Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales  leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5′ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments  In vertebrates, the first step of innate immunity is the detection of microbe-associated molecular patterns (MAMPs) by specific pattern-recognition receptors (PRRs) The precise mechanisms of RIG-I activation are still not fully understood. It has been proposed that RIG-I-mediated activation relies on RIG-I oligomerization via dimerization of RIG-I C-terminal domain (CTD), multiple oligomerization sites within RIG-I, and/or RNA-mediated oligomerization 2.Huh7.5 Moraten-eGFP measles virus was recovered by reverse genetics as described by Radecke et al. ko  cDNA were subcloned into pEF-BOS expression vector using PCR amplification of cDNA fragments and in vitro recombination . HA, Cl25  and Flag tag coding sequences were fused to RIG-I cDNA during the PCR amplification step. RIG-I insert constructs were entirely verified by sequencing (Eurofins).Wild-type human RIG-I and RIG-IGaussia Luciferase-Based Protein Complementation Assay (PCA) , were modified into pCI-glu1 and pCI-glu2 to eliminate the Gateway insert without changing the flanking vector sequence in order to preserve the linker bridging glu domains and inserts. HA-RIG-I and Cl25-RIG-I fragments were subcloned upstream or downstream of gaussia glu1 and/or glu2 domains by InFusion recombination of PCR-amplified fragments. Gcn4 sequence The two original expression vectors used for For immunoblotting the following primary antibodies were used: anti-Flag , Cl25 anti-MeV N  For DNA plasmid transfection, JetPRIME reagent (Polyplus transfection) was used in 293T cells and Transit-LT1 reagent (Mirus) was used in Huh7.5 cells. RNA transfection was performed with Oligofectamine reagent (Invitrogen). Poly(I:C) was purchased from Amersham Biosciences.in vitro T7 transcribed and purified by excising the band after denaturing urea-PAGE Rabies leader 5′ppp-RNA (GGACGCUUAACAACAAAACCAGAGAAGAAAAAGACAGCGUCAAUUGCAAACGAAAAAUGUGC), measles leader 5′ppp-RNA (GGACCAAACAAAGUUGGGUAAGGAUAGAUCAAUCAAUGAUCAUAUUCUAGUACACUUGAAUUC) and Ebola leader 5′ppp-RNA (GGACACACAAAAAGAAAGAAAAGUUUUUUATACUUUUUGUGUGCGAAUAACUAUG) were GGCACCGGGGAACCAAGGCGAACACGGACACGCAACAAACGAGACCGACAACAGACAGGACC). Both 62-mer-5′ppp-ssRNA were made from only three nucleotides to avoid secondary structure and preclude T7 polymerase re-initialization on and copy of the nascent RNA The 62-mer-5′ppp-dsRNA was obtained by annealing two T7 transcribed and purified complementary 62-mer-5′ppp-ssRNA (GGUCCUGUCUGUUGUCGGUCUCGUUUGUUGCGUGUCCGUGUUCGCCUUGGUUCCCCGGUGCC) and . Scattering data was collected for different protein concentrations and from the merged curves the radius of gyration (Rg) was determined from the Guinier plot. Next, the maximal distance in the size distribution function was adjusted, so the calculated Rg from the fit would be in agreement with the experimental value.Cells were seeded into 96-well plates and, 18 h later, transfected with 50 ng DNA of IFN-β luciferase, 17 ng DNA of renilla luciferase and 33 ng DNA of RIG-I plasmid. One day after DNA transfection, cells were transfected with Poly(I:C), synthetic RNA or infected with Moraten-gfp virus at MOI 1. The following day, the luciferase assay was performed using the Dual-Glo system from Promega. Firefly luciferase values were normalized to renilla luciferase to measure transfection efficiency.Gaussia Luciferase-Based Complementation Assay (PCA) For 2, 0.05% NP-40, 25 mM EDTA) or NP-40 buffer  both complemented with Complete (Roche) protease inhibitor cocktail for 20 minutes on ice. The proteins were then separated from cell debris by centrifugation at 7,000×g for 10 minutes. Proteins were denatured by addition of Laemmli 1X buffer and heating at 100°C for 3 minutes before analysis by SDS-PAGE and immunoblotting.For immunoblot analysis, transfected or infected cells were suspended in lysis buffer, either PLB buffer  beads (Sigma). Beads were washed four times with lysis buffer and proteins were eluted by addition of 22.5 µg 3xFlag Peptides (Sigma). Lysates were then analysed by SDS-PAGE and immunoblotting.GCTGCTACTCGGCTGATCTCAC.RNA immunoprecipitated with RIG-I was purified by Trizol/chloroform extraction, then amplified by stem-loop RT-PCR as set up for miRNA detection in vitro was tested by Multi-Angle Laser Light Scattering coupled to Size Exclusion Column (SEC-MALLS) analysis. Purified recombinant hRIG-I was able to bind to synthetic copies of the leader RNAs from three different Mononegavirales families: Ebola (Filoviridae), rabies (Rhabdoviridae) and measles (Paramyxoviridae) viruses (g for RIG-I without RNA appears to be 38.5±0.27 Å and with a short panhandle RNA of influenza virus to be slightly larger 42.2±0.21 Å. This represents an elongation of the molecule but no dimerization. P(R) functions of the scattering curves  protein to bind different 5′ppp RNA  viruses . The incg curves  that werSince RNA sensing by RIG-I results in IFN-β gene expression, the ability of the synthetic RNAs to induce the expression of a luciferase reporter gene under the control of the IFN-β promoter was tested. To avoid any interference of the endogenous innate immune response of the host cell, we selected Huh7.5 cells since they lack TLR3 and MDA5 expression, express a defective T55I RIG-I mutant and exhibit a poor feedback upregulation of RLR genes due to an IFNAR signalling defect in cellula complexes with RIG-I that are stable enough to be detected by immunoprecipitation of RIG-I. This synthetic RNA was transfected in Huh7.5 cells expressing wild type RIG-I or a RIG-Iko construct associating a T55I mutation in the first CARD domain (inhibition of TRIM25 recruitment) with Q229A, T697A, E702A and K888/907A mutations in the helicase and CTD that prevent RNA binding to the corresponding domain ko counterpart  or RNA transfection and finally harvested 18 hours after stimulation for an analysis in a co-immunoprecipitation assay. Cl25-RIG-I could not be co-immunoprecipitated with Flag-RIG-I, since similar trace amounts were detected in the absence or presence of RNA stimulation in Flag-RIG-I eluates  or synthetic dsRNA, we tried to stimulate RIG-I by infecting cells with measles virus Since we did not observe any RIG-I oligomerization Since La Crosse virus (LACV) nucleocapsids that exhibited a triphosphorylated 5′ (5′ppp) terminus as does MeV can be co-immunoprecipitated with RIG-I from infected cells th amount of RNA, the amount of Cl25-RIG-I found in the anti-Flag immunoprecipitate decreased with the 62-mer-5′ppp-dsRNA but increased with the 62-mer-5′ppp-ssRNA. Reducing the amounts of transfected RNA and thus the number of RNA molecules accessible for one RIG-I would increase the chance for RIG-I to oligomerize. Since this assessment was only verified for the non-stimulatory ssRNA, we interpret the co-immunoprecipitation of Cl25-RIG-I with Flag-RIG-I as an experimental artefact likely due to over expression of RIG-I in 293T cells. All of these results were repeatedly observed using various experimental conditions, including the use of different lysis buffers.RIG-I oligomerization was also tested in 293T cells stimulated by transfection of Poly(I:C), synthetic dsRNA or ssRNA, or Moraten-eGFP infection and 18 hours later submitted to the immunoprecipitation assay. Both Flag-RIG-I and Cl25-RIG-I were strongly expressed , inputs.in cellula, Huh7.5 cells were co-transfected with Flag-RIG-I and Cl25-RIG-I constructs, stimulated by either Poly(I:C) transfection or Moraten-eGFP infection and harvested 4 hours after stimulation for a co-immunoprecipitation assay. Both Flag-RIG-I and Cl25-RIG-I were expressed in Huh7.5 cells . PCA has been described to be highly sensitive and have allowed us to detect interactions between monomers in the 0.2–1 µM range Gaussia glu1 and glu2 split domains. All chimeric proteins were strongly expressed in Huh7.5 cells  (We reasoned that the co-immunoprecipitation assay might not be sensitive enough to detect RIG-I oligomerization induced by a cognate RNA. We therefore switched to the .5 cells . However-I-glu1) . It shou-I-glu1) . The add-I-glu1) . Similar-I-glu1) . Moreove-I-glu1) . Correla-I-glu1) .RIG-I oligomerization was proposed to occur during activation by a RNA ligand by two groups in 2007–2008 in vitro analysis by gel filtration of a mixture of pure RIG-I protein and short (from 19 bp to 135 bp) 5′ppp-RNA Mononegavirales leader 5′ppp-RNA (this work) was compatible only with RNA/RIG-I 1∶1 monomer complexes. In agreement with our observations, RIG-I and hairpin duplexes of 10, 20 or 30 base pairs with a single 5′ppp end form 1∶1 complexes as analysed by analytical ultracentrifugation-sedimentation velocity in cellula. This could be explained by an unbalanced molar ratio of RIG-I protein to 5′ppp-dsRNA in the intracellular milieu, a competition with other 5′ppp-RNA binding proteins and/or the highly dynamic interaction of RIG-I with 5′ppp-dsRNA despite a Kd in the 160 pM range The RIG-I oligomerization concept originated from The incubation of very stable 5′ppp- panhandle RNA with dsRNA of variable length with cellular extracts from RIG-I transfected cells allows the observation of RIG-I oligomerization, at least if the dsRNA exceeds 46 bp in length In vivo, RIG-I oligomerization was reported once by pull down assay of Flag- and Myc-tagged RIG-I  and % of GFP expressing cells (right). Figure S2, The anti-Flag immunoprecipitation procedure can detect complex formation between MeV N and FLAG-P proteins. Vero cells were infected with two measles viruses expressing a wt P protein or a Flag-tagged P protein at MOI 0.1. The cell extracts, collected 20 h after infection, were immunoprecipitated with Flag antibody coupled to beads. Proteins eluted with Flag peptide were analyzed by western blot using anti-P 49.21 and anti-N Cl25 monoclonal antibodies. Note the exclusive pull-down of N from cells infected with the Flag-P virus. Figure S3, Ability of RIG-I/glu/gcn4 constructs for self-binding in absence or presence of Poly(I:C) determined by PCA. Luciferase activity was measured 4 hours after transfection or not with Poly(I:C) in 293T cells expressing RIG-I/glu1/2/gcn4 constructs. Figure S4, Accessibility of gcn4 sequence in RIG-I/glu/gcn4 constructs for determination of RIG-I oligomerization by PCA. Luciferase activity was measured 24 hours after transfection of RIG-I/glu1/2/gcn4, gcn4/glu1/2 and MeV Ntail/XD/glu1/2/gcn4 constructs in 293T cells.(PDF)Click here for additional data file.


Highly multiplexed assays for quantitation of RNA transcripts are being used in many areas of biology and medicine. Using data generated by these transcriptomic assays requires measurement assurance with appropriate controls. Methods to prototype and evaluate multiple RNA controls were developed as part of the External RNA Controls Consortium (ERCC) assessment process. These approaches included a modified Latin square design to provide a broad dynamic range of relative abundance with known differences between four complex pools of ERCC RNA transcripts spiked into a human liver total RNA background.ERCC pools were analyzed on four different microarray platforms: Agilent 1- and 2-color, Illumina bead, and NIAID lab-made spotted microarrays; and two different second-generation sequencing platforms: the Life Technologies 5500xl and the Illumina HiSeq 2500. Individual ERCC controls were assessed for reproducible performance in signal response to concentration among the platforms. Most demonstrated linear behavior if they were not located near one of the extremes of the dynamic range. Performance issues with any individual ERCC transcript could be attributed to detection limitations, platform-specific target probe issues, or potential mixing errors. Collectively, these pools of spike-in RNA controls were evaluated for suitability as surrogates for endogenous transcripts to interrogate the performance of the RNA measurement process of each platform. The controls were useful for establishing the dynamic range of the assay, as well as delineating the useable region of that range where differential expression measurements, expressed as ratios, would be expected to be accurate.The modified Latin square design presented here uses a composite testing scheme for the evaluation of multiple performance characteristics: linear performance of individual controls, signal response within dynamic range pools of controls, and ratio detection between pairs of dynamic range pools. This compact design provides an economical sample format for the evaluation of multiple external RNA controls within a single experiment per platform. These results indicate that well-designed pools of RNA controls, spiked into samples, provide measurement assurance for endogenous gene expression studies.The online version of this article (doi:10.1186/s12896-016-0281-x) contains supplementary material, which is available to authorized users. In 2003, the National Institute of Standards and Technology (NIST) hosted a meeting to discuss the need for a universal RNA reference material, which could be used for gene expression profiling assays . As a re20 dynamic range of abundance of controls, and particular controls in the different pools were present in different abundances according to a modified Latin square design. This design provides known relative differences between the pools across a large dynamic range of abundance , each containing a unique set of controls  products representing candidate sequences prior to the release of NIST SRM 2374. The plasmids were designed to produce either “sense” or “antisense” RNA controls [Samples were measured at each test site using the following methods.The NIAID in-house spotted microarrays contain long (70-mer) oligonucleotides designed to hybridize the ERCC transcripts printed on epoxy-coated glass slides (Corning) in quadruplicate using an OmniGrid robot (Genomic Solutions) with 16 SMP3 print tips (Telechem). RNA was reverse transcribed using Oligo dT primer (12–20 mer) mix (Invitrogen) and Superscript II reverse transcriptase (Invitrogen). Fluorescent Cy-Dye-dUTP (GE) nucleotide was incorporated into first-strand cDNA during the reverse transcription. After degradation of the mRNA template strand, labeled single-stranded cDNA target was purified using Vivaspin 500 . Hybridization was performed at 45 C°, for 16 h on a MAUI hybridization station. The arrays were washed twice in 1X SSC and 0.05 % SDS and twice in 0.1X SSC, then air dried. Microarrays were scanned on GenePix 4000B (Axon) at 10 μm resolution. GenePix Pro software was used for image analysis. Median pixel intensity (no background subtraction) was taken for each of the 4 replicate spots, the median of these four values was taken to represent the data.The Agilent microarrays (8x60K Agilent G3 8-pack format with the Design ID 022439) contain 60-mer oligonucleotide probes synthesized in situ onto slides using a proprietary non-contact industrial inkjet printing process. Labeled cRNA for both the one-color and two-color microarray experiments was prepared using the Agilent Low Input Quick Amp Labeling Kit, Two-Color (5190-2306). RNA was reverse transcribed using AffinityScript RT, Oligo(dT) Promoter Primer, and T7 RNA Polymerase. Fluorescent Cy-Dye-dCTP nucleotide was incorporated during cRNA synthesis and amplification. Microarrays were hybridized at 65 °C for 17 h. All microarrays were scanned in one batch in random order using default settings for Agilent C Scanner using a single pass over the scan area at a resolution of 3 μm and a 20-bit scan type. Data was extracted with Agilent Feature Extraction Software (ver. 10.7.3.1) using the default settings for either the one-color protocol or the two-color protocol.The Illumina Human-6 Expression BeadChips contain 50-mer oligonucleotide probes with a 29-mer address sequences attached to beads held in etched microwells. RNA was reverse transcribed using a T7 Oligo(dT) primer containing a T7 promoter sequence. Biotinylated cRNA was prepared using the Illumina TotalPrep RNA Amplification Kit (Ambion). BeadChips were hybridized at 58 °C for 14–20 h, washed, and labeled with streptavidin-Cy3. BeadChips were scanned with the Illumina iScan System. Intensity values are determined for every bead and summarized for each bead type. For more details refer to the Whole-Genome Gene Expression Direct Hybridization Assay Guide .NIST prepared samples of spiked liver total RNA for sequencing analysis with the 5500xl at NIST and the HiSeq 2500 at Illumina. Prior to library preparation samples were depleted of ribosomal RNA. The 5500xl experiment produced an average of 23,866,495 single-ended reads (75 base) per sample and the HiSeq 2500 experiment yielded an average of 48,168,710 paired-end reads (2 x 75 base). For both platforms sequence reads were aligned against a reference sequence consisting of the human genome (hg19) and the ERCC transcript sequences of SRM 2374 (Note: ERCC-00114 is not part of the SRM and not included as part of the reference transcriptome). Alignment and quantification of sequence reads to obtain per transcript counts was performed with the LifeScope bioinformatic analysis suite (Life Technologies) for 5500xl data and the Tophat-Cufflinks suite was used for HiSeq 2500 data [20 range of relative abundance will cover the distribution of the endogenous transcript signals. In the first set of experiments, each microarray platform provider empirically determined in pilot studies its chosen spike-in proportion to add to the total RNA background (not shown). Agilent used 0.144 % (wt/wt) for both one-color and two-color arrays, and Illumina and NIAID used 0.25, and 0.265 %, respectively. For the RNA-Seq experiments, ERCC pools were added to the background at NIST at 0.3 % and shared with the Illumina site. The LifeTech 5500xl and Illumina HiSeq measurements were performed at NIST and Illumina, respectively. The distribution of ERCC signals relative to the endogenous liver background transcripts are shown for all platforms in Table For each of the platforms, if the ERCC spike-in pools are added to the background RNA in the proper proportion, then the 2For each platform, we can determine whether the analytical signal  changes with the concentration of an analyte (the ERCC being measured). For each control, the signal from each pool can be plotted against the corresponding relative abundance Table , produciKd, was determined by fitting the data as follows:For the microarray experiments, a model using the Langmuir isotherm was used , 8. The Imax, and the background, bg, are experimentally derived from the average of the most abundant ERCC in each of the 4 pools and ERCC-00073, a component omitted from the pools, respectively. For the RNA-Seq experiments, a linear fit with a slope of 1 and fitted y-intercept was used as the model. For either model, ERCC controls outside the 99 % confidence interval (CI) were flagged as outliers  corresponds to an ideal dose-response. For the microarray data, the first derivative of the Langmuir function also provides us with a model of the expected slope and the inflection points allow us to demarcate a region of the dynamic range where we should expect a linear response . As a consequence, the ratios do not compress at the upper end of the dynamic range. The RNA-Seq signals in this dataset are derived from counting technical replicates, where the variation can be characterized by a Poisson distribution . In thisThe modified Latin square design provided for simultaneous evaluation of multiple controls with a minimal number of samples. While each individual ERCC control was only tested over a small range of relative abundance, up to 4-fold for the ERCC controls tested at multiple ratios and a single relative abundance value for the 1-to-1 components, in aggregate, they describe the overall measurement behavior of a platform.The spread of the data indicates that differences in signals observed between different RNA species within the same sample may not accurately reflect the relative abundance between different RNA components of the same sample. Some of this dispersion may be due to the complexity of the pools used in these experiments where the distribution of target abundances described in Table erccdashboard, to provide metrics and visualizations for these controls [The ERCC controls did demonstrate that there is a linear region of the dynamic range of each platform where changes in abundance of a particular RNA transcript can produce a proportional change in signal. In this region, the ratios obtained with each platform approach the target ratios of the modified Latin square design. As a consequence, comparisons between samples for any particular RNA species can be expected to be accurate with respect to ratio-based measurements if they fall within this region. A pair of complex mixtures of RNA controls derived from NIST SRM 2374 designed to provide a set of ratios across a similar dynamic range is commercially available (Ambion™ ERCC ExFold RNA Spike-In Mixes). NIST has developed an R-based tool, the controls .The ERCC RNA controls demonstrated utility in four different gene expression microarray platforms and two RNA-Seq platforms. Performance issues with any individual ERCC could be attributed to detection limitations or a target probe issue for particular platforms. The spike-in RNA controls were useful for establishing the dynamic range of relative abundance for a platform as well as delineating a reliable region where ratios can be measured accurately.The composite testing scheme used in this study demonstrated that using well-designed pools of RNA controls provides measurement assurance for endogenous gene expression studies. Pools of RNA controls from this study have been used as spike-ins for RNA-Seq experiments , and comAGL-1, agilent 1-color; AGL-2, agilent 2-color; ERCC, External RNA Controls Consortium; ILM-bead, illumina bead array; ILM-ngs, illumina next-generation sequencing; LifeTech, Life Technologies; NGS, next-generation sequencing; NIAID, National Institute of Allergy and Infectious Diseases; NIST, National Institute of Standards and Technology; PCR, polymerase chain reaction; RNA-Seq, RNA sequencing; SRM, standard reference material


RNA-sequencing (RNA-seq) has a wide variety of applications, but no single analysis pipeline can be used in all cases. We review all of the major steps in RNA-seq data analysis, including experimental design, quality control, read alignment, quantification of gene and transcript levels, visualization, differential gene expression, alternative splicing, functional analysis, gene fusion detection and eQTL mapping. We highlight the challenges associated with each step. We discuss the analysis of small RNAs and the integration of RNA-seq with other functional genomics techniques. Finally, we discuss the outlook for novel technologies that are changing the state of the art in transcriptomics.The online version of this article (doi:10.1186/s13059-016-0881-8) contains supplementary material, which is available to authorized users. Transcript identification and the quantification of gene expression have been distinct core activities in molecular biology ever since the discovery of RNA’s role as the key intermediate between the genome and the proteome. The power of sequencing RNA lies in the fact that the twin aspects of discovery and quantification can be combined in a single high-throughput sequencing assay called RNA-sequencing (RNA-seq). The pervasive adoption of RNA-seq has spread well beyond the genomics community and has become a standard part of the toolkit used by the life sciences research community. Many variations of RNA-seq protocols and analyses have been published, making it challenging for new users to appreciate all of the steps necessary to conduct an RNA-seq study properly.There is no optimal pipeline for the variety of different applications and analysis scenarios in which RNA-seq can be used. Scientists plan experiments and adopt different analysis strategies depending on the organism being studied and their research goals. For example, if a genome sequence is available for the studied organism, it should be possible to identify transcripts by mapping RNA-seq reads onto the genome. By contrast, for organisms without sequenced genomes, quantification would be achieved by first assembling reads de novo into contigs and then mapping these contigs onto the transcriptome. For well-annotated genomes such as the human genome, researchers may choose to base their RNA-seq analysis on the existing annotated reference transcriptome alone, or might try to identify new transcripts and their differential regulation. Furthermore, investigators might be interested only in messenger RNA isoform expression or microRNA (miRNA) levels or allele variant identification. Both the experimental design and the analysis procedures will vary greatly in each of these cases. RNA-seq can be used solo for transcriptome profiling or in combination with other functional genomics methods to enhance the analysis of gene expression. Finally, RNA-seq can be coupled with different types of biochemical assay to analyze many other aspects of RNA biology, such as RNA–protein binding, RNA structure, or RNA–RNA interactions. These applications are, however, beyond the scope of this review as we focus on ‘typical’ RNA-seq.Every RNA-seq experimental scenario could potentially have different optimal methods for transcript quantification, normalization, and ultimately differential expression analysis. Moreover, quality control checks should be applied pertinently at different stages of the analysis to ensure both reproducibility and reliability of the results. Our focus is to outline current standards and resources for the bioinformatics analysis of RNA-seq data. We do not aim to provide an exhaustive compilation of resources or software tools nor to indicate one best analysis pipeline. Rather, we aim to provide a commented guideline for RNA-seq data analysis. Figure A crucial prerequisite for a successful RNA-seq study is that the data generated have the potential to answer the biological questions of interest. This is achieved by first defining a good experimental design, that is, by choosing the library type, sequencing depth and number of replicates appropriate for the biological system under study, and second by planning an adequate execution of the sequencing experiment itself, ensuring that data acquisition does not become contaminated with unnecessary biases. In this section, we discuss both considerations.One important aspect of the experimental design is the RNA-extraction protocol used to remove the highly abundant ribosomal RNA (rRNA), which typically constitutes over 90 % of total RNA in the cell, leaving the 1–2 % comprising messenger RNA (mRNA) that we are normally interested in. For eukaryotes, this involves choosing whether to enrich for mRNA using poly(A) selection or to deplete rRNA. Poly(A) selection typically requires a relatively high proportion of mRNA with minimal degradation as measured by RNA integrity number (RIN), which normally yields a higher overall fraction of reads falling onto known exons. Many biologically relevant samples (such as tissue biopsies) cannot, however, be obtained in great enough quantity or good enough mRNA integrity to produce good poly(A) RNA-seq libraries and therefore require ribosomal depletion. For bacterial samples, in which mRNA is not polyadenylated, the only viable alternative is ribosomal depletion. Another consideration is whether to generate strand-preserving libraries. The first generation of Illumina-based RNA-seq used random hexamer priming to reverse-transcribe poly(A)-selected mRNA. This methodology did not retain information contained on the DNA strand that is actually expressed . In all Another important factor is sequencing depth or library size, which is the number of sequenced reads for a given sample. More transcripts will be detected and their quantification will be more precise as the sample is sequenced to a deeper level . NeverthFinally, a crucial design factor is the number of replicates. The number of replicates that should be included in a RNA-seq experiment depends on both the amount of technical variability in the RNA-seq procedures and the biological variability of the system under study, as well as on the desired statistical power . These two aspects are part of power analysis calculations Fig. ; Box 1.The adequate planning of sequencing experiments so as to avoid technical biases is as important as good experimental design, especially when the experiment involves a large number of samples that need to be processed in several batches. In this case, including controls, randomizing sample processing and smart management of sequencing runs are crucial to obtain error-free data Fig. ; Box 2.The actual analysis of RNA-seq data has as many variations as there are applications of the technology. In this section, we address all of the major analysis steps for a typical RNA-seq experiment, which involve quality control, read alignment with and without a reference genome, obtaining metrics for gene and transcript expression, and approaches for detecting differential gene expression. We also discuss analysis options for applications of RNA-seq involving alternative splicing, fusion transcripts and small RNA expression. Finally, we review useful packages for data visualization.The acquisition of RNA-seq data consists of several steps — obtaining raw reads, read alignment and quantification. At each of these steps, specific checks should be applied to monitor the quality of the data Fig. .k-mers and duplicated reads in order to detect sequencing errors, PCR artifacts or contaminations. Acceptable duplication, k-mer or GC content levels are experiment- and organism-specific, but these values should be homogeneous for samples in the same experiments. We recommend that outliers with over 30 % disagreement to be discarded. FastQC ).Integration of more than two genomic data types is still at its infancy and not yet extensively applied to functional sequencing techniques, but there are already some tools that combine several data types. SNMNMF  and PIMiIn all cases, integration of different datasets is rarely straightforward because each data type is analyzed separately with its own tailored algorithms that yield results in different formats. Tools that facilitate format conversions and the extraction of relevant results can help; examples of such workflow construction software packages include Anduril , Galaxy RNA-seq has become the standard method for transcriptome analysis, but the technology and tools are continuing to evolve. It should be noted that the agreement between results obtained from different tools is still unsatisfactory and that results are affected by parameter settings, especially for genes that are expressed at low levels. The two major highlights in the current application of RNA-seq are the construction of transcriptomes from small amounts of starting materials and better transcript identification from longer reads. The state of the art in both of these areas is changing rapidly, but we will briefly outline what can be done now and what can be expected in the near future.Single-cell RNA-seq (scRNA-seq) is one of the newest and most active fields of RNA-seq with its unique set of opportunities and challenges. Newer protocols such as Smart-seq  and SmarThe small amount of starting material and the PCR amplification limit the depth to which single-cell libraries can be sequenced productively, often to less than a million reads. Deeper sequencing for scRNA-seq will do little to improve quantification as the number of individual mRNA molecules in a cell is small  and only a fraction of them are successfully reverse-transcribed to cDNA , 176; buGAPDH and ACTB, that are expected to be expressed at a detectable level.Single-cell transcriptomes typically include about 3000–8000 expressed genes, which is far fewer than are counted in the transcriptomes of the corresponding pooled populations. The challenge is to distinguish the technical noise that results from a lack of sensitivity at the single-molecule level   SMRT and Oxford Nanopore, that were initially applied to genome sequencing are now being used for transcriptomics and have the potential to overcome this assembly problem. Long-read sequencing provides amplification-free, single-molecule sequencing of cDNAs that enables recovery of full-length transcripts without the need for an assembly step. PacBio adds adapters to the cDNA molecule and creates a circularized structure that can be sequenced with multiple passes within one single long read. The Nanopore GridION system can directly sequence RNA strands by using RNA processive enzymes and RNA-specific bases. Another interesting technology was previously known as Moleculo (now Illumina’s TruSeq synthetic long-read technology), where Illumina library preparation is multiplexed and restricted to a limited number of long DNA molecules that are separately bar-coded and pooled back for sequencing. As one barcode corresponds to a limited number of molecules, assembly is greatly simplified and unambiguous reconstruction to long contigs is possible. This approach has recently been published for RNA-seq analysis .PacBio RNA-seq is the long-read approach with the most publications to date. The technology has proven useful for unraveling isoform diversity at complex loci , and forThree factors determine the number of replicates required in a RNA-seq experiment. The first factor is the variability in the measurements, which is influenced by the technical noise and the biological variation. While reproducibility in RNA-seq is usually high at the level of sequencing , 45, othRNA-seq library preparation and sequencing procedures include a number of steps  that might introduce biases into the resulting data . IncludiMapping to a reference genome allows for the identification of novel genes or transcripts, and requires the use of a gapped or spliced mapper as reads may span splice junctions. The challenge is to identify splice junctions correctly, especially when sequencing errors or differences with the reference exist or when non-canonical junctions and fusion transcripts are sought. One of the most popular RNA-seq mappers, TopHat, follows a two-step strategy in which unspliced reads are first mapped to locate exons, then unmapped reads are split and aligned independently to identify exon junctions , 201. SeMany statistical methods are available for detecting differential gene or transcript expression from RNA-seq data, and a major practical challenge is how to choose the most suitable tool for a particular data analysis job. Most comparison studies have focused on simulated datasets , 209 or As yet, no clear consensus has been reached regarding the best practices and the field is continuing to evolve rapidly. However, some common findings have been made in multiple comparison studies and in different study settings. First, specific caution is needed with all the methods when the number of replicate samples is very small or for genes that are expressed at very low levels , 64, 209


Heterogeneities in nucleotide content distribution along the length of Zaire ebolavirus and Marburg virus genomes have been analyzed. Results showed that there is asymmetric mutational A-pressure in the majority of Zaire ebolavirus genes; there is mutational AC-pressure in the coding region of the matrix protein VP40, probably, caused by its high expression at the end of the infection process; there is also AC-pressure in the 3′-part of the nucleoprotein (NP) coding gene associated with low amount of secondary structure formed by the 3′-part of its mRNA; in the middle of the glycoprotein (GP) coding gene that kind of mutational bias is linked with the high amount of secondary structure formed by the corresponding fragment of RNA negative (−) strand; there is relatively symmetric mutational AU-pressure in the polymerase (Pol) coding gene caused by its low expression level. In Marburg virus all genes, including C-rich fragment of GP coding region, demonstrate asymmetric mutational A-bias, while the last gene (Pol) demonstrates more symmetric mutational AU-pressure. The hypothesis of a newly synthesized RNA negative (−) strand shielding by complementary fragments of mRNAs has been described in this work: shielded fragments of RNA negative (−) strand should be better protected from oxidative damage and prone to ADAR-editing. Viruses from Filoviridae family are known as causative agents of dangerous hemorrhagic fevers. The most known virus from that family is Zaire ebolavirus which recently in 2014-2015) caused a new outbreak [4-2015 caSeparate mRNAs are synthesized on a matrix of a single RNA negative (−) strand, while there is just one site for RNA-dependent RNA-polymerase binding situated at the 3′-terminus of a genome , 5. ThisTranscriptional gradient may be one of the factors leading to differences in mutational pressure directions or intensity along the length of a genome . That isAccumulation of certain nucleotide mutations is associated with specific phases of the viral life cycle . The datThere are such components of nonspecific intracellular antiviral defense as adenosine deaminases able to bind just double-stranded fragments of RNA (ADAR superfamily ) and cytBiases in mutation occurrence rates lead to biases in nucleotide usage , 10. How Zaire ebolavirus and Marburg virus from GenBank. Nucleotide sequences of coding regions from reference genomic sequences  have been used in the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search algorithm. BLAST search has been performed only among the sequences of the corresponding species: Zaire ebolavirus (taxid:186538) and Marburg virus (taxid:11269). Those sets of sequences have been downloaded and processed. Only unique full-length sequences have been left in each alignment: repeated 100% identical sequences, as well as partial sequences, have been deleted. Resulting sets of sequences can be found in the DATAZE.xlsx and DATAMV.xlsx files from Supplementary Material available online at http://dx.doi.org/10.1155/2015/678587.In this study we used all available nucleotide sequences of Zaire ebolavirus) and PRJNA264121 (for Marburg virus). Total RNA in the abovementioned experiment was isolated by using Trizol (Invitrogen) from Vero cells  or differentiated Thp1 cells (human monocytic leukemia cells) infected with EBOV Mayinga (CDC isolate number 808011) or MARV-Ang (CDC isolate number 200501379) at multiplicity of infection of 3 [We used the information on the deep sequencing experiments performed by Shabman et al.  availablFor each of the unique full-length sequences of coding regions from Zaire ebolavirus and Marburg virus secondary structure has been predicted by the CentroidFold algorithm . That alhttp://chemres.bsmu.by). The algorithm calculates nucleotide usage in invariable fourfold and twofold degenerated sites from third codon positions and compares it with average nucleotide usage in all fourfold degenerated and twofold degenerated sites which have no nonsynonymous mutations in the whole alignment (stable sites). If the usage of a nucleotide is higher in invariable sites than in stable sites, we consider this nucleotide to be nonmutable and state that its usage is growing. If the usage of a nucleotide is lower in invariable sites than in stable sites, we consider this nucleotide to be mutable and state that its usage is decreasing [Alignments of sequences for each coding region have been studied with the “VVTAK VarInvar” algorithm (creasing . Sudan ebolavirus (NC_006432), Tai Forest ebolavirus (NC_014372), Bundibugyo ebolavirus (NC_014373), Reston ebolavirus (NC_004161), Lloviu cuevavirus (NC_016144), Vesicular stomatitis Alagoas virus Indiana 3 (NC_025353.1), and Chimpanzee adenovirus type 3 (HC469240.1). To test the significance of the differences in secondary structure amounts for different sequences we used Mann-Whitney U test .Graphs representing nucleotide usage distribution along the length of a genome have been built with the help of MS Excel. We used reference genomes ofP < 0.001).Along most of the length of the Zaire ebolavirus reference genome the usage of adenine (31.87 ± 0.30%) is significantly higher than the usages of other nucleotides see . The aveUsages of adenine and guanine do not exhibit significant variations along the whole Zaire ebolavirus genome. In contrast, the level of cytosine demonstrates three sharp peaks see . The firInterestingly, the first and the third peaks of cytosine usage are associated with the lowest levels of uracil usage . However, the second peak of cytosine usage is not associated with low level of uracil see .According to the mutational pressure theory , there sSudan ebolavirus, Tai Forest ebolavirus, Reston ebolavirus, and Bundibugyo ebolavirus) have patterns of nucleotide content distribution similar to that for Zaire ebolavirus genome (see Genomes of other ebolavirus species (nome see . Howevernome see . For thenome see . They arnome see . Howevernome see . Accordi Ebolavirus and Marburgvirus genuses.In the genome of Marburg virus (the one from the same Filoviridae family) one can observe just a single cytosine content peak associated with the lowest uracil usage see . That si Lloviu cuevavirus [There is a third genus of the Filoviridae family represented byevavirus . In its Because of some reasons fragments of Filoviridae RNA positive (+) strand may be either under AU-pressure or under AC-pressure . There is a hypothesis of transcription-associated mutational pressure  that may−5 to make graphs comparable with each other) for Zaire ebolavirus. The deep sequencing has been performed in two cell lines [R is equal to 0.6886) and THP-1 (R = 0.6202) cell lines with the cytosine content of the sequence (R is −0.6841 for VERO cells and −0.6513 for THP-1 cells). Guanine and adenine usages show much weaker dependence on nucleotide coverage . For C-rich fragments of NP and GP genes there are corresponding peaks of nucleotide coverage in both VERO and THP-1 cell lines (but in VERO cells those peaks are wider), while VP40 mRNA (or its fragments) is not as abundant in THP-1 cells 24 hours after infection as in VERO cells.In ll lines . We usedll lines  as a supll lines . There isequence , while tHigh cytosine usage of the VP40 coding region may be explained by the high expression level of its mRNA in the late period of the infection happening in certain types of cells with weak nonspecific antiviral defense  or two sIn The nucleotide coverage shows that certain fragments of mRNAs are more abundant than others. High abundance of an mRNA fragment may be the consequence of its high expression or of its resistance to degradation by numerous cellular RNAses . There iAnalysis of secondary structure amount (the percentage of nucleotides which make base pairs) showed that RNA fragments highly covered in the deep sequencing experiment  demonstrP < 0.001) amount of nucleotides forming “stems” (33.33%) than part 1 of the same coding region (42.00%) demonstrating decreased nucleotide coverage. The middle part of the GP coding region has lower amount of nucleotides forming “stems” than part 1 and part 2 of the same gene. However, the difference between part 1 and part 2 of GP is not very high , but it is significant  strand. For Zaire ebolavirus NP part 1 and part 2 as well as Marburg ebolavirus GP fragments amounts of stems in negative (−) strand are very close to each other Tables . For MarThe middle fragment of Zaire ebolavirus GP coding region (GP part 2) should be highly covered only in the reference strain  but not in many other strains. Moreover, fragments of GP mRNA corresponding to its part 2 will rarely bind antisense part of the negative (−) strand because of the high amount of stems made by it (49.77%). To clarify the causes of nucleotide content distribution heterogeneities in Zaire ebolavirus and Marburg virus genomes we analyzed current directions of nucleotide mutations in corresponding coding regions and their fragments of interest.The test performed by the “VVTAK VarInvar” algorithm shows which nucleotides are mutable in a given coding region or its fragment. If we assume that in regions with elevated cytosine usage mutations leading to the occurrence of C have higher frequency than mutations leading to its disappearance, the usage of cytosine in invariable fourfold degenerated sites must be significantly higher than its usage in stable fourfold degenerated sites . As one There is also an interesting trend in uracil mutability that can be observed in Adenine usage is increasing throughout the whole RNA positive (+) strand of Zaire ebolavirus. It means that there is a permanent mutational A-pressure, most likely caused by cytosine deamination and guanine oxidation on the negative (−) RNA strand leading to G to A and C to A mutations on the positive (+) RNA strand.Guanine usage in fourfold degenerated sites is decreasing in all coding regions and their fragments in Zaire ebolavirus genome, except NP part 1, GP part 1, and VP30. Those parts of positive (+) strand with nonmutable guanine residues are situated near C-rich fragments. Probably, they form double-stranded fragments together during the time of the full-length positive (+) strand existence. Because of this reason abovementioned fragments may accumulate more A to G transitions and less G to U transversions than other ones.Guanine usage in twofold degenerated sites is growing only in NP part 2 and GP part 2 . This faUnexpectedly, cytosine usage in twofold degenerated sites is decreasing in NP part 2 and in GP part 2 providing evidence that overall cytosine usage in these parts of positive (+) strand is growing due to the higher ratio between A to C and C to A transversions more likely associated with RNA negative (−) strand shielding than with RNA-editing.Adenine and uracil usages in twofold degenerated sites of Zaire ebolavirus coding regions show variable behavior. It is important to highlight that pure mutational AU-pressure (when both A and U are decreasing in both fourfold and twofold degenerated sites) has been found only in Pol coding region.In Marburg virus coding regions and even in their C-rich parts the usage of cytosine in fourfold degenerated sites is decreasing according to the results of the “VVTAK VarInvar” algorithm . The usaIn twofold degenerated sites of Marburg virus coding regions adenine usage is always growing and guanine usage is decreasing, while uracil usage behaves in variable manner . CytosinThe mechanism of cytosine-rich fragments creation and disappearance in genomes of Filoviridae viruses can be described by the following model. There is overall mutational A-pressure in viral genomes caused by C to U and G to U mutations on the negative (−) RNA strand. Adenine usage is growing mostly in fourfold and twofold degenerated sites due to the negative selection eliminating amino acid mutations . HoweverFrom this point of view, three peaks of cytosine usage found in Zaire ebolavirus genome can be described as “active volcanoes” with increasing cytosine content, while the single cytosine usage peak in Marburg virus genome is similar to the “sleeping volcano” which can be awaken by any factor increasing the amount of GP mRNA in cells infected by that virus.Viral genome may accumulate nucleotide mutations in different periods of the life cycle. Below we are going to discuss our results in light of known information about mutational processes. First of all, viruses with RNA negative (−) genomes should accumulate mutations when they are starting replication process (in the beginning of the infection) and when newly synthesized daughter genomes are packing into virions. In both conditions genomes of negative (−) polarity should be single-stranded. However, those single-stranded genomes should form secondary structure elements. The higher the amount of stems in a given part of the RNA negative (−) strand, the higher the rate of its ADAR-editing leading to A to G transitions occurrence and the lower the rate of its APOBEC1-editing, the lower the rate of its contacts with oxidative agents . The conIn this study we are faced with the facts which can be interpreted as evidence of a new mechanism of asymmetric mutational pressure existence. Fragments of mRNAs may bind antisense parts of RNA negative (−) strands at the final steps of viral replication (when RNA negative (−) strands are synthesized, while the amount of partially degraded previously transcribed mRNAs is still high). This binding will lead to double-stranded RNA fragments formation. The outcome of this event will be the same as in the case with extensive secondary structure formation by a fragment of RNA negative (−) strand: the usage of C in the corresponding fragment of RNA positive (+) strand will grow (or will decrease more slowly than in other fragments). There are several factors influencing the success of this RNA negative (−) strand shielding by fragments of mRNAs. First of all, there must be sufficient level of gene transcription to produce enough mRNA fragments. Indeed, the last gene of both Zaire ebolavirus and Marburg virus (Pol) demonstrates very low expression level. This factor affects the nucleotide content of Pol gene: the usage of U is high (and it is keep growing), while the usage of C is low. Unshielded RNA negative (−) strand should show lower rates of ADAR-editing and higher rates of guanine oxidation (both factors lead to cytosine usage decrease and uracil usage increase in corresponding fragments of RNA positive (+) strand). Lloviu cuevavirus  strand shielding, but it does not correlate with two other hypothetical mechanisms of asymmetric mutational pressure. Genomic positive (+) strand of RNA may become a target for oxidative damage during transcription  and durThe information on mutational pressure directions in different coding regions of Zaire ebolavirus is necessary for the creation of viral vector vaccines. Ideally, mutational pressure directions in the viral vector should be the same as in genes from Zaire ebolavirus. Otherwise, significant part of resulting proteins will have amino acid substitutions making vaccine antigens different from natural ones.Some modern viral vectors are based on Vesicular stomatitis virus genome with inserted genes (or their parts) from Zaire ebolavirus . This viAnother popular vector for Ebola vaccines creation is Chimpanzee adenovirus type 3  Figure . That dsFirst six genes of Marburg virus  are under the asymmetric mutational A-pressure, while the last one (Pol) is under the more symmetric mutational AU-pressure.In Zaire ebolavirus the situation with mutational biases is rather complicated. Asymmetric A-pressure can be found in NP part 1, VP35, GP part 1, GP part 3, VP30, and VP24. More symmetric AU-pressure exists in the longest Pol coding region. There is mutational AC-pressure in NP part 2, VP40, and GP part 2. This information can be used for future vaccine design studies.Zaire ebolavirus coding region or its part used in this study can be found in the “DATAZE.xlsx” file. The set of unique nucleotide sequences of each Marburg virus coding region or its part used in this study can be found in the “DATAMV.xlsx” file.The set of unique nucleotide sequences of each


This article describes data related to the research article titled “Functional characterization of importin α8 as a classical nuclear localization signal receptor”  Specifications TableValue of the data•These data are valuable to researchers interested in the molecular mechanisms by which the importin α-cNLS substrate complex dissociates in the nucleus.•These data show that importin α1 and α8 have substantial differences in dimer-forming ability, despite both proteins belonging to the same subfamily.•These data provide a new insight into the function of nuclear-localized importin αs.1To examine whether importin αs can directly bind with other importin α subtypes, FLAG-tagged importin α6, α7, or α8 recombinant proteins were incubated with either GST-importin α8, or GST-importin α1, and analyzed by western blotting A. To inv22.1EcoRI and NotI, and then subcloned into the pGEX6P3 plasmid . The construct integrity of pGEX6P3/FLAG-h-importin α6 and pGEX6P3/FLAG-h-importin α7 was confirmed by DNA sequencing using the following primers: pGEX 5′ sequencing primer: 5′-GGGCTGGCAAGCCACGTTTGGTG-3′, pGEX 3′ sequencing primer: 5′-CCGGGAGCTGCATGTGTCAGAGG-3′, importin α6 sequencing primer: 5′-GCATCTGGAACTTTTCTGCATACC-3′, or importin α7 sequencing primer: 5′-GTACATTACAGTTTGAAGCTGCCT-3′.The N-terminus FLAG-tagged cDNAs encoding full-length human importin α6  or full-length human-importin α7  were amplified from either HEK293 cells or MRK-nu-1 cells  by PCR using the following primers: importin α6 Forward: 5′-CCCGAATTCCGCCATGGACTACAAGGACGACGACGACAAGATGGATGCCATGGCTAGTCC-3′ and importin α6 Reverse: 5′-CCCGCGGCCGCCTCGAGTTAAAGTTGAAATCCATCC-3′ or importin α7 Forward: 5′-CCCGAATTCCGCCATGGACTACAAGGACGACGACGACAAGATGGAGACCATGGCGAGC-3′ and importin α7 Reverse: 5′-CCCGCGGCCGCCTCGAGTTATAGCTGGAAGCCCTCC-3′. The PCR program was as follows: 2 min at 94 °C followed by 40 cycles of 30 s at 98 °C and 15 min at 68 °C. The PCR products were digested with EcoRI and NotI sites of pGEX6P3, and then sequenced using either the pGEX 5′ or pGEX 3′ sequencing primer and the importin α8 sequencing primer: 5′-CAACATCGCTTCAGGGACTTCG-3′.The human-importin α8  cDNA with the FLAG-tag at the N-terminus was amplified by PCR from the pcDNA5/3xFLAG-h-importin α8 plasmid The construct encoding SV40 large T antigen NLS  was subcloned from pGEX2T-SV40TNLS-GFP BamHI and XhoI sites of pGEX6P1, and then verified by sequencing.The human cDNA encoding the tumor protein p53 (NM_000546) was amplified from MCF7 cells by PCR performed using the following primers: p53 Forward: 5′-CACGGATCCATGGAGGAGCCGCAGTCAGATC-3′ and p53 Reverse: 5′-GGACTCGAGTCAGTCTGAGTCAGGCCCTTCTG-3′. The PCR program was as follows: one cycle of 2 min at 94 °C; 40 cycles of 15 s at 94 °C, 30 sec at 64 °C, and 1 min 20 s at 68 °C; and one cycle of 10 min at 68 °C. The PCR product was subcloned into the 2.2Escherichia coli Rosetta, and then the cells were grown at 37 °C in LB medium containing 50 μg/mL ampicillin. Expression was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), followed by incubation at 20 °C for 12 h. The bacteria were lysed in lysis buffer , 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL aprotinin , 1 μg/mL pepstatin , and 1 μg/mL leupeptin (Peptide Institute)) by freeze–thawing twice and passing through a French press . The cell lysates were sonicated using a Sonifier 250 , and centrifuged at 20,400g at 4 °C for 30 min. The resultant supernatant was incubated with glutathione-Sepharose 4B beads  at 4 °C for 12 h. After the GSH beads were washed five times with lysis buffer, GST-tagged proteins were eluted with elution buffer . Cleavage of GST from the GST-fused protein was performed using PreScission protease  with 10 units/mg of fusion protein at 4 °C for 12 h in cleavage buffer . Finally, the purified proteins were dialyzed against dialysis buffer  and concentrated by ultrafiltration using Amicon Ultra centrifugal filter units .Recombinant proteins fused to GST were purified as follows: The expression vectors were transformed into 2.32.4The following antibodies were used for western blotting: anti-FLAG M2 antibody , anti-GST antibody , anti-GFP antibody , anti-p53 (FL-393) antibody , and horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG secondary antibodies 2.5http://rsbweb.nih.gov/ij/).Samples were loaded on a 10% SDS-PAGE gel, and the separated proteins in the gel were transferred onto an Immobilon-P membrane  using a semi-dry transfer blotting system . The membrane was blocked with blocking buffer consisting of 3% skim milk in Tris-buffered saline  with 0.05% Tween (TBS-T) for 1 h. The membrane was probed with primary antibodies diluted in Can Get Signal Immunoreaction Enhancer Solution 1  at 4 °C overnight, and then incubated with the HRP-conjugated secondary antibody diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO) at room temperature (RT) for 1 h. After the membrane was washed with TBS-T, it was developed with Chemi-Lumi One L or Super . The intensity of each western blot signal was quantified by Image J software (


Over the past 20 years, the generation of functional RNAs by in vitro selection has become a standard technique. Apart from aptamers for simple binding of defined ligands, also RNAs for catalysis of chemical reactions have been selected. In the latter case, a key step often is the conjugation of one of the two reactants to the library, requiring suitable strategies for terminal or internal RNA functionalization. With the aim of selecting a ribozyme for deamination of cytidine, we have set up a selection scheme involving the attachment of the cytidine acting as deamination substrate to the 3'-terminus of the RNAs in the library, and library immobilization. Here, we report the synthesis of a bifunctional cytidine derivative suitable for conjugation to RNA and linkage of the conjugated library to a streptavidine-coated surface. Successful conjugation of the cytidine derivative to the 3'-terminus of a model RNA is demonstrated. Tetrahymena thermophilia in 1982 [Since the discovery of the first catalytic RNA in the ciliate  in 1982 –5. Moreo in 1982 , or the  in 1982 . Such ri in 1982 . In the  in 1982 . The devcis-diol, which can be specifically oxidized with metaperiodate [A very useful and often applied technique for the generation of catalytic RNA structures is a variation of the classical SELEX approach  –13. A tyeriodate –28, folleriodate .cis diol of the RNA library molecules, the produced dialdehyde is thought to react with the primary amino group of the cytidine derivative allowing immobilization of the resulting modified RNAs onto a solid surface through biotin–streptavidine interaction. All RNA sequences that can catalyze the desired deamination reaction of cytidine to uridine will be cleaved off and thus released into solution. Upon recovery, beneficial variants can be subjected to the next round of selection. Here we describe the synthetic route to the bifunctionalized cytidine derivative and its successful conjugation to RNA.We set out to select from a random library catalytically active RNAs that support deamination of cytidine to uridine. For this purpose, a bifunctionalized cytidine derivative  was synt1 started from uridine making use of four synthons: a protected hexaethylene glycol linker phosphoramidite 2 bearing a primary amine to be used later for RNA functionalization, protected and suitably activated uridine 3, and a short mono protected diamino linker 4 for the attachment of a biotin moiety 5 in order to immobilize the conjugated RNA molecules onto a streptavidine-coated surface  were fully protected with tert-butyldimethylsilyl (TBDMS) groups and the tris-silylated uridine 7 [3, triazole and triethylamine diethylamine (8) with di-tert-butyl dicarbonate (Boc2O) in dioxane [Among the procedures described for conversion of uridine into cytidine derivatives –34 we ha dioxane   ester, and subsequently the 5'-O-phosphoramidite should be prepared. Unfortunately, preparation of 12 in the suggested one step procedure  at 0 °C. Since cleavage of Boc-groups requires strong Brønsted acids [12 in an alternative way. Apart from protic acids, the tert-butoxycarbonyl moiety of Boc-protected amines can be selectively cleaved with Lewis acids [9 in anhydrous DCM with ZnBr2 delivered the free amine 10 with a yield of 82%  at 0 °C, and solely the 5'-O-deprotected cytidine derivative 12 was obtained in 94% yield chlorophosphoramidite to yield the phosphoramidite 2, which was coupled with synthon 12 in anhydrous THF in the presence of 5-benzylmercapto-1H-tetrazole (BMT) as activator  group was removed by stirring 16 in a 1:1 mixture of concentrated aqueous ammonia and methylamine. Subsequent treatment of 17 with triethylamine trihydrofluoride (TEA·3HF) at 55 °C for 2 h delivered compound 18, which was reacted with neat TFA. Each deprotection step was monitored by MALDI mass spectrometry. The bifunctionalized cytidine derivative 1 as the final product was isolated by reversed-phase chromatography with a yield of 83% over the last three steps  corresponding to the 3'-terminal sequence of the envisaged RNA-library. The RNA was oxidized by treatment with sodium periodate [1. The obtained intermediate was reduced with sodium cyanoborohydride to form the more stable amine as final product , and a new peak (III) indicating the desired RNA–cytidine conjugate, as confirmed by MALDI mass spectrometry (see The RP-chromatogram of the crude product (A) shows two peaks (denoted as I) corresponding to the oxidized and cyanoborohydride reduced RNA (C), peak II corresponding to the cytidine derivative etry see .O-coupled linker carrying an aliphatic amino group allows 3'-terminal conjugation of cytidine to the molecules of an RNA library, whereas the biotinylated linker attached to C4 of the nucleobase allows linking the modified library to a streptavidine coated surface. The suitability of the synthesized cytidine derivative was confirmed by successful conjugation to the 3'-terminus of a model RNA, as analyzed by HPLC and MALDI–MS. This set-up is going to be used for selection of a cytidine deaminase ribozyme supporting the conversion of uridine to cytidine. Active molecules will be cleaved from the solid phase and released into solution, such that those can be collected, reverse-transcribed and amplified to enter the next round of selection.We successfully synthesized a cytidine derivative for the functionalization of an RNA library following a synthetic route over 16 steps. Two linker moieties of different lengths and functionality were attached to the nucleobase and to the sugar residue of cytidine, thus enabling for one coupling of the nucleoside to periodate-oxidized RNAs, and for second linking the functionalized library to a surface. The 5'-1 was synthesized starting from uridine. All reaction steps and the characterization of the obtained products are described in detail in 1 was synthesized on an Applied Biosystems 394 DNA/RNA Synthesizer following the standard protocol for oligoribonucleotide chain assembly. The synthesized RNA was deprotected using aqueous ammonia (32%)/aq methylamine (40%)  at 65 °C for 30 min for removal of base and phosphate protecting groups and cleavage from the support, and TEA·3HF for removal of 2'-O-protecting groups, essentially as described previously [The bifunctional cytidine derivative eviously .cis-diol, 5 nmol of the 20mer RNA substrate were incubated in a total volume of 1 mL 30 mM sodium metaperiodate, 100 mM NaOAc (pH 5.4) for 1 hour at room temperature in the dark. The oxidized RNA was recovered by ethanol precipitation.For the oxidation of the RNA's 3'-terminal 3, 1 mM EDTA and 1 mM cytidine derivative 1. The reaction was carried out at 37 °C. After 2 hours, 25 µL of 50 mM NaBH4 were added, and the reaction mixture was incubated for additional 15 min. Ethanol precipitation yielded the crude coupling product, which was analyzed by reversed-phase HPLC on an Äkta Purifier (Amersham Bioscience). Column: Macherey Nagel EC 250/4 Nucleodur 100-5 C18 ec; Buffers: (A) 0.1 M triethylammonium acetate (pH 7.0), 5% acetonitrile and (B) 0.1 M triethylammonium acetate (pH 7.0), 30% acetonitrile; flow rate 0.5 mL min−1; gradient: 0% → 85% (B) in 14 CV, 85% 4 CV, 85% → 100% (B), 100% (B) 4 CV, 100% → 0% (B) 2 CV. The product containing fraction was collected, RNA was lyophilized and analyzed by MALDI–MS: , 5 mM NaCNBHFile 1Experimental part.


Escherichia coli, the properties of the Rho homologs from most bacteria are not known. The rho gene is unusually large in genus Mycobacterium and other members of actinobacteria, having ∼150 additional residues towards the amino terminal end. We describe the distinct properties of Rho from Mycobacterium tuberculosis. It is an NTPase with a preference for purine nucleoside triphosphates with kinetic properties different from E. coli homolog and an ability to use various RNA substrates. The N-terminal subdomain of MtbRho can bind to RNA by itself, and appears to contribute to the interaction of the termination factor with RNAs. Furthermore, the interaction with RNA induces changes in conformation and oligomerization of MtbRho.Two mechanisms – factor independent and dependent termination – ensure the completion of RNA synthesis in eubacteria. Factor-dependent mechanism relies on the Rho protein to terminate transcription by interacting with RNA polymerase. Although well studied in  Escherichia coli have been studied extensively E. coli Rho (EcRho) is functionally a homohexameric molecule Mycobacterium tuberculosis (Mtb) Termination of transcription of bacterial RNA polymerase (RNAP) is achieved either by intrinsic terminators or protein factor Rho. For factor-mediated termination, Rho binds to the nascent transcript emerging from the ternary elongation complex, translocates along the RNA by ATP-powered steps and finally enforces dissociation of the complex Mtb, the genus Mycobacterium includes some of the well-known Actinobacteria, such as the well-studied model organism Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium leprae, and a large number of Streptomyces species. In recent years, studies on transcription initiation, elongation and termination have been carried out in Mtb and other members. These studies, though not as exhaustive as in E. coli, have revealed considerable differences from the E. coli paradigm. For example, the Mtb and other mycobacterial genomes code for a larger number of sigma factors E.coli and also for the several transcription factors unique to mycobacteria Mycobacterium species In addition to E. coli, it is likely that mycobacterial Rho homologs have also evolved to function differently and optimally for their specific cellular context. Notably, sequence analysis showed that the Rho homologs in Mycobacterium species and other actinobacteria are larger than EcRho mainly due to an ‘extra-stretch’ of ∼150–200 residues in their RNA-binding domains M. tuberculosis Rho (MtbRho) can hydrolyse purine nucleoside triphosphates(NTP) – ATP and GTP-in presence of mycobacterial RNA. The extended N-terminal region of MtbRho, having a distinct RNA-binding ‘subdomain’, can itself interact with RNA and may contribute to the overall interaction. The MtbRho-RNA interactions are stable and the interactions induce changes in the conformation and oligomerization status of the protein.Given the dissimilarities in various steps of transcription between mycobacteria and M. tuberculosis, we expressed recombinant MtbRho in BL21(DE3) cells and purified it to homogeneity. MtbRho has a monomeric size of 65 kDa  site m (75.2 µM) for ATP than EcRho (10 µM) E. coli terminator RNAs had been used E. coli homolog. The superior ability of MtbRho to utilize mycobacterial RNA was further evident when, in presence of sdaA RNA, a specific Mtb RNA, MtbRho hydrolysed ATP at a rate that is 2-fold higher than that reported in presence of the E. coli terminators sdaA RNA for ATPase activity  . Both ceTreating MtbRho with glutaraldehyde, a bifunctional crosslinking agent, showed that crosslinked MtbRho migrates with a mobility corresponding to a mass of ∼400 kDa. Since the MtbRho monomer is 65 kDa, the crosslinked product observed is likely to be hexameric . The funMtbrho construct was used to complement the E. coli AMO14. The AMO14 genome has an inactivated rho gene E. coli rho is supplied on a temperature-sensitive plasmid. The strain is viable at non-permissive temperature (42°C) only when a functional rho allele is supplied in trans. However, the E. coli AMO14 competent cells, transformed with pTrc99C-mtbrho, showed no growth at non-permissive temperature (MtbRho gene which lacked the 158-amino acid region (residues 87–241) also failed to complement(data not shown). The failure to complement was not due to lack of expression  species by glutaraldehyde crosslinking indicates that MtbRho can exist as a hexamer even in absence of its substrate RNA and ATP. DLS and analytical gel filtration studies confirm that at least a fraction of MtbRho exists as hexamer in solution. This hexameric population could provide a ‘readily available’ termination-proficient MtbRho within E. coli strains DH10B and BL21(DE3) were used for cloning and overexpression of proteins respectively. The temperature-sensitive E. coli AMO14 strain was used for complementation studies rho gene was PCR-amplified using M. tuberculosis H37Ra genomic DNA as template, primers MtbRhoF and MtbRhoR  or pTrc99C-rho DNA. Cultures were grown at permissive (30°C) and non-permissive (42°C and 37°C) temperatures. All restriction enzymes and DNA modifying agents were from New England Biolabs. DNA ligase was obtained from Roche Applied Science. MtbRhoR  and Pfu 9C vector. A delet600 0.6, induced with 0.3 mM IPTG for 3 hrs at 37°C and harvested. Cells were resuspended in buffer , lysed by sonication and centrifuged at 100000 g for 2 hrs at 4°C. This was followed by 0.5% polyethyleneimine (PEI)-based precipitation and then 0–40% ammonium sulphate fractionation. The pellet was resuspended in IX TGED buffer  containing 150 mM KCl, dialysed against the same buffer to remove the excess salt and then loaded onto a pre-equilibrated Hitrap Heparin-sepharose column. Fractions were eluted in a salt range from 150 to 1000 mM KCl. The fractions containing MtbRho were pooled, dialyzed with 1× TGED+150 mM KCl and applied onto a Hitrap SP-sepharose column. The purity of the protein was assessed by electrophoresing on 8% SDS-PAGE, silver staining and mass spectrometry. Fractions which contained purified MtbRho were pooled and dialyzed overnight in storage buffer (1× TGED+100 mM K-glutamate+50%glycerol). EcRho and N-229, both with a C-terminal hexahistidine tag, were expressed in BL21(DE3) cells. The respective lysate was passed through Ni-NTA column (Qiagen), eluted with 500 mM imidazole and further purified to homogeneity using Hitrap-Heparin column. Concentration of the proteins was estimated by Bradford method. The Ni-NTA beads were from Sigma, all columns and materials were form Amersham-GE Healthcare. Mycobacterial RNA was purified as described in sdaA RNA, a 330 bp region(genomic coordinates: c76257–c75907) downstream of Mtb sdaA gene(Rv0069c) was PCR-amplified using forward primer, that contains T7 RNAP promoter sequence (sdaARNAfwd) and sdaARNArev. 1.5 µg of this template was used to transcribe sdaA RNA in vitro in presence of 0.5 mM NTP and 20 µCi α-32P-UTP  and T7 RNAP (Fermentas). Unlabeled sdaA RNA was synthesized similarly with only 4 mM ‘cold’ NTPs as precursors. Following DNaseI treatment, the product was purified by phenol-chloroform extraction, alcohol precipitation and passing through Sephadex G-50 minicolumn, and its homogeneity was assessed on denaturing-PAGE. Poly-dC80 oligonucleotide was end-labeled with 15 µCi γ-32P-ATP  by Polynucleotide Kinase(NEB) as per standard protocol and eluted through Sephadex G-25 minicolumn.The recombinant MtbRho was overexpressed from the pET11d-rho clone in BL21(DE3) cells. Cultures were grown till ODM. smegmatis total RNA. To test the effect of bicyclomycin, the MtbRho was pre-incubated with 500 µM bicyclomycin at 37°C for 10 min. For quantitative assays, 40 ng polyC, 1 µg M. smegmatis total RNA and 300 nM sdaA RNA were found to be optimal. Protein concentrations used were with respect to hexameric form of proteins. Reaction was initiated by addition of 1 mM of the ATP/NTP. 100 nCi of γ32P-ATP (NEN) was used as tracer in radiometric assay to detect formation of inorganic phosphate (Pi), while α32P-ATP(NEN) was used to observe if any AMP- or PPi-generating activity was present. The reactions were stopped by adding chloroform. Following centrifugation, the aqueous phase was used to estimate the release of inorganic phosphate(Pi) by resolving on Polyethyleneimine TLC sheets using LiCl (1.2 M) and EDTA(0.1 mM) as mobile phase and visualized with the Typhoon 9200 phosphorimager. For estimating the hydrolysis of the various NTPs in non-radioactive quantitative assays, a plate-based colorimetric assay 2PO4. Reactions were carried till 30–40% of ATP got hydrolysed. Kinetic parameters were obtained by plotting values on a Lineweaver Burk plot. For UV crosslinking, MtbRho was incubated with γ-32P-ATP alone, or in presence of unlabeled ATP or CTP at 37°C for 10 min, followed by crosslinking on ice for 30 min using a handheld UV-torch (254 nm). No RNA was added.The hydrolysis of ATP and other NTPs in presence of different RNA substrates was assessed either by thin-layer chromatography (TLC) or by a Malachite Green-based colorimetric assay. Assays were carried out in T-Buffer  with 100 nM MtbRho and the appropriate RNA in 10 µl reaction volume. Qualitative assays were carried out for 30 min in presence of 5,10 and 20 ng of polyC, polyA, polyU, 1 and 2 µg E. coli RNAP, template containing Trc promoter was amplified using primers FwdTrc and RhoRevpTrc310, using pTrc99C-rho plasmid as template. Reaction was carried out by incubating 50 nM template with 100 nM E. coli RNAP in T-buffer, first on ice and then at 37°C for 10 min each, followed by incubation with 175 nM MtbRho or EcRho for 5 min. Transcription was initiated with 100 µM rNTP and 1 µCi α-32P-UTP and continued for 30 min before terminating by addition of phenol-chloroform. Following centrifugation, equal volume of gel-loading dye  was added to the aqueous phase, heated at 90°C for 2 min, chilled and resolved on 6% denaturing PAGE. To test the effect of bicyclomycin, the MtbRho was pre-incubated with 400 or 500 µM bicyclomycin at 37°C for 10 min before addition to RNAP-DNA complex.For transcription by 32P-labeled sdaA RNA or polydC-80 on ice for 10 min. The 10 µl reactions were carried out in T-buffer. The RNA-protein complexes resolved from free RNA by 4 or 6% native PAGE (30:0.6::acrylamide:bisacrylamide). Protein concentrations used were with respect to hexameric form.For qualititative RNA-binding assays, 100 nM of MtbRho and 100, 200, 400 and 800 nM of N-229 fragment were incubated with 15000 cpm of the E. coli tRNA (USB) or M. smegmatis SN2 total RNA on ice for 10 min and then at 37°C for 5 min, followed by addition of V8 protease and incubation for different time-points. The reaction was stopped by the addition of inhibitor Tosyl Lysine Chlromethyl Ketone  and 6× SDS gel loading dye, resolved on 8% SDS-PAGE and visualized with silver staining method.1 µg MtbRho was incubated in T-buffer with of 2 µg MtbRho was crosslinked by the addition of glutaraldehyde, to a final concentration of 0.05%. The reactions were carried on for 5 and 10 min at 37°C in T-buffer  and terminated by addition of 2 M Tris, followed by resolving products on a 3.5-8% gradient SDS-PAGE.The CD spectra were recorded from 250–200 nm using a JASCO J–810 spectropolarimeter (Japan Spectroscopic Co Inc. Japan) in a 0.2 mm path-length quartz cuvette. T-buffer was used and readings were recorded at room temperature.in vitro experiments described above, except that it contained no glycerol. Protein concentration of 300 ng/ul was found to be sufficient for the assay. A 10∶1 molar ratio of tRNA: Rho was maintained. Data was analysed using the software Omnisize 3.0, associated with the DLS machine.DLS analysis were carried out on a Viscotex 802 DLS instrument in 12 µl volume quartz cuvette. T-buffer used for the assay was identical to that used for Typically, 100 µg MtbRho was loaded onto Superdex S200 column  alone, or after pre-incubating with tRNA (molar ratio 1∶2) and 1 mM ATP, and 1 ml fractions were collected. After electrophoresing fractions on 8% SDS-PAGE, the eluted protein was assessed by silver staining.V.N. is recipient of J.C. Bose Fellowship from Department of Science and Technology, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Figure S1Occurrence and divergence of Rho in bacteria. (A) Phylogenetic distribution of Rho from several bacterial phyla. (B) Alignment of representative Rho homologues from different bacteria. The 150–200 residue-long subdomain present in the N-terminal half of the actinobacterial homologs is enclosed in red. The alignment was done using the MACAW software. Conserved motifs and residues are shown as boxes and shaded regions.(TIF)Click here for additional data file.Figure S2Expression and purification of MtbRho. MtbRho was expressed from pET11d-rho clone in BL21(DE3) cells by induction with 0.3 mM IPTG (lane 2). MtbRho purification was carried as described in (TIF)Click here for additional data file.Figure S3Inefficient binding of N-229 to poly-dC80. N-229 was incubated with the 32P-labelled polynucleotide, and the complexes (RC) resolved from free polynucleotide (pC) by 6% native PAGE with 20% glycerol included as stabilizer both in gel and running buffer. No protein (lane 1); 0.6, 1.2 and 1.8 µM of N-229 was used ; presence of excess unlabeled poly-dC80 competed with binding (lane 5).(TIF)Click here for additional data file.Figure S4Conformational changes of MtbRho upon binding to RNA and ATP. (A) MtbRho was incubated with M. smegmatis RNA and E. coli tRNA were used alone, or with ATP and probed with V8 protease. The presence of RNA  conferred substantial protection against proteolysis, when compared to only MtbRho . The addition of ATP along with RNA  did not result in any additional or altered protection and ATP alone (lane 12) did not protect MtbRho from protease. (B) Changes in secondary structure were monitored at wavelengths between 200–250 nm in presence of RNA (I) or ATP (II). The molar ellepticity of CD spectra are shown for MtbRho alone (black), in presence of RNA(blue) and in presence of ATP (red).(TIF)Click here for additional data file.Table S1Strains, plasmids and oligonucleotides used in this study.(DOCX)Click here for additional data file.


We describe the rare case of a 68-year-old gentleman with the history of a hand-assisted laparoscopic left radical nephrectomy for a T2bN0M1 clear cell renal cell carcinoma (RCC). Seven years after surgery and with clean surveillance imaging for metastasis/recurrence the patient presented with three separate tumors suspicious for malignancy. A bladder lesion was found during workup for hematuria, a stomach lesion during diagnostic endoscopy, and a testicular lesion during self-exam. He underwent transurethral resection of bladder tumor, left inguinal orchiectomy, and upper endoscopic ensnarement. All specimens surprisingly showed RCC by histology and immunostaining. These three sites are rare for RCC metastasis and simultaneous presentation is even rarer, further emphasizing the importance of continuous and careful follow-up in this patient population, despite what could appear as complete remission. Renal cell carcinoma has a strong potential to metastasize to multiple organs . The mosA 68-year-old gentleman with a history of T2bN0M1 clear cell RCC status after hand-assisted laparoscopic left radical nephrectomy (no lymph node dissection), 7 years ago, without evidence of recurrence on surveillance imaging presented with a palpable left testicular mass. During follow-up of his RCC with serial scans, he was found with hematuria and transurethral resection of the bladder specimen was positive for papillary urothelial neoplasm of low malignant potential (PUNLMP). Patient was then followed with serial scans and cystoscopies (and cell cytology) for RCC and PUNLMP, respectively. During follow-up, patient presented to his primary care physician (PCP) with a painless, left testicular mass noted on self-examination. Given his history of RCC, his PCP asked that he be further evaluated by a urologist. Testicular ultrasound showed a 2.1 × 1.7 × 2.0 cm heterogeneous, hypoechoic lesion in the left testicle, which was highly suspicious for a testicular neoplasm . He was During his preoperative workup, he was noted to be anemic and was found to have black tarry stools. A gastroenterologist was consulted and an upper endoscopy showed a friable, ulcerated, and nodular mass in the fundus of the stomach . Two monA cystoscopy, prior to starting the left inguinal orchiectomy, was done (given his history of PUNLMP) and showed a small subcentimeter hyperemic nodular area in the bladder base, just anterior to the trigone, which was biopsied. The radical orchiectomy was performed without any complications after biopsy of the bladder nodule. Pathology reported clear cell RCC with positive CD10, CK8/18 immunochemistry for both testicular and bladder specimens.It is a well-established fact that RCC, like melanoma, is a clinical chameleon as it can metastasize to just about any organ in the body –10. WithTesticular metastasis of RCC often occurs in the ipsilateral left testicle and generally precedes or concurrently presents with primary RCC. Rarely have there been cases of delayed testicular metastasis of RCC , 9. Our RCC (T2bN0M1) metastasis in our patient was a surprise due to the distant nature of the original surgery and it remains significant to note that all three lesions occurred simultaneously and could impact his prognosis. However, this combination presentation of simultaneous multifocal metastasis of RCC has not been reported to the best of our literature search, which was done, on PubMed using search words such as “renal cell carcinoma metastasis,” “gastric metastasis,” “testicular metastasis,” “simultaneous,” and “delayed.” This was an unfortunate patient and it goes to solidify the capricious nature of RCC but most importantly add to the library of case reports of rare testicular metastasis of RCC.RCC metastasis to the testicle, genitourinary, and gastrointestinal tract remains a rarity and simultaneous multifocal RCC metastasis to these sites is even rarer. It goes without saying that urologists and oncologists should anticipate just about any pattern of presentation of RCC metastasis, and this report emphasizes the importance of close follow- up and a high suspicion for odd areas of metastasis.


RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye ) was employed to recognize RNA G-quadruplex structures using UV–Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures. One of the most important types of nucleic acid structure is the G-quadruplex G4), which is formed from four guanine bases by stacking of Hoogsteen bonded G-quartets2, which i5678910111418200 was determined to be as large as 610-, 513-, 500-, 401-, 402-, 406- and 366-fold for ADAM10, BCL-2, ERSI, TRF2, VEGF, C9orf72 and ZIC1, respectively  and other RNA forms, such as transfer RNA (tRNA), mutation sequences , single-stranded , hairpin (HP18), double-stranded (dsAf16), three-way junction (3-WJ) and four-way junction (4-WJ). As shown in ectively . The obsBecause ThT selectively binds to G-quadruplex DNA in the human telomeric motif, we further compared the recognition of the ThT probe for the detection of RNA G-quadruplexes and DNA G-quadruplexes. As shown in Furthermore, the specific binding of ThT with RNA G-quadruplex structures was further demonstrated by the absorption spectra. As shown in 5, (2.93 ± 0.27) × 105, (2.03 ± 0.12) × 105, (0.62 ± 0.61) × 105, (1.24 ± 0.15) × 105 M−1, (1.92 ± 0.05) × 105 M−1 and (0.56 ± 0.22) × 105 M−1, respectively. Additionally, binding constants of (5.51 ± 0.83) × 104, (9.37 ± 1.28) × 104 M−1 (5.53 ± 1.59) × 104, (5.62 ± 1.94) × 104, and (1.43 ± 1.01) × 105 for ThT with tRNA, ssAf20, ssAf22, HP18 and dsAf16, respectively, were also obtained −1) was much lower than that of the groove binding mode . As shown in To explore the mechanisms of ThT binding with the RNA G-quadruplex structure, molecular modelling studies of the 1:1 complex of RNA G-quadruplex with ThT have been performed. In general, the RNA G-quadruplex structures have several sites of interaction with the probes . The molecular docking results indicate that the binding free energy of the end-stacking mode , which is a well-known DNA G-quadruplex binding dye2+NL1G2+NL2G2+NL3G2+ sequences may also form G-quadruplex structures252627According to these results, we discovered that ThT, which is a water-soluble fluorogenic dye with low self-fluorescence, exhibited substantially fluorescence enhancement in the presence of RNA G-quadruplexes compared to the fluorescence enhancement of less than 30-fold that was observed in the presence of non-G4 RNA. The substantial increase in the fluorescence emission was due to restricting the rotation and enforcing the planarization of the dye22Furthermore, TO and ThT are widely used to selectively target DNA G-quadruplex. However, our results indicate that thiazole orange (TO) does not exhibit any selectivity for RNA G-quadruplexes compared to other RNA forms. Therefore, a DNA G-quadruplex probe may not be suitable for detecting or recognizing RNA G-quadruplexes without validation. ThT has been demonstrated to act as a universal probe to recognize RNA G-quadruplex or DNA G-quadruplex structures. However, the G-quadruplex structure formed from human telomeric DNA is different from the G-quadruplex structure formed from RNA. The G-quadruplex in a human telomeric motif is typically a stabilized hybrid structure, and the RNA G-quadruplexes formed a parallel-stranded conformation regardless of the different sequences and the experimental conditionsThe oligonucleotide sequences that are shown in 3,6-Dimethyl-2-(4-dimethylaminophenyl)-benzothiazolium cation (ThT) and the thiazole orange (TO) dyes were purchased from Sigma-Aldrich and used without further purification. The stock solutions of ThT and TO were prepared by dissolving in water. All of the other chemicals were of analytical reagent grade and used without further purification. Ultrapure water, which was prepared using a Milli-Q Gradient ultrapure water system (Millipore), was used in all of the experiments.The absorption spectra were acquired with a UV-1601PC at room temperature using a quartz cell with a path length of 10 mm. The absorption titration experiments were performed by increasing the RNA G-quadruplex concentrations from 0.125 to 8 μM, and ThT was fixed at 2 μM.The experiments were carried out with 96-well microplates from CORNING (Flat Bottom Black Polystyrol). The RNA samples and ThT were mixed at 4 μM and 2 μM final concentrations, respectively, in 20 mM Tris HCl at a pH of 7.0 and 40 mM KCl. The measurements were performed at room temperature. The fluorescence emission was collected at 487 nm with excitation at 440 nm in a microplate reader (PE EnSpire).The fluorescence spectra were acquired with a Hitachi F-4500 spectrophotometer at 25±1 °C, which was equipped with a temperature-controlled circulator. A quartz cuvette with a 10-mm path length was used in all of the experiments. In the fluorescence measurements, both the excitation and emission slits were 5 nm, and the scan speed was 240 nm/min. ThT was titrated with RNA G-quadruplex to measure the binding constants, and the fluorescence intensity at 440 nm was plotted as a function of the RNA concentration. The data were fitted according to a 1:1 binding model. The titration experiments were performed by increasing the RNA G-quadruplex concentrations from 0.125 to 8 μM ThT, which was added to the different RNA concentration solutions with gentle stirring for 10 min, and then, the samples were maintained in darkness for 2 hours prior to the measurements.2 was less than 1.1.The fluorescence lifetime measurements were recorded on a time-correlated single photon counting FLSP920 system. From the measured decay traces, the data were fitted with a multi-exponential decay, and χ7. All of the figures were rendered using the Discovery Studio 3.5 program .The coordinates of the RNA quadruplex (3IBK) structures were retrieved from the RSCB Protein Data Bank. The RNA quadruplex structure was prepared for molecular docking, and the ThT structure was energy minimized (MMFF force field) using the Discovery Studio 3.5 program . The molecular docking studies were performed using the Lamarckian Genetic Algorithm as implemented in the Autodock 4.0 package. The binding site (docking grids) was prepared using 100 × 100 × 100 points with a grid spacing of 0.375 Å. For the Lamarckian Genetic Algorithm based docking, the size of the population was set to 150, and the number of energy evaluations was set to 5.0 × 10How to cite this article: Xu, S. et al. Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes. Sci. Rep. 6, 24793; doi: 10.1038/srep24793 (2016).