Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of the RNA helicase gene family in crop growth and development.RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in  Escherichia coli to humans, viruses and plants. The helicases from all of these organisms represent a large gene family that may have a predominant role in modulating environmental responses. These proteins can be grouped into families based on sequence homologies Helicases have been identified in organisms ranging from Arabidopsis DEAD-box RNA helicases, LOS4, STRS1 and STRS2, were shown to be involved in the stress responses to various abiotic stresses HUA ENHANCER2, encoding a putative DExH-box RNA helicase, was shown to be active in both B and C pathways in the flower and to affect vegetative and inflorescence development in ArabidopsisVDL gene in tobacco was reported to play an important role in chloroplast differentiation and plant morphogenesis Arabidopsis DExH box helicase CAF/DICER-LIKE 1 has been shown to be critical for the biogenesis of microRNAs and plant development Zea mays, MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA metabolism Arabidopsis TEBICHI has been shown to be required for the regulation of cell division and differentiation in meristems ISE2, which is localised to cytoplasmic granules, has been shown to be involved in plasmodesmata function during embryogenesis in ArabidopsisSLOW WALKER3 has been shown to be essential for female gametogenesis as a putative DEAD-box RNA helicase in ArabidopsisThe RNA helicase family in plants is larger and more diverse than in other systems Escherichia coli have been identified and studied extensively, including 5 DEAD-box and 13 DExH-box RNA helicase genes Arabidopsis and Oryza sativa, which contain 113 and 115 RNA helicase genes, respectively, has been reported Despite the diversity of their biological functions and the wide range of organisms in which these proteins have been identified, high sequence conservation has been maintained in the large group of helicases, suggesting that all the helicase genes evolved from a common ancestor. Hence, signature sequences can be used efficiently for the detection and the prediction of new helicases in the genome databases Arabidopsis and rice have been performed in the past ten years, focused on categories such as the RING-finger, receptor-like kinase gene family, MAPK and MAPKK gene family, KT/HAK/KUP potassium transporters gene family, NAC proteins and siRNAs MuDR-related transposable elements, beta-glucosidase gene family, auxin response factor (ARF) gene family and two-component signal system (TCS) genes Zea mays or Glycine max. Extensive genome-wide comparative in silico analysis of the RNA helicase gene family in the completed genomes of Zea mays and Glycine max could identify numerous known or novel gene families associated with defence, photomorphogenesis, gene regulation, development, metabolism, transportation and/or stress tolerance.These studies added more evidence of the role of RNA helicase genes in development and response to abiotic stresses in different species, but related genome-wide resources are limited in two other important crops, maize and soybean. In addition, several whole-genome analysis studies in Arabidopsis, Oryza sativa, Zea mays and Glycine max genomes, respectively, by genome-wide comparative in silico analysis. Each of the different subfamilies, such as DEAD-box, DEAH-box or DExD/H-box, has different rate of alternative splicing in each species. The chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. The phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Additionally, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 RNA helicase genes in Arabidopsis and 13 RNA helicase genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. The comparative genome-wide analysis provides valuable information for understanding the classification and putative functions of the RNA helicase gene family and new insight into the organisation, evolution and functions of the RNA helicase gene family in crop growth and development.In this study, we identified a total 161, 149, 136 and 213 RNA helicase genes in the Arabidopsis, Oryza sativa, Zea mays and Glycine max, we used bioinformatic methods to gather extensive information regarding this family. A total of 161 genes that encode 217 RNA helicase proteins were identified as potential members of the RNA helicase superfamily within the Arabidopsis genome (http://www.tair.org/), whereas 149 genes encoded 199 RNA helicase proteins were identified in the Oryza sativa genome (http://www.phytozome.net/) (Arabidopsis (113) or rice (115) Glycine max and Zea mays have not reported in detail. In our results, we identified a total of 136 and 213 RNA helicase genes in the Zea mays and Glycine max genome (http://www.phytozome.net/), respectively (To identify the members of the RNA helicase gene family in me.net/) . Our preectively .Arabidopsis, Oryza sativa, Zea mays and Glycine max , DEAH-box (40/33/31/48 genes) and DExD/H-box (71/65/50/78 genes) in the four species cine max , respectectively . In Zea Arabidopsis RNA helicase protein genes were analysed first in our study. The chromosomes locations analysis showed that the Arabidopsis RNA helicase protein genes were distributed across all 5 chromosomes with different densities from 9.9% (chromosome 4) to 30.4% (chromosome 1) (Oryza sativa chromosomes from 2.0% (chromosome 12) to 19.5% (chromosome 1) , while chromosomes 6 and 9 contained 7 RNA helicase protein genes (5.1%)  to 10.8% (chromosome 8)  . Second,osome 1) . Only 3 s (5.1%) . Compareosome 8) . To deteectively . It is nArabidopsis DEAD-box RNA helicases, LOS4, STRS1 and STRS2, were shown to be involved in responses to multiple abiotic stresses Arabidopsis LOS4 (At3G53110). In addition, the phylogenetic tree analyses showed that STRS1 (At1G31970) has a high level of identity to Glyma01g01390, Glyma09g34390, LOC_Os07g20580 and GRMAZM2G076484. An analysis of the DEAH-box RNA helicase proteins showed that this subfamily could be further classified into nine subgroups  and AtHELPS (AT3G46960) as LOC_Os02g50560 and Glyma18g07510, respectively. At the same time, the phylogenetic tree analyses revealed that OsBIRH1 (LOC_Os03g01830) has a higher homology to Arabidopsis AT3G06980, which is in accord with preceding research To determine their evolutionary relationship, the phylogenetic relationship of each subfamily of the RNA helicase proteins was examined by aligning their amino acid sequences and implementing the neighbour-joining method in MEGA 5.0. The phylogenetic tree analyses showed that the whole of DEAD-box (332), DEAH-box (192) and DExD/H-box (355) RNA helicase proteins in four species, respectively . We alsoubgroups , whereasubgroups . ISE2 anArabidopsis, 144 genes (89.4%) were expressed in at least one of the development stages tested. More than half of the predicted RNA helicase genes were expressed in ten different development stages with various expression levels, including senescence, mature siliques, flowers and siliques, developed flower, young flower, bolting, developed rosette, young rosette, seedling and germinated seed. AT2G44980, AT3G19760, AT3G53110 (LOS4), AT4G18465, AT5G11200 and AT5G51280 were highly expressed in senescence stage. Our results showed that the most RNA helicase genes were expressed in more than 20 tissues in Arabidopsis with different expression levels and many predicted Arabidopsis RNA helicase genes were expressed in primary cell, seedling, inflorescence, silique, shoot and roots. Forty-four genes were highly expressed in sperm cells and AT5G10370 and AT5G61140 were only highly expressed in primary root tissue  were expressed in at least one of the development stages tested and several genes had high expression levels in the flowering stage  displayed tissue-specific expression patterns, which was not detected in rosette leaf and cauline leaf  database (http://www.arabidopsis.org/) and the Rice Genome Annotation Project (RAP) database (http://rice.plantbiology.msu.edu/) Zea mays and Glycine max. Therefore, the biological functions of a majority of the crop RNA helicases require further investigation.RNA helicases are found in various organisms, ranging from prokaryotes to mammals, and have become a focus of interest in recent years due to their participation in diverse cellular processes Arabidopsis and other crop species genomes by genome-wide comparative in silico analysis, including the gene classification, chromosomal locations, phylogenetic tree and expression profiles in different tissues and development stages under normal growth conditions.In this study, we presented a complete analysis of the RNA helicase gene family in Arabidopsis and 115 members in rice Arabidopsis and rice, respectively. We speculate that this phenomenon may be due to continual updates of the TAIR and RAP database. In addition, we also identified a total of 136 RNA helicase genes in maize and 213 members in soybean. Compared with Arabidopsis (genome size 125 Mb) Arabidopsis genome, 37,500 genes in rice genome, 50,000 genes in maize genome and 66,000 genes in soybean genome), the percentage of the RNA helicase gene family is similar in four species , except higher in Arabidopsis. Although the genome and the total genes number in four species is very different, it still not show differ greatly in the size of RNA helicase gene family. The presence of a large helicase gene family in four species suggests the RNA helicases play important roles in diverse processes. We further compared the number of RNA helicase genes in different subfamilies among Arabidopsis, Oryza sativa, Zea mays and Glycine max (Glycine max than in Arabidopsis (56), Oryza sativa (50), and Zea mays (79), rather than the number of different genes in each of the subfamilies.The RNA helicase gene family has 113 members in cine max . As showArabidopsis thaliana and rice genes can produce multiple diverse mRNA molecules by alternative splicing Arabidopsis thaliana and rice (>25%) was less than the average alternative splicing frequency of whole genome. Our data concerning the alternative splicing frequency of RNA helicase genes in different crop species will not only provide information on mechanisms of gene regulation through alternative splicing in future but also facilitate our understanding of the regulation of RNA helicase genes in crop growth and development. To our knowledge, this is the first report of a genome-wide analysis of the crop RNA helicase gene family. The different gene subfamilies and alternative splicing frequency of the RNA helicases might mirror the diverse functions of these genes in RNA metabolism.Among the total RNA helicase genes in the four species, the percentage of alternative splicing is lowest in soybean (14.11%) and highest in maize (35.81%). The data showed that the phenomenon of alternative splicing of RNA helicase genes is more common in dicot than in monocot. The regulation of alternative splicing is a key step in the control of gene expression, as splicing variants have different biological functions and regulatory features. Alternative splicing is one of the most complex cellular processes in eukaryotes, where information must be processed differently at different times (such as different development stages) or a very high level of diversity is required. Only a small number of alternative splicing events have been reported in plants. Recent progress has occurred in characterising the splicing signals in plant pre-mRNAs, in identifying the mutants affected in splicing and in discovering new examples of alternatively spliced mRNAs. Furthermore, although data from both animals and plants suggest tissue-specific and temporal regulation of alternative splicing Arabidopsis, rice and maize, more than 80% RNA helicase genes were expressed in at least one of the development stages and tissues tested , rice , maize  and soybean  Arabidopsis helicase gene sequences, which were downloaded from the Arabidopsis genome TAIR 9.0 release (http://www.arabidopsis.org/), were used as query sequences to perform multiple database searches against the proteome and genome files downloaded from the Phytozome database (http://www.phytozome.net/). Stand-alone versions of BLASTP and TBLASTN (http://blast.ncbi.nlm.nih.gov), which are available from the NCBI, were used with an e-value cutoff set to 1e-003 http://pfam.sanger.ac.uk/) and SMART (http://smart.embl-heidelberg. de/) with the default cutoff parameters http://pfam.sbc.su.se/). Then, we obtained the sequences with the PF00271 Pfam number, which contained a typical helicase domain, from the genome sequences using a Perl-based script. Finally, all of the protein sequences were compared with known helicase sequences using ClustalX  to verify the sequences were candidate helicases To identify the members of the helicase gene family in http://expasy.org/) The isoelectric points and molecular weights of the proteins were obtained with the help of the proteomics and sequence analysis tools on the ExPASy Proteomics Server (http://www.phytozome.net/) using a Perl-based program and mapped to the chromosomes using the MapDraw and Photoshop tools.The chromosomal locations were retrieved from the genome data downloaded from the Phytozome database  http://www.megasoftware.net/) using the p-distance and complete deletion option parameters The helicase sequences were aligned using the ClustalX program with BLOSUM30 as the protein-weight matrix. The MUSCLE program (version 3.52) was also used to perform multiple sequence alignments to confirm the ClustalX results (https://www.genevestigator.com/gv/) with the Arabidopsis (ATH1∶22 k array), Oryza sativa (OS_51 k: Rice Genome 51 k array), Zea mays (ZM_84 k: Nimblegen Maize 385 k) and Glycine max (GM_60 k: Soybean Genome Array) Gene Chip platform. Then, the identified helicase-containing gene IDs were used as query sequences to perform searches in the Gene Chip platform of Genevestigator.Microarray expression data from various datasets were obtained using Genevestigator  seeds were surface-sterilized and sown on MS plates. Seeds were stratified at 4°C for 2 days and then transferred to 22°C for 2 weeks. One month-old plants grown under a 16-h-light/8-h-dark photoperiod at 22°C with cool white light (120 mmol photons m−2 s−1) were used for sampling. For RNA extraction, the different tissues were frozen and stored in liquid nitrogen immediately after harvest.For maize inbred line Qi 319 , embryos 25 days after pollination was harvested from greenhouse-grown plants in sand under 16 h of light (25°C) and 8 h of dark (20°C), and eight-week-old seedling tissues and organs were harvested for expression analysis. Samples were collected and were immediately frozen in liquid nitrogen for further use. Two biological replicates were performed for each sample.Total RNAs were extracted using Trizol according to the manufacturer’s instructions  from leaves of maize seedlings with different treatments. The first strand cDNAs were synthesised using First Strand cDNA Synthesis kit .Zmactin gene was used as an internal reference for all the qRT-PCR analysis. Each treatment was repeated three times independently. Relative gene expression was calculated according to the delta-delta Ct method of the system. The primers used are described in Real-time quantification RT-PCR reactions were performed in Bio-RAD MyiQ™ Real-time PCR Detection System  using the TransStart Top Green qPCR SuperMix  according to the manufacturer’s instructions. Each PCR reaction , 0.5 µL of each primer, and appropriately diluted cDNA. The thermal cycling conditions were 95°C for 30 s followed by 45 cycles of 95°C for 15 s, 55°C −60°C for 30 s, and 72°C for 15 s. The Figure S1Phylogenetic tree analysis of RNA helicase in Arabidopsis. From left to right are the DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins, respectively. The scale bar represents 0.2, 0.2 and 0.2 substitutions per sequence position, respectively. Sister pairs of paralogous helicase genes were indicated by red shadow, which had very strong bootstrap support (>90%).(TIF)Click here for additional data file.Figure S2Phylogenetic tree analysis of RNA helicase in Oryza sativa. From left to right are the DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins, respectively. The scale bar represents 0.2, 0.2 and 0.2 substitutions per sequence position, respectively. Sister pairs of paralogous helicase genes were indicated by red shadow, which had very strong bootstrap support (>90%).(TIF)Click here for additional data file.Figure S3Phylogenetic tree analysis of RNA helicase in Zea mays. From left to right are the DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins, respectively. The scale bar represents 0.2, 0.2 and 0.2 substitutions per sequence position, respectively. Sister pairs of paralogous helicase genes were indicated by red shadow, which had very strong bootstrap support (>90%).(TIF)Click here for additional data file.Figure S4Phylogenetic tree analysis of RNA helicase in Glycine max. From left to right are the DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins, respectively. The scale bar represents 0.2, 0.2 and 0.2 substitutions per sequence position, respectively. Sister pairs of paralogous helicase genes were indicated by red shadow, which had very strong bootstrap support (>90%).(TIF)Click here for additional data file.Table S1RNA helicase genes in Arabidopsis, Oryza sativa, Zea mays, Glycine max.(DOC)Click here for additional data file.Table S2The primers used in the Real-time quantification RT-PCR reactions.(DOC)Click here for additional data file.


The majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. In this study, mRNA fractions from the cytoplasm and from whole cells  were prepared from three human cell lines and sequenced using massive parallel sequencing.For all three cell lines, of about 15000 detected genes approximately 400 to 1400 genes were detected in different amounts in the cytoplasmic and total RNA fractions. Transcripts detected at higher levels in the total RNA fraction had longer coding sequences and higher number of miRNA target sites. Transcripts detected at higher levels in the cytoplasmic fraction were shorter or contained shorter untranslated regions. Nuclear retention of transcripts and mRNA degradation via miRNA pathway might contribute to this differential detection of genes. The consequence of the differential detection was further investigated by comparison to proteomics data. Interestingly, the expression profiles of cytoplasmic and total RNA correlated equally well with protein abundance levels indicating regulation at a higher level.We conclude that expression levels derived from the total RNA fraction be regarded as an appropriate estimate of the amount of mRNAs present in a given cell population, independent of the coding sequence length or UTRs. The advent of sequence-based assays of transcriptomes (RNA-Seq) has allowed better quantification of mRNA, with less bias and greater dynamic range than microarrays ,2. Howevet al.. The extracted RNA samples were analyzed using an Experion automated electrophoresis system  with the standard-sensitivity RNA chip. All of the total RNA samples displayed a total RNA signature peak; as did one replicate of the cytoplasmic fraction of U-2 OS, which was discarded.Each cell-line was prepared in quadruplicate, with four biological replicates for total RNA and four for cytoplasmic RNA. A total of 3 μg of high-quality RNA (RNA integrity number = 10) per sample was used as input material for the mRNA sample preparations. The concentration and the RNA integrity number of the samples were determined from the run with the standard-sensitivity RNA chip on the Experion automated electrophoresis system . The samples were bar-coded and prepared according to the protocol (Cat# RS-930-1001) of the manufacturer  with the automated platform described previously .The bar-coded libraries were clustered on a cBot cluster-generation system using an Illumina HiSeq single-read cluster-generation kit according to the manufacturer’s instructions. The libraries were pooled together two and two in equal concentration for each lane on the flow cell, and sequenced as single reads to 100 bp on an Illumina HiSeq 2000. All lanes were spiked with 1% phiX control library. The sequencing run was performed according to the manufacturer’s instructions and generated a total of 908 million reads with a median of 40 million reads per sample and replicate that passed the Illumina Chastity filter; these reads were included in the study  correlation of gene expression (read counts) greater than 0.94.All sequences were aligned to the human genome reference hg19 with tophat ,25 versiReads per kilobase of exon per million mapped sequence reads (RPKM) values for features were calculated by rpkmforgenes.py using the parameters: -sam -gffann –readcount. Estimations of intergenic expression levels for each replicate were calculated by rpkmforgenes.py and the R script cut_off.1.0.R .Reads were trimmed to determine the effect of sequencing length on the number of called differentially expressed genes using a custom perl script: trim_length.pl, which is available on github . In addition, the total RNA contains a nucleus-specific peak at roughly 4000 nucleotides (~52 s), marked with an arrow in the upper diagram, which is missing in the cytoplasmic RNA fraction.Click here for fileFigure S2. Scatter plots of gene expression levels between total and cytoplasmic RNA. The median of the gene expression values of total and cytoplasmic RNA. The Pearson correlation coefficient R2 is displayed in each scatter plot. A: U-2 OS, B: U-251 MG, C: A-431.Click here for fileFigure S3. Boxplot showing the length and coding sequence for all three cell lines. Genes detected at a significantly higher level in total RNA than in cytoplasmic RNA (Tot>Cyt), lower level (Tot<Cyt), and genes with no significant differential detection (No Diff). A: Length of 5’ UTRs. B: Length of 3’ UTRs. C: Coding sequence length.Click here for fileFigure S4. Boxplot showing the fold energies of UTRs for all three cell lines. Genes detected at a significantly higher level in total RNA than in cytoplasmic RNA (Tot>Cyt), lower level (Tot<Cyt), and genes with no significant differential detection (No Diff). A: Fold energy of 5’ UTRs. B: Fold energy of 3’ UTRs.Click here for fileTable S1. Spearman correlation for differentially and not differentially detected genes in all three cell lines.Click here for fileTable S2. Summary of information from the sequencing run.Click here for fileTable S3. Reads assigned to features using HTSeq.Click here for fileTable S4. Estimation of intergenic expressions levels within replicates based on RPKM.Click here for file


To analyse this interactions functionally, we transfected fibroblasts with non-replicating, non-translated RNA transcripts from RNA viral genomes with differing degrees of genome-scale ordered RNA structure (GORS). Single-stranded RNA transcripts induced interferon-β mediated though RIG-I and PKR activation, the latter associated with rapid induction of antiviral stress granules. A striking inverse correlation was observed between induction of both cellular responses with transcript RNA structure formation that was independent of both nucleotide composition and sequence length. The consistent inability of cells to recognize RNA transcripts possessing GORS extended to downstream differences from unstructured transcripts in expression of TNF-α, other interferon-stimulated genes and induction of apoptosis. This functional association provides novel insights into interactions between virus and host early after infection and provides evidence for a novel mechanism for evading intrinsic and innate immune responses.RNA viruses infecting vertebrates differ fundamentally in their ability to establish persistent infections with markedly different patterns of transmission, disease mechanisms and evolutionary relationships with their hosts. Although interactions with host innate and adaptive responses are complex and persistence mechanisms likely multi-factorial, we previously observed associations between bioinformatically predicted RNA secondary formation in genomes of positive-stranded RNA viruses with their  Recognition of invading microorganisms and coordinating a defence response are critical steps for host organisms in their perpetual battle with pathogens. Viruses are generally detected initially by host sensors for foreign nucleic acids, including double-stranded RNA duplexes or viral DNA in the cytoplasm and single- or double-stranded RNA or DNA in endosomes ,2. RefleWith their relatively small genome size, RNA viruses, in particular, have become very efficient at making the best use of their seemingly simple genomes beyond merely coding for structural and non-structural proteins required for virion assembly and genome replication. Many RNA viral genomes contain additional genes to inhibit or divert innate cell defences, typically embedded within other viral genes in alternative reading frames , such asFlaviviridae use extensively base-paired internal ribosomal entry sites (IRESs) that recruit ribosomes and initiate translation (cis-acting replication element (CREs) in picornaviruses  and specific phenotypic effects. However, using bioinformatic and biophysical methods we have demonstrated that many genera and families of positive-sense, single-stranded animal and plant RNA viruses exhibit extensive regions of RNA structure extending throughout the genome ,24. We hThe detection of viral infection by hosts relies on sensors termed pattern-recognition receptor (PRR) molecules that detect specific pathogen-associated molecular patterns (PAMP) . As we hWe investigated the possibility that GORS modulates the recognition of viral RNA in a manner that contributes to viral persistence. Genomic RNA sequences from a range of persistent and non-persistent viruses that differed in their degree of RNA folding were transfected into cells and cellular responses recorded. This included downstream induction of IFN-β, the formation of antiviral stress granules (SGs) ,32, acti2. The cell lines A549/pr(IFN-β).GFP, A549/pr(IFN-β).GFP-NS3/4a, A549/pr(IFN-β).GFP-Npro, A549/pr(IFN-β).GFP-PIV5-V and the A549 cells overexpressing shRNAs targeting RIG-I, MDA-5 and PKR were kindly provided by Professor R. Randall, University of St. Andrews  and streptomycin (100 μg/ml) and maintained at 37°C and 5% CO Andrews ,34. CellSupplementary Table S1). After gel purification and subsequent phenol/chloroform extraction of amplified DNA, RNA was transcribed using T7 in vitro transcription with the PCR fragment as template . The integrity of transcripts was verified by gel electrophoresis and on a 2100 Agilent Bioanalyser before transfection. RNA was purified by lithium chloride precipitation, washed with 70% ethanol and re-suspended in nuclease-free water. Cellular RNA was extracted from A549 cells using RNeasy (Qiagen) spin columns according to the manufacturer’s instructions including DNase treatment.For the production of RNA for transfection, template was amplified from viral cDNA clones or viral RNA by PCR using a forward primer that contained a T7 promoter sequence and an antisense primer at ∼4 KB distance from the sense primer binding site : UTP in the transcription mixture. RNA was dephosphorylated using FastAP thermosensitive alkaline phosphatase (Fermentas) as described before ; RNA in 50 μl of DMEM was incubated for 5 min with 1 μl of Lipofectamine 2000, incubated for 20 min and added to wells containing target cells at 90% confluency.Approximately 1.0 × 10Supplementary Table S2 and calculated as fold induction over mock transfected cells. As a result, IFN-β induction levels comparable or even exceeding that of the synthetic dsRNA analogue poly I:C were observed. For each sample ∼1 μg of RNA extracted from the cells was reverse transcribed using M-MLV (Promega) and random hexamers according to manufacturer’s instructions. The cDNA was amplified using either a Step-One-Plus thermocycler (Applied Biosystems) or Rotorgene (Qiagen) using Sensifast Sybr green mastermix (Bioline). The real-time qRT-PCR primers were either published before or designed using qPrimerDepot (http://primerdepot.nci.nih.gov/) and are listed in Supplementary Table S2.Transfected cells were incubated for 8 h and RNA was extracted and isolated using the RNeasy (Qiagen) or Illustra RNAspin kit  according to the manufacturer’s instructions, both including DNase treatment. IFN-β mRNA levels were measured using qRT-PCR using primers listed in http://mfold.rna.albany.edu/?q=DINAMelt/software. For calculation of mean folding energy differences (MFEDs) between native RNA-viral sequences and sequence order-randomized controls, 50 copies of each sequence was generated using the algorithm NDR that preserves dinucleotide frequencies of the native sequence, as implemented in the program Folding Energy Scan in the SSE package  of the pT7:MNV3 clone , 1.3-Kb For immunofluorescence analysis, cells were grown on cover slips in 24-well plates. After treatment, the cells were washed with PBS and fixed with 4% formaldehyde for 30 min at room temperature. After washing in PBS, the cells were blocked and permeabilized with PBS supplemented with 10% foetal calf serum and 0.1% Triton X-100. Subsequent incubation with the primary and secondary antibodies was performed in the same blocking/permeabilization buffer for 1 h at room temperature. Primary antibodies directed against Ras-GAP SH3 domain-binding protein (G3BP) were obtained from BD Pharmingen. Secondary antibodies were anti-rabbit Alexa Fluor 488 (Life Technologies), anti-mouse Alexa Fluor 594 (Life technologies) and anti-biotin CF633 (Sigma).Phosphorylated PKR was detected by Western-blot analysis of cell lysates collected at different time points post-transfection. Cells were lysed in 2% sodium dodecyl sulphate (SDS) in 62.5 mM Tris–HCl, pH 6.8, 5% 2-mercaptoethanol and electrophoresed on a 10% denaturing SDS-polyacrylamide gel followed by immunoblotting. Phosphorylated PKR was detected by specific antibody  followed by an HRP-conjugated antibody (Thermo scientific) and ECL detection.Differential recognition of viral genomic RNA with or without large-scale RNA secondary structure is most likely manifested early after virus entry and before establishment of replication complexes and consequent sequestration of dsRNA replication intermediates in the endoplasmic reticulum. To functionally characterize this initial recognition of viral RNAs, RNA sequences of defined sizes were transfected into A549 cells and expression of IFN-β mRNA monitored by quantitative RT-PCR. Transcripts of ∼4 kb were chosen from a range of representative RNA viruses, avoiding known defined RNA structures (such as CRE-associated stem-loops involved in replication functions) and IRES elements. In addition, transcripts were not capped and could not therefore be translated and their integrity was verified before transfection by gel electrophoresis and using a 2100 Agilent bioanalyser. Transfected cells were incubated for 8 h and RNA was extracted. IFN-β mRNA levels were measured using qRT-PCR and calculated as fold induction over mock transfected cells.Transfections were initially performed using 5 ng of RNA per 200,000 cells (ie. per well of a 24-well plate) derived from viruses that showed low MFED values and that were associated with non-persistent infections  and 2. TSupplementary Table S1) and transcribed in vitro. These were transfected into A549 cells with RNA amounts equal to those used for the 4-kb fragments (10 ng) or amounts equivalent in copy number of those used for the 4-kb fragments. Although IFN-β levels showed some variability between the two viral sequences, IFN-β induction was comparable between different fragment lengths when equal RNA copies were transfected  in RNA recognition were assessed using two approaches. In the first, an A549 cell line overexpressing the HCV protease (NS3/4A) that inhibits MAVS-dependent signalling by RIG-I and MDA-5, was used. The effect of transfecting HPeV and BV RNA sequences on IFN-B responses was assayed in parallel in an A549 cell line that overexpressed the PIV-5 V protein which specifically inhibits MDA-5  Figure A. ExpresFinally, evidence was obtained for a major role of PKR in the viral RNA recognition pathway; 80–85% reductions in IFN-β mRNA induction were observed in cells treated with 2-AP and the imidazolo-oxindole inhibitor, C16 ,44 FiguF. The inPro that inhibits IRF3 signalling . SG formation occurred rapidly post-transfection . In addition to RNA secondary structure, RNA transcripts additionally varied in their base composition, such as frequencies of mono- and di-nucleotides, frequencies of base pairs and ratios (Supplementary Table S3). Weaker associations with IFN-β response were observed for MFE, the frequency of A residues, CpG dinucleotides and G + C content) although these variables also showed compounding associations with each other and with MFED values. To investigate which of these five variables independently influenced IFN-β responses, IFN-β induction values were log transformed to approximate them to a normal distribution and multiple-linear regression was carried out (R2). A weaker association was observed between the presence of GORS and the frequency of SG induction  HCV and MNV sequences of length 1.1–1.3 Kb derived from coding regions of their viral genomes were permuted in base order using an algorithm (CDLR) that retained mono- and di-nucleotide compositions along with coding and codon usage. Mutant sequences (US) with MFED values of −0.4% (MNV) and −1.3% (HCV) induced substantially greater IFN-β responses (3–5 fold) than the native sequences  of transfection D. IntrigSupplementary Figure S2). Both structured  and unstructured (BV) transcripts showed similar 2–4-fold declines in RNA levels between the two time points, thus providing no evidence for differences in their intra-cellular stability that would account for their different induction of IFN-β and SG formation.One explanation for the different cellular response to structured and unstructured RNA sequences is that they differ in stability once transfected into the cytoplasm. To investigate this, RNA levels were compared by quantitative PCR at 4 and 24 h post-transfection  and mouse 3T3 cells using transcripts from GORS (HPgV) and non-GORS (BV) viruses A. The ditrans-inhibition implies that GORS reduces detection by the cell rather than having a specific inhibitory function. Similar results were obtained when HPgV (structured) and BV (unstructured) RNA transcripts were co-transfected (data not shown).To investigate whether possession of genome-scale RNA structure inhibited recognition mechanisms or subsequent signalling pathways or alternatively, whether RNA folding prevented recognition by the cell, structured (HPgV) and unstructured (BV) transcripts were sequentially transfected into cells and IFN-β mRNA induction compared to that of transcripts transfected individually . IFN-β min vivo may therefore be partially or entirely redundant in a cell culture infection model.The association of GORS with virus persistence was originally described through observations that viruses with genomes possessing bioinformatically predicted extensive RNA secondary were those capable of establishing persist infections in their natural (immunocompetent) hosts ,24. We hin vivo replication cycle, but it is one that exerts a minimal phenotype in existing cell culture models. Furthermore, the existence of multiple mechanisms—by an individual virus—for subverting innate responses creates the specific problem of dissecting the phenotypic effects of other contributors to control of cell defences by a wide range of viruses when studied in vitro. Besides recognition, there is some but not entirely consistent evidence that highly structured genomes might enable the virus to escape siRNA or miRNA coupled control mechanisms from the host cells  ,61. The sequence ,65, othesequence ,67. Usinsequence . In anotsequence ,70.Consistent with the current study, a series of 800 base ssRNA transcripts spanning-coding regions of the HCV genome  largely failed to induce IFN-β while the unstructured region in the 3′UTR (polyU/UC) induced IFN-β to levels greater than the poly-I:C control . In a siSupplementary Figure S1) rules out putative roles of IFN-β and interferon-stimulated gene (ISG) expression in their induction, consistent with previous observations . Predicted structures do not obviously resemble those in known inhibitors of PKR. The observation that co-transfection of structured (HPgV) RNA did not prevent the response to unstructured BV RNA (PKR is now known to be able to recognize a much wider range of substrates than dsRNA, including single-stranded RNA containing internal stem-loops ,81,82. An of PKR . Whetherd BV RNA  indeed pet al. (Whether SGs participate in the IFN response  or not , they clet al.  detectedet al. . On the et al. . Our obset al. C and theIn the evolutionary arms race between viruses and their hosts, the relatively simple, small, single-stranded, positive-sense RNA viruses have a potential Achilles heel in that the naked RNA genome is inevitably exposed—in the absence of any immunosuppressive viral proteins and before any are synthesized—early in the replication cycle. Those causing acute infections have evolved to outrun hosts defences. In contrast, we demonstrate here that some that cause persistent infections have evolved extensive RNA structure throughout the genomes which effectively prevents detection by cellular PRRs, in particular PKR. These studies contribute to a broader understanding of both sides of this arms race and further emphasizes the plethora of mechanisms that have evolved to detect, induce and evade innate immune responses.Supplementary Data are available at NAR Online.


The RNA synthesis machinery of vesicular stomatitis virus (VSV) comprises the genomic RNA encapsidated by the viral nucleocapsid protein (N) and associated with the RNA dependent RNA polymerase, the viral components of which are a large protein (L) and an accessory phosphoprotein (P). The 241 kDa L protein contains all the enzymatic activities necessary for synthesis of the viral mRNAs, including capping, cap methylation and polyadenylation. Those RNA processing reactions are intimately coordinated with nucleotide polymerization such that failure to cap results in termination of transcription and failure to methylate can result in hyper polyadenylation. The mRNA processing reactions thus serve as a critical check point in viral RNA synthesis which may control the synthesis of incorrectly modified RNAs. Here, we report the length at which viral transcripts first gain access to the capping machinery during synthesis. By reconstitution of transcription in vitro with highly purified recombinant polymerase and engineered templates in which we omitted sites for incorporation of UTP, we found that transcripts that were 30-nucleotides in length were uncapped, whereas those that were 31-nucleotides in length contained a cap structure. The minimal RNA length required for mRNA cap addition was also sufficient for methylation since the 31-nucleotide long transcripts were methylated at both ribose-2′-O and guanine-N-7 positions. This work provides insights into the spatial relationship between the active sites for the RNA dependent RNA polymerase and polyribonucleotidyltransferase responsible for capping of the viral RNA. We combine the present findings with our recently described electron microscopic structure of the VSV polymerase and propose a model of how the spatial arrangement of the capping activities of L may influence nucleotide polymerization. Using a prototype of the nonsegmented negative strand RNA viruses, vesicular stomatitis virus, we probed the spatial relationship between the RNA dependent RNA polymerase and 5′ mRNA capping and methylation activities of the large polymerase protein. Because the 5′ mRNA processing reactions dramatically impact the nucleotide polymerization activity of the protein, they may function as a quality control step in viral transcription. We developed a means to stall transcription at precisely defined locations following initiation and analyzed the cap status of the stalled transcripts. We show that 30-nt transcripts are uncapped whereas those that are 31-nt long gain are capped and methylated at both guanine-N-7 and ribose-2′-O positions. Combined with our recent work that determined the molecular architecture of the VSV polymerase, this work reveals the spatial relationship within a functional polymerase complex of the polymerase domain and the 5′ mRNA processing domains of the L protein. The RNA synthesis machinery of the non-segmented negative-strand (NNS) RNA viruses contains at its core a large polymerase protein (L) that possesses all the enzymatic activities for genome transcription and replication. During transcription, L catalyzes nucleotide polymerization in vitro. Although L protein was known to possess the enzymatic activities for mRNA cap addition, their identity proved difficult to pin down unambiguously. This is because the enzymatic activities themselves are unusual, in that the cap is added by the action of a polyribonucleotidyltransferase (PRNTase) that transfers pRNA onto a GDP acceptor through a covalent L-pRNA intermediate in vitro and defined a motif GxxT[N]HR that was essential for capping, implicating CRV as the PRNTase Our understanding of the activities of L protein has been largely shaped by studies of a prototype of the NNS RNA viruses, vesicular stomatitis virus (VSV). The NNS RNA virus L proteins are homologous and share six regions of sequence conservation (CRI-VI) Following cap formation, the cap itself is methylated at guanine-N-7 and ribose-2′-O positions in vitro with VSV using either virus in which the membrane is disrupted with detergent in trans by L, but such experiments cannot address at what stage during transcription the RNA chain is modified. In the present study, we established a system to provide a “freeze-frame” view of VSV transcription using templates that lacked sites for UTP incorporation. By stalling transcription reactions at precisely defined chain lengths we identify a minimal length at which the transcript becomes capped. This work reveals the spatial arrangements of the capping activities of the VSV L protein in relation to the RNA dependent RNA polymerase domain during active transcription.Recently, we obtained a first view of the molecular architecture of the VSV L polymerase protein The promoter for VSV mRNA synthesis includes the 3′ leader region and the first 10-nt of the conserved gene-start element in vitro. Briefly, 10 µg of purified virus was incubated with detergent to disrupt the viral membrane and NaCl to liberate the M protein from the RNP core and permit transcription. The detergent-disrupted particles were incubated in transcription buffer containing ATP, CTP, UTP and [32P]-GTP and the products of transcription purified and analyzed by electrophoresis on acid-agarose gels  revealed that the stalled 30-nt transcript was insensitive to cleavage as evidenced by its unaltered mobility, whereas the 40-nt and 50-nt transcripts shifted in mobility by a single nucleotide following cleavage . This inTo more precisely define the point at which the RNA chain gains access to the capping machinery, we constructed a set of recombinant viruses designed to stall transcription at intermediate points between 30 and 40-nt . SpecifiTo definitively determine whether those transcripts contained an mRNA cap structure, we compared the mobility of the stalled transcripts before and after TAP digestion. As expected, the leader RNA, and the +30 RNA were insensitive to TAP cleavage indicating that they lack an mRNA cap structure . By contThe above experiments show that the transcript must be 31-nt long to be capped. To provide further support for this, we took advantage of our previously characterized cap defective polymerase (L-H1227A) 3H]-SAM. The resulting transcripts were purified, analyzed by electrophoresis on a 20% polyacrylamide gel and detected by fluorography  are of sufficient length to be capped when added to VSV L In combination with the recent electron microscopic structures, the present findings provide new insights into the structural and functional organization of the VSV polymerase. EM images of L alone showed the organization of L into a ring domain (90–100 Å) containing the RNA polymerase and an appendage of three globular domains (approximately 45 Å each) containing the cap-forming activities Our findings also have implications for the path the RNA chain takes between the RdRP domain and the capping apparatus. Although, the length of the RNA would permit the transcript to traverse from the core ring domain to the PRNTase appended to the ring itself, as well as the flexible cap methylation apparatus of the same L molecule, we cannot exclude the possibility that the cap formation is catalyzed by an adjacent L molecule. Indeed, our electron microscopic examination of the L-P complex provided evidence for polymerase dimers, and genetic experiments with Sendai virus are consistent with the presence of at least a dimeric polymerase complex. It remains unknown, however, whether cap defective VSV polymerase molecules can complement RdRP defective VSV polymerase molecules. If indeed the RNA chain is capped by an adjacent polymerase molecule, such complementation should be achievable. Additionally, by incorporation of thio substituted nucleotides within the nascent RNA chain, it should now be possible to decipher the path that the RNA chain traverses in its transit from the RdRP domain to the PRNTase domain.During transcription, the capping activities of L are known from biochemical experiments to influence the polymerization activity. Specifically, failure to cap the mRNA leads to premature termination of transcription The length requirement for mRNA 5′ modification is similar to that previously reported for vaccinia virus A second possible explanation for the production of stalled transcripts that vary in their precise point of termination relates to the intrinsic ability of the polymerase to stutter on homopolymeric tracts. The best characterized slippage sequence for VSV is that of the gene-end AUACUUUUUUUG in which the AUAC element regulates the extent of stuttering by L on the U7 tract The ability to stall transcription at specific places on the template, through the use of engineered recombinant templates now provides us an ability to study more precisely the steps of transcription. This methodology should be readily adaptable to probe the length of the nascent RNA chain that is protected by the polymerase during transcription and to determine whether the RNA chain becomes accessible prior to mRNA cap addition. Moreover, combined with our ability to image the L-P complex by electron microscopy, this approach may permit us to examine the polymerase bound to the RNA template at various stages of transcription. Our work also has implications for understanding transcription in other nonsegmented negative-strand RNA viruses in that they likely require a minimal length of transcription prior to cap addition. We anticipate that those lengths will vary for each virus, and as outlined above may perhaps show gene-specific variation. Since the production of triphoshphate RNA itself may serve as an activator of specific cytosolic sensors of the innate immune system, what appears to be a specific and efficient position of mRNA cap addition may also serve to decrease the production of pppRNA transcripts in infected cells. Moreover, since the act of capping serves as a positive regulator of transcriptional elongation this minimal length represents a key check-point in the transcription cycle.in vitro transcription reactions.Plasmid pVSV1(+)60, containing an infectious cDNA clone of the VSV genome with a 60-nt long non-essential gene inserted at the leader-N gene junction, was generated as described previously in vitro as described previously 32P]-GTP  . Total RNA was extracted, purified, and used for secondary manipulations as follows. Where indicated, the RNA cap structure was removed by tobacco acid pyrophosphatase  as previously described Viral RNA was synthesized g, 3.5 h) through a step gradient of 0.25 ml each of 40, 45, and 50% glycerol in TED buffer  supplemented with 0.1 M NaCl. The pellet was resuspended in 0.3 ml of TED buffer plus 10% glycerol and disrupted on ice, except that the Triton X-100 and EDTA concentrations were reduced to 0.05% and 1 mM, respectively. The N RNA was isolated by banding in a 3.6-ml 20 to 40% (wt/wt) CsCl gradient , recovered by side puncture and diluted fourfold with 10 mM Tris-Cl (pH 8.0), 0.1 mM EDTA. The N-RNA was recovered following centrifugation  through a 0.5-ml cushion .The N-RNA template was purified from rVSV(A-)-30, -31, -32 and-33 as described previously Spodoptera frugiperda 21 cells, and P was expressed in BL21 (DE3) as described previously Recombinant L was expressed from recombinant baculoviruses in 32P]GTP  . After 5 h of incubation at 30°C, the RNA was purified by phenol and chloroform extraction and analyzed on 6% polyacrylamide gel and visualized by a phosphorimager . For methylation of the transcripts, the same reaction condition was used as described above with the exception that 20 µM 3H-labeled S-adenosyl-L-methionine (3H-SAM)  was used for labeling the RNA instead of [α-32P]GTP. After 5 h of incubation at 30°C, the RNA was purified by phenol and chloroform extraction and analyzed on 20% polyacrylamide gel followed by autoradiography.Reactions were carried out in the absence of rabbit reticulocyte lysate using 5 µg of N-RNA template, 4 µg of purified L, 2 µg of purified P and nucleoside triphosphates (1 mM ATP and 0.5 mM each of CTP and GTP) as described previously


Burkholderia cenocepacia J2315. The sRNA was found to negatively regulate the hfq2 mRNA, through binding to part of the 5′-UTR region of the hfq2 mRNA, resulting in accelerated hfq2 mRNA decay and reduced protein levels in exponentially growing cells. Both the h2cR transcript and the hfq2 mRNA are stabilized by the other B. cenocepacia RNA chaperone, Hfq. Infection experiments using the nematode Caenorhabditis elegans revealed that down-regulation of Hfq2 by h2cR decreases the B. cenocepacia ability to colonize and persist within the nematode, suggesting a role for h2cR on bacterial persistence in the host.Small non-coding regulatory RNAs (sRNAs) post-transcriptionally affect multiple phenotypes in prokaryotes and eukaryotes, yet most of the underlying regulatory mechanisms and the nature of the target mRNAs remain unclear. Here we report the identification and functional analysis of the novel cis-encoded sRNA h2cR, from the human opportunistic pathogen  In bacteria, sRNAs have been implicated in the response to environmental stress Burkholderia cenocepacia harbors two distinct Hfq-like proteins, Hfq and Hfq2 hfq mRNA reaches maximal levels in the early exponential growth phase, while hfq2 mRNA is maximally expressed in the stationary phase B. cenocepacia have been implicated in the up-regulation of Hfq2 during the stationary phase B. cenocepacia J2315. Data presented indicate that h2cR negatively regulates hfq2 mRNA by interacting with its 5′-UTR. Results obtained upon silencing h2cR in B. cenocepacia J2315 indicate a reduced ability of the opportunistic pathogen to colonize the Caenorhabditis elegans used as a infection model, implicating this sRNA in the persistence of the bacterium at least in this particular host.The soil bacterium B. cenocepacia strains used in this work  using an Agitorb 200 incubator .his work  were maiB. cenocepacia J2315 using the High pure PCR template preparation kit . Plasmids used in this work are described in B. cenocepacia J2315 hfq2, hfq2 5′-UTR, and h2cR was achieved with adequate primers (hfq2 mRNA), pCGR13 (hfq2 5′-UTR), pCGR14 (h2cR sRNA), pCGR28 (nt −220 to −40 of hfq2 5′-UTR), pCGR29 (nt −70 to +30 of hfq2 5′-UTR) and pCGR30 (nt −220 to +30 of hfq2 5′-UTR) were used for in vitro transcription experiments. Silencing of the h2cR sRNA was achieved by expressing a truncated (104 nt) anti-sense h2cR RNA derivative, containing a 3′ proximal CCA extension. Plasmid pCGR26 was constructed by cloning the HindIII/EcoRI fragment of plasmid pSAS5 hfq2 gene fused with a six-histidine tag at the N-terminus, into the HindIII/EcoRI sites of pCGR13. Plasmid pCGR27 was constructed by cloning the fragment obtained from restriction of pCGR26 with XhoI/HindIII into the XhoI/HindIII sites of pBBR1MCS, allowing the expression and translation of the His-tagged Hfq2 from its own promoters. pBBR1MCS and derivatives are fully compatible with pMLBAD sil) was constructed by cloning the EcoRI-XbaI fragment obtained from pCGR14 into the EcoRI-XbaI sites of pBBR1MCS. These plasmids allow the constitutive expression of the cloned genes in B. cenocepacia . All plasmid constructions were confirmed by DNA sequencing.Total DNA was obtained from exponentially growing cells of  primers . PCR prohfq2 mRNA or h2cR sRNA were assessed by Northern blot analysis using 2 µg of total RNA purified from cells of B. cenocepacia J2315 and derivative strains. Total RNA was loaded into 6 or 15 % and 8 M urea polyacrylamide gels for hfq2 and h2cR respectively, and electrophoresed in 1× TBE buffer at 35 mA. After electrophoresis, total RNA was electrotransferred at 20 V, for 16 h at 4°C using 0.5× TBE buffer to a BrightStar Plus membrane . Oligonucleotide probes (The levels of transcripts corresponding to e probes  were labB. cenocepacia J2315 and derivative strains using the Ribopure RNA isolation kit (Ambion) or the mirVana miRNA isolation kit (Ambion), for total RNA or sRNA-enriched fractions, respectively. RNA concentration was estimated in a ND 1000 spectrophotometer (Nanodrop). RNA labeling with biotin was conducted as previously described −1 of rifampicin were added, immediately before the assay, to cell cultures at 24 h of growth for hfq2 assays or at 2 h, 8 h or 24 h for h2cR assays. 2 µg of total RNA were used in each experiment. Films were scanned and the intensity of RNA bands was measured with the ImageJ software. The intensities were plotted in semi-log plots and RNA half-life was calculated using the slope of each plot.Total RNA was obtained from cells of hfq2 coding region, the hfq2 full mRNA and the 5′-UTR of the hfq2 transcript and derivatives were performed as previously described hfq2 full transcript or the hfq2 coding sequence, or increasing amounts of the hfq2 5′UTR derivatives  were incubated in 25 µl of RNA binding buffer hfq2 5′-UTR (2.5 nM), was evaluated by EMSA assays using 0, 0.5, 1, 5 or 10 nM of the hexameric form of His-tagged Hfq (Hfq6), or 0, 0.5, 1, 5, 10 or 50 nM of His-tagged Hfq6, respectively. Non-labelled yeast tRNA (Ambion) was added in excess to each sample to minimize non-specific binding. Incubation, resolution of RNA-protein complexes and detection of band-shifts was performed as described before EMSA assays with the h2cR transcript, the hfq2 mRNA full transcript, the hfq2 5′-UTR, the hfq2 coding region (CDS), the nt −220 to −40 (Start), the nt −70 to +30 (Mid) and nt −220 to +30 (End) 5′-UTR, or the h2cR sRNA, were generated by PCR, using the following primer pairs and DNA templates: T7-TSS-hfq2– hfq2Low, and plasmid pCGR11; 5H2-H –5H2-B, and plasmid pCGR12; Up_hfq2_C – Low_hfq2_C, and plasmid pCGR8; CGRO100– CGRO101, and plasmid pCGR28; CGRO102– CGRO103, and plasmid pCGR29; CGRO100– CGRO103, and plasmid pCGR30, or h2cR-X – h2cR-H2, and plasmid pCGR14. All RNA transcripts were generated from the T7 promoter, using the MEGAshortscript kit (Ambion). The transcripts h2cR (136 nt), 5′-UTR (216 nt), Start (180 nt), Mid (100 nt), and End (250 nt) were purified from 8%−7 M urea polyacrylamide gels, while the hfq2 derivatives CDS (585 nt) and the full transcript (801 nt) where purified from a 1% TBE/agarose gel. RNAs were subsequently ethanol-precipitated and 5′ end-labelled with 11-UTP-biotin (Ambion). RNA 5′end-labelling was achieved after dephosphorylation with 2 U of calf intestinal phosphatase  for 15 min in NEB buffer 2, with T4 polynucleotide kinase . Briefly, 200 pmol of RNA were mixed with 7.5 nmol of 11-UTP-Biotin, in a total volume of 50 µl, containing 1 mM ATP, 5 µl of 10× T4 PNK buffer, and 10 U of T4 PNK. Reactions were carried out at 37°C for 30 min. RNA was phenol-chloroform extracted, ethanol precipitated, and finally cleaned-up with Ilustra NAP-5 columns .The cDNA templates for the B. cenocepacia J2315 cells in the early exponential phase (2 h), exponential phase (8 h) or in the stationary phase (24 h). RNA was isolated from cells using the mirVana kit (Ambion) for h2cR, followed by DNase I treatment with TurboDNase (Ambion). Amplification of the 5′-end of cDNA was carried out using the First Choice RLM Race kit (Ambion), using 20 pmol of the primer 5′R (5′RACE of h2cR)   and 5 µghfq2 fused with a nucleotide sequence encoding a six histidine tag at the N-terminus from each of the hfq2 own promoters, was introduced into B. cenocepacia CJ2 (Δhfq2), yielding B. cenocepacia CJ6. The CJ6 strain was further transformed with plasmids pCGR17 or pCGR18, which allow the h2cR overexpression (strain CJ8) or silencing (strain CJ7), respectively. The cultures were grown for 24 h in liquid LB, supplemented with 650 µg ml−1 trimethoprim and 1% L-arabinose. Culture aliquots of 1 ml were taken, the protein was purified with the Illustra TriplePrep kit , and ressuspended in 100 µl of 2D-DIGE buffer . Protein concentration was estimated in a Nanodrop 1000 spectrophotometer. Aliquots containing 20 µg of total protein were separated by 12% SDS-PAGE and then electroblotted onto a nitrocellulose membrane, using Towbin transfer buffer, for 45 min at 120 mA. The 6xHis-tagged Hfq2 protein was detected using the PentaHis-HRP antibody system  with tetramethylbenzidine  as substrate.The effect of the h2cR sRNA on the levels of the Hfq2 protein was assessed by western blotting. For this purpose, plasmid pCGR27 (pThfq2), which is able to drive the transcription of C. elegans mutant strain DH26, cultured in NGM II medium, supplemented with 2% L-arabinose, when appropriate. Total protein and RNA were also obtained from B. cenocepacia strains CJ6, CJ7 and CJ8, when infecting the nematode C. elegans. Worms were lysed using silica-carbide beads , based on previously described methods hfq2 mRNA, the h2cR transcript, and the 5S rRNA. Total protein was obtained with the Illustra TriplePrep kit , and processed as described above. Protein aliquots were used to assess the Hfq2 levels by western blot, as described above.Nematode killing assays and bacterial colonization of the digestive tract of the nematodes were performed based on previously described methods −50). The h2cR sRNA secondary structure was predicted using the Mfold algorithm hfq2 was achieved using the RNAHybrid software BLAST searches were performed using the Integrated Microbial Genomes (IMG) webserver . Error bars represent the means of the standard deviation. Variance was computed based on ANOVA analysis. Images shown are representative of each the experiments repetitions performed. All experiments were repeated independently at least 4 times, using triplicates, with a minimum of n = 12.An unpaired two-tailed-B. cenocepacia which may interact with the RNA chaperone Hfq, we have co-purified the His-tagged Hfq protein together with B. cenocepacia sRNA fractions (<200 nt) B. cenocepacia J2315 chromosome 1 revealed that the h2cR encoding sequence ranged from nt 1704623 to 1704759 in the reverse strand of the 5′-untranslated region (UTR) sequence of hfq2 .Bioinformatic analyses revealed the presence of h2cR homologues among all members of the Bcc, with an identity of at least 85%. h2cR homologues were also found among other B. cenocepacia J2315 (wt) strain were carried out in liquid LB at 37°C, and total RNA was extracted and used in Northern blot experiments. Results obtained showed that h2cR transcripts were maximal in cells at the early exponential phase of growth, decreasing to undetectable levels in cells at the stationary phase . We have also included in these analyses a B. cenocepacia J2315 hfq mutant (strain CJ1) hfq mutant (Δhfq), hfq2 mRNA levels increased over time, reaching maximal values in cells at the stationary phase, with hfq2 mRNA levels being higher in the wt strain than in the Δhfq strain (hfq (Δhfq+ph2cR) strains led to almost undetectable levels of hfq2 mRNA during the exponential growth phase and high hfq2 mRNA levels in cells at the stationary phase. In contrast, the silencing of h2cR led to higher hfq2 mRNA levels during exponential phase in both the wt (h2cRsil) and the Δhfq (Δhfq+h2cRsil) strains (hfq led to increased amounts of hfq2 mRNA in both strains (wt+phfq and Δhfq+phfq) during the exponential phase of growth, suggesting a possible role for Hfq in hfq2 mRNA expression/stability. The results obtained when assessing the half-life of the hfq2 mRNA on the different genetic backgrounds indicate that silencing of h2cR in the wt strain increased the hfq2 mRNA half-life time to ∼30%, while overexpression of h2cR had almost no impact on the hfq2 mRNA half-life time (reduction of only ∼6%)  . Interestely 25% . Taken thfq2 mRNA levels. The h2cR transcript levels were maximal in the early exponential growth phase in both the wt and the Δhfq strains , was transformed with plasmid pCGR27 (expressing a 6 histidine-tagged hfq2 mRNA from the hfq2 own promoters), yielding strain CJ6. This strain allows the assessment of the levels of Hfq2 protein produced by Western blot using an antibody specific for the histidine-tag of the synthesized Hfq2 , increasing to double the levels observed in cells at the stationary phase of growth  to investigate the formation of complexes between the h2cR sRNA and the hfq2 mRNA. Incubation of biotin-labeled h2cR with increasing concentrations of the complete hfq2 mRNA  resulted in the formation of a slower migrating complex, consistent with an interaction between the two RNA molecules (hfq2 coding sequence (hfq2 CDS) was incubated with the biotin-labeled h2cR, indicating that the 5′-UTR of hfq2 is required for the RNA duplex formation (hfq2 5′-UTR: one RNA molecule spanning nt −220 to +30 (corresponding to the full 5′-UTR), a second RNA molecule spanning nt −220 to −40 (including most of the 5′-UTR), and a third RNA molecule spanning nt −70 to +30 (including the AUG region). h2cR formed RNA duplexes with the RNA molecule corresponding to the complete hfq2 5′-UTR (−220 +30) and with the RNA molecule spanning nt −220 to −40 of the hfq2 5′-UTR, but not with the hfq2 mRNA derivative spanning the −70 to +30 of the hfq2 5′-UTR , compared to the binding to the hfq2 mRNA (Kd  = 9.5 nM) (hfq-depleted genetic background (Next we sought to know whether Hfq forms complexes either with h2cR or with the  9.5 nM) . It is pckground .B. cenocepacia J2315 to colonize and to kill the nematode C. elegans, previously used by our research group as an infection model C. elegans has been successfully used to investigate the biology of a number of human pathogens Since sRNAs have been implicated in the regulation of virulence B. cenocepacia virulence towards the nematodes . The B. cenocepacia wt, and the wt strain transformed with pCGR31 or pCGR32 were used in infection experiments similar to those described above. The results obtained were similar to those shown in With this objective, the silencing or over-expression of h2cR in the wt was achieved by transforming the strain with the plasmids pCGR18 or pCGR17, which express the h2cR anti-sense transcript of h2cR or overexpress the h2cR transcript upon induction with arabinose, respectively. Silencing of h2cR increased the survival of the nematodes, suggesting that h2cR affects ematodes . No signematodes . To chechfq2 null mutant still retained the ability to colonize and kill nematodes, a trait most probably derived from the expression of other Hfq- and/or Hfq2-independent virulence factors B. cenocepacia towards the nematode might result directly from the regulation exerted by the sRNA on the Hfq2. Nevertheless, our results also suggest that h2cR plays an additional role on the bacterium ability to kill the nematodes , while h2cR overexpression led to a significant reduction of about one log in the total CFU per worm . Despiteematodes , besideshfq2 mRNA, and the h2cR transcript in bacteria while infecting the nematodes, worms were harvested after 2, 8, or 24 h post-infection with the strains CJ6, CJ7 and CJ8, and processed to isolate total RNA and total protein from the bacteria thriving inside the nematodes. Results obtained indicate that the Hfq2 protein levels increased over time in the CJ6 strain , evidences point out that several sRNAs are encoded within their genomes hfq2, in the opposite direction of transcription, partially overlapping the hfq2 gene. This chromosomal location does not allow the generation of mutants on h2cR without affecting, at least, the functionality of the hfq2 gene. Therefore, our analysis on the possible biological roles of h2cR was based on the sRNA overexpression or silencing.Recent work from our research group led to the identification of two distinct and functional Hfq-like encoding genes within the genome sequence of the opportunistic human pathogen hfq2 mRNA, exerting its negative regulatory effect by binding to the hfq2 mRNA 5′-UTR region. h2cR was also found to be involved in virulence traits of B. cenocepacia J2315. In fact, results presented show that the sRNA affects the ability of the bacterium to persist inside the nematode's digestive tract. Previous work on the functional characterization of hfq2 indicated a role for the encoded protein in the killing the nematodes and in their persistence in the host digestive tract hfq2 mRNA and, consequently, of the Hfq2 protein. Examples of sRNAs involved in the virulence of bacteria within their hosts are known. For instance, the Streptococcus pyogenes rivX sRNA plays an important role in virulence, linking the CovR and Mga regulatory networks, enabling the pathogen to select from its repertoire the expression of specific virulence determinants in response to a broad spectrum of environments B. cenocepacia J2315 h2cR and the S. pyogenes rivX.Data presented indicate that h2cR is a cis-encoded negative regulator of the hfq-like gene by a sRNA. Interestingly, the results here presented also show that in the mutant in the other B. cenocepacia J2315 RNA chaperone encoding gene hfq, the half-life of hfq2 mRNA was reduced compared to the wt strain, suggesting a role of the RNA chaperone Hfq on the stabilization of the hfq2 mRNA. The role of Hfq-like proteins in the stabilization of RNA molecules has been described in other bacteria, through the protection of the RNA molecules from RNase E degradation Burkholderia organisms, RNase E-like encoding genes are anoted in several Burkholderia genomes To the best of our knowledge, this is the first description of the regulation of an hfq genes are restricted to Escherichia coli, to the intracellular pathogen Legionella pneumophila and to the legume symbiotic bacterium Sinorhizobium meliloti. In the case of E. coli, the hfq gene is part of an operon, being its transcription complex and under the control of several promoters E. coli Hfq synthesis is autoregulated at the translational level, by a mechanism involving the binding of Hfq to 2 distinct sites of the 5′-UTR region of the hfq mRNA, inhibiting the formation of the translation initiation complex S. meliloti Hfq protein was also shown to control its own expression at the translational level, both in its natural host, as well as in the heterologous host E. coliLegionella pneumophila, the hfq gene locates in a genome region only partially resembling that of E. coli. For this pathogen, a regulatory scheme was proposed in which the hfq expression is regulated during the exponential phase of growth by the sigma factor RpoS, while during the stationary phase the gene transcription is turned off, either directly or indirectly by the regulatory protein LetA, independently of RpoS hfq-like genes in the opportunistic pathogens of the Bcc, as well as from bacteria from other genera, it is quite expectable that the involvement of a sRNA in this regulation is restricted to members of Burkholderia genus, since no homologs to h2cR could be found outside the Burkholderia genus. In addition, 2 copies of hfq-like genes are present only among members of the B. genus, Bacillus anthracis, Magnetospirillum magnetotacticum and Novosphingobium aromaticivorans, and in the archaeal species Methanobacterium thermoautotrophicum and Archeoglobus fulgidushfq and hfq2 genes, two important regulators of virulence and survival to stress conditions mimicking those faced by the opportunistic bacteria of the Bcc when infecting the cystic fibrosis host.Despite the numerous studies on Hfq proteins from distinct bacterial species as global regulators of bacterial metabolism and virulence, only a few studies on the regulation of expression of Hfq-like proteins encoding genes are available, none of them implicating a sRNA. These studies on the regulation of


Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA,  The origin of life is undoubtedly a fundamental problem of natural science and attracts extensive attention of specialists in different fields of science. As RNA is the only known macromolecule acting as a genetic material as well as a catalyst, it is popular to think of early evolution of life progressed from an “RNA World”, in which RNA played a central role before protein and DNA emerged Considering the fact that RNA may not serve simply as a molecular scaffold for protein folding but also may influence the function of a protein i.e., Nucleic Acids–Protein Interaction DataBase The NPIDB, Until August 15 2010, 832 structures of RNA–protein complexes are available in NPIDB N) decays with the increase of the number of domains covered by the fold (D) and follows power-law equation N = −baD (P<0.0001). As families are more evolutionarily conserved than domains, we also analyzed the distribution of the folds in family space and it was found that the correlation between the number of folds (N) and the number of families (F) also follows the power-law behavior (As shown in behavior .E. coli) and eukaryotic (yeast or higher) species, while late architectures defined as those owned only by eukaryotic (yeast or higher) species. During the age-assigning process, to guarantee that the conclusion is not confined by the limited three-dimensional structures recorded in PDB, not only the proteins recorded in PDB were checked, but also the corresponding homologous proteins retrieved from Swiss-Prot database The power-law behaviors of protein folds and ligand-protein binding have been successfully explained in terms of preferential attachment principle E. coli) and eukaryotic (yeast or higher) species, which suggests that these folds appeared relatively early. In comparison, 19 folds are only owned by eukaryotic (yeast or higher) species, which implies that these folds originated relatively late. Further analysis showed that early folds cover 2.8 domains on average, in contrast to the late folds that only cover 1.4 domains averagely. These results suggest that preferential attachment principle is indeed applicable to elucidating the power-law behaviors of RNA-binding protein architectures.As shown in et al. and Wächtershäuser proposed that ferredoxins should play an important role in the origin of life and may have been among the earliest proteins formed. Further inspection to the structural information of ferredoxins reveals that they have only iron and inorganic sulphur in their active sites, which is unlike to other electron-transfering proteins, e.g., cytochromes and flavoproteins, employing complex organic molecules as cofactors. Thus, considering the abundance of iron and sulphur in the primitive Earth, ferredoxin should possess inherent advantage than others to emerge readily.Based on the power-law feature of RNA-binding protein architectures and the underlying preferential attachment principle implication that the more widely shared architectures have an earlier origin, we can conclude that the higher occurrence of Ferredoxin-like (d.58) than others in RNA-binding domain space implies that the most ancient RNA-binding proteins were very likely to adopt this fold. This opinion is partially supported by previous studies http://www.manet.uiuc.edu/download/foldAncestryVal2_0.txt) Through a large-scale phylogenomic analysis on 174 proteomes, Caetano-Anollés and co-workers established a chronology for proteins, in which 776 folds are recorded according to their evolutionary order, respectively.  as cofactors in anticodon-plus-amino-acid complexes, in which, cofactors consisted of an amino acid bonded to one or more nucleotide (oligonucleotide) To address this question, the first ten most ancient folds characters were investigated. According to the MANET database i.e., L-amino acids and D-sugars usually being preferred in nature over their respective enantiomers (mirror images) with the precondition of equal production of L- and D-amino acids provided at reaction equilibrium in vitro. Although L- and D-amino acids are proved to possess similar thermodynamic stability The present finding is also helpful for understanding the homochirality conundrum i.e., adenine (A), cytosine (C), guanine (G), and uracil (U) were readily available on early prebiotic Earth. The isolation of adenine and guanine from meteorites can be act as evidence that these substances might have been available as “raw material” on early Earth i.e., A, C, G, U.An RNA world has been widely discussed as a probable stage in the early evolution of life e.g., ATP, NAD, FAD, ADP, SAH, AMP, contain a same base part, i.e., adenine. Considering the notion that early cofactors are vestiges of RNA world, we thus believed that adenine base should be included in the original composition of primitive RNA. Moreover, many modern RNA molecules still contain adenine-rich sequences. For example, the adenine composition in a Sendai virus 18S messenger RNA is as high as 99.1% Ribosomes, a place for protein synthesis, are large macromolecular assemblies consisting of RNAs and proteins, in which RNA plays a catalytic role in the formation of the peptide bond and the key catalytic site is proved to be only an adenine residue in the RNA In summary, as a probable stage in the early evolution of life, RNA world has been wildely accepted because of the duplicate roles of RNA as both genetic material and catalysts. Thus, it is significant importance of tracing the pathway of RNA world to modern world. Although the possibility of random peptides existing in RNA world cannot be excluded, it was suggested that these random peptides usually have no particular function. Through systemic analysis of RNA-protein complexes, we proposed that primitive functional protein emergence is mainly assisted by early primitive nucleotide cofactors, while only a minority induced by RNA itself. The present findings also have significant implications for understanding origin of the homochirality of biomolecules and the composition of primitive RNA.Table S1Folds, domains (number) and ownership of RNA-binding proteins.(DOC)Click here for additional data file.


Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013.DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in  Saccharomyces cerevisiae cells. The resulting profiles contribute to both our understanding of the general properties of hybrid-forming loci and to our knowledge of hybrid-mitigating enzymes. Interestingly, significant DNA:RNA hybrid enrichment was detected at genes associated with antisense transcription. We show that overexpression of RNase H, which degrades the RNA in hybrids, significantly affects the expression of genes associated with antisense transcripts. These findings support a role for DNA:RNA hybrids in regulation of gene expression by antisense transcripts.RNA processing factors are mutated in human cancers, inherited developmental disorders and neurodegenerative syndromes. Defects in RNA processing have been associated with increased levels of mutations and DNA damage in part via the formation of DNA:RNA hybrids. Although it is likely that specific regions of the genome are more prone to DNA:RNA hybrid formation, a map of hybrid-prone regions is not available. In this study, we describe the genome-wide distribution of DNA:RNA hybrids in both normal and mutant  In trans interacting with the promoter for sense transcription Arabidopsis thaliana found an antisense transcript that forms R loops, which can be differentially stabilized to modulate gene regulation To add to the complexity of DNA:RNA hybrid management in the cell, hybrids also occur naturally and have important biological functions S. cerevisiae by DNA:RNA immunoprecipitation followed by hybridization on tiling microarrays (DRIP-chip). We found that DNA:RNA hybrids occurred at highly transcribed regions in wild type cells, including some identified in previous studies. Remarkably, we observed that DNA:RNA hybrids were significantly associated with genes that have corresponding antisense transcripts, suggesting a role for hybrid formation at these loci in gene regulation. Consistently, we found that genes whose expression was altered by overexpression of RNase H were also significantly associated with antisense transcripts. A small-scale cytological screen found that diverse RNA processing mutants had increased hybrid formation and additional DRIP-chip studies revealed specific hybrid-site biases in the RNase H, Sen1 and THO complex subunit Hpr1 mutants. These genome-wide analyses enhance our understanding of DNA:RNA hybrid-forming regions in vivo, highlight the role of cellular RNA processing activities in suppressing hybrid formation, and implicate DNA:RNA hybrids in control of a subset of antisense regulated loci.Here we describe, for the first time, a genome-wide profile of DNA:RNA hybrid prone loci in S. cerevisiae (ArrayExpress E-MTAB-2388) using the S9.6 monoclonal antibody which specifically binds DNA:RNA hybrids, as characterized previously Supplementary Figure S1) and normalized to a no antibody control.DNA:RNA hybrids have been previously immunoprecipitated at specific genomic sites such as rDNA, selected endogenous loci, and reporter constructs Supplementary S2) Supplementary Table S3). These were generally shorter in length than average (p = 4.29e−58), highly transcribed (Wilcoxon rank sum test p = 2.21e−6), and had higher GC content (p = 2.52e−50) . Importantly, despite the correlation between DNA:RNA hybrid association and transcriptional frequency, the wild type DRIP-chip profiles compared to the localization profile of the RNA polymerase II subunit Rpb3 revealed very low correlation , it is not clear from our findings whether GC content or transcriptional frequency contributed more to DNA:RNA hybrid forming potential. Furthermore, we observe that DNA:RNA hybrid prone loci do not encode for mRNA transcripts with particularly long half-lives , suggesting that the act of transcription is vital to DNA:RNA hybrid formation and supporting the notion of co-transcriptional hybrid formation as the major source of endogenous DNA:RNA hybrids.Overall, our DRIP-chip profiles identified several previously reported DNA:RNA hybrid prone sites including the rDNA locus and telomeric repeat regions (Supplementary Table S4). Consistent with our findings at ORFs, the levels of DNA:RNA hybrids correspond well with the known levels of expression of these elements. In general, Ty1 which constitutes one of the most abundant transcripts in the cell has the highest levels of DNA:RNA hybrids. Ty3 and Ty4 that are only slightly expressed have much lower levels of hybrids, and the lone Ty5 retrotransposon which is transcriptionally silent is not enriched for DNA:RNA hybrids  (Supplementary Table S5).Certain DNA:RNA hybrid enriched regions identified by our DRIP-chip analysis such as rDNA and retrotransposons are associated with antisense transcripts −7) (Supplementary Table S5) and tended to have high GC content, similar to DNA:RNA hybrid enriched genes in wild type . However, the genes with increased mRNA levels under RNase H overexpression and the antisense-associated genes enriched for DNA:RNA hybrids in our DRIP experiment both tended towards lower transcriptional frequencies  (Supplementary Table S6) and several of these iron transport genes  are antisense-associated  terms enriched among genes whose expression was changed by RNase H overexpression. Consistent with previous work, genes for iron uptake and incorporation were strongly activated by RNase H overexpression . The rnh1Δrnh201Δ, hpr1Δ, and sen1-1 mutants also exhibited DNA:RNA hybrid enrichment in 1206, 1490 and 1424 ORFs respectively compared to the 1217 DNA:RNA hybrid enriched ORFs identified in wild type (Supplementary Table S4). Interestingly, in addition to the similarities described above, our profiles also identified differential effects of the mutants on the levels of DNA:RNA hybrids. In particular, we observed that deletion of HPR1 resulted in higher levels of DNA:RNA hybrids along the length of most ORFs with a preference for longer genes compared to wild type  (Supplementary Table S7) and this was confirmed by DRIP-quantitative PCR (qPCR) of two tRNA genes in wild type and rnh1Δrnh201Δ . Because tRNAs are transcribed by RNA polymerase III, this observation indicates that Hpr1 is primarily involved in the regulation of RNA polymerase II specific DNA:RNA hybrids while RNase H and Sen1 have roles in a wider range of transcripts. Mutation of SEN1 also led to increased levels DNA:RNA hybrids at snoRNA (two tailed unpaired Wilcox test p = 1.81e−6) (Supplementary Table S8) consistent with its role in 3′ end processing of snoRNAs  and compared to an empty control plasmid p425-GPD  All strains are listed in 600 and grown to 0.7 OD600. Crosslinking was done with 1% formaldehyde for 20 minutes. Chromatin was purified as described previously rnh1Δrnh201Δ. Primers are listed in Supplementary Table S11.Briefly, cells were grown overnight, diluted to 0.15 ODS. cerevisiae microarrays. Samples were normalized to a no antibody control sample (mock) using the rMAT software and relative occupancy scores were calculated for all probes using a 300 bp sliding window. All profiles were generated in duplicate and replicates were quantile normalized and averaged. Spearman correlation scores between replicates are listed in Supplementary Table S10. Coordinates of enriched regions are available in S8. DRIP-chip data is available at ArrayExpress E-MTAB-2388.For DRIP-chip, precipitated DNA was amplified via two rounds of T7 RNA polymerase amplification , biotin Enriched features had at least 50% of the probes contained in the feature above the threshold of 1.5. Only features enriched in both replicates were reported. Transcriptional frequency et al 1998. For GC content transcripts were grouped into four classes according to their GC content obtained from BioMart (CHROMATRA plots were generated as described previously . Relativ BioMart . Average600 of yeast cells using a RiboPure Yeast kit (A&B Applied Biosystems), amplified, labeled, fragmented using a Message-Amp III RNA Amplification Kit (A&B Applied Biosystems) and hybridized to a GeneChIP Yeast Genome 2.0 microarray using the GeneChip Hybridization, Wash, and Stain Kit (Affymetrix). Arrays were scanned by the Gene Chip Scanner 3000 7G and expression data was extracted using Expression Console Software (Affymetrix) with the MAS5.0 statistical algorithm. All arrays were scaled to a median target intensity of 500. A minimum cut off of p-value of 0.05 and signal strength of 100 across all samples were implemented and only transcripts that had over a 2-fold change in the RNase H over-expression strain compared to wild type were considered significant. The correlation between duplicate biological samples was: control (r = 0.9955), RNase H over-expression (r = 0.9719). For statistical analysis, GC content, transcription frequencies and antisense association were analyzed as for DRIP-chip analysis.Gene expression microarray data is available at GEO GSE46652. Strains harboring the RNase H1 over-expression plasmid or empty vector were grown in SC-Leucine at 30°C. All profiles were generated in duplicate. Total RNA was isolated from 1 OD2, pH 7) and digested in spheroplasting solution with 10 mM DTT and 150 µg/mL Zymolase 20T at 37°C for 20 minutes similar to previously described  and spheroplasts were lysed with 1% vol/vol Lipsol and fixed on slides using 4% wt/vol paraformaldehyde/3.4% wt/vol sucrose Click here for additional data file.Dataset S2Wild type replicate 2 enriched region coordinates.(XLSX)Click here for additional data file.Dataset S3sen1-1 replicate 1 enriched region coordinates.(XLSX)Click here for additional data file.Dataset S4sen1-1 replicate 2 enriched region coordinates.(XLSX)Click here for additional data file.Dataset S5hpr1Δ replicate 1 enriched region coordinates.(XLSX)Click here for additional data file.Dataset S6hpr1Δ replicate 2 enriched region coordinates.(XLSX)Click here for additional data file.Dataset S7rnh1Δ rnh201Δ replicate 1 enriched region coordinates.(XLSX)Click here for additional data file.Dataset S8rnh1Δ rnh201Δ replicate 2 enriched region coordinates.(XLSX)Click here for additional data file.Figure S1Spearman correlation scatter plot of wild type replicates.(EPS)Click here for additional data file.Figure S2Box plots comparing the distribution of (A) gene length, (B) transcription frequency, (C) GC content, and (D) mRNA transcript half-life of ORFs enriched for DNA:RNA hybrids versus ORFs not enriched for DNA:RNA hybrids. The p values calculated by the Wilcoxon rank sum test are shown.(EPS)Click here for additional data file.Figure S3(A) Distribution of % GC content of all ORFs sorted by transcriptional frequency. (B) Distribution of % GC content of ORFs enriched for DNA:RNA hybrids in WT sorted by transcriptional frequency. Intervals indicate 95% confidence intervals.(EPS)Click here for additional data file.Figure S4Distribution of % GC content of all genes represented on the expression microarray (n = 5657), and transcripts up- (n = 212) or down-regulated (n = 88) by RNase H overexpression. Intervals indicate 95% confidence intervals. Averages are stated above each sample. The p-value indicates a significant increase in GC content of upregulated genes compared to all microarray transcripts (Wilcoxon rank sum test).(EPS)Click here for additional data file.Figure S5SUF2 tRNA gene and (B) tV(UAC)D tRNA gene detected in WT or rnh1Δrnh201Δ as detected by DRIP-quantitative PCR (qPCR). Error bars indicate standard deviation.Relative quantities of (A) (EPS)Click here for additional data file.Table S1rnh1Δrnh201Δ, hpr1Δ and sen1-1.List of rDNA enriched for RNA:DNA hybrids in wild type, (XLSX)Click here for additional data file.Table S2rnh1Δrnh201Δ, hpr1Δ and sen1-1.List of telomeric repeat regions enriched for RNA:DNA hybrids in wild type, (XLSX)Click here for additional data file.Table S3rnh1Δrnh201Δ, hpr1Δ and sen1-1.List of ORFs enriched for RNA:DNA hybrids in wild type, (XLSX)Click here for additional data file.Table S4rnh1Δrnh201Δ, hpr1Δ and sen1-1.List of retrotransposons enriched for RNA:DNA hybrids in wild type, (XLSX)Click here for additional data file.Table S5Lists of open reading frames (ORFs) and antisense-associated ORFs enriched for RNA:DNA hybrids in wild type or modulated at the transcript level by RNase H overexpression.(XLSX)Click here for additional data file.Table S6GO function sorting of genes modulated at the transcript level by RNase H overexpression.(XLSX)Click here for additional data file.Table S7rnh1Δrnh201Δ, hpr1Δ and sen1-1.List of tRNA genes enriched for RNA:DNA hybrids in wild type, (XLSX)Click here for additional data file.Table S8rnh1Δrnh201Δ, hpr1Δ and sen1-1.List of snoRNA genes enriched for RNA:DNA hybrids in wild type, (XLSX)Click here for additional data file.Table S9Strains used in this study.(XLSX)Click here for additional data file.Table S10Spearman correlation scores.(XLSX)Click here for additional data file.Table S11DRIP-qPCR primers.(XLSX)Click here for additional data file.