Exosomes are nanosized (30–100 nm) membrane vesicles secreted by most cell types. Exosomes have been found to contain various RNA species including miRNA, mRNA and long non-protein coding RNAs. A number of cancer cells produce elevated levels of exosomes. Because exosomes have been isolated from most body fluids they may provide a source for non-invasive cancer diagnostics. Transcriptome profiling that uses deep-sequencing technologies (RNA-Seq) offers enormous amount of data that can be used for biomarkers discovery, however, in case of exosomes this approach was applied only for the analysis of small RNAs. In this study, we utilized RNA-Seq technology to analyze RNAs present in microvesicles secreted by human breast cancer cell lines.Exosomes were isolated from the media conditioned by two human breast cancer cell lines, MDA-MB-231 and MDA-MB-436. Exosomal RNA was profiled using the Ion Torrent semiconductor chip-based technology. Exosomes were found to contain various classes of RNA with the major class represented by fragmented ribosomal RNA (rRNA), in particular 28S and 18S rRNA subunits. Analysis of exosomal RNA content revealed that it reflects RNA content of the donor cells. Although exosomes produced by the two cancer cell lines shared most of the RNA species, there was a number of non-coding transcripts unique to MDA-MB-231 and MDA-MB-436 cells. This suggests that RNA analysis might distinguish exosomes produced by low metastatic breast cancer cell line (MDA-MB-436) from that produced by highly metastatic breast cancer cell line (MDA-MB-231). The analysis of gene ontologies (GOs) associated with the most abundant transcripts present in exosomes revealed significant enrichment in genes encoding proteins involved in translation and rRNA and ncRNA processing. These GO terms indicate most expressed genes for both, cellular and exosomal RNA.For the first time, using RNA-seq, we examined the transcriptomes of exosomes secreted by human breast cancer cells. We found that most abundant exosomal RNA species are the fragments of 28S and 18S rRNA subunits. This limits the number of reads from other RNAs. To increase the number of detectable transcripts and improve the accuracy of their expression level the protocols allowing depletion of fragmented rRNA should be utilized in the future RNA-seq analyses on exosomes. Present data revealed that exosomal transcripts are representative of their cells of origin and thus could form basis for detection of tumor specific markers. In early studies, secreted microvesicles were named based on their cellular origins, e.g., archaeosomes, argosomes, dexosomes, epididymosomes, prostasomes, oncosomes, etc.  allows detection of all RNA subtypes as well as of unannotated transcripts and has a high sensitivity toward identification of low-abundance RNAs. In case of exosomes, this approach was applied only for the analysis of small (20–70 nt) RNAs .In this study, we utilized the RNA-Seq approach to characterize the transcriptomes of exosomes secreted by two metastatic human breast cancer cell lines. We describe optimized computational workflow to analyze data generated by the Ion Torrent semiconductor chip-based technology. We have identified and profiled RNA species present in exosomes and host cells and discuss the utility of exosomal RNA as potential breast cancer-specific biomarkers.® HTB-130™) and MDA-MB-231 (ATCC® HTB-26™) were maintained at 37°C in 5% CO2 and cultured in DMEM/F12 supplemented with 10% FBS. 48 h prior to exosome collection, cells were washed 3 times with PBS and the medium was changed to serum-free CCM5 medium (Thermo Scientific).Human breast cancer cell lines MDA-MB-436  at 25°C. Particle movement was tracked by NTA software  with low refractive index corresponding to cell-derived vesicles. Each track was then analyzed to get the mean, mode, and median vesicle size together with the vesicle concentration (in millions) for each size.Total RNA from exosomes (MDA-MB-436 and 231) and cultured cells (MDA-MB-231) were isolated using the TRIzol reagent (Invitrogen). RNA quality and concentration were assessed with the Agilent 2100 Bioanalyzer (Thermo Scientific). Cellular RNA was analyzed using RNA 6000 Nano Kit (Agilent) and exosomal RNA was analyzed using RNA 6000 Pico kit (Agilent).Two hundreds ng of exosomal RNA and 3 µg of total cell RNA was used as the starting input for RNA-Seq library preparation. Sequencing was performed by AITbiotech company (Singapore). Briefly, total cell RNA was treated with RiboMinus Eukaryote kit Life Technologies) to remove rRNA. Then, exosomal and rRNA-depleted cellular RNAs were fragmented using RNaseIII. Whole transcriptome library was constructed using the Ion Total-RNA Seq Kit v2 (Life Technologies). Bar-coded libraries were quantified with qRT–PCR. Each library template was clonally amplified on Ion Sphere Particles (Life Technologies) using Ion One Touch 200 Template Kit v2 (Life Technologies). Preparations containing bar-coded libraries were loaded into 318 Chips and sequenced on the PGM  was determined for the host cells and exosomal genes expression and normalized to an endogenous control GAPDH relative to a calibrator as 2t−ΔΔC (where ΔC = (Ct of gene of interest) – (Ct of endogenous control gene (GAPDH) and ΔΔCt = (ΔCt of samples for gene of interest) – . Melting curves of each amplified products were analyzed to ensure uniform amplification of the PCR products.Two-step RT-PCR was performed using the QuantiTect Reverse Transcription Kit  according to manufacturer’s protocol. RT-PCR was performed in a Rotor-Gene (Qiagen) using a SYBR Green PCR Master Mix. Primer sequences are provided in Raw reads generated by sequencing were subjected to several quality checks. The low quality reads were removed by read trimming and read filtering. Read trimming included removal of adapter sequences, removal of the 3′ ends with low quality scores and trimming based on High-Residual Ionogram Values. Filtering of entire reads included removal of short reads, adapter dimers, reads lacking sequencing key, reads with off-scale signal and polyclonal reads. Subsequent analysis was performed with high quality reads which passed through the described above filtering steps.Bowtie 2 version 2.1.0 was used to align all high-quality reads to rRNA sequences including 28S (NR_003287.2), 18S (NR_003286.2), 5S (NR_023379.1), and 5.8S (NR_003285.2) rRNA. Reads mapped to rRNA sequences were filtered out while the rest of the reads were mapped to the human genome. The high-quality reads were mapped to hg19 build of the human genome from University of California Santa Cruz (UCSC) genome browser database  using ToThe mapped reads were further manipulated by removing the reads that mapped to multiple locations. In particular, the short aligned reads with the length of <20 nucleotides were eliminated to avoid the alignment errors such as mapping to multiple genomic locations. Further filtering included the removal of the low quality reads which fall below the mapping quality score of 10 (-q 10) using SAMtools. For the coverage search, the BAM file generated by Tophat2 was converted to BED format with option (-split) using BEDtools. The BED file was converted again to BAM format using BEDtools. We then developed python script (using pysam as part of the scripts) to calculate the number of reads and read coverage in exons and protein-coding sequence (CDS) regions consecutively.RNA abundance was estimated with the help of Partek Genomics Suite software  using Reference Sequence Gene (RefSeq Gene) and GENCODE annotation built version 14 of those not overlapped with RefSeq Gene from UCSC genome browser. The Expectation-Maximization (E/M) Algorithm  was usedp-value to demonstrate Gene ontology (GO) and KEGG molecular pathway enrichment, where p-values less than 0.05 after Benjamini multiple test correction are considered to be strongly enriched in the annotation category. We also used a count threshold of 5 and the default value of 0.1 for the EASE (enrichment) score settings. We used more specific GO term categories provided by DAVID, called GO FAT, to minimize the redundancy of general GO terms in the analysis to increase the specificity of the terms.Database for Annotation, Visualization, and Integrated Discovery (DAVID) (version 6.7) was used to identify gene functional annotation terms that are significantly enriched in particular gene lists with the whole Human genes as the background . A list 6 vesicles per cm2 of growth area per 48 h that were predominantly 115 nm in size. MDA-MB-436 cells released 1.04 × 107 vesicles per cm2 of growth area per 48 h that were predominantly 91 nm in size . NTA showed that MDA-MB-231 cells released 4 × 10 in size . The siz in size  typical RNA was isolated from exosomes released by both breast cancer cell lines. Total RNA was also extracted from MDA-MB-231 cell line as a control host cell line that produced exosomes. Bioanalyzer data revealed that exosomes contain a broad range of RNA sizes (30–500 nt) and have very small amount of intact rRNA (5.2% in MDA-MB-231 exosomes and 5.6% in MDA-MB-436 exosomes)  consisteWe utilized the Ion Torrent sequencing technology to profile exosomal RNA produced by MDA-MB-436 and MDA-MB-231 cell lines, as well as RNA obtained from host cell line MDA-MB-231. Based on RNA quality assessment using Bioanalyzer profiling  we perfoRNA-Seq computational analysis workflow is presented in Reads mapped to multiple locations in the genome have been eliminated to obtain uniquely mapped reads. Subsequent downstream analysis was performed with the high quality reads which had the read length greater than 20 bps and mapping quality score of above 10 (see Methods). As a result, about 81, 76, and 70% out of all mapped reads were considered as high quality mapped reads for MDA-MB-231 and MDA-MB-436 exosomal RNA, and MDA-MB- 231 cellular RNA, respectively.Approximately 97% (∼2 M reads) of reads from both exosomal RNA samples were mapped to rRNA sequences  and ∼2% In total, 16,086 transcripts  were detected with a normalized RPKM value of greater than 1.0 at least in one sample. We used Integrative Genomics Viewer (IGV) version 2.1.21  to visuaUsing these criteria, we obtained lower number of annotated transcripts  compared to that when RPKM values were considered . As a reP-value cut-off of p < 0.05 for 3115 host MDA-MB-231 cellular genes: 286 Biological Process (BP) categories and 91 Molecular Function (MF) categories (We performed Gene Ontology (GO) enrichment analysis using the DAVID bioinformatics resource, which employs a Fisher’s Exact Test with Benjamini–Hochberg correction. A total of 377 enriched GO categories were derived using a tegories . In totategories . At the tegories . Since ttegories  of thesetegories . We furttegories . These dNon-coding transcripts could be classified into 13 categories see . Both exBased on RNA-Seq data we evaluated presence and enrichment of several mRNA transcripts in exosomal RNA - RAB13, RPPH1, EEF1A1, FTH1, FTL and RPL28. qRT-PCR analysis showed presence of all selected transcripts in exosomal samples . Figure Until recently, the changes in gene expression during various biological processes have been analyzed using microarray approaches that focus largely on the behavior of protein-coding transcripts. Because microarrays are based on hybridization, they have high background owing to cross-hybridization, they have a limited dynamic range of detection and they rely upon known structures of genes. Development of RNA-Seq technology permitted comprehensive analysis of whole transcriptomes with the single nucleotide resolution allowing quantification of most RNA molecules expressed in the cell or tissue . In thisWe observed a large proportion of reads mapped to rRNA regions in exosomal samples. This was surprising given the fact that intact 18S and 28S rRNA peaks were almost undetectable in exosomal RNA . This obRegardless the biogenesis of rRNA fragments, it is advisable to perform rRNA depletion step even in the absence of visible rRNA peaks on RNA profiles. This procedure would allow obtaining much higher sequencing depth for other RNA species. Our attempt to deplete fragmented rRNA with the popular RiboMinus™Eukaryote Kit failed because of the design of the probes. Because the probes size is short, many fragments of rRNA are not targeted. The use of larger number of longer probes is expected to produce a more efficient way of pulling-down fragmented rRNA. This technical aspect of working with exosomal RNA samples should be certainly considered in the future studies.As a result of large rRNA presence in exosomal samples we observed only 2% of mapped reads to known transcripts using RefSeq and GENECODE gene models. Moreover, with RPKM value >1 we observed a large amount of misannotation due to poor coverage of the reads over transcripts. Therefore, we suggested another approach, namely filtering genes based on reads coverage over protein coding sequence for mRNA or exons for non-coding RNA. This procedure allowed us to achieve more than 90% coverage for protein-coding and non-coding regions which we considered as highly reliable for functional classification. This approach was helpful to reveal highly expressed genes in exosomes which could be potentially used as noninvasive breast cancer markers.We report that exosomes are carrying mRNAs that are highly expressed in the host breast cancer cells . Thus exIn conclusion, here we demonstrated for the first time that fragmented rRNA is a major species of exosomal RNA. Proposed here custom bioinformatics workflow allowed us to reliably detect other, non-ribosomal RNAs under conditions of limited read numbers. Classification and quantification of the RNA-Seq data revealed that exosomal transcripts are representative of their cells of origin and thus could form basis for detection of tumor specific markers. This information can also be used for getting insights in the molecular underpinnings of biological effects produced by these microvesicles. Finding that exosomes bear mRNAs encoding the necessary components to build-on-site ribosomes provides a valuable insight into biological function of these vesicles.10.7717/peerj.201/supp-1Figure S1FTL gene  is selected as an example of alignment comparison. TMAP alignment resulted in poor reads mapping and absence of junctions over exon-exon region. As the same time, TopHat identifies the exon-exon splice junctions and connects the exons through a linker.Click here for additional data file.10.7717/peerj.201/supp-2Figure S2rRNA defined as 5S, 5.8S, 18S, and 28S rRNA sequences. Reads which overlapped with annotated gene models (RefSeq and/or GENCODE) are termed as “known genes”. Reads that placed outside of annotated gene models are termed as “unkown”.Click here for additional data file.10.7717/peerj.201/supp-3Figure S3(A) RNA read density plot represents RNA fragments which fully covers of 5S, 5.8S, 18S, and 28S rRNA sequences from exosomal RNA. (B) 18S and 28S rRNA were major fractions of rRNA species.Click here for additional data file.10.7717/peerj.201/supp-4Figure S4RNA was detected with PicoChip using Bioanalyzer. The depletion procedure has been performed according to the manufacturer’s protocol. Control samples (red) were treated exactly as experimental samples (blue) except they did not contain RiboMinus™ Probe.Click here for additional data file.10.7717/peerj.201/supp-5Figure S5The detection criteria is that gene has more than 1 RPKM in at least one sample, while another approach is that gene has more than 90% coverage over protein-coding or non-coding sequence.Click here for additional data file.10.7717/peerj.201/supp-6Table S1(A) The Partek genomic suite output showing transcripts with RPKM >1 in at least one sample. The read counts from the transcript isoforms were estimated using EM algorithm from Partek genomic suite on RefGene and/or GENCODE v14 gene models. (B) Estimation of read coverage (in percentage) and read count of transcript. The transcripts with >90% coverage for protein-coding sequence and exonic sequence (in case of non-coding transcript) of transcript isoforms on RefGene and/or GENCODE v14 gene models are shown.Click here for additional data file.10.7717/peerj.201/supp-7Table S2(A) Gene lists for GO enrichment analysis. (B) GO enrichment of cellular genes. (C) GO enrichment of exosomal genes. (D) GO enrichment of top 115 expressed cellular genes.Click here for additional data file.10.7717/peerj.201/supp-8Table S3Click here for additional data file.


Here, we present an overview of how proteins interact with RNA molecules in order to achieve properly folded states. In this respect, the discrimination between static and transient interactions is crucial, as different proteins have evolved a multitude of mechanisms for RNA remodeling. For RNA chaperones that act in a sequence-unspecific manner and without the use of external sources of energy, such as ATP, transient RNA–protein interactions represent the basis of the mode of action. By presenting stretches of positively charged amino acids that are positioned in defined spatial configurations, RNA chaperones enable the RNA backbone, via transient electrostatic interactions, to sample a wider conformational space that opens the route for efficient refolding reactions.The RNA folding trajectory features numerous off-pathway folding traps, which represent conformations that are often equally as stable as the native functional ones. Therefore, the conversion between these off-pathway structures and the native correctly folded ones is the critical step in RNA folding. This process, referred to as RNA refolding, is slow, and is represented by a transition state that has a characteristic high free energy. Because this kinetically limiting process occurs  Only one of these conformers  can be aminoacylated and thus serve as a transfer molecule during translation [RNA folding is the crucial process that connects RNA synthesis to RNA function. Many (non)coding RNAs and rocesses . The strnslation , demonstnslation . Riboswinslation . These anslation ,5–7. Becnslation –10. The nslation . RNA folnslation ) or by inslation ,14.Many factors influence the kinetics of RNA folding reactions. Environmental variables, such as temperature or the speed of synthesis and decay of the RNA molecule ,16, are The mechanisms by which proteins shape the RNA folding pathway can be subdivided into two main classes ,21. The The second mechanistic class of protein-assisted RNA folding is characterized by weak, nonspecific interactions. Here, the transient interaction of proteins with the RNA molecule destabilizes misfolded intermediates and lowers the free energy of transition states between conformations. As a consequence, a smoother energy landscape is produced that increases the rate of folding and the probability that a molecule will find its native structure. In this review, we will focus on those proteins that undergo transient interactions with RNA molecules during their folding process or during their assembly into RNP complexes.RNA folding reactions can be modulated either by tight binding to proteins, establishing a functionally static RNA·protein complex, or by transient interactions with proteins that dissociate from the RNA after a stable conformation is established. Generally, transient interactions are most important in reactions where a high turnover is required and the slow folding of one component is detrimental to the assembly of a higher RNP complex (e.g. spliceosome or ribosome). The folding-assisting protein has to dissociate to enable the RNA to function when it has adopted its functional conformation .koff ≥ 0.2 s−1) [To best describe the nature of transient interactions, they are compared with static interactions, as they have an exactly opposite character. Tight complexes have long lifetimes (seconds or longer), whereas RNA-protein complexes based on transient interactions have lifetimes ranging from microseconds to milliseconds. Typically, the characteristic affinities for two binding partners that only interact transiently are found to be in the micromolar to millimolar range, because the off-rates are high (0.2 s−1) . A furth0.2 s−1) , KH doma0.2 s−1) , CCHH-zi0.2 s−1) , dsRBDs 0.2 s−1) , and PAZ0.2 s−1) , the int0.2 s−1) . NonetheGlocal/ΔGglobal ratio is far from unity and thereby unbalanced , then two possible scenarios of how proteins may contribute to the successful achievement of a ΔGlocal/ΔGglobal ratio close to unity can be envisioned – either the protein stabilizes structure elements that are responsible for the formation of the global structure of the RNA (such as tertiary interactions) by recognition and subsequent binding to them, or the protein destabilizes local interactions (which mainly involve secondary structure elements), e.g. by opening base pair interactions.As mentioned above, optimal folding rates of RNA require an energetic balance between local and global interactions within the molecule . If thisWithin the framework of this theoretical consideration, three types of proteins have been found to promote RNA folding: (a) specifically binding proteins, which recognize and bind certain RNAs and thus stabilize the RNA structure, thereby forming a stable RNA-protein complex; (b) proteins with RNA chaperone and annealing activity, which interact only transiently with RNAs without the recognition of a specific structure or sequence, thereby promoting folding via unfolding or via annealing acceleration; and (c) RNA helicases, which accelerate the unwinding of many RNAs under conditions of ATP binding and hydrolysis.Here, we summarize the properties of the three protein classes, with the main focus being on RNA chaperones and annealer proteins.A specific protein cofactor binds to its RNA target through well-defined structural features, thereby stabilizing its native structure. Two scenarios have been shown or postulated – either the protein can bind to the RNA molecule when it has already adopted its correct structure, or the specific binder can interact with the RNA during its folding process and can accelerate folding or even nucleate the folding event. In a distinct mechanism, the protein may capture one specific conformation out of an ensemble of possible structures .Neurospora crassa, which binds specifically to group I introns, thereby stabilizing the three-dimensional structure of the RNA. The protein can display RNA chaperone activity when interacting with nonspecific RNAs [While the functional fold of the RNA molecule has not yet been achieved, the protein can interact transiently with the native RNA substrate. During this first encounter, the protein can perform unfolding activities reminiscent of RNA chaperone activities to support the folding process and to achieve specific binding. Furthermore, specific binders have been shown to exert RNA chaperone activity when encountering RNAs that do not contain the canonical binding motif. A well-studied example is the CBP2 protein from yeast mitochondria, which binds specifically to the bI5 group I intron . The intfic RNAs ,35. In afic RNAs .DEAD-box proteins are RNA helicases that are ubiquitous in all RNA-mediated processes. They use ATP hydrolysis to (mostly sequence-independently) promote conformational changes in RNA molecules, to disrupt RNA structures in a nonprocessive way, and to accelerate structural transitions in RNAs and RNP complexes . DEAD-boDEAD-box proteins have low processivity when unwinding helices shorter than 25–40 base pairs ,43. FollXenopus laevis protein X1rbpa [Escherichia coli protein StpA [RNA annealer proteins are able to accelerate annealing of complementary nucleic acid sequences. RNA chaperones have the ability to destabilize formed RNA structures, which is measurable in strand displacement assays, and may additionally accelerate annealing. The hypothesis that RNA chaperones and annealers interact with their targets in a transient way is founded on four main observations, as follows. (a) By definition, sequence-nonspecific activity is inherent to RNA chaperones ,45. Althn X1rbpa , the tryn X1rbpa , and theein StpA , an inveet al. (2008) [2 concentrations above 2 mm and at NaCl concentrations above 60 mm. Supporting the assumption of ionic interactions between peptide and RNA, the overall charge of the peptide was crucial for the activity, as the replacement of single basic amino acids with alanine resulted in the annealing rate constant decreasing by a factor of 2.3–3 as compared with the wild-type peptide. Thermodynamic calculations regarding the transition state of the reaction explained the importance of the overall charge for the activity – the total peptide charge determines the magnitude of peptide–RNA binding, owing to counterion release from the RNA backbone [1D1H-NMR spectra of a single-stranded RNA showed that, depending on the amount of peptide added, the Tat peptide induced a change in the population of coexisting and interchanging RNA conformations. The lack of intermolecular NOE connectivities indicated a short residence time of the peptide in the RNA-peptide complex, confirming the transient interaction between the molecules [in vivo, such as transcriptional activation of the viral genome, remains to be elucidated.The peptide Tat(44–61) is an 18-residue fragment of the HIV-1 Tat protein. Its sequence-nonspecific annealing activity was first described by Kuciak . (2008) . Because. (2008) . We founbackbone . The resolecules . Taking E. coli genome and thus regulates various genes [in vitro and in vivo. Restricted proteolysis experiments demonstrated a modular architecture of the protein, with two separate structural and functional domains. Most data map the RNA interaction function to the C-terminal domain (CTD) of StpA. Accordingly, this domain alone is able to catalyze RNA folding, as demonstrated in various different assays. In order to exert RNA chaperone activity, both the full-length protein and the CTD must be present in concentrations close to the respective dissociation constants, which are usually in the micromolar range [koff must be high . Interestingly, the StpA G126V mutant shows a dramatically reduced binding affinity, despite being more active in a chaperone assay than the wild-type protein [The nucleoid-associated protein StpA in the form of a heterodimer with its homolog H-NS shapes the structure and organization of the us genes . Besidesus genes . Moreovear range –70. Thisar range . Interes protein . Stressi protein . In all,As StpA and also its CTD alone can promote annealing as well as displacement of complementary RNAs, the question of which changes in the RNA are introduced during the transient interaction arises. Initial results indicate that the protein acts as an electrostatic lubricant that shields repulsive interactions within the RNA molecule. The protein thereby smooths the folding energy landscape. The direction of the RNA folding reaction  is then no longer kinetically controlled, but instead follows the reaction route determined by thermodynamics.From the observations described above, we have delineated a general model for the mechanism of protein-facilitated annealing and strand displacement .To illustrate the mechanism of RNA annealing acceleration, we first consider the annealing of RNA in the absence of any supporting protein . Like otThe strand displacement event resulting in an RNA duplex caused by a third, invading RNA molecule is often closely connected with the process of RNA annealing ,74. RNA A clear advantage of transient interactions between RNA annealers/chaperones and their substrates is the low energy consumption of the reaction, especially in comparison with helicases, which have an ATP-dependent activity. Further advantages of transient interactions are a broad spectrum of substrates and the rapid availability of the protein for subsequent reactions. In order to avoid the general impairment of important cellular RNA structures, stringent regulation of expression and activity of these proteins is necessary. Thus, general RNA annealers and chaperones may be useful additions to the arsenal of specific RNA binders and helicases for the structural remodeling of RNA molecules.


The Ct values of housekeeping genes, 18S rRNA, β-actin, RPLP0 and HPRT1, were as follows: TRI reagent , PAXgene 16.6 ± 1.4, 26.4 ± 1.3, 28.2 ± 1.8 and 33.8 ± 1.1), and NucleoSpin . RNA yield using TRI reagent was 1.7 times higher than that with NucleoSpin and 5 times higher than that with PAXgene. However, the purity and integrity of TRI-extracted RNA was lower than that extracted with PAXgene and NucleoSpin. Moreover, the Ct values of housekeeping genes after extraction with TRI reagent were approximately 1.7-3.8 times higher than those obtained with PAXgene and NucleoSpin. The PAXgene and NucleoSpin kits produced similar results in terms of RNA purity and integrity and subsequent gene amplification. However, RNA yields from NucleoSpin were 2.9-fold higher, compared to PAXgene. Based on these findings, we conclude that NucleoSpin is the most effective kit for extraction of abundant and high-quality RNA from frozen blood.Extraction of high-quality RNA is a crucial step in gene expression profiling. To achieve optimal RNA extraction from frozen blood, the performance of three RNA extraction kits- TRI reagent, PAXgene blood RNA system (PAXgene) and NucleoSpin RNA blood kit (NucleoSpin)- was evaluated. Fifteen blood specimens collected in tubes containing potassium ethylenediaminetetraacetic acid (EDTA) and stored at −80°C for approximately 5 years were randomly selected. The yield and purity of RNA, RIN (RNA integrity number) values and cycle threshold (Ct) values were assessed. Mean RNA yields with TRI reagent, PAXgene and NucleoSpin were 15.6 ± 8.7 μg/ml, 3.1 ± 1.7 μg/ml and 9.0 ± 5.5 μg/ml, respectively. Mean A Human blood specimens are routinely collected by biobanks for medical applications, scientific research, and diagnostic purposes  is based on this technology and rapidly inhibits RNase activity, making it a complete, ready-to-use reagent for total RNA extraction , facilitates extraction of RNA from blood samples collected in EDTA blood collection tubes.In the current study, three different RNA extraction kits, specifically, TRI reagent, PAXgene, and NucleoSpin, were compared in terms of RNA yield, purity, and integrity, with a view to achieving optimal high-quality RNA extraction from frozen whole blood. RNA extracted with the three kits was additionally assessed for use in real-time PCR assays.260/A280 ratios are thus used to indicate RNA sample purity. The mean RNA purity (A260/A280) was evaluated as 1.7 ± 0.1 with TRI reagent, 2.0 ± 0.1 with PAXgene, and 2.0 ± 0.0 with NucleoSpin. Generally, samples with ratios in the range of 1.8 ~ 2.0 indicate higher levels of RNA purity. The A260/A280 ratios for PAXgene and NucleoSpin extractions were within the desired range, while the ratio for TRI reagent was somewhat lower, indicative of protein or DNA contamination with this kit. As a secondary measurement of RNA purity, A260/A230 ratios were additionally evaluated for extracts with TRI reagent (1.4 ± 0.4), PAXgene (1.1 ± 0.3), and NucleoSpin (1.8 ± 0.2) , PAXgene, 3.1 ± 1.7 μg/ml , and NucleoSpin, 9.0 ± 5.5  μg/ml  values. As shown in Figure 18S rRNA, 30.6 ± 1.8, 26.4 ± 1.3 and 27.2 ± 1.3 for β-actin, 29.9 ± 1.4, 28.2 ± 1.8 and 27.0 ± 1.6 for RPLP0, and 36.3 ± 1.3, 33.8 ± 1.1 and 32.9 ± 1.6 for HPRT1, respectively. The Ct values for RNA extracted using TRI reagent were ~1.7 to 3.8 times higher than those evaluated with the PAXgene and NucleoSpin kits, clearly indicating that RNA extracted with TRI reagent is of lower quality.To further validate the quality of RNA obtained using the three different extraction kits, real-time PCR analysis was performed. The expression patterns of four housekeeping genes, 260/280 ratio of 1.7) and integrity (RIN 3.2) were lower, compared to RNA extracted using the PAXgene  and NucleoSpin  systems. Earlier, Fleige and co-workers reported that RNA with low RIN values shows reduced PCR performance due to the presence of more highly fragmented molecules, leading to inaccurate gene expression evaluation , transferred to Axygen microtubes , and stored at −80°C. After approval of the Institutional Review Board, 15 frozen blood specimens stored for approximately 5 years were randomly selected.Frozen blood specimens were thawed on ice and RNA extracted using three different kits  according to the respective manufacturer protocols.For TRI reagent extraction, 200 μl of each blood sample was incubated with 800 μl TRI reagent for 5 min at room temperature, followed by supplementation with 200 μl chloroform. After vigorous mixing and centrifugation at 12,000 g for 15 min at 4°C, the upper layer was transferred to a new tube. An aliquot of 500 μl isopropanol was added and the resulting mixture incubated for 10 min at room temperature, followed by centrifugation at 12,000 g for 15 min at 4°C to pellet RNA. The pellet was washed with 1 ml 75% ethanol and air-dried for 5 min. RNA was dissolved in 40 μl RNase-free water.For PAXgene extraction, 300 μl of each blood sample was dispensed into 830 μl PAXgene reagent at a blood:reagent ratio of 2.5:6.9, which was the same as in the PAXgene Blood RNA Tubes . RNA purity was calculated from the ratio of absorbance at 260 nm and 280 nm, and integrity assessed using the Eukaryote Total RNA Nano assay on the Agilent 2100 Bioanalyzer . The RNA integrity number (RIN) was calculated using the Agilent 2100 Bioanalyzer and accompanying software.18S rRNA (Hs99999901_s1), β-actin , RPLP0 (Hs99999902_m1) and HPRT1 (Hs99999909_m1) genes were amplified and quantified. Each reaction mixture (20 μl) was transferred to a 96-well plate, which was loaded onto the 7500 Real Time PCR System . Plates were heated to 50°C for 2 min and 95°C for 10 min, and subjected to cycles of 95°C for 15 sec and 60°C for 1 min. Data were expressed as cycle threshold (Ct) values.cDNA was reverse-transcribed from total RNA using SuperScript II RNase transcriptase  following the manufacturer’s guidelines. In total, 200 ng of total RNA was used as template. The reaction mixtures were incubated in a 2720 Thermal Cycler . TaqMan Gene Expression Master Mix was used to perform real-time PCR . t-test. Asterisks (***p < 0.001) represent statistically significant differences.Experimental data are expressed as mean values and standard deviation. The performance of the three RNA extraction kits was compared with the Student’s


In fission yeast, centromeric heterochromatin is necessary for the fidelity of chromosome segregation. Propagation of heterochromatin in dividing cells requires RNA interference (RNAi) and transcription of centromeric repeats by RNA polymerase II during the S phase of the cell cycle.dh and dg repeats and for the silencing of reporter genes inserted in centromeric heterochromatin. Mutations in the Med8-Med18-Med20 submodule did not alter Mediator occupancy at centromeres; however, they led to an increased recruitment of RNA polymerase II to centromeres and reduced levels of centromeric H3K9 methylation accounting for the centromeric desilencing. Further, we observed that Med18 and Med20 were required for efficient processing of dh transcripts into siRNA. Consistent with defects in centromeric heterochromatin, cells lacking Med18 or Med20 displayed elevated rates of mitotic chromosome loss.We found that the Med8-Med18-Med20 submodule of the Mediator complex is required for the transcriptional regulation of native centromeric Schizosaccharomyces pombe centromeres. In wild-type cells this submodule limits RNA polymerase II access to the heterochromatic DNA of the centromeres. Additionally, the submodule may act as an assembly platform for the RNAi machinery or regulate the activity of the RNAi pathway. Consequently, Med8-Med18-Med20 is required for silencing of centromeres and proper mitotic chromosome segregation.Our data demonstrate a role for the Med8-Med18-Med20 Mediator submodule in the regulation of non-coding RNA transcription at  Saccharomyces cerevisiae Mediator was the first to be characterized but Mediators have since then been described in many other species. A comparative genomics approach of approximately 70 eukaryotic genomes shows that although its exact subunit composition varies, Mediator is conserved across the eukaryotic kingdom  [+ due to the interallelic complementation between ade6-M210 and ade6-M216. They form white colonies on media containing low concentrations of adenine. Loss of Ch16m23::ura4+-Tel[72] results in red colonies or sectors. White and sectored colonies were counted following plating of the strains of interest on yeast extract plates to which no adenine had been added. The rate of minichromosome loss was determined as the number of colonies with a red sector equal to or greater than half the colony size  divided by the number of white or sectored colonies.Mitotic chromosome loss was assayed as previously described  using ce-Tel[72] . Cells w32P]-dCTP . The template for random priming was a dh repeat PCR product amplified from genomic DNA with the dhH-siRNA and Cen-dh-FOR2 primers. Northern blots detecting the dg and dh repeats were obtained following electrophoresis of 10 μg total RNA prepared by a hot phenol protocol from the strains of interest. The gels used were 1% agarose in MOPS buffer with 6.7% formaldehyde. RNA was blotted onto a Hybond-XL membrane . The dg and dh repeats were detected by a random-primed [α-32P]-dCTP radioactively labeled probe made on PCR products amplified from genomic DNA using p30F and p30R (dh repeat) or p33F+p33R (dg repeat). Hybridizations were performed overnight at 42°C in PerfectHyb PLUS hybridization buffer .For siRNA Northern blots, total RNA was isolated with Tri Reagent  and 20 μg RNA was run on a 17.5% polyacrylamide/7 M urea gel and blotted onto a positive nylon membrane . siRNA were detected using a random-primed probe radioactively labeled with [α-dh repeat, oMiT142 and oMiT143 for the dg repeat, or oMiT127 and oMiT128 for the putative promoter.ChIP was performed according to standard procedures. Antibodies used to immunoprecipitate RNA Pol II and H3K9me2 were ChIPAb RNA Pol II  and histone H3 (dimethyl K9) antibody ChIP Grade ab1220 , respectively. Protein G Dynabeads were used to pull down the antibody captured proteins. Rabbit Anti-Mouse Immunoglobulins  were covalently coupled to the surface of Dynabeads with the Dynabeads Antibody Coupling Kit  and these beads were used to pull down the Mediator complex through a TAP-tagged Med7. Presence of RNA Pol II, Mediator or dimethyl H3K9 was detected by qPCR using the primers dhA q-PCR FW and dhA q-PCR RV for the ChIP: Chromatin immunoprecipitation; RNA Pol II: RNA polymerase II; RT-PCR: Reverse transcription PCR; RT-qPCR: Quantitative reverse transcription PCR.The authors declare that they have no competing interests.MT, HH and MV carried out the research. MT, GT and SH wrote the manuscript. GT and SH provided guidance in experimental design and interpretation of data. All authors read and approved the final manuscript.


Escherichia coli RNA polymerase core enzyme obtained through a classic purification procedure forms stable (α2ββ'ω)2 complexes in the presence or absence of short DNA probes. Multiple control experiments indicate that this self-association is unlikely to be mediated by RNA polymerase-associated non-protein molecules. We show that the formation of (α2ββ'ω)2 complexes is subject to regulation by known RNA polymerase interactors, such as the auxiliary SWI/SNF subunit of RNA polymerase RapA, as well as NusA and σ70. We also demonstrate that the separation of the core RNA polymerase and RNA polymerase holoenzyme species during Mono Q chromatography is likely due to oligomerization of the core enzyme. We have analyzed the oligomeric state of the polymerase in the presence or absence of DNA, an aspect that was missing from previous studies. Importantly, our work demonstrates that RNA polymerase oligomerization is compatible with DNA binding. Through in vitro transcription and in vivo experiments (utilizing a RapAR599/Q602 mutant lacking transcription-stimulatory function), we demonstrate that the formation of tandem (α2ββ'ω)2–DNA complexes is likely functionally significant and beneficial for the transcriptional activity of the polymerase. Taken together, our findings suggest a novel structural aspect of the E. coli elongation complex. We hypothesize that transcription by tandem RNA polymerase complexes initiated at hypothetical bidirectional “origins of transcription” may explain recurring switches of the direction of transcription in bacterial genomes.In this work, using multiple, dissimilar physico-chemical techniques, we demonstrate that the  Escherichia coli, the chromosomal DNA is replicated by the DNA polymerase III complex, which contains two copies of the pol III core connected through a single clamp loader complex 2ββ'ω subunits transcribes the same DNA In E. coli RNA polymerase was initially isolated and characterized, multiple, independent studies reported self-association of RNA polymerase in vitroWhen the E. coli RNA polymerase core enzyme, obtained through a purification procedure similar to that developed by Hager et al.2ββ'ω)2 complexes in the presence or absence of short DNA probes. Multiple control experiments indicate that this association is unlikely to be mediated by accessory nucleic acids or other RNA polymerase-associated non-protein molecules. Furthermore, we show that the formation of these complexes is subject to regulation by RapA, NusA, and σ70. Our data demonstrate a physical interaction between individual α2ββ'ω complexes in vitro and the likely functional significance of this association for the transcriptional activity of the polymerase. We have also analyzed the oligomeric state of the polymerase in the presence or absence of DNA, an element that was missing from previous E. coli RNA polymerase studies. Our findings suggest a novel structural aspect of the E. coli elongation complex that warrants further studies addressing the functional significance of E. coli RNA polymerase self-association in vivo.In this work, using multiple, dissimilar physico-chemical methods, we demonstrate that the Escherichia coli RNA polymerase on a regular basis utilizing a protocol reminiscent of that developed by Hager et al. E. coli core RNA polymerase eluted before the RNA polymerase holoenzyme. It ran as a single peak with an apparent molecular mass of 780–800 kDa, which is approximately double the predicted molecular mass of an RNA polymerase core enzyme ‘monomer’ and well within the column's separation range (>8 min after the column's void volume) (2ββ'ω)2 core RNA polymerase complexes.Due to our interest in the function and mechanism of the RNA polymerase-associated SWI/SNF homolog RapA  volume) . The mob2ββ'ω complexes. To remove such  accessory material, we subjected core enzyme from the final purification step in the protocol described in 32P] ATP. Likewise, these experiments showed no detectable radioactivity co-migrating with the core enzyme in gel-filtration (data not shown).We questioned whether the core RNA polymerase purified in this manner could have retained small accessory molecules – such as nucleic acid fragments or lipids – which could have led to the self-association of individual α32P 5′ end-labeled DNA probe with the indicated secondary structure  an alternative technique not relying on gel-permeation chromatography to compare the molecular masses of the core RNA polymerase and the RNA polymerase holoenzyme under non-denaturing conditions and (b) to test the effect of DNA on the stability of the observed core RNA polymerase complexes. For this purpose, we used a short (30-nt), tructure  and comptructure . There wtructure . These rtructure , suggestE. coli RNA polymerase 2ββ'ω)2 complexes. We observed a significant shift in the core enzyme's electrophoretic mobility in the presence of RapA, which was consistent with its conversion to the ‘monomeric’ state in the presence of RapA , we again sought evidence for the existence of two distinct forms of the core enzyme in the absence of DNA. To this end, we analyzed the content of three key Mono Q peaks by native PAGE in the presence of various detergents. This set of experiments also tested potential contributions of small RNA polymerase-associated hydrophobic molecules (such as lipids) to RNA polymerase oligomerization. Excess deoxycholate (DOC) (the detergent used during the extraction stage in the RNA polymerase purification procedure) did not preclude the formation of (α2ββ'ω)2 complexes (2ββ'ω and (α2ββ'ω)2 forms toward single-copy complexes 2 complexes is compatible with the presence of duplex DNA templates. To this end, we took advantage of a set of extra-short – yet transcriptionally active – DNA templates that were recently constructed in connection with our study of the function and catalytic mechanism of RapA. In the hairpin-shaped DNA template selected for this analysis, shown schematically in E. coli RNA polymerase DNA footprints) also supported the formation of (α2ββ'ω)2–DNA transcription complexes  transcriptional activity of the polymerase. As seen from 2ββ'ω)2 complexes were efficiently dissociated by the RNA polymerase auxiliary SWI/SNF subunit RapA , we monitored the initial rates of non-promoter-specific RNA synthesis in reactions containing sequentially diluted core enzyme  complexes formed on more conventional DNA templates carrying previously characterized promoters could ‘dimerize’. To this aim, we used two dissimilar model systems/DNA templates, one of which contained the well-characterized 32P-labeled dsDNA templates containing the tac promoter, the addition of rNTPs shifted the equilibrium in the direction of ‘dimeric’ complexes  of (a) We demonstrate that the E. coli RNA polymerase core enzyme obtained in a purification procedure based on that described by Hager et al. 2ββ'ω)2 complexes. Multiple control experiments indicate that this oligomerization is unlikely due to mediation by RNA polymerase-associated accessory, non-protein molecules. (i) Gel-exclusion chromatography-, (ii) native PAGE-, and (iii) electron microscopy-based analyses yielded evidence supporting the formation of (α2ββ'ω)2 complexes. The RNA polymerase concentration corresponding to 50% oligomerization  Our work suggests that DNA-bound RNA polymerase may retain the ability to form oligomeric complexes. We show that (α2ββ'ω)2 complexes can be formed in the presence of transcriptionally active DNA templates. It is clear that if RNA polymerase were to elongate RNA transcripts in tandem in vivo – even only under specific conditions – this could have a significant impact on our understanding of the general mechanisms of transcription and its regulation in Eubacteria.(c) Importantly, we show that (α2ββ'ω)2 complexes can be dissociated into single-copy (α2ββ'ω) complexes by known protein interactors of the core enzyme, such as RapA, NusA, or σ70. We therefore found no evidence for oligomerization of the RNA polymerase holoenzyme under our experimental conditions, contrary to previous reports (d) Our results may explain the efficient separation of the E. coli core RNA polymerase and RNA polymerase holoenzyme during Mono Q chromatography. Our data indicate that this separation is likely due to differences in the oligomeric states of the two forms of the polymerase  We demonstrate that the oligomerization of RNA polymerase may be functionally significant. Our finding that RapA triggers the dissociation of putative tandem complexes likely explains previously reported (but never conclusively explained) observations that RapA, while capable of significant stimulation of multi-round transcription, actually reduces the initial rates of RNA synthesis E. coli RNA polymerase have previously reported the concentration-dependent ‘activation’ of the polymerase. For example, a 2003 study by Epshtein and Nudler reported such a phenomenon cis’ (within a transcription complex formed on a single DNA template). Transcriptional activation resulting from the interaction of RNA polymerase with a DNA-binding protein (other than RNA polymerase) is also well known and is, in fact, a basis for the bacterial version of a two-hybrid system. Our work, therefore, points to the possibility that such activation can be achieved through contact with another RNA polymerase molecule transcribing from a different template.(f) Utilizing DNA templates containing either tac or slyD promoters, we demonstrate ‘dimerization’ of the E. coli elongation (DNA–RNA polymerase–nascent RNA) complexes (omplexes  and S2.(a)–(f) warrant a hypothesis in which the E. coli core RNA polymerase carries out the elongation stage of its catalytic cycle in tandem, while the (sigma-assisted) initiation stage of the transcription cycle is carried out by a single-copy holoenzyme species (2ββ'ω)2 complexes. This alternative mode of binding predicts the existence of (2+n) core enzyme complexes , which, had they been present, should have been apparent during our gel-exclusion chromatography-based and native PAGE-based experiments (see comment ‘d’ above). The lack of experimental evidence for ‘head-to-tail’ stacking, therefore, supports the mode of interaction shown schematically in 2ββ'ω complexes. Even though at present we cannot entirely exclude the possibility that the core enzyme obtained in our purification procedure may contain deeply embedded nucleic acid fragments, extensive trials (involving T4 PNK labeling in the presence of [γ32P] ATP) carried out under varied experimental conditions consistently failed to detect the presence of exogenous nucleic acids that could contribute to the oligomerization of RNA polymerase. Furthermore, RNase A and DNase I digests of the ‘dimeric’ complexes formed by the core enzyme failed to shift the ‘monomer’–‘dimer’ equilibria  with either tethered or surface/grid-bound DNA molecules . However, we are not aware of any study in which analysis of the oligomeric states of the DNA-bound polymerase was carried out in a ‘soluble’ system. If E. coli RNA polymerase is indeed able to synthesize RNA and track DNA in tandem, this could potentially open new insights into the biophysics of transcription. It is possible to speculate that the energy needed for translocation of the polymerase along DNA could be at least partially derived from conformational changes of the nucleoid. . Discrepancies between the in vivo and in vitro transcription elongation rates may indirectly support this hypothesis. We also speculate that transcription by tandem RNA polymerase complexes initiated at hypothetical bidirectional ‘origins of transcription’ may explain recurring switches of the direction of transcription in bacterial genomes.It is generally believed that in E. coli RNA polymerase was purified as described et al.Gel-filtration-based studies were carried out using a Superose 6 10/300 column  integrated into an AKTA FPLC system . Samples were injected manually; the typical sample volume was 40 µl. Chromatography was carried out in 0.01 M Tris–HCl (pH 7.5), 5% glycerol, 0.2 mM EDTA (TGED buffer) containing 100 mM NaCl, at a flow rate of 0.5 ml/min. Elution times of the protein peaks were determined using Unicorn software . The core RNA polymerase utilized in this set of experiments (samples A1–A3) was isolated as described Electrophoretic Mobility Shift Assays (EMSA) were carried out using a Gibco BRL SA-type vertical electrophoresis apparatus. Polyacrylamide gels were cast using 30% Protogel ; 10× TBE  stock was diluted tenfold for the gel mix; 0.5× TBE was used as a running buffer. Unless indicated otherwise in Figure Legends, EMSA gels contained no detergents or chaotropic agents. Purified DNA probes were end-labeled using T4 polynucleotide kinase (Sigma) and [γ32P] ATP . 32P 5′ end-labeling and gel-purification of nucleic acid probes was carried out as previously described 32P-labeled DNA, and purified proteins; protein concentrations are specified in Figure Legends.Electron microscopic experiments were carried out essentially as previously described In vitro transcription experiments were carried out as previously described 2, 100 mM NaCl, 1 mM dithiothreitol (buffer B) or 1× buffer D. Synthetic DNA templates were obtained from Invitrogen. DNAs were purified either by cartridge or gel extraction (Qiagen). In kinetic experiments, enzyme–DNA mixtures were pre-incubated at room temperature for 15 min, and the transcription reaction was initiated by the addition of rNTPs.DNA templates containing tac and slyD promoters were constructed as follows. The 153-nt DNA containing the tac promoter was constructed as previously described E. coli chromosomal DNA using DNA primers MS722 (5′-AATTCTGTTGACAATTAATCATCGGCTCGTATAATGTGGGAATTGTGAGCGGATAACAATTTCACACAGGAAACACGCGAAGCGACTGAAGAAGAAC) (this primer introduced the tac promoter into the amplified DNA) and MS731 (5′-GTCGTGGTCGTGATCGTGGTGGTGATC) ; end-labeling and gel-purification of 32P-labeled DNAs were carried out as previously described mfd gene was amplified from MG1655 E. coli chromosomal DNA using MS781 (5′-GCCTATCCCGGGGGATCCTCATGCCTGAACAATATCGTTATACGCTGCCC) and MS782 (5′-GCCAAACCCGGGAAGCTTTTATTACCATTAAGCGATCGCGTTCTCTTCCAG) DNA primers and the Expand High FidelityPLUS PCR kit ; reactions were conducted as suggested by the manufacturer. Amplified linear DNA was gel-purified using the Qiagen Gel Extraction kit, digested with BamHI/HindIII and ligated into pQE32 vector DNA (Qiagen) digested with BamHI/HindIII . Expression of a full-length protein from the resulting construct was confirmed by SDS-PAGE.Plasmid pQE32Mfd used in the experiments described in Figure S1E. coli RNA polymerase in the presence of NusA. Reactions were similar to those in Disruption of the putative tandem complexes formed by the (TIF)Click here for additional data file.Figure S2in vitro transcription. Design and purification of the 965-nt linear DNA template containing the slyD promoter 70/mol core . When indicated, rNTPs were present at 0.2 mM. Following a 3-min incubation at 37°C, the reaction products containing 32P-labeled nucleic acids were fractionated by agarose gel electrophoresis. Purified RNA polymerase was present at either 15.9 nM (lanes 2 and 4) or 79.6 nM (lanes 6 and 8). Single-copy RNA polymerase-DNA (M) and putative tandem elongation complexes (D) are indicated. Panel at the left: the purified 32P-labeled DNA probe is shown on an ethidium bromide-stained 1% agarose gel next to a 1-kb DNA ladder (New England Biolabs).Formation of putative tandem elongation complexes during (TIF)Click here for additional data file.


The RNA structures specifying rapid or slow decay of different small cellular RNAs remain unstudied. We searched for RNA structural features that determine the short lifetime of 4.5SH in comparison with the long lifetime of 4.5SI RNA. The sequences of genes of 4.5SH and 4.5SI RNAs were altered and human cells (HeLa) were transfected with these genes. The decay rate of the original and altered RNAs was measured. The complementarity of 16-nt end regions of 4.5SI RNA proved to contribute to its stability in cells, whereas the lack of such complementarity in 4.5SH RNA caused its rapid decay. Possible mechanisms of the phenomenon are discussed.Two RNAs (4.5SH and 4.5SI) with unknown functions share a number of features: short length (about 100 nt), transcription by RNA polymerase III, predominately nuclear localization, the presence in various tissues, and relatively narrow taxonomic distribution . It was reported that 4.5SH RNA turns over rapidly, whereas 4.5SI RNA is stable in the cell, but their lifetimes remained unknown. We showed that 4.5SH is indeed short-lived (t Short lifetime of such mRNAs is due to the need to quickly change the cellular level of proteins encoded by them . The rapid degradation is usually due to the presence of AU-rich elements in mRNA Most types of RNA in mammalian cells are stable: their lifetime is estimated at hours or even days. However, some mRNAs are short-lived and their half-life  were discovered in the 70 s of the last century The most studied among them is BC1 RNA that is synthesized by RNA polymerase III (pol III) in the nervous tissue and testes of (presumably) all rodents, but not other mammals The two RNAs studied here, also belong to the stenoRNA group. 4.5SI RNA is present in rodents of only three related families: Muridae , Cricetidae , and Spalacidae  3H]uridine incorporation in 4.5SH RNA and concluded that this RNA has a rapid turnover in murine erythroleukemia cells. However, the lifetime of 4.5SH RNA was not estimated in those experiments. We have not found reports on 4.5SI RNA lifetime, although Ro-Choi et al. Schoeniger and Jelinek B), as well as the replacement of 3′ end (construct C), 5′ end (construct D) or central (construct E) regions of 4.5SH RNA with corresponding regions of 4.5SI RNA did not significantly change the stability of these RNAs, in comparison with natural 4.5SH RNA (construct A). However, the simultaneous replacement of 3′ and 5′ end regions in 4.5SH RNA (construct F) by the corresponding regions of 4.5SI RNA essentially increased the lifetime of the chimeric RNA. Its half-life was estimated as 2 h . The half-life of the RNA obtained was estimated as 2 h  resulted in disruption of the end complementarity (1/2∼20 min) (K) recovered the complementarity and rendered RNA stable  . On the A stable .Thus, the secondary structure (rather than the nucleotide sequence of 5′ and 3′ ends) is crucial for stability of 4.5SI RNA in the cell. Short lifetime of 4.5SH RNA is a result of the lack of the complementarity between its end regions, whereas other mechanisms seem not to be involved in stabilizing this RNA in the cell.1/2∼18 min in KAC cells). In human, chimpanzee, and gorilla (but not in other primates), a 117 nt RNA, SnaR , was found 4.5SH RNA belongs to the rare type of small RNAs that is characterized by short lifetime (tAAUAAA) are rapidly degraded in the cell (t1/2∼40 min), whereas polyadenylated B2 transcripts are stable Pol III transcripts of SINEs (that are unable for polyadenylation) are also short-lived small RNAs. Pol III transcripts of murine SINE B2 without a polyadenylation signal , apparently due to the large number (700–800) of genes encoding this RNA in the mouse genome 1/2∼22 h). These RNAs demonstrate a number of similar traits: length, synthesis by pol III, predominately nuclear localization, the presence in various tissues, relatively narrow taxonomic distribution, and the evolutionary relationship with SINEs. A priori the great difference between lifetimes can be due to the difference of nucleotide sequences of these two RNAs. For example, 4.5SH RNA could contain degradation signals, much as short-lived mRNAs carry AU-rich elements. On the other hand, 4.5SI RNA could contain nucleotide sequences that can specifically bind proteins preventing rapid degradation of the RNA.Here we searched for RNA structural features which determine the short lifetime of 4.5SH, as compared with the lifetime of 4.5SI RNA (tG). It was found that the lifetime of this RNA is also 6-fold longer than that of the original 4.5SH RNA. This result proves that it is the complementarity of the ends of the RNA, rather than primary structure per se, that increases the lifetime of the RNA.We found that replacing the nucleotide sequences of 4.5SH RNA with the corresponding regions of 4.5SI RNA did not significantly increase the lifetime of the chimeric 4.5SH RNA. These results argue against the hypothesis that the primary structure of some regions determines the lifetime of these two RNAs. On the other hand, the simultaneous replacement of 22 nt at 5′ end and 12 nt at 3′ end (construct F) increased the RNA lifetime by 6-fold. According to the secondary structure prediction, this double replacement results in the formation of a double-helical stem (16 bp with a single mismatch) that includes both termini of the RNA. A similar double-helical stem (but without the mismatch) can be predicted for 4.5SI RNA H. We su1/2∼2 h), and not many times more? Perhaps, other elements of secondary structure (in addition to 16-bp stem) also contribute to the long lifetime of 4.5SI RNA. In other words, probably only the entire secondary structure of genuine 4.5SI RNA ensures its long lifetime (t1/2∼22 h). One can speculate that some proteins recognize the RNA shape formed by its secondary structure and prevent its decay. However, this complex issue remains unexplored. At the same time, by changing 11-nt end regions to mutually complementary sequences, we clearly showed the importance of complementarity of ends in determination of 4.5SI RNA lifetime. The change of just one end (constructs I and J) resulted in drastic decrease of the lifetime of the modified 4.5SI RNA (t1/2∼20 min), whereas the replacement of both ends preserved the RNA stability at the level similar to that of native 4.5SI RNA.A question may arise: why in these experiments, the lifetime of RNA is increased by 6 times (tH). Labuda and Zietkiewicz Here we have not studied the secondary structure of RNA by enzymatic methods. There are no reports on the enzymatic study of 4.5SI RNA folding. We believe that this lack of knowledge is not very significant in the context of this study: computer prediction of the secondary structure of this RNA is quite reliable as it contains a long (16 bp) perfect stem  and exosomes 7 per ml) were gently stirred at 37°C in a vial with a tightly closed cap. Inhibitor of transcription, actinomycin D (5 µg/ml), was added to medium and 5 ml aliquots were taken in different time periods. Cells were collected by centrifugation and total cellular RNA was isolated using the guanidinium thiocyanate method Krebs ascites carcinoma II cells in vitro Transfection Reagent  following the manufacturer's protocol. Actinomycin D (5 µg/ml) was added to cells 20 h post-transfection. Then total cellular RNA was isolated in different time periods (0 to 180 min). Following ethanol precipitation, RNA was treated with 100 µg/ml RNase-free DNase I at 37°C for 30 min and purified by chloroform extraction. Ethanol-precipitated RNA was dissolved in 40 µl 0.1% SDS and its concentration was measured by NanoDrop 1000 Spectrophotometer.Hela cells were grown to 80% confluency in 60 mm Petri dishes using DMEM with 10% fetal calf serum. Cells were transiently transfected with 4.5 µg of plasmid DNA applying TurboFect D, E, F, and I, two-round PCR was used. The list of PCR primers is shown in Original 4.5SH RNA gene construct contained natural genomic 50 bp upstream sequence 32P-labeled probe obtained by PCR Equal amounts of cellular RNA were separated by denaturing electrophoresis in 6% PAAG and transferred onto Hybond-XL membrane  by semidry electroblotting at 5 V for 1.5 h. The 4.5SH RNA, 4.5SI RNA and their chimeras were detected by hybridization with Secondary structure was predicted for small RNAs using the mFold 3.2 web server with default parameters Figure S1Position of the PCR primers used in the preparation of templates.(DOC)Click here for additional data file.Table S1Primers used for PCR preparation of 4.5SH and 4.5SI-derived constructs.(DOC)Click here for additional data file.


Beet black scorch virus (BBSV) is a small single-stranded, positive-sense RNA plant virus belonging to the genus Necrovirus, family Tombusviridae. Its capsid protein (CP) contains a 13 amino acid long basic region at the N-terminus, rich in arginine and lysine residues, which is thought to interact with viral RNA to initiate virion assembly.in vitro. These experiments allowed us to identify two key basic amino acid residues in this region that are responsible for initiating virus assembly through RNA-binding. Proper assembly of BBSV particles is in turn needed for efficient viral systemic movement.In the current study, a series of BBSV mutants containing amino acid substitutions as well as deletions within the N-terminal region were generated and examined for their effects on viral RNA replication, virion assembly, and long distance spread in protoplasts and whole host plants of BBSV. The RNA-binding activities of the mutated CPs were also evaluated We have identified two basic amino acid residues near the N-terminus of the BBSV CP that bind viral RNA with high affinity to initiate virion assembly. We further provide evidence showing that systemic spread of BBSV in infected plants requires intact virions. This study represents the first in-depth investigation of the role of basic amino acid residues within the N-terminus of a necroviral CP. The capsid proteins (CPs) encoded by viruses play important roles at various stages of viral multiplication cycles . One of Necrovirus of Family Tombusviridae encompasses some of the smallest (+) RNA viruses with monopartite genomes and icosahedral virion symmetry. Among them, the Toyama isolate of Tobacco necrosis virus (TNV) was the first necrovirus for which the crystal structure of assembled virions was determined [Olive latent virus 1 (OLV-1) [Beet black scorch virus (BBSV) [The genus termined . Analysitermined . Similartermined , Olive l (OLV-1) , TNV-A [ (OLV-1) ,18, and s (BBSV) . HoweverMost plant viruses also require CP for their long distance spread in the infected plants. Indeed, a number of viruses have been found to access long distance movement in the form of virions -24. HoweNecrovirus. It was first identified in sugar beets grown in China [Nicotiana benthamiana[BBSV is a single-stranded (+) RNA virus of the genus in China ,28, and in China -31. Its in China . The thrin China ,33. The in China ,27,32-34N. benthamiana for their competence in replication, particle assembly, as well as systemic spread. The mutated CPs were also examined in vitro for their RNA-binding capacities. We conclude from these experiments that the lysine (K) and arginine (R) residues at the amino acid positions 4 and 5 of BBSV CP are essential for efficient CP-RNA interactions, and that intact virions are required for viral systemic spread.In the current report, we document a systematic analysis of the N-terminal basic region of BBSV CP through the creation of a series of deletion and single amino acid substitution mutants within this region. The resulting mutants were exhaustively examined in protoplasts, local, and systemic leaves of 4KRNKGGKKSR13, in the N terminus of BBSV CP that mediated the nuclear localization of CP, leading to the speculation that this K/R-rich motif could play an important role in BBSV long distance movement and symptom development [N. benthamiana protoplasts. As shown in Figure N. benthamiana single cells.In a previous study, we have identified a motif rich in K and R residues, elopment . To furt4KR5 → AA) that are absent in other mutants, thus suggesting a critical role of these two amino acid residues in CP stability.We also examined the levels of BBSV CP in these protoplast cells. As shown in Figure E. coli expression system. The yield of purified proteins was first confirmed by Coomassie blue-stained SDS-PAGE gels, as well as Western blot with BBSV CP antibody. As shown in Figure The reduced stability of BM1 and BM2 CPs could be due to their inability to form stable virions, thus exposing the unassembled CPs to protein degradation processes in the infected cells. To test this possibility, we decided to assess the ability of various CP mutants to interact with BBSV viral RNA, which is considered to be the first step of virion assembly. The WT and mutant CPs were expressed and purified using an 4KR5 → AA changes, were able to bind only trace amounts of BBSV RNA . The purified virions were first examined with electron microscopy (EM). Surprisingly, only BM3 and BM4 produced virions at sufficiently high concentrations to permit the detection by EM  in the initiation step of virion assembly through RNA-binding.In this report, we have investigated an N-terminal K/R-rich motif of BBSV CP for its role in high affinity binding of viral RNA, virion assembly, protection of viral RNA, as well as viral systemic movement. A set of six mutants was generated, each altering this motif to a different extent. All of the mutants were able to replicate normally in single cells, thus ruling out a significant role for this motif in genome replication. However, while all mutants showed varying degrees of defect in terms of viral systemic movement and virion integrity, it is the BM1 and BM2 that showed the most severe debilitation in all four sets of testing. These experiments implicated the first two basic residues of BBSV CP  was used to engineer both deletions of a desired group of amino acids, and substitutions of specific amino acids within BBSV CP Figure . The mutin vitro transcription, infectious clones containing wild-type (WT) and mutant cDNAs of BBSV were linearized with SmaI. The run-off transcription was carried out at 37°C for 1.5 hours with an in vitro transcription kit equipped with T7 RNA polymerase (Promega). The concentration of transcripts was estimated with UV spectrophotometry and gel electrophoresis.Prior to N. benthamiana leaves as previously described [6 of freshly isolated protoplasts were transfected with 20 μg of in vitro transcripts with a PEG-calcium chloride-mediated transfection procedure. The transfected protoplasts were incubated for 18 hours before being subjected to total RNA extraction using the Trizol reagent (Invitrogen). The replication efficiency of various BBSV RNAs was evaluated with Northern blot hybridizations with BBSV-specific probes. For mechanical inoculation of intact plants, in vitro transcripts were diluted to 1 μg/μl with the inoculation buffer , and rubbed gently onto young leaves of the plants to be tested. The inoculated plants were placed in a greenhouse room set at 18°C. At 7–8 dpi, total RNA and protein were extracted from both the inoculated and systemic leaves [32P-labeled riboprobe complementary to the 3’ terminal 300 nt of the BBSV genome was used to detect positive-stranded BBSV RNA progeny. For Western blotting, specific antibody to BBSV CP was used to detect the accumulation of BBSV CP [Protoplasts were prepared from escribed . Approxic leaves , and use BBSV CP .N. benthamiana leaves were used for particle purification and RNase-sensitivity analyses. BBSV particle purification was performed according to a previously published protocol with minor modifications [6,000, 10% NaCl. After being incubated at 4°C with stirring for 2 h, the mixture was centrifuged at 13,000 rpm for 20 min at 4C. The pellet was resuspended with 0.2 M sodium phosphate buffer (pH 7.0) and centrifuged at 14,000 rpm for 90 min at 4°C. Virus particles in the clarified supernatant were sedimented by ultracentrifugation through a 5 ml 20% sucrose cushion using a Beckman Type 70Ti rotor . The pellet was resuspended with 1 ml ddH2O. The purified virions were used for Western blotting analysis.Mechanically inoculated ications . BrieflyThe stability of virus particles formed by mutant CPs was assayed using an RNase-sensitivity assay as previously reported . 0.2 g iBamHI and SalI to acquire GST fusion proteins. The derived constructs were transformed into Escherichia coli BL21(DE3) pLysS cells. 0.2 mM Isopropyl â-D-thiogalactopyranoside  was used to induce protein expression. The induced E. coli cells were harvested and lysed by sonication for 15 min on ice in the GST suspension buffer . After clarification, the lysate was loaded onto a GST affinity column. The column was washed with five column volumes of GST buffer. The CPs were then released from the column by incubating overnight at 4°C with a solution containing 0.5 mM Tris–HCl, 150 mM NaCl, 2.5 mM CaCl2, 0.1% β-mercaptoethanol and 0.5 U Thrombin protease , and then eluted from the column with 10 column volumes of elution buffer . The fractions of interest were pooled and purified through ion exchange chromatography and gel filtration chromatography (Bio-Rad). The purified proteins were detected by Western blotting with a BBSV CP antibody.cDNAs of WT and mutant BBSV CPs were cloned into the pGEX-KG vector  using BaE. coli-expressed, purified WT and mutated CPs were separated on a 15% SDS-PAGE gel, and transferred onto a piece of nitrocellulose membrane. The similarly expressed green fluorescent protein (GFP) was used as a negative control. A non-radioactive North-western blot procedure was used to assess the RNA-binding activities of CPs bound on the membrane [in vitro transcribed digoxigenin-11-UTP-labelled BBSV RNA as the probe. The binding reaction was allowed to proceed for 2 h in the binding buffer containing 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.05% Triton X-100, and 1X Denhardt’s reagent. The bound RNA was then detected using the anti-digoxigen conjugated Fab-alkaline phosphatase , and the substrate solution containing nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate .The membrane , using iAny experimental research that is reported in our manuscript was performed with the approval from Ethics Committee of China Agricultural University. No any experimental research on humans and animals in our manuscript.The authors declare that they have no competing interests.X Zhang, D Li, C Han, and J Yu conceived the study. X Zhang and X Zhao carried out the experiments. X Zhang, F Qu, and D Li wrote the manuscript. Y Zhang, S Niu and Y Zhang provided various reagents and valuable advices throughout the study. All authors read and approved the final manuscript.


Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs.The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of whole-genome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs , some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene  Mature messenger RNA (mRNA) transcripts include a non-templated poly adenosine (polyA) tail at their 3′-ends conferring stability and supporting nuclear export and translation of the RNA transcript to protein . As mRNAUnlike the PA fraction, the NPA fraction has diverse RNA characteristics. One group is actually protein-coding, despite the lack of a polyA tail: the replication dependent, or canonical histone genes, which encode for histone proteins, are intensively transcribed in S-phase of the cell cycle and are not subject to standard RNA processing mechanisms such as polyadenylation and RNA splicing. Instead, they are stabilized by a stem-loop binding protein, which binds to a conserved stem-loop element at their 3′-ends . As histConversely, there are many non-coding RNAs (ncRNA), which do not undergo translation, but function as RNA transcripts. Long ribosomal RNA (rRNA) and transfer RNA (tRNA) act as core functional elements in the process of mRNA translation. Short ncRNAs include small nuclear RNAs (snRNAs) that participate in the RNA-splicing process as part of the core spliceosomal machinery; small nucleolar RNAs (snoRNA) and small Cajal Body RNAs that have roles in the post-transcriptional processing of rRNA and snRNAs; piwi-interacting RNAs (piRNAs) that are associated with transcriptional silencing of retrotransposons; and micro RNAs (miRNAs) that regulate transcriptional levels and translation of target mRNAs. An additional handful of other singleton NPA ncRNA examples carry out critical cellular functions such as Telomerase, which lengthens telomeres in rapidly dividing cells such as ESCs and in many transformed malignant cell types. Some of these ncRNA families may be initially transcribed with a PolyA tail, but undergo regulated RNA processing leading to their functional, NPA form ,19. SomeStill, in comparison with the standard protein-coding genes, few ncRNA genes have fully characterized function; yet, it has been hypothesized that ncRNA may partially explain differences in complexity among organisms  and coulin vitro differentiation  derived from ESCs by ntiation  into an ntiation . We findMouse R1 male ESCs were grown and differentiated into 7-day ESC-derived NPCs ,16: ESCsGapdh and Oct4 for the PA fraction and of histone H4 and Snord32 for the NPA fraction. We ensured that the NPA transcripts cannot be amplified when reverse transcribed with oligo-d(T) primers, only when random primers were used.We extracted total RNA from undifferentiated ESCs and 7 day NPCs. Total RNA was divided into poly(A+) and poly(A−) enriched fractions using three rounds of a modified version of Oligotex extraction (Qiagen). Briefly, after the hybridization of the total RNA to oligo(dT) beads, supernatants were saved rather than discarded and then subjected to isopropanol-ethanol extraction to obtain the Poly(A−) fraction. Enrichment of PA transcripts in the poly(A)+ RNA and of NPA transcripts in the poly(A)− RNA was assessed using primers specific for Samples were prepared and labeled as described . Brieflyet al.  above noise threshold and (ii) greater than the normalized fluorescent levels in the reciprocal PA samples. Only probes that fulfilled these criteria in each biological replicate separately were called ‘positive’ and used in transfrag analysis. Transfrags (continuous fragments of transcribed units) were assembled from these ‘positive’ probes as described in Kampa et al. . BrieflyRefseq mm9 genes were parsed into exonic and intronic regions. Transfrags were considered exonic if completely or partially intersecting exonic regions, intronic if entirely intersecting intronic regions and otherwise intergenic. For normalization purposes, the total exonic and intronic proportions of the genome were calculated based on the sums of all non-redundant Refseq exons’ or introns’ sizes divided by total genome size, which was inferred from the chromosome lengths in the UCSC ChromInfo table. Significance of enrichment was calculated using Fisher’s exact test. A 5′ or 3′ bias of transfrag overlap within genic regions was measured by dividing exons into three categories: 5′ exons are exons located upstream of the median gene length, 3′ exons located downstream of the median gene length or exons of genes that harbor only one exon.et al. . Briefly, rRNA was removed using one round Life technologies’ RiboMinus Eukaryote kit (A1083708). RNA was fragmented by RNaseIII . Adaptors were hybridized and ligated directly to the fragmented RNA, and reverse transcription was performed on the ligation product. Size selection was done on 4% agarose gels: 150–250 bp fragments were excised and purified using Qiagen MinElute Gel extraction Kit (28604). Library was then amplified by 15 cycles of polymerase chain reaction (PCR). In all, 1400 pmole of each library was amplified on magnetic beads (ePCR) and sequenced on 1/8 sequencing slide, on SOLiD 3 machine.9 × number of reads in region/. Of 30 million reads, >9 million reads were mapped for each ESCs and NPCs sample. Boxplot quality scores for the first 1 million reads for both ESCs and NPCs are shown .Reads of 50 nt were aligned to the mm9 genome using Bowtie . Seed seTen micrograms of total RNA in loading buffer was denatured at 65°C for 15 min and loaded onto 15% PAGE containing 7 M urea. The small RNAs were then transferred to a Nylon membrane and immobilized under UV. The membrane was pre-hybridized for 2 h at 65°C in prehybridization buffer ). Hybridization was carried out overnight at 42° by using [α-32P] labeled DNA probe. The membrane was then washed three times in 3× SSC/0.1% SDS at room temperature and exposed to a PhosphorImager screen. U6 was used as loading control. Oligonucleotide sequences used in this study were as follows: m-snora61: 5′-AGTCTGGCCTGCTGCTCTAC-3′, U6: 5′-GCCATGCTAATCTTCTCTGTATC-3′.15 primer (Promega). PCR reactions were carried out in 20 µl volumes using 2× DreamTaq Green PCR Master Mix (Fermentas) and 1 µl of 1:10 diluted cDNA template. Parallel samples without RT enzyme were prepared to detect DNA contamination. PCR conditions were 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 60°C for 20 s, 72°C for 30 s. Primer sequences for validations can be found in Supplementary Table S1.RNA was extracted with RNeasy (Qiagen). RNase-free DNase (Qiagen) was applied to the column to remove traces of genomic DNA according to protocol. Reverse transcription (RT) was performed on 1 µg of total RNA with High capacity cDNA reverse transcription kit (Applied Biosystems) with random hexamer primers or with oligo-dTQuantitative (qRT-PCR) reactions were performed with ABI PRISM 7900HT Sequence Detection System in 15 µl volume containing 7.5 µl Power SYBR Green PCR Master Mix (Applied Biosystems). PCR conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. Melting curve analysis was used to confirm the homogeneity of each PCR product. Standard curves including 5 serial dilutions were carried out for each primer pair. Each experimental sample was tested in duplicate for three biological replicates of ESCs and ESC-derived NPCs.Snora61 was designed according to its secondary structure obtained with Kinefold (Snora61 sequence. Oligos were targeted at ssRNA regions of the newly detected 5′ portion overlapped by the NPA transfrag. 2′-O-Methyl modified RNA/DNA chimera oligos were used in this study. The sequences of the ASOs are as follows (stars represent the phosphorothioate bond between nucleotides):GFP ASO: 5- mU*mC*mA* mC*mC*T* T*C*A* C*C*C* T*C*T* mC*mC*mA* mC*mU -3Snora61-ASO: 5- mC*mC*mA* mA*mG*A* C*C*A* G*C*G* T*C*A* mA*mG*mA* mU*mC -3Antisense oligo (ASO) specific to Kinefold  based onR1 cells were seeded in mouse ES cell media 1 day before transfection in a 6-well plate. Cells were transfected with 100 nM ASO by using Lipofectamine-2000 according to manufacturer’s protocol. After 4–6 h post-transfection, media was replaced with fresh ES cell media and cell growth conditions were maintained for up to 48 h.Gapdh, 18S and Lamin B was used to normalize sample Ct values. Splicing efficiency values were calculated as the ratio of  spliced mRNA to unspliced pre-mRNA. Primers for Drap1, Gars and Psmc4 were designed based on Pessa et al.  under accession number GSE37338.Supplementary File S1.UCSC custom tracks representing the validated NPA transfrags and mapped sequencing reads can be easily uploaded in the genome browser by copy-pasting lines from Genome-wide, PA and NPA transcriptomes were obtained for murine ESCs and ESC-derived NPCs: RNA was extracted from these cell types in three biological repeats, and then fractionated into PA and NPA fractions via oligo(dT) columns. Each fraction was independently hybridized to a repeat-masked Affymetrix whole mouse genome tiling array at 30-bp resolution . The normalized intensity signal from the NPA sample clearly correlates with exons of a replication-dependent histone cluster in both cell types, which have been extensively characterized as the only protein coding genes in the eukaryotic genome to encode for mRNAs that do not contain a poly A tail at their 3′-end  Figure B. ReplicSupplementary Figure S1), obtaining validation rates of 83 and 75% for ESCs and NPCs, respectively.Although some studies have deemed expressed regions that show both PA and NPA transcription signals as ‘bimorphic’ (producing both transcripts with polyA tails and without polyA tails) ,38,39, tSupplementary Figure S2). The intersection of transfrag intervals from the two groups showed almost 1000 NPA regions common to the two cell types, but many more are cell-type specific with a clear excess of ranges in NPCs   B.FigureGapdh, Oct4), for the NPA histone H4 mRNAs and for Snord32. In all cases, we found a significant NPA enrichment spanning an order of magnitude. We also verified that the NPA transcripts do not amplify (by RT-PCR) when reverse transcribed with oligo-d(T) primers, but only when random primers are used.The significant overlap with exons of known PA genes piqued our curiosity as to whether the NPA transfrag transcription was coming from the same strand as the overlapping host gene or in the antisense orientation to it. Widespread antisense transcription has been detected in mammalian organisms , and manSupplementary Table S3). We inferred the orientation of each validated NPA transfrag according to the overlapping mapped reads’ strands and compared the direction of the NPA transcription with that of the host gene (inferred from the annotation). Surprisingly, only 3% of validated NPA transfrags were antisense to their overlapping host genes  . From this observation, it seems unlikely that these exonic NPA transfrags represent full-length mRNA (with none or short polyA tails), as equal representation of all exons would be expected in this case. There was no corresponding enrichment in 5′ intronic transfrags over 3′ intronic ones (data not shown), suggesting that these exonic transfrags represent partially degraded mature mRNAs and not nascent pre-mRNA transcripts. Additionally, the tiling array probe intensity profile over a multi-exonic gene shows clear correlation of high probe intensities with annotated exons, whereas probe levels representing intronic regions are mostly at noise level . This suggests that the NPA RNA fraction as obtained by oligo-d(T) fractionation does not contain a high percentage of nascent RNA transcripts, although this option cannot be completely ruled out, as pre-mRNA splicing can occur co-transcriptionally   orientation to the host gene, represent? We noticed that, in most cases, exonic NPA transfrags overlap only a subset of the exons within a protein-coding gene with an apparent bias toward exons at the 5′-end. We validated the perceived bias by categorizing PA Refseq exons into three subcategories: (i) 5′ exon of a multi-exon protein-coding transcript, (ii) 3′ exon of a multi-exon protein-coding transcript, and (iii) exon of a protein-coding transcript with an exon count of 1 (Single Exon). The enrichment of overlap with 5′ exons is three times greater than that of 3′ exons in ESCs and over five times greater in NPCs  D, as theSNORA61 (starred arrow), an snoRNA gene, and resides within the intron of the Snhg12 (snoRNA hostgene 12) gene. This transfrag location is close to the mouse Snora61 gene of ‘provisional’ status according to Refseq . Northern blots using probes against both the NPA transfrag and the annotated mouse Snora61 show a single RNA transcript of ∼120 nt, which is more abundant in ESCs than differentiated cell types, and qPCR experiments confirm a significant decrease in expression during differentiation .A small, 70 nt, intronic region on chromosome 4 had the highest number of reads per transfrag of 66 236 in the ESC sample, accounting for 1.5% of all mapped reads and corresponding to almost 1000× coverage. The number of reads on the opposite strand for this transfrag was zero, indicating the highly significant strand-specific nature of the data A. This No Refseq A, arrow,Rmrp  are even higher than the NPA transfrags in the exonic category, which we examined previously  genes, such as Rmrp B, Rpph1 p   , which i(fRNAdb) B. This i(fRNAdb) ,49, 4.5S(fRNAdb) B, right.We defined intronic and intergenic transfrags with the highest potential to be real novel ncRNAs as those with higher RPKM levels than a threshold of 1 in log scale. This threshold represents the fifth percentile of RPKM levels of transfrags overlapping histone gene exons, meaning that by only looking at NPA transfrags above this threshold, we would preserve our 95% recall rate of histone genes (our positive control) A and B. P < 10−10, Kolmogorov–Smirnov test)  C. These We sought to gain insight into the transcriptional regulation properties of the novel, intergenic snacRNAs. The search for RNA polymerase II-associated epigenetic marks, i.e. H3K4me3/H3K36me3 genomic domains, greatly facilitated the discovery of another novel class of functional ncRNAs, termed long-intergenic-ncRNAs (lincRNAs) . Yet, liSupplementary Figure S6A). This may indicate that these RNA genes are regulated and transcribed differently than protein-coding genes. We looked for evidence of Pol III binding . Several snacRNA ranges had typical ‘K4-K36’ signature of RNAPII transcribed genes , which is transcribed by RNAPII as a 7 kb polaydenylated transcript but undergoes cleavage and processing by RNaseP to produce a shorter NPA transcript, mascRNA .The intergenic snacRNA transfrags clustered into three distinct epigenetic patterns. The largest cluster included snacRNA regions that had neither K4 nor K36 signal A IV, 7E. binding , which t binding , but onled genes AV and F. mascRNA , leading mascRNA AVI and GSupplementary Figure S8, et al. when comparing human ESCs to HeLa cells , which was detected by an overlapping NPA transfrag in NPCs, but not in ESCs. U11 is relatively well characterized as a fundamental RNA component of the minor spliceosomal complex, along with U12, U4atac and U6atac. The minor spliceosome splices out special introns, termed ‘minor’ or ‘U12’ introns, with unique branchpoint sequences and polypyrimidine tracts, and often with non-canonical AT-AC splice sites . The minor spliceosome is considered less efficient than the major one, which perhaps acts as a rate-limiting step in the expression of U12 genes , which was significantly downregulated in NPCs compared with ESCs , displayed altered splicing efficiency between ESCs and NPCs . The splicing efficiencies of Eed’s U12 and U2 control intron were both increased ∼2-fold in NPCs . This is particularly interesting, as Eed is a component of the PRC2 complex  . A similar effect was observed in somatic fibroblasts. This shows that Snora61 is an essential RNA, although its exact role remains to be investigated.Finally, as a proof of concept for the essential role of some of the ncRNAs, we selected the most highly expressing ncRNA we identified, Snora61 A for knoin (GFP) . We founIn this study, we characterized the NPA transcriptome in ESCs and ESC-derived NPCs, by using two complimentary methods for genome-wide transcription analysis. Using a whole mouse genome tiling array, we took advantage of the robustness of the DNA microarray platform, which we increased with the application of threshold criteria separately to three biological repeats. But tiling arrays suffer from cross-hybridization effects and the usage of suboptimal probes, which also introduce false positives; therefore, we also applied RNA-seq to further validate the regions of transcription defined by the array and provide additional data regarding their strand specificity. In all, 30–40% of transfrags contained at least one mapped read, representing 25% of total mapped reads. Similar levels of intersection between the results of the tiling array and the RNA-sequencing data were also perceived in previous studies ,70 largeSupplementary Figure S11) strengthens this claim, as these transcripts would be more likely to take longer to be transcribed or degraded and could be detected by our methodology. Based on the high similarity of the probe intensity profile of the NPA fraction to that of the PA fraction , the percentage of nacsent transcription in the NPA fraction is probably minimal, and the 5′ bias is more likely a product of RNA degradation. A similar profile was found by the study of NPA transcription in human HeLa cells by Yang et al.  finds mostly canonical histone genes in all cell types as expected from their conservation and ubiquitously expressing nature. A small number of other protein-coding genes exist in that same category (see table). In addition, there is a significant number of common genes to both the hESCs and mESCs and to NPCs and HeLa cells . The level of intersection is also greatly underestimated, as it is done across species. Some protein-coding genes are common to other previous studies of protein-coding genes with short polyA tails such as Actin-b . The biological importance of this trend, which contrasts the ESC pervasive transcription paradigm (based on PA transcription) is still not clear. We predict that the functional characterizations of new ncRNA groups such as snacRNAs as well as better characterization of RNA biogenesis and processing properties will shed light on this observation and the importance of its impact on stem cell biology.Curiously, the trend of more pervasive NPA transcription is apparent starting from the tiling array analysis and persisting through the RNA-seq validations even with different thresholding criteria. The trend is stable even when looking at subcategories of the genic territories where NPA transfrags reside: overlapping exons or introns of annotated protein coding genes, as well as the intergenic regions between them and even the overlap with annotated ncRNA genes ; the Abisch-Frenkel Foundation (to E.M.) and the European Research Council [ERC-281781 to E.M.].Funding for open access charge: Conflict of interest statement. None declared.