Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.Members of the  Secoviridae, Potyviridae, Luteoviridae families and Sobemovirus genus possess VPg proteins of 2–24 kDa in size, covalently linked to the 5′-terminal nucleotide of the viral RNA via tyrosine, serine or threonine residues within VPg  at 37 °C for 1 h prior to the addition of SDS-PAGE sample buffer and separation by 15% SDS-PAGE.The F9 strain of FCV and the CW1 isolate of MNV were grown in Crandell-Reese feline kidney cells (obtained from ATCC) and RAW264.7 cells (obtained from ATCC) respectively. For each preparation, at least five 170 cmE. coli as previously described (Untagged derivatives of FCV and MNV VPg proteins were expressed and purified in escribed .http://spectrumviewer.org) and images were prepared using Inkscape (http://www.inkscape.org). The levels of viral RNA present in the samples used for mass spec were not routinely quantified, however yields from identical preparations were typically in the range of ∼107 genome equivalents per μg of total RNA.VPg, covalently bound to the RNA, was trypsin digested and the RNA was subsequently hydrolyzed in 10% trifluoroacetic acid for 48 h at room temperature. 10 μg of total RNA, measured by an absorbance-based quantitation (NanoDrop), was used per analysis. The samples were then dried under vacuum, purified with StageTips  and analWe initially compared the VPg sequences from a number of representative caliciviruses to identify amino acids that were highly conserved across the genera . CalicivAlignment of the 5′ end sequences of representative calicivirus genomes has demonstrated that almost all genomic and subgenomic RNAs start with a GU dinucleotide  and 1C. E. coli without any exogenous non-viral amino acids were used to confirm the expected mass of the VPg proteins. FCV VPg was readily visible following Coomassie staining of RNase digested RNA; however MNV VPg, because of its larger mass, was obscured by the RNase present within the sample. Western blot analysis, and subsequent mass spectrometry (see below) was used to confirm the isolation of the MNV VPg protein.In order to identify the nucleotide and the amino acid involved in the covalent linkage of calicivirus RNA to VPg, a source of viral VPg-linked RNA that would yield sufficient nucleotide-linked VPg was required. Attempts were initially made to purify sufficient quantities of VPg-linked viral RNA from infectious virions, however the yields were insufficient to allow the robust detection of VPg peptides by mass spectrometry. As an alternative approach, we isolated total RNA from infected cells as we have previously demonstrated that RNA isolated in this way is covalently linked to the mature form of VPg only, that the RNA is infectious when transfected into permissive cells, that the VPg-linked RNA is translationally competent and that the linkage to VPg is essential for the infectivity of the viral RNA . Taken tInitial attempts were made to analyse the RNase treated and trypsin-digested VPg-linked RNA preparations as a method to identify the nucleotide and amino acids involved in the covalent linkage; however, this approach failed to produce spectra that allowed for the detection of the nucleotide-linked VPg peptides. As an alternative approach, we used tryptic digestion of VPg-linked RNA preparations followed by acid hydrolysis of the RNA-linked peptides as described previously . The RNAUsing this approach we determined that the FCV VPg is linked to RNA through the tyrosine residue at position 24 (Y24) and the corresponding modification was pGp, as assigned by the modification delta mass and the corresponding fragmentation spectrum . This isThe significance of VPg phosphorylation in the FCV life cycle clearly warrants further study using reverse genetics and mutagenesis. VPg is a multifunctional protein; it functions in viral RNA synthesis via an interaction with NS7, in viral protein synthesis via an interaction with translation initiation factors, and possibly in viral encapsidation via an interaction with VP1 . TherefoThe MNV VPg protein was identified as also linked to RNA through a tyrosine residue at position 26 and the modification was also pGp . SurprisPrevious studies on caliciviruses VPg nucleotidylylation have examined the ability of the viral RdRp to uridylylate VPg by leading to the formation of a VPg-pUpU(OH) moiety . As all In picornaviruses, the VPg protein is removed immediately upon viral RNA release into the cytoplasm by the host cell enzyme TDP2 , but theIn conclusion, this work has provided experimental confirmation that at least for FCV and MNV, the covalent linkage of the VPg proteins to the 5′ end of the viral genome occurs specifically via a highly conserved tyrosine residue to the 5′ G nucleotide. The identification of potential phosphorylated sites in the FCV VPg protein may provide a mechanism by which the function of VPg is temporally regulated during the viral life cycle.


Small RNAs, between 18nt and 30nt in length, are a diverse class of non-coding RNAs that mediate a range of cellular processes, from gene regulation to pathogen defense. They guide ribonucleoprotein complexes to their target nucleic acids by Watson–Crick base pairing. We report here that current techniques for small RNA detection and library generation are biased by formation of RNA duplexes. To address this problem, we established FDF-PAGE  to prevent annealing of sRNAs to their complement. By applying FDF-PAGE, we provide evidence that both strands of viral small RNA are present in near equimolar ratios, indicating that the predominant precursor is a long double-stranded RNA. Comparing non-denaturing conditions to FDF-PAGE uncovered extensive sequestration of miRNAs in model organisms and allowed us to identify candidate small RNAs under the control of competing endogenous RNAs (ceRNAs). By revealing the full repertoire of small RNAs, we can begin to create a better understanding of small RNA mediated interactions. Genome regulation by small RNAs (sRNA) is a feature of all eukaryotes, including plants, animals and fungi. There are three main types of small RNAs: (i) microRNAs (miRNAs), which target genes and have important roles in development and in stress-responses –3, (ii) sRNAs exert control over the genome at the transcriptional, post-transcriptional and translational levels, yet the mechanisms regulating the sRNAs themselves remain poorly understood. Recently, a regulatory mechanism was discovered  in whichTo investigate the diverse cellular functions of sRNAs, it is essential to gain an accurate profile of sRNA abundance in the cell. However, researchers studying sRNAs typically begin by extracting total ribonucleic acid from a tissue sample, resulting in a sample that contains both the sRNAs of interest and their complementary transcripts. It is possible that hybridization to these complementary RNAs could interfere with small RNA detection. Indeed, addition of synthetic RNA as a miRNA inhibitor can affect the detection of complementary miRNAs  and a moin vitro. We show here that complementary RNA reduced the small RNA signal by northern blotting in as little as 10-fold molar excess and reduced sRNA representation in sequencing libraries. To overcome this problem we developed FDF-PAGE, which results in full denaturation and signal recovery even in the presence of 1000X fold excess complementary RNA. FDF-PAGE therefore provides a more robust representation of the sRNA profile than previous methods and will be a useful technique for routine analysis of sRNA. In addition, by comparing sRNA cloning and high-throughput sequencing from FDF-PAGE and non-denaturing methods we were able identify known and novel candidate sRNAs that are under the control of ceRNAs in Arabidopsis thaliana, Mus musculus, Drosophila melanogaster and Caenorhabditis elegans. These results demonstrate the importance of full denaturation during the analysis of endogenous sRNA profiles.To fully understand the effect of complementary RNAs on sRNA detection, we mixed synthetic RNAs F: RNA was mixed in a 1:1 ratio with 2× FDE ), incubated at 65°C for 5–10 min then snap cooled on ice before loading , 7.5 ul of de-ionized formamide, 0.75 ul of 10× MOPS buffer ) and incubated at 55°C for 15 min. 2 ul of dyes was added at room temperature before loading . Glyoxal/DMSO: RNA in 4 ul volume was mixed with 1.5 ul of TBE , 7.5 ul of DMSO and 2 ul of de-ionised glyoxal. Samples were incubated at 50 for 1 h and snap cooled on ice. 2 ul of dyes was added before loading  and boiled at 98°C for 5 min before loading.directly . FDF: RN loading . Note: F loading . 8.5M urPre-loading treated RNA samples were loaded in to 0.75 mm polyacrylamide–urea gels , 700 mg/l ammonium persulphate and 350 ul/l tetramethylethylenediamine). Gels were run in a 0.5× TBE (or 0.5× MOPS buffer if using FDF treated RNA) running buffer at up to 150 V until the bromophenol blue reached the bottom of the gel.After visualization of nucleic acid by SYBR Gold (Life Technologies) staining on a dark reader, polyacrylamide slices containing the small RNA band were excised and placed into a shredder (0.5 ml microcentrifuge tube with three 21 G needle holes punctured into the bottom within a 2 ml nuclease free microcentrifuge tube) and spun at 10 000 × g. Three volumes of 0.3 M NaCl (pH 7.0) was added per volume of gel and rotated at 4°C overnight. NaCl + gel was transferred into a SpinX column (Costar) and spun at 17 000 × g for 2 min at 4°C. Flow through was transferred to a nuclease free siliconised 1.5 ml tube and ethanol precipitated, including two washes of the pellet in 80% ethanol. RNA was re-suspended in nuclease free water.were performed as described by (32P-ATP prior to mixing with RNA (as indicated) and separated by denaturing PAGE. The gel was exposed directly to a phosophor imager plate and then was stained by SYBRgold for total nucleic acid visualization.21_S sRNA oligonucleotide was 5′ end labeled with γ-Arabidopsis total RNA and competitor oligonucleotide (as indicated) were subjected to the illumina small RNA cloning protocol up to and including cDNA synthesis. qPCR was performed with the primers indicated using SYBR green (Thermo Scientific) mastermix.For small RNA qPCR the 21_S sRNA oligonucleotide was first 5′ phosphorylated using PNK forward reaction in the presence of ATP. The phosphorylated oligonucleotide was mixed with 1ug of 5–10 ug of total RNA was used as the starting material for all small RNA libraries. All libraries were generated and sequenced according to the Illumina TruSeq small RNA cloning protocol (RS-200-9002DOC) with any modifications indicated.A. thaliana Col-0 floral tissue, mixed sex adult D. melanogaster Oregon-R strain, C. elegans N2 strain and M. musculus testis 25 days post-partum (25 dpp) were extracted by TRIzol reagent (Invitrogen). CymRSV infected Nicotiana benthamiana total nucleic acid was extracted by standard protocol based on  was identified.Total RNA from based on . HPLC puA. thalinana (genome and annotation) from The Arabidopsis Information Resource (TAIR10); N. benthamiana from the Sol Genomics Network (v0.42); M. musculus annotation and genome (mm9) from ensemble.org and transposon annotation from hgdownload.cse.ucsc.edu; D. melanogaster from ftp.flybase.net (Release 5.51); C. elegans genome and annotation for version WS236 from ftp.wormbase.org. Drosophila piRNAs datasets were obtained from piRNABank (http://pirnabank.ibab.ac.in/) and Mouse piRNAs from piRBase as of 14.8.2013 (http://regulatoryrna.org). miRNAs were identified by matching against mature miRNAs from miRBase release 20. piRNAs were identified in the same manner, cross matching sRNAs to the respective piRNA resources. MA Plots: Normalized reads (library size estimate TMM method) from FDFSS and NSS libraries were analyses; y-axis (M) shows log2 fold change [log2(FDFSS/NSS)] and x-axis (A) shows average abundance [2(FDFSS*NSS)]. Bioinformatic scripts are available on request from S.Y.M. Libraries are available for download at arrayexpress E-MTAB-2733.sRNA reads were trimmed and size selected (between 15nt and 30nt) and aligned by PatMaN without mismatches to the respective genomes. Multi-mapping reads files were selected for downstream analysis. Reads were normalized with the TMM method as implemented by the ‘segmentSeq’ package (BioConductor). Differential expression analysis was performed using baySeq. Genomes: Cymbidium ringspot virus (GenBank accession NC_003532.1); Northern hybridization is a standard and widely used approach to identify, validate and quantify the levels of sRNAs in plant and animal extracts ,26. CurrA. thaliana , glyoxal/DMSO and formaldehyde treatment  and from samples subjected to F-PAGE or FDF-PAGE and size-selection treatments   genomes that are targeted by the host antiviral RNA silencing machinery, generating vast quantities of viral derived small RNAs (vsRNAs) . These v) Figure .equally to both strands. This marked difference in strand ratio between treatments indicates that the increased strength of denaturation results in decreased artifactual bias to positive strand vsRNAs. The likely explanation of these data is that the highly abundant (+) sense viral RNA genome  viral genome ,36. Howein vivo. An illustration of how sequestration has affected other datasets is with CymRSV . However, the 21nt sRNAs were relatively more abundant in the FDFSS treated samples. The proportion of miRNAs was roughly ten fold higher in FDFSS than NSS samples, with the FSS samples being intermediate. This finding suggests that miRNAs may be sequestered by complementary endogenous RNAs in the NSS samples.The small RNAs that map to the A. thaliana , D. melanogaster (whole), C. elegans (whole) and M. musculus . The samples were prepared directly from total RNA (NSS) or after FDF-PAGE treatment and size selection (FDFSS) as these treatments have the largest difference in denaturation efficiency  in the FDFSS libraries could be largely  attributed to a 25-fold enrichment of the highly expressed miR166 and miR165 . However, some small RNAs may only be sequestered in vivo when in complex with Ago and therefore might not be identified using this approach. miRNAs under sequestration have not yet been described for D. melanogaster or C. elegans but our results indicate that miR-956 and miR-90, respectively, might be good candidates to investigate in these organisms (Supplementary Data).These findings with We wondered whether miRNAs that were released from the long RNA pool might sequester their cognate miRNA* strands and reduce their cloning efficiency. To investigate this possibility we picked the top five most significantly FDFSS enriched miRNAs from each organism and determined the representation of their miRNA* strands under FDFSS or NSS. There were no consistent patterns of enrichment, with some miRNA* increasing under FDFSS and others decreasing . It is likely therefore that miRNAs do not re-associate with their star strand after FDFSS or, if they do, that the 2nt 3′ overhangs from the dicer cleavage may be sufficient to allow ligation of the 3′ adapter.To find out whether FDFSS treatment introduces a sequence composition bias in sRNA data  we firstA. thaliana libraries indicated a sequence bias due to the FDFSS treatment. Using baySeq  that releases the full suite of sRNAs from any complementary RNAs in the sample. This technique eliminates a bias in vsRNA distribution and it may be useful to identify lowly expressed or cell type specific sRNAs as it effectively uncouples sRNA representation from the abundance of complementary transcripts. Comparison of FDF-PAGE to non-denaturing methods is a good first step in the identification of sRNAs under the control of ceRNAs.Supplementary Data are available at NAR Online.


Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100–400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicase•zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans. All helicases are divided into six superfamilies (SFs) based on amino acid sequence and structure. The eukaryotic RNA helicases are monomeric and belong to SF1 and SF2. The ring-forming helicases  belong to SF3-to-6. SF1 and SF2 helicases are further divided into several protein families and subfamilies. The DEAH-RHA subfamily helicases are placed within SF2 and are found in all eukaryotes. These proteins are named after the sequence Asp-Glu-Ala-His (DEAH), which is also known as the Walker B motif or motif II, and after the RNA helicase A (RHA), which is also a member of SF2. Several excellent recent reviews have described in detail the function and architecture of RNA helicases in general and DEAH-RHA RNA helicases in particular –4.RNA and their ribonucleoprotein complexes (RNPs) in nearly all aspects of gene expression are remodeled by helicases, which are nucleoside triphosphate dependent molecular motors that unwind double helical nucleic acids –7. This processing involves site-specific insertion and deletion of uridylates at many mRNA sites in protein-catalyzed reactions directed by small guide RNAs (gRNAs). The gRNAs exhibit complementarity to fully edited mRNA through canonical and G•U base pairs. In Trypanosoma brucei, the causative agent of human African trypanosomiasis or sleeping sickness , the holo-editosome targets most mitochondrial mRNAs and consists of over 30 proteins, including two RNA helicases, one of which is a DEAH-RHA helicase , 7, 9–11. Ribosome biogenesis in yeast and humans requires the largest number of different helicases, including several DEAH-RHA proteins. The spliceosome, which comprises over a hundred proteins, also requires a large set of RNA helicases, including four different DEAH-RHA proteins in yeast , 12, 13.Highlighting the wide distribution of DEAH-RHA helicase-dependent processes is the participation of these proteins in a unique process of RNA editing in the single mitochondrion of kinetoplastid protozoa . The single DEAH-RHA helicase in kinetoplastid RNA editing does not have a typical G-patch protein cofactor, but instead it binds a zinc finger (Znf) protein cofactor. Only two other DEAH-RHA helicase•Znf cofactor systems have been reported so far. One system participates in the control of embryogenesis in nematodes and the other system participates in antivirus protection in humans , 16. The presence of a DEAH-RHA helicase•Znf cofactor partnership in trypanosomes, nematodes, and humans suggests that this partnership is widespread in eukaryotes.DEAH-RHA helicases participate in almost every kind of RNA processing reaction in RNA biology and are specifically regulated by a class of protein cofactors that typically contain G-patch domains. The characterized G-patch protein cofactors have been reviewed in detail This review compares the sole DEAH-RHA helicase in kinetoplastid RNA editing with other subfamily members and examines the available studies and functional models for the known DEAH-RHA helicase•Znf cofactor systems. It is feasible that DEAH-RHA helicase•Znf cofactor partnerships share mechanistic features despite their taxonomic distance and their participation in very different RNA processes.. This cluster includes a winged helix motif, a ratchet motif, and an oligonucleotide-binding (OB-fold) domain. Some DEAH-RHA proteins have additional domains on the N-terminal side of the catalytic core of the helicase. These N-terminal domains are thought to be crucial for facilitating the recruitment of specific cofactors and the RNA target, and for modulating the function of the helicase activity. The kinetoplastid RNA helicase termed REH2 (RNA Editing Helicase 2) has all of the conserved features of DEAH-RHA proteins  of Tb927.4.3020 remains undetermined; initial studies using RNAi-inducible down-regulation were unable to link this protein to RNA editing . Only the RHA helicase in humans (DHX9) is known to carry two dsRBDs, while DHX30 and DHX29 each carry a single predicted dsRBD. Other RNA binding sites have been experimentally determined (DHX36)  or predicted (DXH8)    . It is p, 14, 24. REH2 participates in RNA editing in kinetoplastid mitochondria. Additional roles for REH2 are feasible but not established yet. An early study of the REH2-associated RNP speculated that this complex serves as an “organizer” of mitochondrial genome expression . Indeed, isolations of REH2 are enriched in mitoribosomes and other mitochondrial complexes known to participate in mRNA 3′ maturation and stability , 9. Among the DEAH-RHA proteins in kinetoplastids, a few additional putative auxiliary domains with relatively low E-values can be detected. These domains include a RWD domain   in Tb11.01.3930 that is also observed in the human DHX57 protein (The N-terminus of both kinetoplastid REH2 and human RHA (DHX9) helicases carry two dsRBDs; however, the distance in sequence that separates the dsRBDs is very different between the two proteins . The pre protein  and 4. AT. brucei includes the multi-subunit RECC (RNA editing core complex) enzyme and a large number of auxiliary proteins. Most auxiliary proteins are found in ribonucleoprotein subcomplexes with mRNA and gRNA , 20, 26. One of these RNPs, the REH2-associated protein subcomplex (REH2C)  associates with variants of a gRNA-associated RNP (gRNP)  of REH2 with the editing site of the substrate , 19. A model of editosome assembly proposes that mRNP basepairing with gRNPs leads to the formation of mRNA-gRNA hybrid substrates. The first editing site (ES1) in the mRNA is the position just 5′ of a short initial duplex between mRNA and gRNA (termed the anchor duplex) , 28. The ES2 is established as the anchor duplex incorporates correctly edited ES1 sequence. This cycle is repeated one site at the time as the editing machinery advances along the mRNA in a 3′-to-5′ direction. Transient addition of the RECC enzyme to preassembled substrate-loaded multi-RNP scaffolds would establish higher-order catalytic holo-editosomes . REH2 and one of its cofactors (H2F1) with eight predicted zinc fingers are required for efficient editing in vivo  or in dsRBD2 . Indeed, a loss of mRNP-RECC association is observed upon depletion of gRNA in mitochondria . Thus, formation of complete multi-RNP scaffolds enables the transient association of the RECC enzyme with the helicase mRNP. This finding also supports the concept of RECC transient addition through its binding to preassembled mRNA-gRNA duplexes in the multi-RNP scaffolds . A H2F1 knockdown prevents the normal association of the REH2 helicase with gRNPs and the RECC enzyme. H2F1 is a proposed regulator of REH2 that controls both the docking of this helicase with gRNPs and the addition of the RECC enzyme to holo-editosomes . Consistent with the proposed REH2•H2F1 direct interaction in vivo, recombinant versions of REH2 and H2F1 bind directly with each other to form a stable complex in vitro. Yet, additional studies are needed to establish how H2F1 and REH2 act in concert during the assembly of substrate-loaded holo-editosomes.The holo-editosome in  (REH2C)  includesP (gRNP) . The mRN in vivo . This wan dsRBD2 . MutatioC. elegans can exist as sequential hermaphrodites. Sperm production occurs during development and oogenesis occurs in adults. The switch from sperm production to oogenesis requires a controlled posttranscriptional repression of fem-3, a sex-determining gene that promotes male development –32. The switch from sperm to oocyte production is controlled at different levels including by three critical nuclear DEAH-RHA helicases: MOG-1, MOG-4 and MOG-5 (“MOG” stands for Masculinization Of the Germline) . These proteins are the orthologs of the DEAH-RHA helicases Prp16, Prp2, and Prp22 in yeast. A cofactor of these MOG proteins, MEP-1 (for MOG-interacting and ectopic P-granules) (in vitro-translated proteins. Disruption in the expression of MEP-1 or any of its MOG partners prevents the required repression of fem-3 mRNA at the translation level. This results in the masculinization of the germline (the “mog phenotype”) . Because MOG and MEP-1 are nuclear proteins, it has been unclear how the cytosolic inactivation of fem-3 mRNA takes place. A MEP-1 homolog of unknown function was identified in Drosophila but not in yeast, suggesting that MEP-1 evolved among metazoans. Recombinant MEP-1 binds RNA non-specifically in vitro, although the fem-3 mRNA is the only known cognate target of MEP-1 . MEP-1 alone or with its helicase partners or other cofactors may provide the required RNA specificity in vivo. Prp16, Prp2, Prp22, and their human orthologs control steps of pre-mRNA assembly with several snRNPs. Thus, the MOG•MEP-1 system could control similar steps. Yet, splicing defects were not observed in mog-1 null mutant animals . It was speculated that the MOG•MEP-1 system mediates epigenetic effects that are coupled with splicing. One such effect is the deposition of the exon junction complex (EJC) near exon-exon junctions of mRNAs. This post-splicing accumulation of EJCs by the spliceosome machinery is known to affect the fate of mRNAs such as nuclear export, degradation, subcellular mRNA localization, and translational yield . Consistent with this idea, two core components of EJC in C. elegans are also required for the sperm⇒oocyte switch , 36. The precise mechanism of action of the MOG•MEP-1 system remains to be determined.ranules)  is also ranules) . Binding, 38. ZAP has four predicted zinc fingers  is a host factor that inhibits the replication of a broad spectrum of important viruses, including HIV, Ebola virus and Sindbis virus  fingers , and itsal dsRBD . ZAP and. The small N terminal fragment of ZAP with all four zinc fingers binds DXH30 and leads to the same antiviral activity as the full-length ZAP. Each of the four zinc fingers in ZAP may be an RNA binding unit because mutation of any of the zinc fingers reduced ZAP’s activity to some extent. ZAP also binds directly with the exosome component hRrp46p through a 30 amino acid binding region. This ZAP-exosome interaction is relevant because depletion of the exosome subunits hRrp41p or hRrp46p significantly reduced ZAP’s antiviral activity . Thus, ZAP is a hub with distinct binding surfaces that bring together the DXH30 helicase, target viral mRNAs, and the RNA processing exosome. It has been proposed that the ZAP antiviral activity involves the removal of secondary structure in bound target mRNAs via DXH30-catalyzed unwinding to facilitate the exosomal nucleolytic degradation of relatively disentangled RNA conformers . The few identified ZAP-responsive RNA fragments have no known sequence motifs. The only common feature in these fragments is that they are at least ~500 nucleotides long, so the source of binding specificity for these RNAs has been elusive. A crystal structure of a N-terminal fragment (residues 1–225) with all four fingers in ZAP provided insights into RNA target recognition by ZAP . The structural features of ZAP (discussed in more detail below) suggest that the target RNA is recognized by its tertiary structure rather than its base sequence. This explains why only a few ZAP-responsive RNAs are known and why they are at least 500 nucleotides long and have no easily identified sequence motifs.The zinc finger motifs of ZAP are required for RNA binding and antiviral activity . G-patch cofactors typically establish direct contacts with the C terminal domains in DEAH-RHA helicases. The G-patch and OB-fold domains act in concert and are thought to provide specificity in RNA binding and activation of RNA-dependent NTPase and unwinding activities.Eukaryotic DEAH-RHA helicase are known to bind a group of regulatory factors termed G-patch proteins, which carry one or more conserved G-patch motifs  were identified in kinetoplastid protozoa, nematodes and humans. All three helicase•Znf protein systems impact the functional or regulatory fate of specialized mRNPs. In trypanosomes, the mitochondrial REH2•H2F1 system was proposed to modulate the assembly and remodeling of mRNA-gRNA hybrids that are targeted by the editing enzyme . In the worm germline, the nuclear MOP•MEP-1 system is thought to control epigenetic remodeling of mRNP complexes that modulate the translation fem-3 mRNAs, and thereby, the switch from spermatogenesis to oogenesis. The location of EJCs near exon-exon junctions by the spliceosomal machinery could be used to mark fem-3 transcripts for downstream cytosolic inactivation . In humans, the cytosolic DXH30•ZAP system specifically binds viral mRNA targets and promotes their exosomal degradation , 38. The Znf cofactors modulate the function of their helicase partners in RNA editing, RNA splicing related processes, and the destruction of viral RNAs. It is likely that Znf cofactors can modulate DEAH-RHA helicase in additional RNA processes where they are present.A second group of DEAH-RHA helicase cofactors are proteins that have multiple zinc-fingers. Three DEAH-RHA helicase•Znf cofactor systems in organisms that diverged 100–400 Ma A critical question that applies to these three helicase•Znf cofactor systems is how they achieve RNA target specificity. The problem is complicated by the fact that multiple related substrates must be recognized with efficiency and specificity. Moreover, substrate specificity may not always involve consecutive sequence-dependent cis-elements. For example, most mitochondrial mRNAs in trypanosomes require editing, and many viral mRNAs are specifically targeted by exosomes. Also, in the lifetime of mRNAs, the mRNPs undergo constant remodeling including the addition and removal of EJC at positions of variable sequence near splice junctions in mRNAs. Thus, neither, the editing substrates, viral mRNAs, or splice junction sites appear to carry sequence-specific motifs that are easy to identify.. Structural and functional analyses of residues 1–225 in ZAP identified an RNA binding surface involving multiple positively-charged residues. The four Znf motifs in the ZAP structure are positioned to flank two sides of a positively charged cleft that likely binds folded RNA. Model structures of the ZAP-RNA interaction suggested that the target RNA should have a specific tertiary structure to precisely fit into the three-dimensional RNA-binding cleft of ZAP. The same principle may apply to other Znf cofactors, with the level of complexity in RNA recognition influenced by the number in zinc finger domains involved. Thus, protein dimerization, as in ZAP and MEP-1 , 16, would provide a larger platform to coordinate complex RNA features. Moreover, the protein may bind several related RNA targets if each zinc finger contributes differently to the binding of the distinct RNAs. This would meet the need for specific recognition of a large set of diverse editing substrates, viral mRNAs, or splice sites. Thus, in an analogy to ZAP, the tandem zinc finger array in other proteins, including H2F1 and MEP-1, could be arranged to build up specialized modules for the binding of complex RNA tertiary structures rather than specific sequences.The crystal structure of ZAP provides important clues as to how relatively small protein regions with a tandem array of zinc finger domains may achieve complex specificities in RNA recognition , 2. The proposed role of ZAP in removing double-helical structure of 3′ ends in bound mRNAs via DXH30-catalyzed unwinding for exosome-mediated degradation serves as a guide to propose a common function for other zinc-finger cofactors. In kinetoplastid RNA editing, H2F1 could recruit mRNA targets for REH2-catalyzed unwinding of structure that may interfere with annealing of a gRNA next to the editing site. Interestingly, a photo-crosslinking experiment with a synthetic RNA substrate containing a photo-reactive thio-U base detected a close-proximity (≤ 4 Å) contact of REH2 with the editing site. This localized REH2 at the catalytic center of the editosome, and suggests that the helicase could contribute to local unwinding of the substrate including at the editing site, the abutting 3′ residues for gRNA annealing, or both . Finally, MEP-1 may assist in modulating RNA structure of bound sites via MOP-1 catalyzed unwinding. This unwinding may facilitate engagement by spliceosome components including during EJC deposition. In these three systems, the zinc-finger protein alone could specifically bind to the RNA target. Its helicase partner could then catalyze cycles of local structure remodeling on the bound RNA, thus facilitating access and activity of the components of the RNA editing enzyme, exosome, or spliceosome.A second critical question is whether or not DEAH-RHA helicase•Znf cofactor systems share a common function. Monomeric DEAH-RHA helicases appear to require a 3′ single-stranded extension, and they are not highly processive. Instead, they promote both local 3′-to-5′ unwinding of discrete structures and RNP assembly in vivo. For DXH30, the ZAP-binding region includes a dsRBD but not the catalytic core. In ZAP, the DXH30-binding region has all zinc fingers and renders full virus protection . The direct interaction of MEP-1 with MOG helicases, examined in yeast and with in vitro-translated proteins, only required the N-terminus of MEP-1 including its zinc fingers. The MOG helicase requirements for interaction were not examined, so the MEP-1 binding to MOG helicases may involve one or more zinc fingers. Alternatively, determinants outside the zinc fingers may be responsible for the protein-protein interaction. Nonetheless, a recombinant REH2 helicase fragment (residues 1261–2167) and H2F1 form a stable complex in vitro. That tested REH2 fragment lacked dsRBD1 and dsRBD2, so the helicase core and the C terminal domain cluster were sufficient to bind H2F1. Only full length H2F1 was tested in that study. Overall, the N-terminus of DXH30 and MOG were shown to bind ZAP and MEP-1, respectively. In contrast, the C-terminus of REH2 sufficed to bind H2F1 in vitro. This suggests that these helicases differ in their binding mechanism to their cognate Znf cofactors. An alternative model could be that both N-and C-termini in the RNA helicases engage in direct contacts with their Znf cofactors in vivo. Yet, either terminus of the helicase may suffice to detect an association in the applied assays in vitro or in yeast.Furthermore, a better understanding of the functional synergy between RNA helicases and their cofactors during RNA substrate recognition will involve identification of relevant domains and specific amino acids required for intra-protein and inter-protein contacts in the helicase•cofactor complexes. The requirements for Znf cofactor binding to cognate helicases may exhibit important differences. The N-terminus in both DXH30 and ZAP suffices for association between these proteins . However, a few contacts between sites outside the G-patch motif and N-terminus helicase positions were also observed. Potentially, contacts of cofactors with both N- and C-termini of the helicase may lead to higher binding affinities between the two proteins. Finally, binding of the Znf cofactors to the helicases could induce changes in the conformation and function of their helicase partners. The aforementioned structural proteomic study of Prp43 found major rearrangements in the OB-fold domain on binding of the G-patch protein Ntr1 . G-patch and zinc-finger cofactors are structurally very different; however, both proteins appear critical in directing DEAH-RHA helicases to specific substrates and processes. A DEAH-RHA protein could employ either a G-patch or a zinc-finger cofactor. Alternatively, the same helicase could use both cofactors in the same or different processes, but no G-patch cofactor has been identified with the discussed helicases. For example, MOG-4 in nematodes (the homolog of Prp2 in yeast) could also use a G-patch protein, whereas Prp2, which binds the G-patch protein Spp2, could also have a zinc-finger cofactor. A recent structural study of the MLE  helicase, a RHA homolog that controls the IncRNA-mediated assembly of the ribonucleoprotein dosage compensation complex during activation of X-linked genes in Drosophila, showed a stable core with inter-domain contacts between RecA2, dsRBD2 and the OB-fold domain . The structure may represent a transition “on” state showing how DEAH-RHA helicases couple ATP hydrolysis and RNA translocation. Coupling of a cofactor with its helicase partner may modulate the intricate inter-domain contacts in the helicase core and thereby its functions in specific RNA or RNP recognition and remodeling.Structural proteomics of a recombinant complex between Prp43 and its G-patch cofactor Ntr1 mapped primary contacts between the G-patch motif and the helicase C-terminal domains, namely, the winged helix, the ratchet, and the OB-fold domains in vivo? Crystal structures are available for several DEAH-RHA helicases, and for ZAP, but not for any G-patch proteins nor for any DEAH-RHA helicase•Znf cofactor complexes. Structural information of a complex between a helicase with its Znf cofactor will be very important to further understand how these emerging protein partner systems function.Several questions need to be addressed to better understand the mode of action of DEAH-RHA helicase•Znf protein systems in their targeted RNA processes, and their impact on diseases associated with these processes. How do Znf regulators specifically target their monomeric DEAH-RHA helicase partners? How do the helicase and Znf proteins interact and cooperate to enable specific RNA or RNP recognition and remodeling? Is the assembly of the helicase with its Znf cofactor regulated H2F1 cofactor with eight zinc fingers. The long and independent evolutionary history of the RNA processes discussed in this short review suggests that analogous helicase•Znf systems are widespread across biology. Studies of ZAP, an antivirus protein, provide a guide to suggest potential mechanistic parallels between helicase•Znf protein systems. For example, the Znf cofactor may be a hub that brings together the RNA substrate, the RNA helicase and either exosome, spliceosome or editosome components. Also, structural studies of ZAP suggest a mechanism of RNA target recognition that may be used by other Znf cofactors. That is, a tandem array of zinc fingers creates a three-dimensional RNA-binding surface that fits complex spatial determinants in the RNA targets, not continuous sequence elements. Variations in the number and contribution of each zinc finger in the array to RNA binding may allow specific recognition of a set of related substrates. Thus, Znf proteins may directly recruit RNA substrates and thus enable modulation of local RNA structure of bound sites via helicase catalyzed unwinding. These proposed basic properties of multi-Znf cofactors would meet the needs of mitochondrial RNA editing, nuclear EJC remodeling, cytosolic viral mRNA degradation, and potentially other RNA processes involving DEAH-RHA helicases.Multi-Znf proteins are an emerging class of DEAH-RHA helicase regulators. A previous class of DEAH-RHA helicase cofactors includes G-patch proteins. Three DEAH-RHA helicase•Znf protein partnerships have been identified so far in taxonomically distant species. The recently discovered system in trypanosome RNA editing includes REH2, the largest known DEAH-RHA subfamily member, and its


Phytomonas serpens . The properties of PserNV1 permit assignment to the genus Narnavirus (Narnaviridae), the first reported from a host other than fungi or oomycetes.We describe here a new RNA virus (PserNV1) from the plant protist parasite  Narnaviridae are composed of a positive single-stranded RNA segment (2.3 to 3.6 kb) encoding a single protein, the RNA-dependent RNA polymerase (RDRP) .Viruses within the family e (RDRP) –3. Narnaestans 3–. Here, wP. serpens isolate 9T (4-CCCGTCGTTTGCTGGCTCTTT-NH2) was added to the eluted RNA band with T4 RNA ligase (NEB), amplified using primers complementary to the adapter and virus, and sequenced to determine the termini.Total cellular RNA from olate 9T  was extrolate 9T . A promiP. serpens nuclear genome  . In the future, studies of NV1-positive and -negative lines of Phytomonas may establish potential roles of this viral element in plant pathogenicity (Members of the genus il palms , which mspecies. . Some daPhytomonas serpens 9T strain was deposited in GenBank under the accession no. KU882057.The full-length viral genomic sequence of PserNV1 from


Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device. Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device. Here, we present a simple, effective configuration for rapidly obtaining purified RNA from low concentration cell medium. This Total RNA Extraction Droplet Array (TREDA) utilizes an array of surface-adhering droplets to facilitate the transportation of magnetic purification beads seamlessly through individual buffer solutions without solid structures. The fabrication of TREDA chips is rapid and does not require a microfabrication facility or expertise. The process takes less than 5 minutes. When purifying mRNA from bulk marine diatom samples, its repeatability and extraction efficiency are comparable to conventional tube-based operations. We demonstrate that TREDA can extract the total mRNA of about 10 marine diatom cells, indicating that the sensitivity of TREDA approaches single-digit cell numbers. Currently, typical bench-top purification methods for nucleic acids are performed in either tubes or wells in microplates using solid phase extraction (SPE). These tubes or wells contain purification columns with large surface areas whose affinity to the targeted nucleic acids can be manipulated. These conventional bench-top purification processes are usually labor-intensive. Robotic workstations that use 96-well microplates exist; however, they are typically not accessible to general biologists and utilize liquid volumes larger than 10 μL. In addition, the cost of reagents and operation of large-scale robotic purification is high, and sensitivity for detecting low abundant targets is limited because of low template concentration.1 For general applications, a separate step for nucleic acid extraction and purification is still required.Generally, purified nucleic acids are a prerequisite for performing downstream analysis, such as quantitative PCR (qPCR), reverse transcriptase quantitative PCR (RT-qPCR) and DNA/RNA sequencing. Some platforms such as Fluidigm's C1 , a commercial system which provides parallel pre-qPCR preparation for single mammalian cells, adopt all-in-one qPCR/RT-qPCR processes to eliminate DNA/RNA purification. These processes typically use lysis master mixtures that inhibit DNase or RNase activity to prevent degradation of the nucleic acids, and the components in the mixtures do not inhibit PCR/RT-PCR. However, some non-mammalian cells such as diatoms and most gram-positive bacteria have stronger cellular membranes, thus the necessary lysis buffer is no longer compatible with downstream PCR.4 porous polymers,10 or sol–gel matrices.11 These devices have complicated channel networks and on-chip valves to direct various buffers to the columns during the purification process. Therefore, the devices in the literature are limited in the number of purification reactions performed on one chip. Zhang et al.12 developed a microwell system to perform RNA purification based on liquid phase extraction. This system does not need on-chip columns so it can perform multiple purification reactions simultaneously, but sophisticated liquid/air control valves are needed.Initial efforts to perform on-chip nucleic acid extraction and purification incorporated purification columns with packed beads,13 Application of an external magnetic field causes the magnetic beads to form a bead aggregation in a section of a microchannel.14 The remaining purification processes are very similar to those of on-chip columns. For example, Quake's group uses on-chip pneumatic fluidic control valves to deliver lysis buffer, wash buffer, and elution buffer, to the retained magnetic beads and to purify mRNA from single mammalian cells.15 Lee's group also makes similar devices with integrated electromagnets to manipulate magnetic beads.17 RNA purification devices using magnetophoresis with continuous flow are also demonstrated.20 In these cases, RNA binding magnetic beads in continuous flow pass by an adjacent magnet, so the trajectories of the beads are deflected into a branch channel and separated from the main stream. However, the use of continuous flow is not compatible with applications that favor liquid segmentation .Purifying DNA/RNA using magnetic beads to retain and then elute nucleic acids can be compatible with microfluidic platforms and operate in parallel. These beads are commercially available with a variety of affinity options. Beads bound with nucleic acids can be easily immobilized by a nearby magnet. Therefore, the magnetic bead-based method eliminates the need for fabricating structures for on-chip columns required for each purification reaction and reduces the complexity of fabrication.et al.21 use an array of planar electromagnets to move and merge magnetic bead-containing droplets on a Teflon-coated surface, and employ hydrophilic/hydrophobic patterning to facilitate droplet splitting. Because magnetic fields do not interfere with static electric fields, magnetic separation has been integrated with electrowetting on dielectric (EWOD) droplet manipulation.23 Among these EWOD devices, Ohashi et al.23 demonstrated that magnetic beads can move between two stationary droplets which are held by two sets of circular electrodes. A drawback of these EWOD-magnetic hybrid devices is that the electrodes require special surface coatings and electrical circuitries to manipulate the motion of the droplets, making the fabrication process more complicated. An alternative droplet-based method to EWOD is to use specially designed structures that can hold droplets but let magnetic beads traverse through gaps in the structures. Gu et al.24 use external magnets to drag magnetic beads through a series of stationary picoliter droplets which contain different solutions for DNA purification in capillary tubing. The authors successfully purified the DNA of single mammalian cells. Similar approaches to move magnetic beads through an array of droplets in a chip format have also been demonstrated, where the droplets were held by microfabricated obstructions  with small openings for beads to go through.26 mRNA extraction at the single-cell level has been achieved.26Combining magnetic beads and microdroplets is gaining researchers' interest. Magnetic beads can penetrate through the oil/droplet interface through the use of an external magnetic field, so this class of devices does not need extensive microchannel networks for liquid manipulation. Simple structures reduce the cost needed for the extraction process and increase the number of reactions on a chip. Lehmann 29 our device focuses on using hydrophilic spots to hold stationary droplets to facilitate extraction steps. These novel features enable rapid and inexpensive device fabrication, as well as provide efficient total RNA extraction. We demonstrate that this approach efficiently purifies RNA directly from a single digit number of marine diatom cells based on serial dilution. This improvement facilitates higher RNA recovery efficiency and leads to single cell level sensitivity.Although the introduction of droplets significantly reduces the complexity of the device, the solid structures that hold droplets may also obstruct magnetic beads during manipulation. In this paper, we present a Total RNA Extraction Droplet Array (TREDA) that uses hydrophilic patterns to constrain aqueous solutions without physical obstruction. This approach not only reduces the fabrication processing required, but also removes the physical obstruction  that might also prohibit magnetic beads from moving, and easily performs RNA extraction in parallel for different samples. TREDA is similar to recently published devices that used magnetic beads to drag liquids through hydrophilic spots to perform complicated liquid manipulations,The key components are a TREDA chip, a movable Plexiglas plate embedded with two linear arrays of sixteen “extraction magnets”  below the TREDA, and another movable Plexiglas bar with one linear array of eight “mixing magnets”  above the TREDA . The TRE31 to quantify the RNA extracted/purified by the TREDA chip  were used to extract RNA in this study. The beads carry positive charges when the surrounding pH is lower than 6.0, and negative charges when the pH is higher than 8.0. Therefore, naturally charged RNA can be bound or released from the surface of the beads based on the surrounding pH. A three-step purification method was used in this study. First, the cell medium, lysis buffer, binding buffer and beads were mixed together for cell lysis and RNA binding. The cells were lysed, and then the total RNA was bound onto the surface of the beads since the binding buffer has a low pH value. Second, the washing buffer was mixed to wash out the unwanted substances on the beads. Third, the low-salt, high pH elution buffer was mixed with the RNA-bound beads to release total RNA from the surface of the beads. After elution, the beads were removed, leaving purified total RNA in the elution buffer.32 The fabrication process is to make a hydrophilic pattern on silanized glass coverslips to confine the aqueous droplets. In this study, such patterns were generated by Microscale Plasma Activated Templating (μPLAT), a technique that employs a stencil to expose air plasma only to designed areas to increase the hydrophilicity of the surface.33 The TREDA chip was made on a no. 2, 76 mm × 89 mm, cover glass . The hydrophilic pattern was determined using a 1 mm-thick PDMS stencil, which had an array of 8 × 6 1/8 inch-diameter holes, with a 9 mm pitch between centers to be compatible with standard 96-well microplates. The stencil was adhered on the coverslip and placed in a plasma cleaner  for plasma exposure with 6.8 W RF-power for one minute. The areas exposed to the plasma became hydrophilic while the unexposed areas remained hydrophobic. Then the stencil was removed, leaving an array of hydrophilic patterns on a more hydrophobic background. The produced coverslip can hold 48 droplets. To confine oil during operation, a PDMS frame  was plasma bound on the edges of the chip to surround the array.The procedure of producing a hydrophilic array on the TREDA chip is derived from our previous work.Thalassiosira pseudonana (CCMP1335) cells were grown in f/2 medium35 with constant light exposure. Artificial seawater was used for medium preparation based on the formula of Kester et al.36 Cells were harvested and directly counted by using a Petroff Hausser counting chamber . A serial dilution with 1× PBS was performed to generate around 10 000 cells μL–1, 1000 cells μL–1, 100 cells μL–1, 10 cells μL–1 and 1 cell μL–1, respectively. 1 μL of each concentration level was used as the starting material for on-chip RNA extraction.Marine diatom –1 proteinase K every 500 μL lysis buffer (L16). A 1 μL cell culture was directly lysed with this lysis mix and incubated at 60 °C for 15 min. 100 μL of magnetic beads and 200 μL of binding buffer (B9) were added into the cell lysates and gently mixed by using a pipette. A magnetic rack was used to form a tight pellet of beads and then the supernatant was removed by gently pipetting. 750 μL of wash buffer (W13) was added to wash the beads and followed by another washing step using 750 μL of wash buffer (W14). Supernatant was removed by using a magnetic rack. Final elution was achieved by adding 5 μL elution buffer (E7).Bench-top RNA extraction was performed according to the protocol provided by the manufacturer. In short, we freshly prepared lysis mix, which contained 5 μL 0.5M DTT and 5 μL 20 mg mLSixteen 4 μL droplets and thirty-two 5 μL droplets were pipetted onto the TREDA chip. There were sixteen sets of droplets. Each set contained three droplets. The first droplet in each group contained a 4 μL mixture of 1 μL lysis Mix, 1 μL binding buffer, 1 μL cell medium and 1 μL suspension of magnetic beads. The second and the third droplets contained 5 μL washing buffer and 5 μL elution buffer, respectively. After pipetting the droplets onto the TREDA chip, 2.5 mL mineral oil  was loaded to cover the droplets inside the PDMS frame. The oil was utilized to prevent the droplets from evaporation and contamination. The RNA extraction process followed the description in the experimental setup section. The liquid in the third droplet was pipetted to a conventional PCR tube for RT-qPCR to validate RNA extraction efficiency.T. pseudonana: forward primer (5′-TGCCAGTAGTCATACGCTCGTCTCA-3′) and reverse primer (5′-CCTTCCGCGAACAGTCGGGTATT′). Express SYBR GreenER qPCR Supermix Universal kit (Life Technologies) was used in qPCR. Total reaction volume was 10 μL, which contained 1 μL of each primer with the concentration of 4 μM, 5 μL of EXPRESS SYBR GreenER qPCR SuperMix Universal, 0.1 μL ROX Reference Dye, 0.9 μL DEPC treated water and 2 μL cDNA. qPCR was performed on a StepOne Real-Time PCR System  instrument. The negative controls were DEPC-treated RNA-free water. The thermal cycling program for qPCR was as follows: 95 °C for 5 min, 40 cycles for 95 °C for 15 s, 60 °C for 60 s and 75 °C for 10 s , followed by melt curve analysis according to the default program of the instrument. Three replicates for each RNA sample extracted by the TREDA chip were performed.SuperScript VILO cDNA Synthesis kit was used for synthesis of cDNA  from RNA. Total reaction volume was 10 μL, which contained 1 μL 10× SuperScript Enzyme Mix, 2 μL 5× VILO Reaction Mix, 4 μL of DEPC treated water (Life Technologies) and 3 μL of eluted RNA. The thermal program for cDNA synthesis was as follows: 25 °C for 10 min, 42 °C for 2 hour and 85 °C for 5 min. For qPCR, primers were designed by using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and manufactured by Invitrogen  which amplified a 184 bp of 18S of T. pseudonana cells. The PCR primers target 18S RNA. The blue and green amplification curves represent the use of TREDA with and without the mixing process, respectively. In this experiment, we performed three purification experiments for each purification approach  so the triplicate results indicate the technical variations of purification. Each purified droplet was further partitioned into three Eppendorf tubes for RT-qPCR. The triplicate RT-qPCR results of the same purification indicate the technical variations of PCR. Therefore, each approach has 9 curves and 1 curve for negative control. The average Cq, quantification cycle represents the number of cycles needed to reach a set threshold fluorescence signal level. The average Cq value of the conventional approach was 26.5, and for our approach with the mixing process Cq was 27.5, and for our approach without the mixing process Cq was 34.6. Standard deviations of the conventional approach, our approaches with and without the mixing process were 0.39, 0.53 and 0.88, respectively. The results indicate the comparable quantity and quality between our approach and the conventional approach and the necessity of the mixing.We demonstrated that cell lysis, RNA extraction and purification on a TREDA chip with mixing were as efficient as conventional approaches which used vortexing or pipetting. Cq value of the mixing curves is much higher than that of the no mixing curves. The negative control shows amplification in the late stage due to random nonspecific amplifications after extended thermocycling. The melting curve of the negative control can be easily distinguished from the ones with specific amplifications.In RNA extraction without the mixing process, magnetic beads attached to the chip surface in all processes due to the extraction magnet. When utilizing the mixing process, the beads are attracted from the chip surface to the top of the droplet. The mixing increases the contact surface between the beads and liquid which improves the mixing efficiency. Therefore, the average –1) were achieved by serial dilution from bulk cells. There were triplicates for each cell concentration level. We used these triplicate results to identify variations in the purification process. In addition, we partitioned each purified RNA into three tubes for RT-qPCR. We further used the triplicate RT-qPCR results to identify the variation of the PCR process. The small graph in the upper right corner represents the average Cq value and variation in each set. The average Cq values from low concentration to high concentration are 33.3, 26.4, 22.5, 19.4, 15.3, respectively. Negative controls were not detected. The standard deviations from a low number to a high number of cells are 0.73, 2.75, 0.30, 0.30 and 0.40, respectively. The result shows our approach has high sensitivity to obtain RNA from a single cell. In addition, the result shows our method has a wide working range since there is a high linear relationship between the average Cq value and the cell number. However, compared to higher cell concentrations, the standard deviation increases significantly at 10 cell and single cell levels, which have higher Cq values. Since PCR becomes more stochastic in the presence of fewer templates, the higher variation seen with higher Cq values is an inherent characteristic of the PCR process.31The efficiency, sensitivity, and working range of the method using the TREDA chip with the mixing procedure have been evaluated. As shown in 32 Our team is developing a similar chip with a droplet array for performing RT-qPCR, which can be readily integrated with TREDA. TREDA can be integrated with other RT-qPCR platforms, e.g., loading the extracted RNA into tubes then perform RT-qPCR on commercially available thermocyclers. Additionally, this device is able to provide high quality purified RNA for other downstream analysis, such as RNA-Seq, regardless of the initial cell concentration. This property will assure the reliability of RNA-Seq results, especially for single cell analysis.We presented a novel, magnetic bead-based RNA purification chip called TREDA with a series of surface tension-confined droplets. We demonstrated this device can efficiently purify RNA from raw diatom cells ranging from 10 000 cells to single cells. The RT-qPCR reactions performed on the purified RNA show consistent, sensitive data with low variation which confirmed the high quality of RNA purified by our TREDA chip. The constructional and operational simplicity of the chip makes it a robust tool for RNA purification. It will save significant time and minimize human errors due to reduced handling steps. This droplet-based purification format can be extended to measure quantitative gene expression if one of the droplets in the reaction contains RT-qPCR reagents. The design is an ideal format for coupling with the qPCR arrays that utilize surface-adhering droplets.


Poliovirus is the etiologic agent of poliomyelitis, an acute flaccid paralysis affecting 1%–2% of infected patients and, on rare occasions, causing death by paralyzing muscles that control the throat or breathing. A striking feature of infection is lifelong disabilities that may affect survivors of the acute disease. Transmitted by the fecal—oral and oral—oral route, this virus (three serotypes) was one of the most feared pathogens in industrialized countries during the 20th century affecting hundreds of thousands of children every year, via outbreaks during warm summer months. Although there are highly effective vaccines to control poliomyelitis, it remains endemic in a few countries, from which spread and outbreaks continue to occur throughout the world. Since its discovery in 1908, poliovirus has been intensively studied to better understand and control this formidable pathogen. The history of poliovirus is not, however, limited to the fight against the disease. Poliovirus replication studies also have played important roles in the development of modern virology since poliovirologists and, more generally, picornavirologists have been pioneers in many domains of molecular virology. Poliovirus was, for example, the first animal RNA virus to have its complete genome sequence determined, the first RNA animal virus for which an infectious clone was constructed, and, along with the related rhinovirus, the first human virus that had its three-dimensional structure solved by X-ray crystallography. Indeed, the history of over half a century of poliovirus replication studies is marked by major discoveries, many of which are summarized here and illustrated in In 1949, John Enders, Thomas Weller, and Frederick Robbins performed a landmark study showing that poliovirus could be propagated in cultured, non-neural human cells that did not correspond to the tissues infected during the disease . John HoThe first evidence for an RNA-dependent RNA polymerase used by the virus for RNA genome replication was reported in 1962 by Baltimore and Franklin . Until tIn the late 1960s, Summers and Maizel ,8, showepol to the 5'-terminal uridylic acid of the virus genome is the key player in protein-primed initiation of poliovirus RNA synthesis [pol. Their functions appear to be strand-specific, since the cre is required for positive-strand RNA synthesis but may not be essential for negative-strand RNA synthesis. A cre was first found in the capsid coding region of human rhinovirus (HRV) RNA and later in the 2C coding region of poliovirus RNA [This period corresponds to several major advances in understanding the mechanisms of translation and replication of poliovirus genomic RNA. In 1977, a small protein of 22 amino acids, called VPg, was discovered to be covalently linked to the 5' end of poliovirus RNA . Its absynthesis . Experimynthesis ,14. Sincynthesis ,16, the irus RNA ,18. SincThe complete sequence of poliovirus genomic RNA was reported in 1981 by two different groups . PolioviTranscribed viral RNA from a plasmid-derived cDNA was also used in vitro for the de novo synthesis of infectious poliovirus, a first for an animal RNA virus. Published in 1991 by Eckard Wimmer’s group, "Life in a Test Tube" resulted from the addition of viral RNA to a crude cytoplasmic extract of uninfected HeLa cells. These extracts contain all essential elements for poliovirus replication, including cytoplasmic membranes and components required for virion assembly . ImplemeBased upon X-ray crystallography studies, poliovirus and its relative, HRV, were the first animal viruses for which the three-dimensional virus structure was solved in 1985 by the groups of Hogle and Rossmann ,25. And Numerous studies carried out during this period uncovered the ability of poliovirus to usurp cellular components and structures for its own benefit, reflecting the necessity for this virus with a very limited coding capacity to hijack cell resources during infection. Relocalization of nuclear proteins in the cytoplasm during poliovirus infection was first reported in 2000–2001. Cleavage of nuclear pore complex components such as Nup153 and p62 by the viral 2A proteinase led to the accumulation in the cytoplasm of virus-infected cells of a number of proteins normally present in the nucleus ,29. ThesPoliovirus replication sites on cellular membranes (first described in 1969) were also shown to be the result of viral hijacking of components of cellular membrane metabolic pathways, leading to intracellular membrane remodeling and generation of specialized sites distinct in protein and lipid composition from that of the host cell. Belov, Altan-Bonnet, and colleagues demonstrated that viral proteins promoted membrane-binding of members of the Arf family of small guanosine triphosphatases (GTPases), leading to the formation of phosphatidylinositol-4-phosphate lipid-enriched organelles that are essential for poliovirus RNA replication , 34. Indhttp://www.polioeradication.org/). Moreover, vaccine-associated paralytic poliomyelitis resulting from the use of a live, attenuated vaccine complicates any poliovirus endgame strategy [Despite a dramatic reduction in the number of cases of poliomyelitis , as well as of polio endemic countries (125 countries in 1988 versus 3 countries in 2014), poliovirus is not yet eradicated from our planet and is still the target of a massive worldwide vaccination campaign (strategy . Finally


Model evaluation is a necessary step for better prediction and design of 3D RNA structures. For proteins, this has been widely studied and the knowledge-based statistical potential has been proved to be one of effective ways to solve this problem. Currently, a few knowledge-based statistical potentials have also been proposed to evaluate predicted models of RNA tertiary structures. The benchmark tests showed that they can identify the native structures effectively but further improvements are needed to identify near-native structures and those with non-canonical base pairs. Here, we present a novel knowledge-based potential, 3dRNAscore, which combines distance-dependent and dihedral-dependent energies. The benchmarks on different testing datasets all show that 3dRNAscore are more efficient than existing evaluation methods in recognizing native state from a pool of near-native states of RNAs as well as in ranking near-native states of RNA models. RNA molecules play different biological roles besides messengers between DNA and protein ,2, e.g. et al.  is involved in 3dRNAscore. The dihedral-based energy can describe the flexibility of RNA molecules more efficiently ,25. FurtThe steps for building 3dRNAscore Figure  are as fOur knowledge-based potential 3dRNAscore is composed of two parts: the first part is based on distance between any two non-bonded heavy atoms located at different residues in the molecule, and the second part is based on the backbone torsion angles. Among them, the distance-dependent part is constructed as a mean force potential  derived R represents the value of a ‘reaction coordinate’. The reaction coordinate may be any convenient measure of properties of the molecule. When using the distance d between atoms i and j of types a and b as the reaction coordinate, the assumption can be expressed as:T is the absolute temperature and is set to 298 K, a and b in native RNA structures and a and b in reference state structures.Samudrala  pointed entclass1pt{minimaThe third assumption is the thermodynamic hypothesis : the lowd between two atoms of types a and b. N is the total counts. d regardless of atom types. a and b in whole distance region.The probability The RASP  and KB  by the sum of the total densities m is actually In 1990, Sippl developed a method to address the problem of small data sets . He apprcument}:\document8 atom pairs in the dataset of 317 structures. Hence, there are averagely ∼1090 samples (5.25 × 108/(85 × 85 × 20/0.3)) located in each distance bin. The atom distances are ignored when we are counting the total samples for each nucleotide pair type (one of 85×85 types). There are averagely 39 030 samples for each nucleotide pair type. Thus, both the average number of samples in each bin (∼1090) and the average number of samples in each nucleotide pair type (∼39 030) are large enough to avoid the sparse data problem. Furthermore, we do not use the statistics in the 0–3 Å regions where sparse data most likely appear. Instead, we utilize a penalty to substitute it. That is to say, once the distance between two atoms is <3 Å, it will give a penalty to the total energy score. In physical sense, the penalty corresponds to the Van der Waals exclusive force between two atoms. These analyses show that 3dRNAscore has no need of using the method proposed by Sippl to address the issue of sparse data.In order to avoid sparse data, we checked all the training parameters. There are totally about 5.25 × 10We used not only distance-dependent potential, but also a dihedral-dependent potential, involving seven RNA dihedral angles . First, we calculated their statistical distribution over the training set Figure .T is the absolute temperature that is set to 298 K, i  of θ degree (0–360) and type a in the database of experimental RNA structures and i of θ degree and type a in reference state.Once we get their statistical distributions, just like the distance-dependent potential, it can be assumed that all of these angles obey Boltzmann statistical distribution, and once again the mean force potential  is used:Then, we could also get a total score In 3dRNAscore the two energy terms are combined together to get the final total energy:et al. . RMSD depicts the global geometry differences between two RNA 3D structures but it is usually difficult to describe the hydrogen bond networks of RNA molecules. Hence, some metrics accounting for hydrogen-bonding networks intramolecular in RNA have been proposed to assess RNA structures. One of the commonly used metrics specifically devised for RNA is DI (deformation index) proposed by Parisien . The DI n of MCC ,45:(15)In this work, we use both RMSD and DI to measure how well a RNA model recapitulates the corresponding experimental structure in the benchmark of the performance of 3dRNAscore and other scoring functions.An important function of scoring methods is to identify the native-like tertiary structure of target RNA in a pool of structural decoys correctly. To compare such ability of 3dRNAscore with three existing RNA knowledge-based potentials: RASP , KB 18)18) and RTwo tests are done on test set I and test set II, respectively  is eentclass1pt{minimaentclass1pt{minimaNon-canonical base pairs occur frequently in RNA tertiary folds and functional motifs –51. The Most statistical potentials for RNA structures are based on distance distribution of intermolecular atom pairs or residue pairs. 3dRNAscore incorporated an additional energy contribution based on the distribution of backbone dihedrals (torsion angles) in the light of molecular force field. This will be discussed in the ‘Discussion’ section.To verify the contribution of the dihedral-based energy, we tested the performance of single distance-dependent energy, single dihedral-dependent energy and combined total energy according to Equation . The resith residue and the (i + 2)th residue. The third peak is at the distance of 8.7 Å, and it results from the base-pairing interaction between adenine and uracil.Capriotti and coworkers have found that all-atom version of RASP, RASP-ALL, could well capture base-pairing and base-stacking interactions in RNA. We observed the same phenomenon using 3dRNAscore. It seems that base-pairing and base-stacking interactions are implicitly contained in all-atom distance-dependent statistical potential. For example, Figure Figure Evaluation function is used to select proper structure models. Here, we give an example of how to choose a structure model in a robust way during RNA tertiary structure prediction. We firstly use the RNA 3D structure building program 3dRNA  to prediTable The native structure tends to have the lowest free energy according to the thermodynamic hypothesis proposed by Anfinsen . So precA significant difference between 3dRNAscore and other RNA statistical potentials is the combination of the conventional distance-dependent energy with a dihedral-dependent energy. Thus, in principle, 3dRNAscore can take account of both overall shape and base pairs. The results above indicate a better performance of this combined potential. It is known that molecular force fields are the basis of molecular dynamics simulation to study conformations of biomolecules. Many force fields in use today consist of four components: bond stretching, angle bending, the rotation of bonds and non-bonded interactions. The distance-dependent statistical potential corresponds to non-bonded interactions part of a force field while the dihedral-dependent potential corresponds to bond rotation part. The latter can efficiently describe the flexibility of RNA molecules, which is one of the major features of RNA structures that are different from that of protein structures. Originally, we planned to design four different statistical potentials corresponding to the four components of a force field. However, we found that the potentials corresponding to bond stretching and angle bending have no significant effect because all of the decoy sets don't involve any variation of bond length and bond angle in the process of their generations.RNA backbone is rotameric ,53, and The Rosetta RNA scoring function (FARFAR)  is more Test set I is generated by Gaussians on distances and torsions and Test set II is generated by molecular dynamics in a relative short period of time, hence decoys in these two datasets have low diversity and are distributed around a local minimum free energy state  in the sense of free energy landscape. Results in these two sets show that 3dRNAscore is quite qualified for identifying local minimum free energy state and ranking structures around the local minimum. Test set III is a more real-world decoy set which is generated by RNA modelling with FARFAR , so decoOther limitations exist for knowledge-based statistical potentials at present. The limited number of RNA tertiary structures of the training set is still the major problem for developing knowledge-based potentials –56. AlmoIn this paper, we have developed a novel RNA knowledge-based potential for identifying native RNA structures and ranking predicted structures and functional structural motifs with non-canonical base pairs. We used a non-redundant RNA training set to train the parameters by combining the distances of paired atoms and torsion angles to construct our statistical potential. The benchmark tests show that our method could identify not only appropriate tertiary folds, but also tertiary motifs with the non-canonical base pairs. Although some limitations, 3dRNAscore performs consistently better than existing methods in both two cases.http://biophy.hust.edu.cn/download.html.Supplementary Data are available at NAR Online.


Streptomyces coelicolor is a model for studying bacteria renowned as the foremost source of natural products used clinically. Post-genomic studies have revealed complex patterns of gene expression and links to growth, morphological development and individual genes. However, the underlying regulation remains largely obscure, but undoubtedly involves steps after transcription initiation. Here we identify sites involved in RNA processing and degradation as well as transcription within a nucleotide-resolution map of the transcriptional landscape. This was achieved by combining RNA-sequencing approaches suited to the analysis of GC-rich organisms. Escherichia coli was analysed in parallel to validate the methodology and allow comparison. Previously, sites of RNA processing and degradation had not been mapped on a transcriptome-wide scale for E. coli. Through examples, we show the value of our approach and data sets. This includes the identification of new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor and the identification of host-encoded leaderless mRNA and rRNA processing associated with the generation of specialized ribosomes in E. coli. New regulatory small RNAs were identified for both organisms. Overall the results illustrate the diversity in mechanisms used by different bacterial groups to facilitate and regulate gene expression. Streptomyces coelicolor serves as an important model for studying the biology of streptomycetes, which are the source of numerous antibacterial, anticancer, immunosuppressive, anthelmintic and antifungal agents  and another that provides a ‘global’ view of transcript abundance and the boundaries of transcription (gRNA-seq) . M145 was grown until the mycelia became visibly pigmented. This point was chosen as it is known to coincide with exit from exponential growth and the onset of secondary metabolism , presumably because of the high prevalence of stable secondary structures. In the original and most widely used dRNA-seq approach to date, TSSs are identified by their resistance to treatment with TEX  scatterplots of the reads obtained before and after TAP treatment Fig. . For botcf. Fig. A and B, S. coelicolor and E. coli, we identified 1147 and 728 sites, respectively, that were associated with leading edges of transcription as well as being enriched following TAP treatment (Class I). The higher number associated with S. coelicolor probably reflects its larger gene content (7.8 versus 4.3k genes). TSSs were identified for all types of functional RNA: mono- and poly-cistronic mRNAs, ribosomal RNAs, transfer RNAs and small RNAs . For both organisms, we also identified, as described previously for P. acnes . This extends the association between lmRNA and the regulators of mobile genetic elements in E. coli, which has been a major bacterial model for the study of ‘leaderless’ translation  and the RhlB helicase. The former is involved in phospholipid biosynthesis , reverse transcription (RT) PCR . The start of the RhlB protein has been confirmed by N-terminal sequencing  PCR . MoreoveE. coli lmRNAs, or at least those generated by 5′ processing under conditions of stress, are translated by specialized ribosomes from which the last 43 nt of the 3′ end of 16S rRNA have been removed endonucleolytically by MazF, the toxic component of a toxin-antitoxin (TA) system  as the RT primer. A nested primer was used for the PCR step  to allow detection of 5′-hydroxylated ends.Recently, it has been shown that mazF strain during exponential growth produced a pattern similar to that of the wild-type strain in the absence of PNK treatment. However, in contrast, no substantial increase in the abundance or number of amplicons was detected following treatment with PNK. This suggested that in cultures in which the bulk of the cells are growing exponentially much of the cleavage that occurs at the −43 site and others in its vicinity are dependent on MazF. For RNA isolated from the ΔmazF strain during stationary phase, the detection of the 88 bp amplicon was dependent on PNK treatment. This suggests that during stationary phase the RNase that generates downstream products with a 5′-monophosphate group is not as active and an RNase other than MazF can cleave at the −43 site to produce downstream products with a 5′-hydroxyl group. In other words, processing of the 3′ end of 16S rRNA may represent a hub for the integration of signals from multiple regulatory paths. The identity of the amplicons corresponding to the −43 site and positions immediately upstream was confirmed by cutting the products of RLM-RT-PCR with BstEII . The origins of these cleavage sites are not known, as far as we are aware. We note that the reads associated with cleavages at the 5′ end of 23S rRNA  were substantially lower than those of cleavages at the 5′ end of 16S and 5S rRNA precursors. This could be due to differences in the efficiency of their cloning (as discussed later) or the actual abundance of the corresponding fragments being skewed as a result of mRNA enrichment or both. Nevertheless, the above analysis indicates that our dRNA-seq approach provides good coverage of the multiple steps involved in rRNA processing.Sites at the precise 3′ ends of 23S and 5S RNA were not detected as these are generated primarily via 3′ exonucleolytic trimming , an endonuclease with dual 5′ to 3′ exonucleolytic activity , which has dual endo/3′ to 5′ exonucleolytic activity  tRNA , which presumably adds CCA to tRNAs that are not transcribed with this motif  and tRNase Z (SCO2547), which could cut on the 3′ side of tRNAs. These cleavages presumably allow 3′ to 5′ exonucleolytic trimming of the tail prior to addition of the CCA by tRNA nucleotidyltransferase.In contrast to the situation in RNA Fig. D, which e 3′ end . S. coelrpsT  and another downstream of the RNase III sites in adhE that has been mapped previously, but not analysed , which might facilitate 3′ to 5′ exonucleolytic attack. Thus, our RNA-seq approach not only confirms, but extends knowledge of events controlling the activity and longevity of mRNA in E. coli. We also detected numerous processing sites within S. coelicolor mRNA. This included sites upstream of the coding segment of pnp (panel F), which are cut by RNase III to initiate a mechanism that autoregulates the cellular level of PNPase activity , an endoribonuclease with dual 5′ to 3′ exonuclease activity  suggesting that 5′ to 3′ exonucleolytic decay does not dominate bulk mRNA degradation in the former, even though it contains a ribonuclease with 5′ to 3′ exonucleolytic activity.With regard to mRNA, we were able to detect endonucleolytic sites known to be involved in both the degradation and processing of mRNA Fig. . This in Condon, , the preS. coelicolor and E. coli revealed a number of short transcripts of high abundance relative to the background. A proportion of these transcripts mapped to palindromic sequences, which are the signatures of intrinsic transcriptional terminators . Thus, as others have indicated, RNA-seq data can aid the accurate prediction of the 5′ ends of protein-coding regions  against the number of global RNA-seq reads obtained over the regions covered by the oligonucleotide probes. This revealed reasonable congruence , undecylprodigiosin (Red) and the calcium-dependent antibiotic (CDA) respectively , Leu (TAG) and Arg (CCG) (data not shown). Transcription of the biosynthetic genes for Act, Red and CDA was barely detectable suggesting secondary metabolite production may have been awaiting the triggering of the stringent response  that conceivably has a regulatory role . Regardless of the underlying molecular biology, the results of this study add to the growing body of evidence that bacteria differ substantially in the extent to which they use lmRNAs  in vivo. TEX treatment would remove the majority of the de-pyrophosphorylated species thereby allowing those with nascent 5′ triphosphorylated ends to be detected. The mapping of TSSs could also be improved in E. coli at least by analysing strains deficient in RppH; however, this might not always be desirable given the resulting changes in gene expression and presumably cell physiology  strain M145  from the Keio Collection , and E. coli K-12 strain MG1655 (seq) from the E. coli Genetic Stock Center . Both S. coelicolor and E. coli were cultivated with shaking (100–200 r.p.m.) in 250 ml Erlenmeyer flasks containing 50 ml of media. The flask for growing the former was fitted with a spring baffle to aid dispersed growth of the mycelia , a 1/8th volume of stop solution  was added to inhibit cell metabolism , as described previously for this organism .RNA for RNA-seq analysis was isolated from et al., et al., in vivo, was counted. This was done separately for each of the two libraries and the counts compared. It should be noted that as described previously  strain M145 and E. coli K-12 strain BW25113 were AL645882 and U00096.2 respectively. The RNA-seq data have been deposited in the GEO archive  as a service that included the construction of cDNA libraries before and after treatment with TAP, and the alignment of RNA sequence reads to the corresponding genome positions  was grown in Luria–Bertani (Amresco) as well as M9 minimal media (Sigma) supplemented with glucose  at 37°C with shaking (100 r.p.m.) until an OD600 of ∼ 0.5 at which point RNA was isolated as described previously , denatured by incubation at 90°C for 90 s, chilled on ice, and analysed along with other samples by denaturing electrophoreses using a 6% sequencing-type gel . Fractionated RNA was electro-transferred to a Hybond-N+ membrane (Amersham) using 20× saline-sodium citrate (SSC) buffer at 11 V for 1 h, and subsequently fixed to the membrane by UV cross-linking.RNA for northern blotting was isolated as described for the RNA-seq analysis, with the exception that the mRNA was not enriched, from a second batch of cultures. E. coli transcripts were probed using complementary oligonucleotides (see 32P using T4 polynucleotide kinase (Thermo Scientific) and γ-32P-ATP . The labelling reaction was carried out at 37°C for 30 min and stopped by the addition of EDTA, both as described by the vendor of the enzyme (Thermo Scientific). The radioactively labelled probes were precipitated by ethanol as described above, and resuspended in 20 μl of RNase-free water. The membrane was pre-hybridized with 3 ml of ULTRAhyb-Oligo Hybridization Buffer (Ambion) at 42oC for 30 min. Radiolabelled probe, which had been denatured by incubation at 90°C for 90 s and chilled on ice, was added to the hybridization tube. Hybridization was done at 42°C overnight. The membrane was washed twice with 20 ml of preheated washing buffer [5× SSC containing 0.5% (w/v) SDS] at 49°C for 30 min and exposed to Imaging Screen-K (Bio-Rad). The image was captured by Molecular Imager FX (Bio-Rad), and further processed using Quantity One (Bio-Rad) and GeneSys (Syngene) software.Specific ides see  labelledS. coelicolor transcripts were probed using riboprobes generated by in vitro transcription. The primers used to construct the templates for the riboprobes are listed in 32P UTP , 5 μM UTP, 0.5 mM rATP, rGTP and rCTP, 1 U yeast inorganic pyrophosphatase (Sigma), 80 U RNaseOUT™ (Invitrogen) in 1× T7 RNA polymerase buffer: 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine-(HCl)3, 25 mM NaCl, and 5 mM DTT. Reactions were incubated at 37°C for 3 h. Unincorporated nucleotides were removed by adding molecular biology-grade water (Sigma) to 50 μl and passing reaction products through G-25 spin columns, as per manufacturer's instructions . Hybridizations were carried out overnight in the PerfectHyb™ Plus Hybridization buffer (Sigma-Aldrich) at 68°C followed by 2 × 20 min washes in high stringency buffer  at 68°C. The hybridized blots were then exposed to a phosphoimager plate (Fuji) and read with a FLA 5000 scanner (Fuji).Specific S. coelicolor A3(2) strain M145 and E. coli K-12 strain BW25113 were AL645882 and U00096.2 respectively. The RNA-seq data have been deposited in the GEO archive (Barrett et al., For both types of RNA-seq, the reference genomes for The authors declare that they have no competing interests of a financial or non-financial nature.S. coelicolor microarray analysis, supervised by CPS. DRA, AHH and OR-L analysed individual transcripts, supervised by LK and VRK. LM performed the global RNA sequencing. GPvW provided valuable input. KJM, DRA and AHH wrote the article. All authors read and approved the final manuscript.KJM, Y-fL and DRA designed the transcriptome study. KJM, DRA and AHH performed the bulk of the RNA-seq analysis, with the assistance of MU. GB and EEL performed the