The study of two examples of endogenous genes with coding or non-coding natural antisense transcript partners provides evidence against the involvement of RNAi in the natural antisense-mediated regulation of mammalian gene expression. Antisense transcription, yielding both coding and non-coding RNA, is a widespread phenomenon in mammals. The mechanism by which natural antisense transcripts (NAT) may regulate gene expression are largely unknown. The aim of the present study was to explore the mechanism of reciprocal sense-antisense (S-AS) regulation by studying the effects of a coding and non-coding NAT on corresponding gene expression, and to investigate the possible involvement of endogenous RNA interference (RNAi) in S-AS interactions.We have examined the mechanism of S-AS RNA base pairing, using thymidylate synthase and hypoxia inducible factor-1α as primary examples of endogenous genes with coding and non-coding NAT partners, respectively. Here we provide direct evidence against S-AS RNA duplex formation in the cytoplasm of human cells and subsequent activation of RNAi.Collectively, our data demonstrate that NAT regulation of gene expression occurs through a pathway independent of Dicer associated RNAi. Moreover, we introduce an experimental strategy with utility for the functional examination of other S-AS pair interactions. Trans-NAT are complementary RNA transcribed from different chromosomal locations [cis-NAT have a tail-to-tail format with a 3' overlap, while 15% have a head-to-head format with a 5' overlapping region. The remaining molecules have intronic or coding sequence overlaps [Naturally occurring antisense transcripts (NAT) have been reported for 20% of the human genome -3. Recenense RNA ,7. By deocations . Chimeriocations . Over 70overlaps . Many NAoverlaps -13.The interaction between antisense and corresponding sense transcript partners does not follow a unified and predictable pattern . Here, wCaenorhabditis elegans, RNAi is an evolutionarily conserved, post-transcriptional gene silencing mechanism. The dsRNA is processed by the RNase III enzyme called Dicer into small duplex RNA molecules of approximately 21 to 22 nucleotides, termed small interfering RNA (siRNA). The siRNA molecules then interact with a multi-protein complex, termed RNA-induced silencing complex (RISC), resulting in sequence specific association of the activated RISC complex with the cognate RNA transcript. This interaction leads to sequence-specific cleavage of the target transcript [One of the most exciting findings in genome biology in recent years has been the discovery of RNA interference (RNAi), which has been proposed as a possible mechanism by which NAT may regulate gene expression ,21. RNAianscript ,23.In summary, NAT have been proposed to regulate gene transcription, RNA splicing, polyadenylation, editing, stability, transport, and translation . The aimin situ hybridization method was used to assess the simultaneous presence of both endogenous TS and rTSα. HeLa cells were grown on the surface of slides, fixed and treated with DNase . First strand cDNA was synthesized and subjected to in situ hybridization using strand specific intron spanning probes  of the vectors, made artificial RNA duplexes and then transfected them into the cells. To investigate the presence of RNA duplexes in transfected and untreated cells, cytoplasmic lysate was isolated and subsequently treated with RNAse A and T prior to separation on a polyacrylamide gel. Existing RNA duplexes were detected with radiolabeled probes for the S-AS overlap regions. As expected, S-AS duplexes were detected in cells transfected with IVT dsRNA and in cells transfected with the third vector (hairpin vector) designed to make a synthetic hairpin RNA duplex. Additionally, endogenous S-AS single-stranded RNA, as well as vector based RNA, were detected in RNAse negative samples. In cells overexpressed with sense, antisense or cells expressing endogenous levels of NAT, RNA duplexes were not detected . Although HeLa cells endogenously express both sense and antisense mRNA, we constructed three vectors that produce sense, antisense or consecutive S-AS overlapping mRNA in eukaryotic cells Figure . For twod Figure . RNA dupd Figure . These dIt is possible that putative RNA duplexes in the living cells are transient and labile and are processed to endogenous siRNA or other intermediate products rapidly. To investigate this possibility, we designed a Northern blot analysis with radiolabeled probes spanning the overlap region of the S-AS mRNA. These randomly designed probes, which can potentially detect S-AS sequences of any length from full length RNA to less than 20 base-pair (bp) Dicer products, were used to search for the presence of processed RNA. The hypothesis was that, if RNA duplexes are present, they should ultimately be processed by Dicer into the 21 bp RNA oligonucleotides. HeLa cells were transfected with the same vectors used in the previously described experiment, which produced sense, antisense, or hairpin RNA. The RNA duplexes from the S-AS overlap region produced by IVT served as a positive control and were transfected into the cells. Dicer products were only present in cells transfected with IVT dsRNA or cells transfected with a hairpin vector, which produced internal hairpin dsRNA Figure . As seenin vitro transcribed dsRNA from HIF or TS but were unchanged in cells with overexpressed S-AS transcripts. OAS2 levels were also up-regulated, by about 500-fold, only in the cells with IVT duplex RNA transfection. These data indicate that cytoplasmic RNA duplexes with S-AS mRNA are unlikely to form; nevertheless, it is possible that the IFN pathway may be unresponsive to intracellular RNA duplexes.The interferon signaling cascade is part of the cell's antiviral defence mechanism and can be triggered by dsRNA. Interferon (IFN)-β and 2',5' -oligoadenylate synthetase-2 (OAS2) mRNA levels were measured in cells overexpressing S-AS transcripts Figure . IFN-β mTaken together, the present results suggest that NAT do not form cytoplasmic RNA duplexes that activate RNAi mechanisms. Overlapping transcripts in an antisense orientation, be they protein coding or non-coding, have the potential to form dsRNA, a substrate for a number of different RNA-modification pathways. One prominent route for dsRNA is its breakdown by Dicer enzyme complexes into small RNA. We used several experimental approaches to identify the presence of RNA duplexes in the cytoplasm of cells, and to detect Dicer products involved in processing of dsRNA. Our results, using synthetic S-AS constructs as well as endogenous NAT, did not support the presence of cytoplasmic RNA duplexes or engagement of the RNAi mechanism.in silico predicted NAT candidates can be ruled out on this criterion alone. Expression levels of S-AS pairs are also important, as these could predict the mode of regulation. High levels of S-AS pairs in a single cell, as suggested from our experimental model, argue against RNAi involvement. However, another explanation for this phenomenon is a translation block or other kind of RNA mediated regulation of gene expression, without alteration of mRNA levels. Expression assessment and evaluation of mRNA levels would be recommended as a first step in studying other predicted S-AS candidates.The concomitant presence of both sense and antisense mRNA is one requirement for NAT regulation and many Alterations in antisense transcript levels can affect the sense mRNA level; however, S-AS changes are not necessarily reciprocal. Recently, we showed that antisense transcript knock down elevated sense transcript levels but the reverse interaction was not observed . This obOverall, the above observations are consistent with the conclusion that RNAi mechanisms are not engaged by S-AS gene regulation. Indeed, further support is derived from two other observations. First, small RNA molecules were not detected even for highly expressed S-AS pairs, implying Dicer-independent RNA processing. Second, the IFN cascade was not activated by NAT. Indeed, it may have been expected that, if at least 70% of mammalian genes have NAT and the mechanism is through RNA duplex formation, there would be a cumulative IFN response. Our studies show a dramatic IFN-β and OAS2 mRNA induction with dsRNA transfection, but not in cells overexpressing S-AS pairs, indicating the absence of duplexes of NAT.To date, there are no reports of endogenous mammalian siRNA derived from NAT in the literature . It is pet al. [Arabidopsis thaliana. Additionally, endogenous siRNA has been defined for plants; however, only 11 pairs of NAT had siRNA sequences mapped uniquely to the overlapping region of NAT, which substantiates the notion that RNAi is not involved in the processing of S-AS pairs [Consistent with data in the present investigation, Jen et al.  pursued AS pairs . In otheOur data suggest that antisense expression is not linked to transcript degradation pathways. However, our methods do not completely exclude the formation of RNA duplexes in the cell nucleus, or any proposed functions for NAT regulation of gene expression, such as editing, nuclear retention, splicing or transport. Although many different functions and mechanisms have been suggested for NAT, no systematic approaches for the classification or prediction of these mechanisms have been suggested to date. Our study could be a start for a functional approach to NAT studies that could lead to a categorization of NAT based on their unique bioinformatic features. Our methodology could also be expanded to provide a systematic approach to natural antisense mediated regulation of gene expression.2, 10 mM dNTP mixture, 10 pM random hexamers, 100 units RNase inhibitor, and 500 units of reverse transcriptase in a final volume of 200 μl. The reverse transcription (RT) reactions were completed using the following conditions: 30 minutes at room temperature, 3 hours at 42°C, and 5 minutes at 95°C. For in situ hybridization, the cells were incubated at 65°C for one hour in blocking buffer . After blocking, the cells were hybridized at 70°C for one hour with 10 μM of specific intron spanning probes . The slides were then washed two times with pre-warmed PBS. Hybridization of the probe directly to the RNA was done under the same conditions without RT.HeLa cells were grown on the surface of silane-coated slides overnight and fixed with 4% paraformaldehyde (pH 7.4) for 4 minutes. After air drying of the slides, a chamber was utilized for easy treatment of the attached cells with DNase at 37°C for 16 hours. DNase Master Mix contained 10× TurboDNase Buffer , 100 units DNase1, 100 units of TurboDNase, and 100 units of Suprasin in a final volume of 200 μl. The cells were then washed with 1× phosphate-buffered saline (PBS) and subsequently incubated at 95°C for 5 minutes. First strand cDNA was synthesized with an RT-Master Mix of 10× RT buffer , 2.5 mM MgClThe HeLa cultures were diluted to a few cells in each bright field. RNA was extracted from 15 individual cells that were picked under the guide of a confocal microscope. First strand cDNA synthesis was made from the RNA by using SMART and CDS III 3' oligonucleotides and Powerscript reverse transcriptase from Clontech  according to the manufacturer's instructions. The first strand cDNA was then used for PCR amplification using the LD primer, DSIII PCR primer, and Advantage2 Polymerase mix from the Clontech cDNA library kit.g for 5 minutes at 4°C. Cell pellets were washed three times with ice-cold PBS, pH 7.2, and lysed for 10 minutes on ice in three packed cell volumes of lysis buffer  Triton X-100). Nuclei were isolated by centrifugation at 5,000 g for 10 minutes at 4°C. The supernatant contained the cytoplasmic extract and was immediately used for RNA extraction with Trizol  Nuclear extracts were prepared by washing the pellet once in lysis buffer and twice in 1× PBS, pH 7.2. Nuclear RNA was then collected using Trizol reagent. Purity (>98%) and integrity of nuclei were determined microscopically.Cytoplasmic extracts were prepared from HeLa cells transfected with different vectors. Cells were harvested after 24 hour transfections and centrifuged at 1,000 Using the Direct Protect Lysate RPA kit from Ambion, cytoplasmic lysate was treated with RNase cocktail buffer and incubated with RNase A and T cocktail at 37°C for 30 minutes. Nucleases were removed by incubation with sodium sacrosyl and proteinase at 37°C for 30 minutes. RNA was precipitated using 99% ethanol and glycogen blue and subsequently DNase treated with TurboDNase (Ambion) prior to separation on a 5% denaturing PAGE/8 M urea. RNAse negative samples were treated exactly the same, except for addition of RNAse A and T to assess the specificity of the probe and efficacy of RNAse treatment.32P-labeled nucleotide and the Amersham random priming kit. All membranes were washed one time with low stringency and two times with high stringency buffer, each for 1 hour, and signal was detected with a Typhoon (Amersham) phosphor-imaging instrument.Total RNA was collected using Trizol (Invitrogen) and precipitated with 99% ethanol. Total RNA (30 μg) was loaded per lane and separated out on a 10% PAGE/urea gel. The RNA was then transferred onto a nylon membrane  and blocked with salmon sperm DNA for six hours. The blocked membrane was hybridized overnight with radiolabeled S-AS probes spanning the overlap region of the TS and rTSα genes. The probe was made by random priming of overlap DNA using HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells in logarithmic growth were transfected with plasmids containing the luciferase gene with either the sense or antisense overlap region or both. At 24 hours post-transfection, cells were used for further applications. The pGL3 control vector  was used for making all S-AS constructs. We engineered Pst1 and EcoR1 restriction sites downstream of the firefly luciferase for cloning. A BamH1 sequence was used to form a hairpin between overlap regions and to construct a vector with a consecutive S-AS sequence . The same vector was used as a template for IVT of S-AS overlap mRNA, using a MEGAscript transcription kit (Ambion). For IVT of HIF, transcript primers with a flanking T7 promoter sequence were designed and the PCR product then used for synthesizing duplex RNA.Real-time PCR was carried out with the GeneAmp 7000 machine (Applied Biosystems). The PCR reactions contained 20 ng cDNA, Sybrgreen or Universal Mastermix (Applied Biosystems), 300 nM of forward and reverse primers, and 200 nM of probe in a final reaction volume of 50 μl . The primers and probe were designed using PrimerExpress software (Applied Biosystems). They were strand specific for each S-AS pair and the probe covered exon boundaries to eliminate the chance of genomic DNA amplification. The PCR conditions for all genes were as follows: 50°C for 2 minutes and 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The results are based on the cycle threshold (Ct) values. Differences between the Ct values for the experimental genes and the reference gene  were calculated as ΔΔCt.in situ hybridization, cloning of Ts and rTSα and for in vitro transcription of HIF.The following additional data are available with the online version of this paper. Additional data file 1 is a table containing sequence information for all the primers and probes used in this study. Primers included were used for real-time PCR, in situ hybridization, cloning of Ts and rTSα and for in vitro transcription of HIF.Primers included were used for real-time PCR, Click here for file


Science and Immunity, respectively, have provided some of the first direct evidences that RNA silencing is an important component of antiviral defence in vertebrate cells. In addition to demonstrating RNA silencing to be critical to vertebrate innate immunity, these studies also highlight the potential of utilising virus-infection systems as models to refine our understanding on the molecular determinants of RNA silencing in vertebrate cells.In recent years, RNA silencing, usage of small double stranded RNAs of ~21 – 25 base pairs to regulate gene expression, has emerged as a powerful research tool to dissect the role of unknown host cell factors in this 'post-genomic' era. While the molecular mechanism of RNA silencing has not been precisely defined, the revelation that small RNA molecules are equipped with this regulatory function has transformed our thinking on the role of RNA in many facets of biology, illustrating the complexity and the dynamic interplay of cellular regulation. As plants and invertebrates lack the protein-based adaptive immunity that are found in jawed vertebrates, the ability of RNA silencing to shut down gene expression in a sequence-specific manner offers an explanation of how these organisms counteract pathogen invasions into host cells. It has been proposed that this type of RNA-mediated defence mechanism is an ancient form of immunity to offset the transgene-, transposon- and virus-mediated attack. However, whether 1) RNA silencing is a natural immune response in vertebrates to suppress pathogen invasion; or 2) vertebrate cells have evolved to counteract invasion in a 'RNA silencing' independent manner remains to be determined. A number of recent reports have provided tantalizing clues to support the view that RNA silencing functions as a physiological response to regulate viral infection in vertebrate cells. Amongst these, two manuscripts that are published in recent issues of  Caenorhabditis elegans [RNA silencing was originally recognised as post-transcriptional gene silencing in plants (PTGS) , co-supp elegans  and fung elegans ,6. The gWhen these small RNAs are generated from double-stranded RNA, they are known as small interfering RNAs (siRNAs); however, if these small RNAs are produced from within the cell as natural RNAs that fold into imperfect hairpin structures, they are referred to as microRNAs (miRNAs). siRNAs are produced by cleavage mediated through a ribonuclease-III (RNaseIII)-related enzyme known as Dicer, which gives rise to the siRNA duplex. This siRNA duplex is then unwound by RNA helicase and assembled into the RNA induced silencing complex (RISC). It is believed that the RISC then directs the siRNA to the target mRNA via sequence specificity which leads to cleavage of the target mRNA and silencing of the target gene. In contrast with siRNAs, miRNAs are first produced as ~70 nucleotides pri-microRNAs (pri-miRNA) in the nucleus, which are then cleaved by a RNaseIII-like enzyme known as Drosha to generate the pre-miRNA. The pre-miRNA is then transported into the cytoplasm aided by Exportin 5. Once the pre-miRNA has reached the cytoplasm, Dicer cleaves the pre-miRNA to generate the miRNA duplex. The miRNA duplex is subsequently unwound by RNA helicase and assembles with RISC. As seen with siRNA, some of the miRNA will be guided by RISC for cleavage and degradation of the target mRNA, however, most miRNA will suppress translation of the target mRNA by binding to the 3' untranslated region (3'UTR) of the target.C elegans can be spread to other uninfected cells in the organism to prevent further infection. This spreading of RNA silencing relies on an RNA-directed RNA polymerase (RdRP) to amplify the RNA silencing targeting sequences. Interestingly, no RdRP was identified in either Drosophila melanogaster or human genomes when a BLAST search was performed on the nearly completed genomes for these two species [Drosophila melanogaster and human genomes to amplify the siRNA signals as part of their immune responses could suggest that the existence of RNA silencing machineries in these species represents a 'molecular fossil' of an ancient innate immunity, and both Drosophila and vertebrates have since evolved to counter viral invasion through an RNA silencing independent mechanism.Because plants and invertebrates lack the protein-based adaptive immunity that is found in higher vertebrates, it is believed that the sequence-specific RNA silencing mechanism is important for the host cell to fight off invading-viruses, -pathogens and -nucleic acids . Similar species . The lacRecent studies have provided a number of indirect and tantalising clues to support the participation of RNA silencing in viral infection of vertebrates. Using a heterologous system, it has been shown that some of the mammalian virus-encoded proteins, such as influenza viral protein NS1 and vaccinia viral protein E3L, have a negative regulatory role on RNA silencing in both plant and insect cells, providing circumstantial evidence to illustrate the potential involvement of RNA silencing of these mammalian viral proteins in their natural vertebrate target hosts -11. OtheLecellier et al. were unable to identify viral derived small RNA that suppressed the propagation of PFV-1 in the host cell, they noted that PFV-1 infection promoted the non-specific accumulation of cellular derived miRNAs as a means to interfere with the miRNA regulatory pathway [Bennasser et al. found that human immunodeficiency virus type 1 (HIV-1) contains a rare siRNA precursor within its genomes, which can be utilised by the host cell to regulate HIV-1 infection [Bennasser et al. further demonstrated that the transcriptional activity of Tat is not essential for this SRS activity of HIV-1 Tat [Direct evidence of the importance of RNA silencing in vertebrates to control viral invasion has recently emerged from studies using human retroviruses. In the first study, primate foamy virus type 1 (PFV-1) was used to infect mammalian cells . While L pathway , a situa pathway . In contnfection . It is nnfection ,17. UsinIV-1 Tat ,17. ThisThe author(s) declare that they have no competing interests.


It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template.To characterize the biochemical properties of JEV RdRp, we expressed in in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the  Japanese encephalitis virus (JEV) is the most common cause of epidemic viral encephalitis worldwide, with approximately 50,000 cases and 15,000 deaths annually throughout a wide geographical range ,3.Flaviviridae family, which consists of the genera Flavivirus , Pestivirus , and Hepacivirus . JEV is an enveloped, positive-stranded RNA virus whose genome consists of a single-stranded RNA molecule of approximately 11 kb. The RNA genome of JEV consists of 98-nucleotide (nt) long 5' untranslated region (UTR) with the type-1 cap structure at its 5' terminus, a single open reading frame (ORF), and a 585-nt long 3'-UTR with no poly(A) tail at its 3' terminus  ATP and all four cold ribonucleotides (data not shown). The limited amount of [γ-32P] ATP (0.33 μM) and cold ATP (1 or 5 μM), which might be lower than the Km value for ATP required for efficient initiation of RNA synthesis, and less sensitive 5'-end labeling of the internally initiated RNA products with single radiolabeled ribonucleotide than the internal labeling at multiple sites with [α-32P] UTP, may explain this failure. Therefore, instead of using the [γ-32P]-ATP incorporation assay, we performed RdRp assays with insertion and deletion mutants of the 83-nt RNA, namely 83(Δ1) (deletion of U residue from the 3'-end of the 83-nt RNA), 83(+U) (addition of extra U residue to the 3'-end of the 83-nt RNA), and 83(+ST2) (addition of single base pair at the lower stem-region of the 83-nt RNA)  and 83(+U), still directed the synthesis of an 81-nt RNA product co-migrating with the RNA product synthesized from the wild-type template  and determined the exact size of the RNA product by running in parallel with RNA size markers. The result showed that the size of the major product from the 83-nt RNA template is 81-nt long, shorter than the template RNA Figs. , lane 3,NA) Fig.  to confiE. coli have an intrinsic TNTase activity; TNTase activity was not an activity of any cellular enzyme co-purified with the recombinant RdRps but an inherent function of the RdRps [32P] UTP and cold UTP (10 μM) or in the presence of [α-32P] UTP as a sole ribonucleotide substrate. Under these conditions, recombinant JEV NS5 synthesized template size and bigger-than-the template products but not the internally initiated 81-nt RNA product  and by de novo initiation with the RNA templates derived from JEV genome. Several reports showed that the TNTase activity is a common intrinsic property of many RdRps of Flaviviridae. The recombinant RdRps of HCV and BVDV have been shown to possess a TNTase activity under certain reaction conditions  UTP . The reaction mixture was incubated with 200 ng of RNA template for 2 hr at 30°C. For RdRp reaction with poly(A) template, 1 μg of RNA was added to the reaction mixture as described above, except that 10 μM UTP was included in the reaction with 2 μCi of [α-32P] UTP. The RdRp reactions were performed in the absence or presence of 10 pmol of oligonucleotide (U)20. For the TNTase assay, 10 μCi of [α-32P] UTP mixed with or without 10 μM of cold UTP was used as a single ribonucleotide in the RdRp reaction mixture with the 83-nt RNA as a template. [γ-32P]-ATP incorporation assay was performed as described above for the standard RdRp assay, except that the reaction mixture contained cold ribonucleotide mixture  and 0.33 μM [γ-32P]-ATP .RdRp assays were performed with 500 ng of purified JEV NS5 RdRp in a total volume of 25 μl containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 25 mM potassium glutamate, 1 mM MgCl32P-UMP incorporated into the products were measured in a LS-6500 multi-purpose scintillation counter (Beckman).RdRp reaction products were precipitated and resuspended in a denaturing loading buffer as described previously . After h4, 5% glycerol) containing either a low (50 mM) or high (500 mM) concentration of NaCl.RdRp products were digested with 4 units of nuclease S1 per ml (Promega) at 37°C for 30 min in nuclease S1 digestion buffer , and then phosphorylated with T4 polynucleotide kinase (New England Biolabs) in a final reaction volume of 50 μl containing 50 μCi [γ-32P] ATP and 0.1 mM cold ATP. After incubation at 37°C for 30 min, free nucleotides were removed using a Sephadex G-25 spin column, and the labeled RNA was purified from the denaturing polyacrylamide gel as described previously [32P-labeled 83-nt RNA was partially digested by incubation in Na2CO3/NaHCO3 buffer (pH 10) for 10 min at 90°C.For 5'-end eviously . The 32PRNA secondary structures were analyzed by the Mfold program . The strFor Western blot analysis, protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes (Amersham Biosciences). The samples were blocked using 5% skim milk in TBST , then reacted with an anti-His antibody (Qiagen) at a 1:1,000 dilution, followed by incubation of goat anti-mouse lgG conjugated with peroxidase (Sigma-Aldrich). The immunoblots were washed and then immunoreactive protein bands were visualized using enhanced chemiluminescence . Silver staining of gels and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis were performed as described previously .YGK, JSY, and CMK purified JEV NS5 protein and characterized its biochemical properties. JHK made the original clone of JEV NS5. YGK and JSY prepared the initial draft of the manuscript. JWO conceived of the study, coordinated all the components of the project, and prepared the final manuscript. All authors have read and approved the final manuscript.


C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo.Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which ~1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons.Fluorescence Activated Cell Sorting (FACS) was used to isolate C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system.We have described a microarray-based method, MAPCeL, for profiling gene expression in specific  C. elegans is a useful model system for addressing this issue. The C. elegans hermaphrodite nervous system is composed of exactly 302 neurons. The morphology and connectivity of each one of these neurons has been defined at high resolution ..unc-4::G mutants ,8. It isunc-4::GFP cells is presented in Fig. C. elegans embryonic cells were cultured for 24 hr to allow differentiation of GFP-labeled motor neurons. unc-4::GFP cells are rarely observed in freshly dissociated preparations but constitute about 2% of all cells after 1 day in culture. The delayed appearance of unc-4::GFP cells in culture is consistent with the developmental timing of unc-4::GFP expression in vivo; unc-4::GFP motor neurons are normally generated after morphogenesis is initiated .-55.C. el synapse . The enrs effect ,58. Simi neurons . Dopamindop-3), transcripts for all of the known components of these pathways are either enriched  or detected as EGs . Lack of enrichment of some of these components is consistent with the widespread utilization of G-protein signaling pathways in C. elegans neurons and muscle cells  are also expressed in these cells [ric-19 encodes a novel arfaptin-related protein that is believed to function in the Golgi in the generation of neurosecretory granules and may through this activity and subsequent neuropeptide signaling exert an indirect effect on ACh release from conventional synaptic vesicles [ric-19 is highly enriched in cholinergic motor neurons could be indicative of autocrine neuropeptide modulation of ACh secretion at the neuromuscular synapse. Consistent with this idea is our finding that ida-1, a conserved membrane component of the dense core vesicles in which neuropeptides are typically packaged, is an EG [UNC-31 (CAPS), a known facilitator of dense core vesicular release, is enriched [Our studies have revealed that a surprisingly large number of neuropeptide genes are transcribed in UNC-4 motor neurons. These results indicate that UNC-4 motor neurons are likely to exhibit significant neurosecretory activity. This conclusion is consistent with our finding that proteases required for neuropeptide processing and activation [se cells -75. Othevesicles ,77. Our is an EG . Finallyenriched . Plasma enriched  most of npr-2 and F59D12.1, results in locomotory defects that could be indicative of specific functions in DA motor neurons [npr-2 is a close relative of npr-1 (not detected) which has been shown to control social feeding behavior in response to the FLP-21 (not detected) peptide [F59D12.1 is most closely related to melatonin receptors but its in vivo ligand is unknown. Neuropeptides are believed to modulate secretion of classical neurotransmitters [Ascaris ventral nerve cord have been reported [C. elegans indicates that acetylcholine release at the neuromuscular junction is enhanced by neuropeptide activity [qα protein [In addition to secreting neuropeptides, UNC-4 motor neurons are also likely to respond to neuropeptide signaling. Transcripts for nine putative neuropeptide receptors are enriched. Table . RNAi of neurons . npr-2 i peptide . F59D12.smitters . Neuropereported . Geneticactivity  and that protein   A majority (21/27) of genes with known expression in unc-4::GFP neurons are detected in our microarray data set; (2) ~80% (15/18) of GFP reporters constructed for transcripts enriched in UNC-4 motor neurons are expressed in these cells in vivo  and function  are highly represented in our data sets. These findings parallel earlier studies showing that cultured C. elegans neurons and muscle cells adopt apparently normal morphological and physiological characteristics [C. elegans embryonic cells after an initial phase of inductive signaling events [C. elegans embryonic cells. In some cases, however, differentiation of a given neuron is likely to depend on specific intercellular signals that primary cultures will not provide. Thus, in every instance, it will be necessary to confirm microarray data by independent methods as described here.We have described MAPCeL, a microarray-based strategy for fingerprinting specific vations:  A majori2) ~80% 1/18 of GFeristics  and are g events . We haveC. elegans cells. Comparisons of microarray data from mutant animals with either supernumerary or absent sensory neurons in the male tail, have revealed genes that are preferentially expressed in these cells [C. elegans touch neurons [C. elegans embryonic cells [Previous studies have described other methods for cataloging transcripts from specific se cells . However neurons . Howeveric cells ,86.C. elegans body muscle cells and ciliated sensory neurons [C. elegans larvae . Thus, in principle, it should now be possible to obtain an accurate gene expression profile for virtually any C. elegans cell throughout development.MAPCeL cannot be used for postembryonic cells because these apparently do not arise in culture . Microar neurons ,88. We hdop-1 transcript and also detected elevated levels of transcripts encoding GPCRs for acetylcholine and serotonin, additional neurotransmitters known to modulate cholinergic motor neuron activity via G-protein signaling pathways [unc-4::GFP motor neurons express elevated transcript levels for nine different GPCRs with significant homology to insect or mammalian neuropeptide receptors. This signaling complexity is further compounded by the enrichment of transcripts for 18 members of the serpentine GPCR-like family in unc-4::GFP neurons  release at the DA neuromuscular junction is presumptively triggered by excitatory input from command interneurons. The strength of the DA cholinergic signal, however, may be strongly modulated by other cells that release neurotransmitters from distal locations. For example, dopamine is produced by 8 neurons, none of which are presynaptic to DA motor neurons . Dopaminpathways ,55. Enripathways ,81. We hunc-4::GFP motor neurons could be modulated by extracellular signals  and 2/3 of EGs (4050/6217) detected in unc-4::GFP neurons have human homologs (BLAST ≤ e-10)  equipped with a 488 nm argon laser. Emission filters were 530 ± 30 nm for GFP fluorescence and 585 ± 22 nm for PI fluorescence. The machine was flushed with egg buffer  prior tounc-4::GFP cells stained with PI were gated  and either plated on peanut lectin-coated slides for visualization [Immediately prior to sorting, supernatant from the 24 hour cultures was removed and discarded. 1 ml of egg buffer was added to the chamber coverglass. Cells are loosely adherent to poly-L-lysine and can be easily dislodged with gentle pipetting. 3 ml of egg buffer + cells were drawn into a 3 cc syringe and the suspension filtered with a 5 μm Durapore syringe filter. Propidium Iodide (PI) was added to the cell suspension at a final concentration of 5 μg/ml prior to sorting. Autofluorescence levels were established by flow cytometry of cells isolated from wildtype (i.e. non-GFP) embryos See Fig . Next, wated Fig  using thated Fig  was empilization  or used unc-4::GFP cells for comparison to RNA from the wildtype reference strain (N2). Cells were pelleted using low-speed centrifugation (300 × g). The supernatant was removed and RNA was extracted with a micro-RNA isolation kit (Stratagene) using the recommended volumes for 1 million cells. Typical yields were 1 pg total RNA/cell. 100 ng of total RNA was subjected to 2 rounds of amplification, as described in the Affymetrix GeneChip Eukaryotic Small Sample Target Labelling Protocol, with the following modifications. 100 ng (5 pmol) of T7-dT primer (5'-GGCCAGTGAATTGTAATAC GACTCACTATAGGGAGGCGG-(dT)24-3') was used as opposed to the recommended 100 pmol. RNA cleanup was achieved using the RNeasy mini kit (Qiagen); 300 μl of 100% ethanol  was added to the sample prior to absorption to the column matrix. Eluate was passed through the column 2× prior to washing to improve yields. The BioArray High Yield RNA Transcript Labeling Kit (Enzo) was used to biotinylate the sample in the second round of amplification. 10–15 μg of labeled aRNA (amplified RNA) was fragmented and hybridized to the Affymetrix C. elegans chip according to the Affymetrix Expression Analysis Technical Manual. The Agilent Bioanalyzer was used to assess RNA quality prior to labeling and to confirm fragmentation (<200 bp) before hybridization.RNA was prepared from FACS-isolated C. elegans Affymetrix array was used for all experiments. This chip was designed using the December 2000 genome sequence. All probe set information is available at  as well as . unc-4::GFP neurons were profiled in triplicate; baseline data  were obtained from four independent experiments with wildtype embryonic cells. Hybridization intensities for each experiment were scaled in comparison to a global average signal from the same array  as determined by Affymetrix MAS 5.0 (see below)  available through GeneTraffic (Iobion) in which the MM values are not considered  [unc-4::GFP cells vs the wildtype reference  of non-GFP cells contaminating this preparation (see above). Therefore, we excluded all 729 of these wildtype-enriched transcripts from the list of present calls in the unc-4::GFP motor neuron data set  ,99. A tweference . Althougdata set . Finally neurons .  and cosmid names are input into the script which then searches the wormbase mirror and matches Affy ID/cosmid name to a specific transcript. Cosmid names are used for this search when Affy IDs have not been mapped in wormbase. This information is used to acquire other linked annotations .A Wormbase mirror was established by downloading code and databases from . The keywords "DA motor neurons" generated a list of 68 citations, a similar search was conducted using the keywords "I5 pharyngeal neuron" and "SAB neurons" that detected an additional 21 citations. Expression patterns on wormbase were also searched using the "Cell identity" function to identify genes with documented expression in DA, SAB or I5. A list of 27 genes with documented expression in DA motor neurons, the I5 pharyngeal neuron and the SAB neurons was compiled from this information (An extensive literature search was performed using Textpresso ormation .Nematode strains were maintained at 20–25°C using standard culture methods . The wiltwk-30::GFP (25 ng/ul) was microinjected with the myo-3::dsRed2 marker (25 ng/ul) [[unc-119 (ed3) animals. For each construct, 1 'shot' was performed using a 1550 psi rupture disk at 28 inches of Hg vacuum. After a 1 hr recovery period, animals were washed from the plates with 7 ml M9 buffer and transferred to 7 NGM plates (1 ml/plate). Animals were grown at 20°C for 1 week. To pick transgenic animals, one-half of the plate was 'chunked' and added to a new 100 mm NGM plate; animals with wildtype movement were picked to 60 mm NGM plates and allowed to self. Worms derived from separate plates were considered independent lines; at least 2 lines were obtained for each construct.5 ng/ul) . Other t ng/ul) [. Micropa ng/ul) [ in a BioTransgenic animals and cultured cells were visualized by differential interference contrast (DIC), or epifluorescence microscopy using either a Zeiss Axioplan or Axiovert compound microscopes. Images were recorded with CCD cameras . Some images were recorded on a Zeiss 510 META confocal microscope.unc-4::GFP microarray experiments, analyzed microarray data, generated/scored GFP reporters, and helped draft the manuscript. SEV helped develop RNA amplification and bombardment protocols, generated/scored GFP reporters and helped draft the manuscript. SJB generated the reference dataset. CS and KO wrote Perl scripts used to annotate datasets, make comparisons, and identify Present genes. JM offered advice on statistical methods. DD and MV provided unpublished GFP reporter constructs as part of the Promoterome project. DMM collected images of GFP lines in the confocal microscope, oversaw all aspects of the project and helped draft the manuscript.RMF developed the FACS protocol, RNA amplification methods, performed promoter::GFP fusions Strain names, source and references for GFP reporters used to validate microarray data.Click here for fileIntensity Values Following Global Normalization (Affymetrix MAS 5.0) Raw intensity values were scaled against a global average intensity value calculated for each chip using Affymetrix MAS 5.0. Column A lists Affymetrix IDs for 22,548 features on the chip. These are indexed by rows with normalized intensity values for each independent hybridization and its Present/Absent (P/A) call. Columns labeled DMR 28, 30, 32, 34 include intensity values for hybridizations using RNA from all N2 (wildtype) cells. Columns labeled DM39, 51, 59 include the experimental hybridizations using RNA from FACS-isolated unc-4::GFP neurons.Click here for fileIntensity values after RMA normalization. Raw data from C. elegans chip. Affy IDs are listed in column A. Columns C-I show RMA normalized values for individual replicates. DMR 28, 30, 32, 34 are the baseline hybridizations using RNA from all N2 (wildtype) cells. DM39, 51, 59 are the experimental hybridizations using RNA from FACS-isolated unc-4::GFP neurons.Click here for fileN2 (wildtype) Expressed Genes (EGs) 9103 genes were called "Present" by MAS 5.0 in 3 out of 4 N2 hybridizations and are listed here as EGs. Affymetrix ID, Cosmid Name and common names (Columns A-C respectively) are given for each gene. Column D contains KOG descriptions and Column E indicates the dataset in which an individual gene is present.Click here for fileunc-4::GFP Present Genes 6828 genes were called "Present" by MAS 5.0 in 2 out of 3 unc-4::GFP hybridizations and are listed here.Click here for fileunc-4::GFP Present Genes minus N2 Enriched Genes The list of unc-4::GFP "present" calls in unc-4::GFP Present calls  RMA normalization ignores the MAS 5.0 P/A calls and therefore does not discard some of the "absent" genes that are excluded from unc-4::GFP Present Genes). As a result, the unc-4::GFP enriched genes (unc-4::GFP enriched genes were therefore restored to the list of unc-4::GFP Present Genes in unc-4::GFP cells. These unc-4::GFP Expressed Genes (EGs) are listed with Affy ID, Cosmid Name, Common Name and KOG description.ed genes  identifiClick here for fileExpressed Genes (EGs) EGs from N2  ≤1% were considered enriched. 1012 transcripts met these criteria and are listed according to statistical rank (Column A). Annotation includes Affy ID, Cosmid Name, Common Name and KOG description (Columns B-E); SAM score, Fold Change and q-value are listed (Columns F-H). We attribute the high ranking (2) of dpy-20 (T22B3.1) to its use as a coselectable marker in the generation of the unc-4::GFP transgenic line [nic line .Click here for fileN2 Enriched Genes 1586 genes are enriched 1.7 fold (FDR ≤1%) in N2 cells compared to unc-4::GFP neurons. These are listed according to statistical rank (Column A). Annotation includes Affy ID, Cosmid Name, Common Name and KOG description (Columns B-E); SAM score, Fold Change and q-value are listed (Columns F-H).Click here for fileunc-4::GFP neurons. Genes previously known to be expressed in Published literature and wormbase  were searched to identify 27 genes that are expressed in embryonic neurons that also express unc-4::GFP .Click here for fileunc-4::GFP motor neuron enriched genes with human homologs. 537 Enriched genes (-10.ed genes  and humaClick here for fileunc-4::GFP EGs with human homologs. 4050 Expressed genes (-10.ed genes  and humaClick here for fileunc-4::GFP enriched and expressed (EGs) transcripts in selected categories. Summary of This file is an expanded version of Table ). Statistical rank is indicated for each enriched transcript and "EG" designates transcripts that are EGs.Click here for fileunc-4::GFP neurons. Genes Expressed only in Affy ID, Cosmid Name, Common Name and KOG description for 968 genes that are expressed in unc-4::GFP neurons. These genes are not detected in the reference (N2) dataset.Click here for fileunc-4::GFP neurons. Gene families represented in A comprehensive description of neuronal transcripts organized according to gene family.Click here for fileLogic tree of data analysis methods.Click here for file


RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs. A novel in vitro method to study transcription and splicing leads to the proposal that linking transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs. Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intronic sequences to assemble exons into mature mRNA prior to export and translation . ThroughThe generation of pre-mRNAs by RNA polymerase II (Pol II) has recently been demonstrated to influence pre-mRNA splicing patterns –8. ThesWhile the evidence for a physical link between transcription and splicing is not as substantial as for capping and polyadenylation factors ,11, sevIndependent of the mechanism, the connection between transcription by Pol II and pre-mRNA processing appears to provide a significant advantage to achieve high levels of gene expression. To gain insights into the biochemical and kinetic nature that govern the temporal link between transcription and pre-mRNA splicing, we have optimized an in vitro system that simultaneously carries out transcription and splicing. We demonstrate that integrating the two events, pre-mRNA splicing and transcription by Pol II, ensures efficient processing. This is achieved because the increased local concentration of the splicing machinery protects the newly synthesized pre-mRNA from degradation. Therefore, the link between Pol II transcription and RNA splicing ensures that the vast majority of pre-mRNAs enter the RNA processing pathway.β-globin minigene, the adenovirus major late (AdML) promoter for Pol II, and the T7 promoter for T7 RNA polymerase  chase protocol along with the in vitro transcription/splicing assay. After incubation at reaction conditions for 20 min, an excess of uridine triphosphate (UTP) was added to quench the synthesis of radiolabeled pre-mRNAs. The fate of the labeled RNA that was generated during the initial pre-chase conditions was then evaluated independent of new pre-mRNA synthesis. The addition of excess UTP therefore uncouples splicing from RNA synthesis. The reactions were initiated with polymerase-specific DNA templates of theinigene A. As illl event . As expel event . HoweverFurther inspection of the splicing profiles shows that the majority of the Pol II transcripts can be accounted for by their conversion into intermediate lariat or spliced product B and2D.Pol II transcripts are capped at the 5′ end by the sequential action of the capping enzyme Hce1 and the cap methyltransferase Hcm1. Thus, increased stability of Pol II-generated transcripts may be caused by protection from 5′ exonucleases. However, transcript stability may also be increased due to spliceosomal activity. To evaluate the contribution of splicing on transcript stability, we made use of the nucleotide chase protocol to measure the degradation profiles for various Pol II transcripts coding for the wild-type (wt) minigene, minigenes with an abolished 5′ or 3′ splice site A, or an An additional approach was taken to evaluate the influence of splicing on the stability of the newly synthesized pre-mRNA. Rather than mutating the splice sites of the test substrates, the activity of the splicing machinery was targeted. Antisense oligonucleotides were designed to interact with either U2 snRNA  or U5 snspl of splicing is 0.03/min, independent of a link to transcription. In addition, the active concentration of spliceosomes at the conditions used was estimated by independent titration experiments to be ∼5 nM . ToIn higher eukaryotes, splice sites and regulatory sequences, such as exonic splicing enhancers and silencers, are not highly conserved ,39. TheWhen Pol II links pre-mRNA splicing to transcription, we observed robust intron removal. However, when the identical RNA was generated by T7 polymerase, splicing efficiency markedly dropped . These rThe results obtained from the analysis of alternative 3′ splice-site choice demonstrates that pre-mRNAs generated by Pol II are more likely to select the proximal splice site compared to pre-synthesized RNA substrates . These oPhoto-crosslinking studies and crystallography have shown that the nascent RNA exits the polymerase in the vicinity of the CTD ,44. In Chironomus tentans, upstream introns are excised while transcription is in progress; yet, downstream introns were removed post-transcriptionally [Recent cross-linking/immunoprecipitation experiments have described the co-transcriptional assembly of splicing components onto nascent pre-mRNAs, demonstrating that the assembly of the spliceosome, and possibly intron excision, occurs before Pol II terminates transcription ,37. Conionally . Given tThe results presented here underscore the importance of integrating gene expression processes, spatially and temporally. They support the growing concept that nuclear processing events are not independent and compartmentalized, but rather linked in a continuous trajectory. In this scenario, processing factors recruited at the DNA level are handed off to the newly synthesized RNA. It will be of particular interest to determine whether the link of transcription and pre-mRNA splicing merely increases the concentration of splicing factors or whether a higher order gene expression complex selectively deposits splicing factors onto the pre-mRNA. No doubt, the in vitro transcription/splicing assay described here will be a valuable tool in addressing these important questions.β-globin was excised at the 5′ end withPvuII and at the 3′ end withEcoRV and inserted into the SP73 plasmid, downstream of the T7 transcription start site. The sameβ-globin minigene fragment was inserted into thePflmI site (downstream of the RNA Pol II transcription start site) of the pMLP-U-A plasmid containing the AdML promoter (courtesy of Brenton Graveley). DNA templates containing the promoter and encoding regions of the RNAs were generated by PCR from these constructs and subsequently digested withEcoRI orMluI. To make the constructs used to evaluate the 5′ splice site, a PCR-generated product beginning immediately downstream of the 5′ splice site of exon one continuing through exon two ofβ-globin, was inserted downstream of a test 5′ splice site containing one of two 5′ splice sites, strong (5′-CAG|GTAAGTCA-3′) or abolished (5′-GTA|CTTCTACA-3′). This fragment was blunt-ended and inserted into thePflmI site of the same pMLP-U-A AdML promoter plasmid. Constructs used to evaluate the 3′ splice site, the SP73β-globin minigene constructs containing tandem repeats of the 3′ splice site, were described previously [PvuII andEcoRV and inserted intoPflmI site of the pMLP-U-A plasmid. DNA templates containing the promoter and encoding regions of the RNAs were generated by PCR from these constructs and subsequently digested with eitherEcoRI orHindIII.To generate the constructs used in the transcription/splicing assays, a fragment containing exons one through two ofviously . These mβ-globin minigenes were generated by PCR using a T7 forward primer, which anneals at the transcription initiation start site (of RNA Pol II) in the pMLP-U-Aβ-globin minigene constructs and a SP6 reverse primer, which anneals downstream of the minigenes. PCR products were subsequently digested withEcoRI, MluI, orHindIII. Pre-synthesized32P-labeled RNA transcripts were synthesized with T7 RNA polymerase in the presence of 2 mM m7G (5′) ppp (5′) G cap analog and resolved and purified on 6% denaturing PAGE [DNA templates containing the T7 promoter and encoding regions of theng PAGE .32P-UTP, 1.0 mM ATP, 20 mM creatine phosphate, 4 mM MgCl2, and incubated at 30 °C for a determined time course indicated in figures. To approximate the same RNA yield, T7 RNA polymerase was added to reactions containing DNA templates at a final concentration of 0.07 units/μL (˜3 nM). The amount of T7 polymerase added to the reactions was determined by titration experiments (unpublished data). Following incubation, reactions were digested with proteinase K, phenol chloroform extracted, ethanol precipitated, resolved on 8% denaturing PAGE, and analyzed by PhosphorImager analysis as previously described [Reactions were carried out in 50% nuclear extract  containi32P-labeled RNA Pol II transcript. Time points were taken immediately after the addition of the chase (defined as the zero chase time point) and as indicated inReactions were carried out as described for the transcription/splicing assay, except that after a 20-min incubation, 3 mM UTP was added to chase the32P-labeled RNAs were used as a splicing template and32P-labeled UTP was not added.Conditions were the same as described for the in vitro transcription/splicing assay except that pre-synthesized2, 40 μM anti-snRNA DNA oligonucleotides, and 93% nuclear extract at 30 °C for 1 h. This treatment resulted in splicing deficient nuclear extract (unpublished data). The depleted extracts were then used to carry out splicing reactions at identical experimental conditions as described above. The sequences of DNA oligonucleotide used were 5′-GGCCGAGAAGCGAT-3′ (against U2 snRNA) and 5′-TTAAGACTCAGAGTTGTTCC- 3′ (against U5 snRNA).Selective targeting of U2  or U5 sn5 to 3 × 105 cells/well 20–24 h before transfection in 24-well plates. 0.5 μg of theSMN2 minigenes were transfected using LipoFectamine 2000 and incubated for 24 h. Total RNA was extracted and then isolated as previously described [HeLa cells were grown in MEM supplemented with 10% (vol/vol) FBS and 2 mM glutamine and plated 1 × 10scribed . ReactioTM [m, kcat). They are free of charge to academic, United States government, and other nonprofit organizations.The model described above, kMech and Cellerator, are available at the Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California, United States  Diagrams of the intronless DNA template and the pre-synthesized intronless RNA transcript. (B) Autoradiogram of the transcription/splicing UTP chase experiment of intronless RNA generated from the Pol II template (left lane) compared to the degradation profile of a pre-synthesized transcript (right lane). The UTP chase was initiated after 20 min by adding saturating amounts of unlabeled UTP. The migration of the intronless RNA transcript is indicated on the right. (C) Quantitation of the data in (B) showing the degradation profile of the intronless transcripts over time.(495 KB PDF)Click here for additional data file.Figure S2SMN2 minigene, indicating the site of nucleotide insertion. (B) Representative autoradiogram displaying the exon 7 inclusion ratio forSMN2 constructs containing none or multiple 500 nucleotide intron 7 inserts. Band representing exon 7 inclusion or exclusion are indicated on the right. The fraction exon 7 inclusion is shown below each lane. (C) Quantitation of the data shown in (B) as a function of insert length.(A) Diagram of the(753 KB PDF)Click here for additional data file.Figure S3To estimate the concentration of active spliceosomes, increasing amounts of pre-mRNA (0.2–200 nM) were incubated at standard reaction conditions. The splicing rates determined from each of the time course reactions are plotted against the concentration of pre-mRNA added. Saturation of active spliceosome is indicated by the drop in the observed rate of intron removal, corresponding to ∼5 nM.(169 KB PDF)Click here for additional data file.Table S1(59 KB DOC)Click here for additional data file.


Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside ∼50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of ∼10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of ∼100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)–infected  Whereas cells store and replicate their genomes as DNA, most viruses have RNA genomes that replicate by using virus-specific pathways in the host cell. The largest class of RNA viruses, the positive-strand RNA viruses, replicate their genomes on intracellular membranes. However, little is understood about how and why these viruses use membranes in RNA replication. The well-studied flock house virus (FHV) replicates its RNA on mitochondrial membranes. We found that the single FHV RNA replication factor and newly synthesized FHV RNA localized predominantly in numerous infection-specific membrane vesicles inside the outer mitochondrial membrane. We used electron microscope tomography to image these membranes in three dimensions and found that the interior of each vesicle was connected to the cytoplasm by a single necked channel large enough to import ribonucleotide substrates and to export product RNA. The results suggest that FHV uses these vesicles as replication compartments, which may also protect replicating RNA from competing processes and host defenses. These findings complement results from other viruses to support possible parallels between genome replication by positive-strand RNA viruses and two distinct virus classes, double-stranded RNA and reverse-transcribing viruses. Drosophila cells provides insight into the replication process.All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements; here, a three-dimensional analysis of these complexes in flock house nodavirus-infected  Positive-strand RNA [(+)RNA] viruses contain messenger-sense, single-stranded RNA in their virions; they represent over a third of known virus genera; and they include many important human, animal, and plant pathogens . A commoFor many (+)RNA viruses—including alphaviruses , other mTo resolve these and other issues central to the mechanism of RNA replication, we used EM tomography (EMT) to provide the first, to our knowledge, three-dimensional (3-D) ultrastructural study of the membrane-bound RNA replication complexes of a (+)RNA virus. EMT generates high-resolution, 3-D images or tomograms by digitally processing a series of 50–100 electron micrographs collected as a specimen is tilted in 1–2 ° increments on an axis perpendicular to the electron beam . SimilarNodaviridae, as a (+)RNA virus with advantageous features for such studies. FHV has been used as a model to study RNA replication  templates RNAs appear to function only as RNA replication intermediates and are completely membrane-associated . (−)RNA thus is a key measure of a minimal RNA replication complex, because every mature RNA replication complex, active in (+)RNA synthesis, must contain at least one (−)RNA template. Therefore, the number of (−)RNAs gives an estimate of the maximal number of replication complexes per cell. (−)RNA1 accumulation plateaued by 8 hpi at ∼16,000 copies per cell D. (−)RNAThe number of protein A molecules plateaued by 8 hpi F, which Drosophila S2 cells at 12 hpi, processed them for transmission EM, and imaged 25 randomly sectioned cell profiles. All spherules in each imaged cell section were counted and divided by the cell section volume, which was calculated by measuring the cell area using ImageJ  and multiplying by the effective section thickness RNA molecules per cell. To measure the number of spherules per cell, we collected FHV-infected ness see . The numS2 cells  RNA viruses and the intracellular membranes on which they replicate their RNA genomes, we combined 3-D ultrastructural imaging with quantitative biochemical data and other results to model the architecture and organization of a nodavirus RNA replication complex. Immunogold labeling identified virus-induced membranous spherules as the sites of accumulation of the sole FHV RNA replication protein, protein A, and of FHV RNA synthesis  and 2. E3, based on the protein A molecular weight of 112 kDa [2. Thus, the average spherule interior membrane surface area of 6,000 nm2  or two dsRNA copies of all three FHV RNA species. Given this maximal occupancy, the estimate from biochemical data of an average of one (−)RNA1 and two (−)RNA2 templates per spherule , then the ratios of (−)RNA1 and (−)RNA2 to total spherules  imply eaMembrane spherules similar to those of FHV are induced by many other (+)RNA viruses including alphaviruses , other mIn addition to the many (+)RNA viruses whose RNA replication is associated with spherules, other (+)RNA viruses induce various, apparently distinct membrane rearrangements ,27,75,76Based on results with BMV, Schwartz et al. identified potential parallels between the assembly, structure, and function of membrane-associated RNA replication complexes and the cores of reverse-transcribing and dsRNA virus virions, including the sequestration of genomic RNA templates within a virus-induced compartment for replication ,19. The Drosophila S2 cells were grown at 28 °C in Gibco Drosophila serum-free media (SFM). Cells were dislodged by gentle scraping, pelleted, and resuspended at 107 cells/ml. FHV was added at a multiplicity of infection of 10 for all experiments. The cells and virus were incubated at 26 °C on a rotary shaker at 1,000 revolutions per minute (rpm) for 1 h to let the virus attach. After the hour incubation, the cells were plated onto a tissue culture dish and further incubated at 28 °C.Drosophila cells as described by Echalier [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid , 1 mM EGTA, and a protease inhibitor cocktail . After a 10 min incubation at room temperature, an equal volume of double isotonic buffer was added that consisted of the hypotonic buffer plus 0.5 M mannitol. The cells were lysed for 7 min using a pre-chilled Potter-Elvehjem homogenizer fitted with a Teflon pestle  and attached to a stirrer motor spinning at 250 rpm. The lysate was transferred to a Dounce homogenizer fitted with a type B glass pestle (Kimble-Kontes) and disrupted manually for 100 strokes on ice. Unbroken cells and nuclei were removed by two 10 min centrifugation steps at 500g at 4 °C. Mitochondria were pelleted by centrifugation at 3700g for 10 min at 4 °C, resuspended in an isotonic buffer containing 0.25 M mannitol, and washed by a second centrifugation at 7000g. BrUTP incorporation on the isolated mitochondria was performed at 28 °C for 1 h as described previously [Mitochondria were isolated from Echalier . Brieflyeviously .Drosophila cells were infected with FHV as above. At 8 hpi, cells were treated with 20 μg/ml actinomycin D for 30 min. FuGENE 6  was diluted 10-fold in phosphate buffered saline pH 7.4 and mixed with BrUTP and actinomycin D to final concentrations of 10 mM and 20 μg/ml, respectively. The FuGENE/BrUTP/actinomycin D mix was incubated for 15 min at room temperature then added to the cells and incubated at 4 °C for 15 min. After the 4 °C incubation, the cells were moved to 28 °C for a 15-min labeling period and then immediately fixed and processed for EM.Mouse monoclonal antibodies against FHV protein A have been described previously . MAb clohttp://www.aurion.nl), and incubated for 1 h with an anti-BrU antibody , diluted 1:100 in an incubation solution containing 100 mM phosphate-buffered saline pH 7.4 and 0.1% BSA-c (Aurion). Grids were washed six times in incubation solution without antibody, then incubated for 2 h with a goat-anti-mouse antibody conjugated to an ultrasmall gold particle (Aurion) that was diluted 1:100 in incubation solution, and washed six times again with incubation solution. Silver enhancement was performed for 30 min using R-GENT SE-EM (Aurion). Protein A immunogold EM was performed in the same manner using the mouse monoclonal antibody at a dilution of 1:100. Background labeling was determined using uninfected control cells. Labeling density was determined by calculating the surface area of spherules, mitochondria, and cytoplasm using the point-hit method [BrUTP immunolabeling fixation was performed as described previously , except t method . Specifihttp://www.mdyn.com/).Northern blotting was done as described previously . The numEscherichia coli as described previously [6 tag, and purified by talon column  affinity chromatography. To further purify protein A, we performed preparative electrophoresis using a BioRad mini-prep cell. A 6%, 9.5-cm gel was run at 200 V for 9 h with an elution speed of 150 μl/min. Fractions containing the purified, truncated protein A standard were collected and quantitated based on comparison with known standards of bovine serum albumin and β-galactosidase. We quantitated protein levels with Lumi-imager software (Roche).Western blotting was done as described previously . The numeviously . To puriFor conventional transmission EM, cells were fixed and embedded as previously described . For eleDrosophila S2 cells were collected at 12 hpi, processed for transmission EM, and sectioned into 70-nm-thick slices. For each of 25 randomly selected cells imaged in these sections, we then counted all observable spherules with diameters larger than 20 nm. The number of spherules counted for each cell then was divided by the relevant section volume, which was calculated by measuring the cell area using ImageJ  and multiplying by the effective section thickness. The effective section thickness is a correction used to avoid overcounting spherules with centers outside of the 70-nm physical section, which would otherwise be counted twice if adjacent sections were analyzed. As previously used to calculate synaptic vesicles per cell [Drosophila S2 cells [To calculate the total number of FHV-induced mitochondrial spherules per cell, FHV-infected per cell , this efS2 cells   was examined using a JEOL 1200FX electron microscope. 3-D reconstructions of portions of the cell containing FHV-infected mitochondria were generated using current techniques of electron tomography [Three separate FHV infections produced samples for six independent tomograms. To survey the preservation quality and FHV-infection efficiency of the aterial ∼ nm thickmography .Double-tilt tomography was performed by first collecting two tilt series of the same cellular region around orthogonal axes. After the first tilt series was complete using an angular increment of 2 ° from −66 ° to +66 °, the specimen grid was rotated 90 °, and the second tilt series was acquired from −60 ° to +66 °. The IMOD software suite was used for fiducial mark tracking and alignment. The positions of 30 gold particles were tracked in both tilt series. After alignment, the tomographic reconstruction was generated by a projective algorithm .http://www.tgs.com).Volume segmentation was performed by manual tracing in the planes of highest resolution with the program Xvoxtrace . The mitVideo S1(8.1 MB MOV)Click here for additional data file.Video S2z-series.Note how the morphology of the mitochondrion near the center changes during the progression through the (1.3 MB MOV)Click here for additional data file.Video S3y-axis and the green arrow points along the x-axis of the tomogram. The boundary of the total tomographic volume is outlined by the orange bounding box.Blue indicates outer mitochondrial membrane, white indicates FHV spherules, yellow indicates inner mitochondrial membrane. The red arrow points along the (5.8 MB MOV)Click here for additional data file.Video S4(1.0 MB MOV)Click here for additional data file.


Escherichia coli is a RNA binding protein which can translocate along the RNA and unwind the RNA:DNA hybrid using the RNA-dependent ATPase activity. In order to investigate the involvement of each of these functions in releasing RNA from the elongation complex, we have isolated different termination defective mutants of Rho by random mutagenesis, characterized them for their different functions and established the structure–function correlations from the available structural data of Rho. These mutations are located within the two domains; the N-terminal RNA binding domain  and in the C-terminal ATP binding domain  including the two important structural elements, the Q-loop  and R-loop (G324D). Termination defects of the mutants in primary RNA binding domain and Q-loop could not be restored under any conditions that we tested and these were also defective for most of the other functions of Rho. The termination defects of the mutants , which were mainly defective for secondary RNA binding and likely defective for translocase activity, could be restored under relaxed in vitro conditions. We also show that a mutation in a primary RNA binding domain (Y80C) can cause a defect in ATP binding and induce distinct conformational changes in the distal C-terminal domain, and these allosteric effects are not predictable from the crystal structure. We conclude that the interactions in the primary RNA binding domain and in the Q-loop are mandatory for RNA release to occur and propose that the interactions in the primary RNA binding modulate most of the other functions of Rho allosterically. The rate of ATP hydrolysis regulates the processivity of translocation along the RNA and is directly correlated with the efficiency of RNA release. NusG improves the speed of RNA release and is not involved in any other step.The transcription termination factor Rho of  Distinguished by their mechanism and structural features, there are two types of terminators in the Escherichia coli genome. (a) Intrinsic terminators characterized by a GC-rich inverted repeat followed by a oligo(dT) stretch that induce RNA polymerase to disengage RNA; and (b) the factor-dependent terminators, which depend on an essential protein factor, called Rho, for termination.Once the process of transcription is initiated, RNA polymerase (RNAP) makes a stable elongation complex with the DNA and the nascent RNA.rut) site.Rho is a homo-hexameric RNA/DNA helicase or translocase that dissociates RNA polymerase from DNA template and releases RNA. RNA-dependent ATPase activity of Rho provides free energy for these activities.rut siteRho-dependent termination involves a series of sequential events. At first Rho binds to the C-rich regions of the nascent RNA, called the rho gene, isolated the termination defective mutants, characterized each of them for their defects in other functions and established a structure–function relationship of the effects of these mutations based on the recent closed ring structure of Rho.in vitro experimental set-up to measure the Rho-mediated RNA release from a stalled EC, uncoupled from the transcription elongation process. This set-up enabled us to find the conditions under which some of these mutants regained the efficiency of RNA release and to understand the specific roles of ATP and NusG in the process of Rho-dependent termination. We concluded that the mutations in the N-terminal primary RNA binding domain can exert allosteric effects in the distal C-terminal domain, and this domain may act as a “master switch” that governs all the other functions of Rho. The primary RNA binding domain and the Q-loop are the most crucial structural elements for the RNA release function. Altered interactions in the secondary RNA binding domain only reduce the rate of ATPase activity which most likely slows down the translocation rate of Rho along the RNA. The rate of ATP hydrolysis is directly correlated with the efficiency of RNA release and involvement of NusG is restricted only to the final step of RNA release from the EC.Here, we investigated the involvement of different functions of Rho in releasing the RNA from the elongation complex. We randomly mutagenized the rho gene and screened for mutants defective for termination at two Rho-dependent terminators (see nutR/TR1 terminator is described below) suggest that the defect is not specific for a particular terminator. We also observed that mutants other than G51V, Y80C and Y274D caused a growth defect at 42 °C.We have randomly mutagenized the whole tors see . After slacZ reporter fused to a Rho-dependent terminator (Plac-H-19B nutR/TR1-lacZYA) to that without the terminator (Plac-lacZYA) was measured (read-through efficiency=%RT). This Rho-dependent terminator is derived from the nutR-cro region of a lambdoid phage H-19BnutR/TR1 of λ phage. These reporter fusions were present as a single copy prophage  of each of the mutants for a 34-mer DNA oligonucleotide, (dC)34. Occupancy of a DNA oligomer only in the primary RNA binding site of the Rho crystalThere are two distinct types of polynucleotide interaction sites in Rho.Kd) for oligo(dC)34 of different Rho mutants were measured by both gel retardation and filter binding assays 34 except Y80C, which did not show significant binding in both the gel retardation and filter binding assays. In order to check whether this defect is specific for a DNA oligo, we checked the efficiency of binding of a radiolabeled RNA oligo, rC10 to Y80C by UV cross-linking. This mutant showed significantly reduced cross-linking efficiency even at 20 μM of rC10 and did not show any gel-shift compared to WT Rho (data not shown). The binding defect of this mutation is consistent with the fact that this amino acid directly stacks against the base of the oligonucleotideThe dissociation constants (34 compared to WT. Therefore the stability of the oligonucleotide-bound complexes of these two mutants in the presence of both single-stranded DNA and RNA as competitors was tested. Rho complexed with end-labeled oligo(dC)34 was challenged with increasing concentrations of either unlabeled oligo(dC)34 10 (10-mer) and rC25 (25-mer) required to elicit half-maximal ATPase activities in the presence of excess ATP for estimating the binding efficiency to this site. For the oligos, rC10 and rC25, measurements were done in the presence of oligo(dC)34 at the primary site, because short RNA oligos (rC5–8) can elicit ATPase activity by binding to the secondary site only in the presence of oligo(dC).Interaction of RNA in the secondary site is mandatory to activate ATP hydrolysis.10 and rC25, to assess the binding defects in the secondary site. All the mutants, except I382N, failed to show significant amounts of ATP hydrolysis even at a very high concentration of the oligo, rC10  as substrate 34 in the primary site, might have failed to elicit the allosteric effect in the secondary site. Earlier, the reported tight binding mutant, G99V, also had a similar defect in binding to oligo rC10.Most likely, the defect in binding of oligo (dC)Kd,app) of ATP for WT and mutant Rho proteins in the absence of the RNA cofactor by using the UV-cross-linking technique. The apparent affinity of ATP for mutants Y274D, P279S and G324D was observed to be reduced by five-to eightfold, whereas it was comparable to WT for mutants N340S, G51V and I382N  as cofactor and compared with the WT Rho   the mutaBesides impairing the primary RNA binding, the Y80C mutation also caused significant defect in ATP binding. This led us to hypothesize that this change may cause conformational changes in the distal (∼ 40 Å) C-terminal domain allosterically. To test this hypothesis, we probed the conformations of WT and mutant Rho by limited proteolytic cleavages, fluorescence anisotropy and fluorescence quenching.Ksv) (Ksv increases as the tryptophan becomes more surface accessible. We observed that this tryptophan in the Y80C mutant is more surface accessible compared to the WT.We monitored the surface accessibility of the single tryptophan residue (W381), located ∼ 15 Å away from the ATP-binding site, by the fluorescence quenching technique using acrylamide as a neutral quencher.r) of the Tb-GTP moiety upon binding to the ATP binding pocket. The anisotropy (r) of a fluorophore gives the measure of the rotational freedom of the species and reports the conformational flexibility of the surroundings.r means less rotational freedom of the fluorescent probe. A lower value of r for Tb-GTP bound to Y80C compared to that obtained for WT Rho  defective for these Rho mutants we followed the time-course of RNA release from the stalled EC in the presence of different concentrations of ATP. We varied the concentrations of ATP to change the translocation rate. We preformed stalled EC, immobilized on magnetic beads, removed all the NTPs by extensive washing and WT or different mutant Rho proteins were added in the presence of 0.02 mM, 0.1 mM (data not shown) and 1 mM ATP. The time-course of RNA release in the supernatant was followed for 30 min, both in the absence  can affect functions and induce conformational changes in the distal C-terminal domain, which is not predictable from the structure of Rho. We did not observe any severe in vitro defect in I382N, which is not consistent with its in vivo phenotype. Probably a modest in vitro defect can be amplified under more stringent in vivo conditions.Here we report the isolation and characterization of the termination defective mutants of Rho from a random mutagenesis screen and attempt to find the involvement and importance of different functions of Rho in releasing RNA from the EC in light of the recent crystal structure of Rho.rut site, had been envisioned earlier.rut site once it starts tracking the nascent RNA. Based on the properties of these two point mutations in the primary RNA binding domain we propose that this domain of Rho not only works as an “eye” to find the rut site but also exerts allosteric control over the downstream interactions upon binding to RNA. Therefore, mutations in this domain block the subsequent steps in the process of RNA release. We have recently found that Psu, an inhibitor of Rho, which inhibits Rho-dependent termination by slowing down the rate of ATP hydrolysis, does not interact with the Y80C mutant.Importance of the polynucleotide interactions in the primary RNA binding domain of Rho in modulating other activities of Rho, apart from recognizing the What is the role of Q-loop? The Q-loop is a structural element, which is located at the entry point of secondary RNA binding domain and protrudes into the central hole of the Rho hexamer. A comparison of the open and closed ring structures of Rho reveals that this loop undergoes major conformational changes upon ring closureIt is known from the previous works that ATPase or NTPase activity of Rho is required for transcription terminationin vivo conditions where the RNA release has to be performed within a small time window. The RNA release step, in addition to their translocation defect, must be severely compromised in case of the mutants Y274D, G324D and N340S and therefore they are highly dependent on NusG. The final RNA release step may involve a direct interaction of Rho with the EC, so it is possible that the region of Rho defined by these amino acids is involved in the interaction with the EC.On the other hand, RNA release kinetics experiments  suggest via the Q-loop and the closed ring structure is formed. (3) This activates ATP hydrolysis and onsets the translocation of Rho along the mRNA, which brings Rho in the vicinity of the EC. (4) Rho then releases RNA possibly by pushing the EC or by pulling the RNA out of the EC by its translocase activity. It is also possible that Rho specifically interacts in the RNA exit channel and exerts allosteric effects on the stability of the EC. As NusG is only involved in increasing the rate of RNA release at this step, it is possible that NusG in the presence of Rho makes the EC more prone to termination.Based on the properties of different termination defective mutants of Rho and in the light of open and closed ring hexameric structures complexed with nucleic acids in primary and secondary RNA binding domains we sum-up the sequential steps that lead to the RNA release from the EC. (1) The primary RNA binding domain binds RNA and forms an open ring hexameric Rho. This interaction also involves conformational changes in the C-terminal domain. (2) RNA gets channeled into the secondary RNA binding domain in vivo? All the termination defective mutants of Rho that we have isolated are viable. Among them, Y80C is defective for most of the functions of Rho. Surprisingly, these apparent severe defects did not cause lethality to the cells. This raises the question, that how important is the process of Rho-dependent termination when the cells are growing exponentially and why Rho is essential to E. coli. We speculate that due to the multi-functional nature of Rho, it may very well have other essential function(s), which is more important than transcription termination.How important is Rho-dependent termination 32P]ATP  and [α-32P]CTP (3000 Ci/mmol) were purchased from Amersham. Antibiotics, IPTG, lysozyme, DTT and BSA were from USB. Primers for PCR, oligo(dC)34, rC10, rC25, galactose, Protein kinase A, acrylamide and TbCl3 were obtained from Sigma. Restriction endonucleases, polynucleotide kinase and T4 DNA ligase were from New England Biolabs. WT E. coli RNA polymerase holoenzyme was purchased from Epicenter Biotechnologies. Taq DNA polymerase was from Roche. Staphyloccocal aureus V8 protease and Sub-maxillary protease (Arg-C) were from Pierce. CNBr was obtained from Fluka.NTPs, poly(C), ATP (6000 Ci/mmol) and was stopped after 15 min by formic acid. Release of Pi was analyzed by exposing the TLC sheets to a Phosphorimager screen for 5 min and subsequently by scanning using Typhoon 9200 (Amersham) and quantified by Image QuantTL software. The concentration of poly(C) corresponding to half-maximal ATPase activity was determined by fitting the plot of amount of Pi release against concentration of poly(C) to a sigmoidal curve. To determine the concentration of rC10 and rC25 corresponding to half-maximal ATPase activity (34. The concentration of ATP was 1 mM. Other conditions were similar to those described above.ATPase activity of the WT and mutant Rho proteins was measured from the release of inorganic phosphate (Pi) from ATP after separating on the polyethyleneimine TLC sheets (Macherey-Nagel) using 0.75 M KHKm values of ATP for WT and Y80C mutant of Rho, in the same reaction mixture as described above, 5 nM of Rho was incubated with 10 μM of poly(C) at 30 °C for 10 min and ATP hydrolysis was initiated by the addition of different concentration of ATP (10 μM –100 μM). Aliquots were removed and mixed with 1.5 M formic acid at various time points. The product formation was linear in the time range (up to 5 min) that we used for calculating the initial rate of reaction. The initial rates of the reaction were determined by plotting the amount of ATP hydrolyzed versus time using linear regression method. Then the Km values were determined from the double-reciprocal Lineweaver–Burk plots.To calculate the The rate of ATP hydrolysis in the presence of poly(C) was measured in the same reaction mixture as described above. The concentrations of Rho, ATP and poly(C) were 10 nM, 1 mM and 20 μM, respectively. Reactions were performed at 30 °C and aliquots were removed at different time points  and mixed with 1.5 M formic acid.Kd,app) of ATP, UV cross-linking of labeled ATP was performed with 50 nM Rho and varying concentrations of [γ-32P]ATP (30 Ci/mmol) in a 10 μl reaction mixture containing 25 mM Tris–HCl (pH 8.0), 50 mM KCl, 5 mM MgCl2and 1 mM DTT. The samples were irradiated for 5 min at room temperature in CL-1000 Ultraviolet cross-linker from UVP. This method of determining the apparent dissociation constant was used earlier.10 to WT and Y80C mutant of Rho were performed. The samples were separated by SDS–PAGE, followed by scanning the gels in the phosphorimager Typhoon 9200 and quantified by Image-Quant software.To determine the apparent dissociation constant (KSV) was obtained using Stern-Volmer equation: (F0/F)350 = 1+ KSV[Q], where, F0 is the initial fluorescence intensity and Q is the concentration of acrylamide.All the fluorescence experiments were performed in the buffer containing 10 mM Tris–HCl (pH 7.0) and 100 mM KCl at 25 °C in a Perkin Elmer LS 55 Luminescence spectrometer. Changes in the tryptophan (W381) fluorescence at 350 nm of WT and Y80C Rho were monitored upon exciting at 295 nm. Fluorescence quenching was measured in the presence of different concentrations of a neutral quencher, acrylamide. Normalized emission was plotted against increasing concentrations of acrylamide and the quenching constant  complex upon excitation at 295 nm gives rise to an emission signal characterized by two emission peaks at 488 nm and 545 nm.32P]ATP (3000 Ci/mmol) using protein kinase A. The labeling reaction was done in buffer containing 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2 and 10 μM ATP. 30 μg of Rho were incubated with 60 units of the kinase for 2 h at 21 °C. After labeling, 0.8 μM of labeled Rho were incubated with 0.05 μg of V8 protease in transcription buffer supplemented with 1 mM ATP for the indicated time at 37 °C. The reactions were stopped by adding 6X SDS-loading dye and were stored on ice. Samples were heated to 95 °C for 3 min, prior to loading onto a SDS–12.5%(w/v) PAGE. Gels were exposed overnight to a phosphorimager screen and were scanned using Phosphor-imager Typhoon 9200 and analyzed by Image-Quant software. Molecular weight markers of end-labeled Rho were generated by cyanogen bromide (CNBr) and Sub-maxillary protease (Arg-C) mediated cleavages. Methionine-specific cleavage reactions using CNBr contained (pH adjusted to pH2 with 1 M HCl) 0.8 μM of labeled Rho, 1 M CNBr and 0.4%(w/v) SDS in a 10 μl reaction. Reaction mixtures for arginine specific cleavage contained 0.8 μM of labeled Rho, 2 μg of Sub-maxillary protease and 0.1%(w/v) SDS in a 10 μl reaction. Reactions were terminated after 10 min at 37 °C by addition of 6X SDS-loading dye followed by boiling.The conformational changes induced by the Y80C mutation were probed from the limited proteolysis pattern generated by V8 protease. For the purpose of end-labeling the protein, we used heart muscle protein kinase (HMK) tagged WT and Y80C Rho. The tag was introduced at the C terminus by PCR amplification using appropriate primers and was cloned in pET21b vector. The resultant Rho proteins have both His and HMK-tags at the C-terminal end. They were purified in a similar way as described above and the termination assays showed that these two tags did not interfere with the function of the WT Rho. HMK-tagged Rho proteins were radiolabeled with CTP (3000 Ci/mmol) was added to the reaction to label the EC23. The complex was chased with 20 μM NTPs in the presence of 10 μg/ml of rifampicin for 5 min at 37 °C. 50 nM WT Rho or Rho mutants and 200 nM NusG were added to the chase solution as indicated. The reaction was stopped by adding 20 μl of phenol after 5 min of incubation at 37 °C and the released RNA was phenol extracted. RNA was fractionated in a 10% sequencing gel. For the reactions with B8 RNAP, the enzyme concentration was 100 nM and the EC was chased with 100 μM NTPs.trp t' template with the lac operator sequence at position 161 was used to study RNA release from stalled elongation complex (RB) by WT and Rho mutants. In order to make the stalled roadblocked complex on streptavidin beads, lac repressor was added before addition of the chasing solution. EC23 was made first and then was chased with 20 μM NTPs and 10 μg/ml of rifampicin for 2 min to make the RB. This was followed by addition of 50 nM of either WT or mutant Rho proteins in the presence or absence of 200 nM NusG. The reaction was incubated at 37 °C for 5 min and the supernatant was separated from streptavidin beads on a magnetic stand to measure the released RNA from the EC (m the EC .trp t' template as described above, except that EC23 was chased with 50 μM NTPs. The RB was then washed extensively to remove the unincorporated NTPs and was re-suspended in T buffer supplemented with different concentrations of ATP (For the kinetics of RNA release from the RB, under different concentrations of ATP both in the presence or absence of NusG, RB was at first formed at position 161 of the s of ATP . Followi