chosen item, F = 1.15, p>.30, η2 = .06, .The last fixation data were also similar to Experiment 1a such that the proportion of final fixations on the chosen object  was greater than those on the unchosen object , F = 12.89, p<.005, ηOverall, the results are consistent with those of Experiment 1a. These findings provide further evidence that visual fixations were reliably related to arbitrary subjective decisional processes and not to the aesthetic preferences.Visual fixations have a long history of being used to assess internal mental representations, and among these are studies that have used looking times as proxy for preference In Experiment 1, we found that the object chosen as more aesthetically pleasing was not always given a higher aesthetic rating when viewed alone. This result was similar to previous findings in which choice decisions were influenced by factors beyond the personal value of objects To further test the relationship between fixation parameters and choice processes, dimensions of choice and preference were dissociated in Experiment 2: participants chose which of the two objects looked more “organic.” Organic choices are completely unrelated to aesthetic preferences, but similar to an aesthetic choice; an “organic” decision is arbitrary and devoid of any direct consequences . We found that, consistent with Experiment 1, participants spent more time fixating the object chosen as being more “organic.” There were no reliable effects of aesthetic ratings on fixation durations.Overall, the current data suggest that preferences play little role in controlling visual fixations when a choice decision is being made. However, it is possible that this may have been partly due to the fact that our simple geometric stimuli lacked emotional content In conclusion, our data demonstrated that cumulative fixation durations indexed the evolution of comparative processes that preceded a choice decision, even when the choice was entirely subjective. The results enhance understanding of how distinct parameters of visual fixations are related to preferences and choice processes


Synechococcus elongatus PCC 7942, generated by combining three high-resolution data sets: RNA sequencing, tiling expression microarrays, and RNA polymerase chromatin immunoprecipitation sequencing.Previous molecular and mechanistic studies have identified several principles of prokaryotic transcription, but less is known about the global transcriptional architecture of bacterial genomes. Here we perform a comprehensive study of a cyanobacterial transcriptome, that of We report absolute transcript levels, operon identification, and high-resolution mapping of 5' and 3' ends of transcripts. We identify several interesting features at promoters, within transcripts and in terminators relating to transcription initiation, elongation, and termination. Furthermore, we identify many putative non-coding transcripts.We provide a global analysis of a cyanobacterial transcriptome. Our results uncover insights that reinforce and extend the current views of bacterial transcription. This approach to analyzing the bacterial transcriptome may not only help reinforce the progress made from traditional molecular and mechanistic studies, but may also identify new global features in transcription that have previously been underappreciated.Over the past few decades considerable progress has been made in understanding the mechanisms and regulation of bacterial transcription. However, relatively few studies have attempted to identify the prevalent features of bacterial transcription Synechococcus elongatus PCC 7942 - integrating data from RNA sequencing, tiling expression microarrays, and RNA polymerase (RNA pol) ChIP sequencing.The advent of next-generation sequencing allows for a complete characterization of bacterial genomes that was previously not possible. RNA sequencing gives unprecedented insights into transcription unit architecture, while RNA polymerase chromatin immunoprecipitation (ChIP) sequencing reveals the flow of information into the transcriptome. We provide a comprehensive analysis of a cyanobacterial transcriptome - that of S. elongatus PCC 7942 is a genetically tractable model organism for prokaryotic photosynthesis [S. elongatus is built on a three-protein central oscillator that controls the global rhythmic expression of the majority of the genome [S. elongatus as a model organism.The unicellular cyanobacterium ynthesis , bioenere genome ,4. Our tS. elongatus. RNA was isolated at 4-hour intervals from circadian free-running cells grown in constant light conditions and RNA from a pool of circadian timepoints was sequenced . Strand-specific RNA sequencing was performed on the Illumina platform yielding over 22 million uniquely mappable non-rRNA reads and over 620 million nucleotides of coverage, strand-specifically covering each nucleotide of the approximately 2.7 Mb genome an average of approximately 115 times [We used RNA sequencing, tiling expression microarrays, and RNA pol ChIP sequencing to interrogate transcription in the cyanobacterium S. elongatus is rather dense with very small inter-transcript regions  predicted ORFs have negligible transcription (less than a mean of two reads per nucleotide across the ORF), and the remaining ORFs have absolute expression distributed over a dynamic range of nearly 10,000. In this study we only sample standard exponential growth conditions during circadian free-run in constant light conditions; both transcription density and the number of expressed ORFs are likely to be higher if multiple growth conditions are sampled.The RNA sequencing and RNA pol ChIP sequencing profiles demonstrate that the transcription landscape in s Figure . Assumina priori knowledge of JGI ORF, tRNA, and rRNA annotations . A total of 1,473 transcription units were identified, 1,415 of which were designated as mRNA transcripts as they are devoid of tRNA or rRNA and contain at least one JGI annotated ORF. 5' and 3' ends were determined for all transcripts and all subsequent analysis is performed on the subset defined as mRNA transcripts . The HIP1 palindrome is highly over-represented in many cyanobacteria, including S. elongatus - it appears 185 times more frequently in the S. elongatus chromosome than expected for a random 8-mer sequence, but its function is unknown [RNA sequencing affords high-resolution determination of the 5' and 3' ends of each transcription unit. Transcription units were defined using  unknown . It is kProchlorococcus MED 4 - allow for rapid transcriptome turnover. The distribution of mRNAs per cell appears approximately log-normal with a dynamic range of almost 10,000. Most mRNAs fall within a smaller dynamic range of approximately 100, with a tail of higher expressed transcripts. The bottom part of the distribution was cut at 2-4 because transcripts below this level are almost undetectable at our sequencing coverage . The highest expressed KEGG (Kyoto Encyclopedia of Genes and Genomes) [P-values of 2.6e-20, 1.3e-20, and 0.001, respectively (two-sided Wilcoxon rank sum test). The lowest expressed KEGG categories include mismatch repair, homologous recombination, and nucleotide excision repair - ORFs that may not be expressed in standard growth conditions . Absolute transcript levels are generally correlated  with RNA pol occupancy   categoriy Figure , suggestS. elongatus may be used to infer the operon structure in other cyanobacterial genomes. The median operon size is 1,320 nucleotides  have more than one ORF per transcript Figure . Most mRs Figure , approxiSynechocystis PCC 6803 and 25 experimentally determined promoters in Prochlorococcus MED4 [Prochlorococcus MED4 and in the psbA transcripts of many cyanobacteria [Identification of the 5' ends of all mRNAs allows for more detailed characterization of the promoter and initial steps in transcription. When we align all mRNAs by their 5' transcription start and average their AT content, we observe an increase at the -10 element, also known as the Pribnow box ,15 Figu. At thiscus MED4 -18  of thosbilosome   bit score 97.96) [S. elongatus.We find that most non-coding transcripts are at least partially anti-sense to an mRNA transcript Figure . These tPCC 6803 . This 17omponent . We finde 97.96) . This trTo identify if any other known RNA families are present within our set of non-coding RNAs, we queried the RFAM database . In addiS. elongatus, we queried our set of 5' UTRs against RFAM and identified metabolite-binding riboswitches for thiamine (vitamin B1) and coenzyme B12 (vitamin B12). The 5' leader of the thiC mRNA in S. elongatus contains a 'thi box' riboswitch domain that undergoes a structural change that has been shown to cause both a reduction in translation and transcription when bound to thiamine or its pyrophosphate derivative [Synpcc7942_1373) contains the cobalamin riboswitch domain, which represses expression in the presence of coenzyme B12 [S. elongatus of 30 nucleotides. Although most 5' UTRs are small, 12% are longer than 100 nucleotides and 6% are longer than 150 nucleotides. Transcripts with long 5' UTRs may be good candidates for riboswitches or RNA-based regulators. Interestingly, both riboswitch-containing mRNAs show large RNA pol occupancy peaks near the riboswitch domain in the 5' UTR, suggesting that these riboswitches - likely when in their bound configuration - can cause RNA pol pausing or termination. These peaks in RNA pol are qualitatively similar to the peaks we observe globally, although mechanisms likely differ, as most RNA pol peaks are situated within the beginning of the ORF.To extend our analysis of potential RNA-based regulators in rivative . Similarzyme B12 . Both ofS. elongatus transcriptome. We report absolute transcript levels, operon identification, and high-resolution mapping of 5' and 3' transcript ends. At the 5' end of transcripts, we characterize promoter sequence and find widespread peaks in RNA pol occupancy. At 3' ends we observe significant Rho-independent transcription termination and occasional incomplete termination resulting in interesting transcriptional structures. In addition, we find extensive non-coding transcription, suggesting a larger role for these non-coding RNAs in bacteria, and cyanobacteria in particular, than previously anticipated. The presence of numerous non-coding RNAs and 5' proximal pausing of RNA pol suggest that post-transcriptional regulation - regulation after binding of RNA pol at the promoter - may be more widespread in bacteria than expected. We hope this work will serve as a catalog and primer for further studies of bacterial and cyanobacterial transcription.Here we combine three high-resolution data sets - RNA sequencing, tiling expression microarray, and RNA pol ChIP sequencing - to present a characterization and analysis of the S. elongatus , and subsequently diluted with modified BG-11 lacking antibiotics. Cells were exposed to surface flux of approximately 25 μmol photons m-2 s-1 cool white florescent light, bubbled with 500 ml/minute 1% CO2 in air, maintained at 30°C, and stirred at one rotation per second. Constant optical density (OD750 0.15) and volume are achieved via a two state controller. OD does not fluctuate greater than 8% during an experiment. Cells are exposed to two 12-hour light-dark cycles for entrainment before release into continuous light.Cyanobacteria were cultured as previously described . A conti AMC 408 ): psbAI:n in NS1   to a final concentration of 2 units/μl and depleted of 23S and 16S ribosomal subunits using the MICROBExpress Bacterial mRNA Enrichment Kit  according to manufacturer's instructions.2, pH 9.2. Fragmentation reactions were immediately precipitated in 300 mM NaOAc, pH 5.2, glycogen, and isopropanol. Fragmented RNA was resuspended in RNA loading buffer , briefly denatured, and loaded in a 15% TBE-Urea polyacrylamide gel  for size selection. Gels were stained with Sybr Gold (Invitrogen) and a 25- to 30-nucleotide band was excised using a synthesized 28-nucleotide RNA and denatured 10-bp DNA ladder (Invitrogen) as standards. The gel slice was physically disrupted and RNA was recovered in 300 mM NaOAc, 1 mM EDTA, 0.1 units/μl SUPERase·In (Ambion) overnight at room temperature. Solution was transferred to a Spin-X cellulose acetate filter (Corning) to remove gel debris and precipitated with glycogen and isopropanol. Size selected fragmented RNA was denatured briefly and dephosphorylated in a 30 μl reaction with 1 × T4 polynucleotide kinase buffer without ATP , 20 units SUPERase·In, and 15 units T4 polynucleotide kinase (NEB) at 37°C for 1 hour. The reaction was precipitated, resuspended, briefly denatured, and poly-(A) tailed in a 25 μl reaction with 1 × poly-(A) polymerase buffer (NEB), 5 units SUPERase·In, 1 mM ATP, and 1.25 units E. coli poly-(A) polymerase (NEB) at 37°C for 10 minutes. Reactions were quenched with 80 μl of 5 mM EDTA and precipitated.RNA sequencing libraries were prepared from total RNA depleted of 16S and 23S rRNA with modifications to a previously described procedure . RNA  for 1 hour at 60°C, and then heat-inactivated for 10 minutes at 80°C.Reverse transcription was carried out from the introduced poly-(A) tail anchor of denatured RNA using primer oNTI255 [S. elongatus genome with Bowtie [CP000100, CP000101, and S89470 were used to align reads to the chromosome and endogenous plasmids. Uniquely mappable reads with a maximum of three mismatches were mapped to the genome and extended by the length of the individual read.Circularized cDNA template (1 μl) was amplified using Phusion Hot Start High-Fidelity enzyme (NEB) and primers oNTI230 and oNTI231  to creath Bowtie . StrippiA total of 22,375,035 uniquely mappable reads were mapped to the genome with approximately 624 million bases of sequences covering each nucleotide strand-specifically an average of approximately 115 times. These uniquely mappable reads exclude any reads from rRNA since multiple copies of each rRNA exist in the genome. Technical replicates showed very high Pearson correlation coefficients (r > 0.99). RNA sequencing data are displayed and analyzed as coverage per nucleotide - defined as the number of times a given nucleotide position was observed in all the sequencing reads. Absolute transcript levels are assumed to be equal to the average coverage per nucleotide across the length of the transcript. All analysis was performed on the chromosome, although raw data for both endogenous plasmids are available.CP000100, CP000101, and S89470 - which define the chromosome and two plasmid sequences, respectively.Expression was measured using two separate custom designed two-color 244 k microarrays - one for the forward strand and another for the reverse strand . Arrays were designed using eArray software (Agilent). Forward and reverse strand sequence is as defined by GenBank i and probei+1 overlapped by 48 nucleotides. A 6-nucleotide offset of the tile between strands allows for 6-nucleotide resolution of double stranded targets and 12-nucleotide resolution for strand-specific targets. In addition, each array included four temperature matched probes (80°C) against each JGI predicted ORF, luxA through luxE, and Arabidopsis spike-in controls (Ambion). These additional probes are identical to those in Agilent Array ID 020846, as previously described [All tiling probes were 60 nucleotides in length with 12-nucleotide spacing between probe starts such that probeescribed .cDNA was prepared for each individual timepoint (foreground channel) as well as for a pool of all timepoints (background channel). The background channel consisted of a pool of samples collected at 4-hour intervals from 24 to 84 hours after release into continuous light. The foreground channel consisted of individual timepoints 60, 68, 72, and 80 hours after release into continuous light. The same samples were analyzed by non-tiling microarray in . Spike-iN-hydroxysuccinimide-ester cyanine 3  or cyanine 5   in 0.1 M sodium bicarbonate pH 9.0 for 6 hours. Labeled cDNA was purified (Microcon 30) in preparation for hybridization. Each array was hybridized with approximately 750 ng Cy3 and approximately 750 ng Cy5 labeled cDNA and rotated (five rotations per minute) at 60°C for 17 hours in SureHyb chambers (Agilent). Arrays were subsequently washed in 6.7 × SSPE and 0.005% N-lauryl sarcosine buffer for at least 1 minute, 0.67 × SSPE and 0.005% N-lauryl sarcosine buffer for 1 minute, and then Agilent drying and ozone protection wash for 30 seconds at room temperature . The arrays were immediately scanned using an Axon 4000B scanner at 5-μm resolution. The median intensity of the Cy3 and Cy5 florescence at each spot was extracted using GenePix software . For calculation of logarithmic ratios, Loess and quantile normalization were performed in succession using the MATLAB  bioinformatics toolbox.First-strand cDNA was labeled with 750 0.15) with 1% formaldehyde for 15 minutes and then quenched them with 125 mM glycine for 5 minutes at room temperature. Cells were collected by centrifugation and washed twice with cold phosphate-buffered saline buffer, pH 7.4. The cell pellet was snap frozen in liquid nitrogen and stored at -80°C. Samples were collected 32 to 52 hours after release into continuous light at 4-hour intervals. At the same time, samples were collected and processed for non-tiling microarray as described in [We crosslinked 250 ml of cells from continuous culture  and incubated for an additional 2 hours at 4°C. After incubation, sepharose beads were washed in cold buffer at room temperature: 2 × 5 minutes lysis buffer A; 1 × 5 minutes lysis buffer B ; 1 × 5 minutes wash buffer ; 1 × 5 minutes TE buffer . Protein-DNA was eluted from beads by incubation of samples at 65°C for 1 hour in elution buffer . Crosslinks were reversed in supernatant by incubation of samples at 65°C overnight in elution buffer. Western blotting of supernatant of mock versus immunoprecipitation shows 45% efficiency pull-down of the β subunit and 25% co- immunoprecipitation of the β' subunit in the immunoprecipitation using Neoclone antibodies WP023 and WP001, respectively. Proteins were digested with 0.2 mg/ml proteinase K for 2 hours at 37°C. Nucleic acid was then purified with phenol/chloroform extraction and precipitated with ethanol and LiCl. Nucleic acid was re-suspended in TE buffer and RNA was digested in 20 μg/ml RNase and subsequently phenol/chloroform purified. For input control, 5% of the volume of cell lysate was removed after sonication and used to prepare the input DNA. The ChIP DNA concentration was estimated with the Pico-green DNA detection kit (Invitrogen).ChIP was performed in a manner similar to that previously described ,51. CellkaiC and purF by microarray. Mock ChIP sequencing libraries were prepared for an equal mix of lysate from ZT 32 through ZT 52 . A total of six sequencing libraries were prepared (Table ChIP sequencing libraries were prepared for samples zeitgeber time (ZT) 32 (subjective dusk) and ZT 44 (subjective dawn) as these timepoints showed maximal/minimal gene expression for canonical circadian mRNAs ed Table .S. elongatus genome with Bowtie [CP000100, CP000101, and S89470 were used to align reads to the chromosome and endogenous plasmids. Uniquely mappable reads with a maximum of three mismatches were mapped to the genome. Reads were then extended 150 bp to cover the average length of insert DNA between sequencing adaptors as determined by the Agilent Bioanalyzer.Sequencing libraries were prepared from 10 ng DNA following the Illumina ChIP protocol (revision A) and libraries sized between 200 and 300 bp were selected for amplification. Libraries were assayed with the Agilent Bioanalyzer and 8 pM of template was used for cluster generation. Libraries were sequenced using Illumina primers on an Illumina Genome analyzer II, and each sequence tag was aligned to the h Bowtie . GenBankA comparison of change in RNA pol ChIP versus change in gene expression (measured by non-tiling microarray) at timepoints ZT 32 and ZT 44 is shown in Figure S3 in Additional file S. elongatus chromosome was calculated by imposing a coverage cutoff for transcription of two reads per nucleotide. If a nucleotide is expressed at or over this cutoff it is regarded as transcribed.The percent of transcription along the This conservative cutoff indicates that only approximately 84.7% of the nucleotides within annotated JGI chromosomal ORFs are transcribed. Of the approximately 15.3% of nucleotides within annotated ORFs that do not pass this cutoff, approximately 41.4% are within an ORF that has an average number of reads per nucleotide of less than 2, which corresponds to approximately 1 RNA per 15 cells when we assume a total of 1,500 mRNAs per cell .Using this cutoff we find 54.7% of each strand is transcribed and 88.0% of the chromosome is transcribed on either the plus or minus strand. That is, on any given strand and at any given chromosomal position, there is a 54.7% chance that the nucleotide is transcribed. Similarly, at any given chromosomal position, there is an 88% chance that the nucleotide is transcribed on either the plus or minus strand. Eighty-two percent of non-coding regions are transcribed on either the plus or minus strand.a priori knowledge of translation start and stop positions. Of 2,665 chromosomal ORFs (and rRNA and tRNA), 2,420 had an average number of reads per nucleotide of ≥2. For every predicted translation start, we searched for the first upstream nucleotide (i - 1 is upstream of i) on the same strand i that was not within a JGI predicted ORF and that satisfied one of the following three criteria: (1) binomialcdf  < 0.01 and readsi/readsi-1 ≥ 2; (2) binomialcdf  < 0.01 and readsi/readsi-2 ≥ 2; and (3) readsi-1 <2.The 5' and 3' ends of all JGI predicted ORFs (and rRNA and tRNA) with an average coverage of at least two reads per nucleotide were identified using a probability-based approach using cdf  is the probability of getting at least k success in n trials when p is the success probability of each trial. This i was designated the 5' transcription start site. The distance of predicted 5' ends to those published in previous studies is reported in Table S4 in Additional file i that was not within a JGI predicted ORF and that satisfied one of the same criteria. This i was designated the 3' transcription end. 5' Ends tend to be better defined than 3' ends, possibly related to the biology of transcription termination. ORFs that shared the same 5' transcription start site were defined as being on the same operon. We observed 43 cases of multiple transcription start sites - the presence of a 5' transcription start within another transcript. All identified transcripts are reported in Table S1 in Additional file Where binomialNon-coding transcripts were identified using a multi-tiered approach that first identifies transcribed regions and then estimates their 3' and 5' positions.First, 15,000 nucleotide intervals of the chromosome  were optimally segmented into 30 segments of approximately constant signal, yielding a total of 8,070 segments per strand. Segmentation was performed in MATLAB to minimize the cost function:yi is the log2(1 + readsi) at nucleotide i, 2(1 + reads) along segment s, t1, ..., ts and are segment boundaries [where undaries -54. ThisNext, all segments that correspond to non-transcribed regions - mean coverage less than two reads per nucleotide - were removed. Segments that overlapped with an annotated transcript (see previous section) were removed and the remaining segments were consolidated. The exact 5' and 3' end of each segment was determined using the same algorithm described in the previous section except 5' and 3' ends were not allowed to overlap with an annotated operon. A total of 1,579 non-coding transcripts were detected using this method. All non-coding transcripts are reported in Table S2 in Additional file Tiling microarray ratios were utilized to identify a set of high-confidence non-coding transcripts. We took advantage of the fact that transcripts have high Pearson cross-correlation among internal probes  across all circadian timepoints . That isCircadian transcripts corresponding to annotated JGI ORFs have been previously described ,4. To idP < 0.1). Those peaks that are not enriched over mock appear to be actual peaks in RNA pol ChIP, but these RNA pol ChIP peaks are smaller than the mock background, which is elevated with respect to the ChIP background after both data sets are normalized for the number of reads . Z-scores were calculated as the difference in mean of native and dinucleotide shuffled sequences divided by the standard deviation of dinucleotide shuffled sequences and P-value was calculated using the two-sided Wilcoxon rank sum test. This suggests that a particular stem-loop feature, likely associated with transcription termination, is present at the end of transcripts.At the 3' end of transcripts, a dip in minimum free energy was not observed in the dinucleotide shuffled sequences, but was observed in native sequences ; column C, first ORF on transcript; column D, last ORF on transcript; column E, predicted 5' transcription start site; column F, predicted 3' end; column G, length of transcript; column H, is the transcript an mRNA? ; column I, the number of ORFs per transcript; column J, length of 5' UTR; column K, length of 3' UTR; column L, mean of the raw RNA sequencing reads over the full transcript; column M, number of transcripts per cell assuming a total of 1,500 mRNAs per cell. Table S2 - all non-coding transcripts: column A, non-coding transcript ID number; column B, predicted 5' transcription start site; column C, predicted 3' end; column D, strand ; column E, mean of the raw RNA sequencing reads over the full non-coding transcript; column F, length of non-coding transcript; column G, percent overlap that a non-coding transcript has with an ORF that was designated as not transcribed (designated when mean RNA sequencing coverage of ORF is less than two reads per nucleotide); column H, percentage of non-coding transcript that is antisense to an annotated transcript; column I, does the non-coding transcript pass the high confidence criteria? ; column J, does the non-coding transcript pass the circadian criteria? ; column K, the difference in gene expression of the non-coding transcript in the dusk versus dawn circadian timepoints calculated by tiling microarray ; column L, RFAM homology. Table S3 - all RNA polymerase peaks: column A, peak ID number; column B, start of peak; column C, end of peak; column D, position of peak maximum; column E, total ChIP reads at peak maximum ; column F, P-value for enrichment of reads in ChIP sample versus mock immmunoprecipitation. Table S4 - comparison of literature 5' versus RNA sequencing 5': column A, JGI ID for ORF; column B, common name for ORF; column C, strand ; column D, translation start position of ORF; column E, literature-based 5' transcription start site; column F, alternative 5' transcription start site from literature; column G, 5' transcription start site estimate from our RNA sequencing; column H, difference between our 5' transcription start site estimate and the closest literature estimate; column I, method of 5' transcription start site determination used in the literature reference; column J, literature reference. Table S5 - expression of all JGI predicted ORFs: column A, JGI ID for ORF; column B, Synpcc7942 ORF ID; column C, start of ORF ; column D, end of ORF ; column E, strand ; column F, mean of the raw RNA sequencing reads over the full ORF.Click here for fileSupplementary Figures S1 to S8. Figure S1: examples of 5' determination from RNA sequencing. (a) 5' Determination of the ntcA transcript. A sharp drop in RNA sequencing reads is observed at the 5' end of the mRNA. 5' end determination by RNA sequencing and traditional methods [(b) 5' determination of the purF transcript. The RNA sequencing estimate is over 80 nucleotides different from that derived by traditional methods [purF transcript contains the RNA sequencing 5' end but not the literature 5' end. A more complete comparison of RNA sequencing and traditional transcription start determination is provided in Table S4 in Additional file (a) RNA sequencing data. Positive strand transcription is shown in blue (positive y-axis), and negative strand transcription in red (negative y-axis). ORFs on the positive and negative strands are indicated by horizontal black lines. RNA pol peaks significantly enriched over the mock immunoprecipitation (P < 0.1) are indicated with vertical green lines and those that are not (P ≥ 0.1) are indicated with vertical pink lines. Large RNA pol peaks tend to be located near the 5' end of transcripts, although there are many peaks in the middle of transcripts potentially caused by RNA pol pausing. (b) RNA pol ChIP and mock. RNA pol ChIP (black) and mock immunoprecipitation (green) are normalized such that the genome average is 200 reads per nucleotide. Almost all RNA pol peaks are enriched over the mock immunoprecipitation. A complete listing of RNA pol peaks and their enrichment is provided in Table S3 in Additional file (c) RNA pol ChIP normalized by input. Normalization of RNA pol ChIP by input does not qualitatively change the data  Changes in RNA pol occupancy at two separate times during the circadian cycle (dusk and dawn). Changes in RNA pol are reflective of changes in transcript level by microarray . The probability of getting a correlation as large by random chance  is 2.2286e-197. Figure S4: characteristics of transcription start. (a) Melting temperature at transcription start. The melting temperature of 10-nucleotide fragments from -200 to +200 of all mRNAs was averaged . A drop in the melting temperature is observed at the promoter. (b) Nucleotide content at transcription start sites. Nucleotide content of all mRNAs aligned by transcription start. (c) Zoomed in nucleotide content at transcription start. Nucleotide content of all mRNAs aligned by transcription start. Preference for adenine at the +1 position and a -10 element can be observed. Figure S5: comparison of minimum free energy changes with that of dinucleotide-shuffled sequences. (a) Minimum free energy change at RNA pol peaks. The minimum free energy of 60-nucleotide RNA fragments with 10-nucleotide spacing was calculated and averaged for all mRNAs . A drop in minimum free energy slightly prior to the position of the RNA pol peak is observed. To prevent sequence features of the transcription terminus or promoters from interfering with this analysis, a subset of 183 RNA pol peaks satisfying the following criteria were used: (1) RNA pol peak must be closer to a 5' end than a 3' end; and (2) RNA pol peak must be +100 to +300 relative to the 5' end. Since RNA pol ChIP does not specify the strand being transcribed, the strand of transcription was inferred from RNA sequencing data. Dinucleotide shuffled sequences show a qualitatively similar trend to native sequences, suggesting that there is no specific secondary structure at this transition . (b) Sequence changes near RNA pol peaks. A sequence content change from low to high GC content can be observed near the position of the RNA pol peaks. The same subset of RNA pol peaks are used here as in Figure S5a in Additional file (c) Minimum free energy change at transcription terminus. Minimum free energy was calculated as above after aligning all transcripts by transcription terminus. Dinucleotide-shuffled sequences do not resemble native sequences, suggesting that a discrete hairpin-like structure exists at the terminus of transcripts . (d) Minimum free energy change at transcription start. Minimum free energy was calculated as above after aligning all transcripts by 5' transcription start. A drop in minimum free energy occurs globally within transcripts and may be related to our observation of global RNA pol pausing. Dinucleotide-shuffled sequences show a qualitatively similar trend to native sequences . Figure S6: enrichment in RNA sequencing at 5'. (a) Increased RNA sequencing signal at 5' ends. An increase in RNA sequencing signal can be observed at the 5' end of mRNAs. Several biological phenomena may account for this enrichment, but one intriguing possibility is the existence of many partial or nascent transcripts caused by pausing of RNA pol near the 5' end of the transcript. (b) RNA pol pausing at 5' ends may contribute to RNA sequencing enrichment at 5' ends. A slight but significant correlation exists between the retention ratio of RNA pol and the enrichment of RNA sequencing prior to the RNA pol peak. The same subset of RNA pol peaks was used as in Figure S5a in Additional file P-value) is 6.2879e-11. Figure S7: the phycocyanin operon - a functional case of partial transcription termination. (a) Partial transcription termination controls the stochiometry of cpcβ and cpcα to rod linker mRNA at approximately 6:1. This stochiometry reflects the organization of the phycobilisome - a hexameric α-β double disc with an associated linker [cpcβ and cpcα coding region because it is not unique in the genome . The position of predicted terminators (from TransTermHP) is indicated in green, and the position of JGI predicted ORFs is indicated in black. Figure S8: circadian gene expression of putative non-coding RNAs. (a) Gene expression by tiling microarray of high-confidence circadian non-coding RNAs. Gene expression of non-coding RNAs with potential for circadian gene expression are plotted by non-coding transcript ID (Table S2 in Additional file  methods  differ o methods . Subsequ methods  have shod linker . RNA seqClick here for file


During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer–TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer–dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are 21–24 nucleotide (nt) non-coding sequences that regulate gene expression by targeting mRNAs. During miRNA biogenesis, primary RNA transcripts in the nucleus are cleaved by the Microprocessor complex to produce pre-miRNAs that are exported to the cytoplasm. The endoribonuclease Dicer then catalyzes further cleavage of these pre-miRNAs to produce mature miRNAs, which regulate the translation or degradation of specific mRNAs. Dicer can also generate short interfering RNAs (siRNAs) by cleaving long double-stranded RNA (dsRNA) precursors , which is required for efficient pre-miRNA cleavage during miRNA biogenesis. Dicer-2, the other Dicer isoform, binds to Loqs-PD and R2D2, which contribute to efficient pre-siRNA processing and siRNA loading onto Ago2 during siRNA biogenesis, respectively  family of endonucleases and with specific double-stranded RNA-binding proteins (dsRBPs) . In Drosectively . In orgato DCR-1 ,7. In huto DCR-1  and tranto DCR-1 ,4.in vitro reconstituted system to investigate the roles of dsRBPs in different dsRNA substrate processing reactions. The results of these experiments show that despite their similar size and three-domain architecture, TRBP and PACT affect Dicer-catalyzed substrate processing differently. TRBP and PACT have distinct activities both in determining cleavage sites to produce different sized miRNAs (isomiRs) and in distinguishing between pre-miRNA and pre-siRNA substrates for Dicer. In particular, PACT containing Dicer complexes disfavor pre-siRNA substrates compared with pre-miRNA substrates much stronger than TRBP containing Dicer complexes. Using multiple hybrid forms of TRBP-PACT proteins swapping and deleting domains and/or linker region, we found that the two N-terminal dsRNA-binding domains of TRBP and PACT confer these differential behaviors of Dicer–dsRBP complexes. These results suggest a model in which TRBP and PACT function as key regulators of miRNA and siRNA biogenesis by Dicer in substrate recognition, binding, processing and RISC loading.In association with Dicer, TRBP contributes to RNA substrate binding, product length determination and the assembly of RNA-induced silencing complexes (RISCs) . However6-MBP instead of a Ni-nitrilotriacetic acid column. The bound PACT protein was eluted from the HiTrap-SP column at 350–400 mM KCl during a linear gradient run from 150 mM KCl to 1 M KCl and subsequently loaded onto a Superdex-200 gel filtration column. Chimeric proteins P12T3 and TRBP-PL were expressed and purified using the same methods as the TRBP purification, and PACT-TL was expressed and purified using same methods as the PACT purification. For the T12P3 purification, a 5 ml HiTrap-Q column was used before the HiTrap-SP column, and the unbound flow-through fraction was loaded to 5 ml HiTrap-SP column for the next steps, which were same as the PACT purification method. Chimeric proteins were designed as follows: T12P3 has the combined sequence of TRBP residues 1–232 and PACT residues 197–313. P12T3 has the combined sequence of PACT residues 1–196 and TRBP residues 221–366. TRBP-PL has the combined sequence of TRBP residues 1–96 and 159–366, connected by PACT linker residues 97–125. PACT-TL has the combined sequence of PACT residues 1–96 and 126–313, connected by TRBP linker residues 93–158. The sequences of the chimeric proteins are available in the Supporting Information.Dicer, TRBP, Dicer–TRBP and Dicer–TRBP–Ago2 complex were prepared as reported before . PACT, Din vitro transcription using T7 RNA polymerase, and the transcribed RNAs contained one ribozyme at each end for homogeneous RNA production. After in vitro transcription, RNAs were gel purified and end-labeled for use in the processing assay. For 5′ end labelling, RNA was incubated with T4 polynucleotide kinase (New England Biolabs Inc.) and [γ-32P] ATP for 1 h at 37°C. For perfect dsRNA substrates, RNA oligos were ordered from IDT with a 5′ phosphate, and gel purified. One strand of each complementary RNA oligo pair was Calf Intestinal Alkaline Phosphatase (CIP)-treated before 5′ end labelling with [γ-32P] ATP. Equal amounts of each complementary RNA oligo were incubated at 65°C for 10 min and slow cooled to room temperature for annealing. The sequences of RNA substrates used in this study are as follows: pre-let-7a, 5′-UGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUACC-3′; pre-miR-200a, 5′-AUCUUACCGGACAGUGCUGGAUUUCCCAGCUUGACUCUAACACUGUCUGGUAACGAUGU-3′; pre-miR-34c, 5′-AGGCAGTGTAGTTAGCTGATTGCTAATAGTACCAATCACTAACCACACGGCCAGG-3′; dsRNA W1, 3′-UUACCGGAACCCGUUGCCAUUAAGCCCUAAUGGCUCUGGC-5′-Biotin, 5′-UGGCCUUGGGCAACGGUAAUUCGGGAUUACCGAGACCG-3′; dsRNA W2, 3′-UUAAUGGCAACGGGUUCCGGUAAGCCCUAAUGGCUCUGGC-5′-Biotin, 5′-UUACCGUUGCCCAAGGCCAUUCGGGAUUACCGAGACCG-3′.Pre-miRNA substrates were prepared by 2, 25 mM NaCl, 1 mM dithiothreitol and 1% glycerol]. Each time point sample was prepared by being quenched with 1.2 volumes of loading buffer . Samples were heated at 70°C for 10 min before loading on a 12–15% denaturing 7 M urea polyacrylamide gel electrophoresis. The gels were dried and the amount of pre-miRNA and pre-siRNA substrates and miRNA and siRNA products were quantified with a phosphorimager .Labeled RNA substrates were processed with the indicated amount of Dicer or Dicer–dsRBP complexes in dicing buffer  (in part). Funding for open access charge: HHMI.J.A.D. is a Conflict of interest statement. None declared.


Advances in sequencing technologies have opened a new era of high throughput investigations. Although RNA-seq has been demonstrated in many organisms, no study has provided a comprehensive investigation of the bovine transcriptome using RNA-seq.in vitro were used as models to study early embryonic development in cattle. RNA amplified from limited amounts of starting total RNA were sequenced and mapped to the reference genome to obtain digital gene expression at single base resolution. In particular, gene expression estimates from more than 1.6 million unannotated bases in 1785 novel transcribed units were obtained. We compared the transcriptomes of embryos showing distinct developmental statuses and found genes that showed differential overall expression as well as alternative splicing.In this study, we provide a deep survey of the bovine embryonic transcriptomes, the first application of RNA-seq in cattle. Embryos cultured Our study demonstrates the power of RNA-seq and provides further understanding of bovine preimplantation embryonic development at a fine scale. Arabidopsis [de novo to recover structural models of annotated and unannotated genes and/or infer expression. Due to its attractive features and continually decreasing cost, RNA-seq is quickly replacing traditional technologies such as microarrays for high throughput transcriptomic studies.The recent developments in high throughput sequencing technologies allow for surveys of transcriptomes at unprecedented completeness and resolution. The 'next generation' RNA sequencing technology (RNA-seq) has several advantages, such as the ability to detect unannotated transcriptional activity, to differentiate between different transcriptional or splicing isoforms, and to provide digital measurements at single base resolution. Recently, the efficacy of RNA-seq has been demonstrated in humans  and modebidopsis ,4, and mbidopsis . An RNA-in vitro. Because early embryonic loss accounts for the majority of cow infertility [in vitro fertilization (IVF) system to recapitulate the early development of cattle and allow systematic investigations of abnormities during the very early stages of development [The recent sequencing and analysis of the bovine genome have provided opportunities for further post-genomic investigations of this important livestock species . Despiteertility , we haveelopment . The traelopment .Expression microarrays, however, rely on existing genome annotations and lack the ability to detect more complex regulations in gene expression such as changes in alternative splicing. Therefore, in this study, we seek to take advantage of RNA-seq to identify transcriptomic changes associated with abnormal bovine early embryonic development, especially for transcriptional activities that have not been annotated before and differential alternative splicing that could not be detected by expression microarrays. Although the importance of alternative splicing in regulating complex traits has been long recognized and extensively studied, there is limited information on the pattern of alternative splicing in cattle or its roles in bovine embryonic development.Herein, using RNA-seq, we estimated and compared digital gene expression in IVF embryos. In addition, unannotated transcribed regions in the bovine genome and novel splice junctions were discovered. Finally, we show that alternative splicing is widespread in the bovine embryonic transcriptome, and we have identified alternative splicing events associated with abnormal bovine embryonic development. This study is the first application of RNA-seq in cattle and provides fine-scale insights into bovine preimplantation embryonic development.To overcome the scarcity of RNA present in individual embryos, we linearly amplified RNA  from twoSequencing reads were mapped to the reference genome (Btau4) using the software package Tophat . ApproxiAmong the uniquely mapped fragments, 14,121,649 and 10,748,218 were mapped to annotated genes (exons and introns) in blastocysts and degeneratives, respectively, including 2,174,923 and 1,268,315 fragments mapped to annotated exon-exon junctions  terms. Genes that showed a nominal significance of p = 0.05 and had GO annotations (n = 330) were selected and tested against the background set of all genes with GO annotations (n = 8139). We found several GO terms significantly enriched (FDR = 0.01) for differentially expressed genes, among which were GO processes related to developmental processes Table .One important feature of RNA-seq is its independence from gene annotations, which allows investigating unannotated transcriptional activities. Uniquely mapped alignments were assembled using the software package Scripture , and thehttp://genome.ucsc.edu/) and compared to novel TUs. About two thirds of the novel TUs in both blastocysts and degeneratives were supported by EST alignments. In addition, phastCons scores [We then looked for evidence to support the novel TUs found by our RNA-seq experiment from external sources. EST alignments were downloaded from UCSC genome browser  gene showed a significantly higher inclusion ratio  in degeneratives than in blastocysts  was upregulated by about eight fold in degenerative embryos significantly , it became apparent that deeper sequencing was required to detect subtler changes in alternative splicing with sufficient statistical power.In summary, we demonstrated RNA-seq in cattle, providing for the first time a high resolution map of transcriptional and alternative splicing activities in IVF embryos. The results provided further systematic understanding of mammalian embryonic development at a fine scale.IVF embryos were collected as previously described . BrieflyThe alignment of sequencing reads to the bovine reference genome (repeat masked assembly Btau4 downloaded from UCSC genome browser) comprised three stages. In the first stage, sequencing reads from blastocysts and degenerative embryos were trimmed to 60 bp and mapped independently using the software package Tophat v1.0.13 . Tophat For digital gene expression measurements, we first obtained constitutive exons that are present in all isoforms of each of the genes, and then filtered out exons that overlapped between genes. These non-overlapping constitutive exon sets were then used to calculate overall gene expression with assistance from SAMtools  and BEDTThe alignments were assembled using Scripture, which assembles transcripts at disjoined genomic locations by finding paths through a connectivity graph representation of the alignments . After cTo identify novel transcriptional activities that have not been annotated, we required that for a locus that was farther than 1000 bp away from known genes, at least one assembled transcript was at least 500 bp long and covered by at least 40 sequencing fragments. Our sequencing fragments had a mean insert size of 122 bp, this threshold for expression was about five fragments per base. Transcripts that were overlapped by repeats for more than 50% of the sequences were discarded. The Cuffcompare utility in the Cufflinks package was used for comparing transcripts to known annotations . RegionsWe estimated and classified alternative splicing events from reads that mapped to exon-exon junctions . We consWH and HK designed the study and wrote the manuscript. WH performed experiments and analyzed the data. Both authors read and approved the final manuscript.List of differentially expressed genes and novel TUs identified by DESeq.Click here for fileRNA-seq is more sensitive than microarrays. Histogram of RNA-seq gene expression was plotted according to MAS5 Present/Marginal/Absent calls for blastocysts (a) and degeneratives (b).Click here for fileTest for DNA contamination in aRNA samples.Click here for fileIntronic read fraction as a function of pre-mRNA fraction. In this hypothetical example, exons were assumed to constitute 1/20 of the bases of genes. As pre-mRNA fraction increases, intronic read fraction also increases at a faster rate.Click here for fileBioanalyzer quality check of RNA extracted from pools of embryos. Approximately 5 ng of total RNA was analyzed on a RNA Pico6000 chip.Click here for file