Small ncRNAs (sncRNAs) offer great hope as biomarkers of disease and response to treatment. This has been highlighted in the context of several medical conditions such as cancer, liver disease, cardiovascular disease, and central nervous system disorders, among many others. Here we assessed several steps involved in the development of an ncRNA biomarker discovery pipeline, ranging from sample preparation to bioinformatic processing of small RNA sequencing data.A total of 45 biological samples were included in the present study. All libraries were prepared using the Illumina TruSeq Small RNA protocol and sequenced using the HiSeq2500 or MiSeq Illumina sequencers. Small RNA sequencing data was validated using qRT-PCR. At each stage, we evaluated the pros and cons of different techniques that may be suitable for different experimental designs. Evaluation methods included quality of data output in relation to hands-on laboratory time, cost, and efficiency of processing.Our results show that good quality sequencing libraries can be prepared from small amounts of total RNA and that varying degradation levels in the samples do not have a significant effect on the overall quantification of sncRNAs via NGS. In addition, we describe the strengths and limitations of three commercially available library preparation methods: (1) Novex TBE PAGE gel; (2) Pippin Prep automated gel system; and (3) AMPure XP beads. We describe our bioinformatics pipeline, provide recommendations for sequencing coverage, and describe in detail the expression and distribution of all sncRNAs in four human tissues: whole-blood, brain, heart and liver.Ultimately this study provides tools and outcome metrics that will aid researchers and clinicians in choosing an appropriate and effective high-throughput sequencing quantification method for various study designs, and overall generating valuable information that can contribute to our understanding of small ncRNAs as potential biomarkers and mediators of biological functions and disease.The online version of this article (doi:10.1186/s12920-015-0109-x) contains supplementary material, which is available to authorized users. There is significant interest in the prediction and early detection of disease through the analysis of biological markers, or biomarkers, which have the potential to significantly improve clinical outcomes , 2. BiomOver the years, non-coding RNAs (ncRNAs) have become the focus of biomarker research, an approach that has been favorably used in the investigation of response to treatment for several medical conditions. There are several types of ncRNAs, of which microRNAs (miRNAs) are the best known and the most frequently assessed for their potential role as biomarkers. MiRNAs have been proposed as molecular biomarkers in cancer , liver aO-Methylation of ribosomal RNA (rRNA) [Other ncRNA species such as PIWI-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs) and long non-coding RNAs are also gaining support as key components of cellular regulation , 25, andA (rRNA) . SnoRNAsA (rRNA) . In addiA (rRNA) –36. LongA (rRNA) –40. TheyA (rRNA) . LincRNAA (rRNA) . FinallyA (rRNA) . T-UCRs A (rRNA) .While any ncRNA is a putative biomarker, miRNAs have received the most attention because they possess several features that render them especially powerful : (1) they are highly conserved, and evolutionary complexity correlates with miRNA complexity, which suggests an important biological function; (2) there are a relatively small number of individual miRNAs with a large dynamic range of expression; (3) they are secreted into circulation and can be measured in all biological fluids; (4) they are not easily degraded and are thus highly stable in clinical samples; (5) they are involved in pathway regulation, as one miRNA can target many genes, and a single gene can be regulated by many different miRNAs; (6) miRNAs show tissue and cell specific expression profiles; and (7) there is a large body of literature supporting their role in the pathophysiology of disease .in situ hybridization, or microarray techniques. These methods have several strengths, but also contain some important limitations. These include: the number of miRNA molecules that can be analyzed simultaneously, the amount of RNA required for the analysis of multiple targets, the quality and source of the RNA, the sensitivity of detection, and the need for previous knowledge of targets [Most ncRNA quantification studies performed to date rely on qRT-PCR,  targets . Next ge targets .et al. provides a comprehensive review on sample collection and processing for miRNA quantification [In terms of clinical utility, blood is a reliable and non-invasive source of biological tissue that reflects different stages of disease. Blood samples are relatively easy to collect and can be stored over long periods of time without having a significant effect on the levels of miRNAs and other ncRNAs in whole-blood, plasma or serum . As biomfication .The objective of this study is to provide researchers with general guidelines for quantification, data processing and analysis of miRNA, and other small non-coding RNAs (sncRNAs), from human clinical samples using NGS. Here, we test critical, alternative library preparation steps based on the ubiquitously used Illumina TruSeq small RNA sequencing methodology, as well as the effects of total RNA input and quality. Additionally, we describe methods for data processing, data analysis, and downstream validation techniques. Finally, we provide expression patterns and distribution of miRNAs and other sncRNAs from human whole-blood, brain, heart, and liver samples. This study provides tools and outcome metrics that will aid researchers and clinicians in choosing an appropriate quantification method, processing large amounts of data efficiently, and overall generating valuable information that can contribute to our understanding of small non-coding RNAs as potential biomarkers and mediators of biological functions and disease.N = 32) obtained at a community outpatient clinic at the Douglas Mental Health University Institute from healthy anonymous volunteers; 2) postmortem, prefrontal cortex brain tissue (N = 4), which was obtained in collaboration with the Quebec Coroner’s Office and the Douglas-Bell Canada Brain Bank ; 3) commercially available, human brain (N = 1), human heart (N = 4), and human liver (N = 4) (Ambion). Ethics approval for this study was obtained from the Institutional Review Board of the Douglas Mental Health University Institute, and written informed consent was obtained from volunteers or family members, as appropriate.A total of 45 biological samples were included in the present study, and include 1) peripheral blood samples . PAXgene tubes were frozen using a sequential freezing process. This involves storing tubes at room temperature for 3 h, transferring to 4 °C overnight, followed by 6–8 h at −20 °C and then final storage at −80 °C. Total RNA (including the miRNA fraction) was isolated from whole-blood using the PAXgene Blood miRNA Kit , according to manufacturer’s instructions. Furthermore, total RNA was isolated from frozen brain, heart and liver tissues using the miRNeasy Mini Kit protocol  with no modifications. RNA and miRNA yield and quality were determined using the Nanodrop 1000  and Agilent 2100 Bioanalizer .Comparison of small RNA library preparation methods : The Pippin Prep system (PPS) allows automatic selection of specified cDNA products. 25 μl of amplified cDNA from samples A4-A7 were loaded into a Pippin Prep machine. Furthermore, in order to test variability between machines, samples A4 and A5 were loaded into PPS1, while samples A6 and A7 were loaded into PPS2. Size selection was automated for products between 125 and 180 nt . This ratio allows for optimal selection of all products higher than 100 nt.In order to compare three different small RNA library purification methods, we prepared 11 libraries starting with 1 μg of good quality total RNA (RIN >8). All libraries were prepared using total RNA extracted from peripheral blood of a single individual Fig. . The RNALibraries were validated and quantified using an Agilent 2100 Bioanalyzer High Sensitivity DNA chip and qRT-PCR with the KAPA library quantification kit . Sample C1 (control-human brain) was not sequenced. All additional samples (A1-A10), as well as sample AC (control-no purification method), were sequenced.Next, we tested the optimal amount of total RNA input required to prepare small RNA libraries from peripheral blood samples. As previously done, we split total RNA from the same individual into 5 aliquots and each was used as a technical replicate. We prepared 5 additional libraries, starting with different amounts of RNA: A11 (1 μg), A12 (0.5 μg), A13 (0.25 μg), A14 (0.1 μg), and A15 (0.05 μg) Fig. . All 5 lWe also explored the effects of RNA integrity on library preparation for small RNA sequencing. To address this issue, we selected peripheral blood samples from 15 healthy volunteers. These samples were collected and processed following the same protocols as previously described, but were selected based on varying RNA integrity number (RIN) values. These values represent the level of RNA degradation in the sample, where 10 and 0 are the highest and lowest quality scores, respectively. The 15 samples were split into 5 groups with average RIN values of 9, 7, 5.4, 2.2 and 0 Fig. . Small RNext we tested how sequencing depth affects the amount of information obtained from whole-blood samples. We prepared small RNA libraries using total RNA from an additional 12 healthy volunteers, as previously described. All 12 libraries were pooled and sequenced on both a HiSeq2500 and MiSeq Illumina sequencers Fig. .To characterize the expression and explore tissue specificity of small ncRNAs in human biological samples, we prepared 16 additional libraries from human whole-blood, brain, heart and liver tissues Fig. . Brain, GSE69825.Samples were sequenced at the McGill University and Genome Quebec Innovation Centre  and the European Molecular Biology Laboratory (EMBL), Genomics Core Facility , using the HiSeq2500 or MiSeq Illumina sequencers with 50 nt single-end reads. All sequencing data were processed using CASAVA 1.8+  and extrSmall RNA sequencing data was validated using qRT-PCR. Total RNA samples were reverse transcribed using TaqMan RT-PCR microRNA assays (Applied Biosystems) according to the manufacturer’s instructions. Real-time PCR reactions were run in quadruplicate using the ABI 7900HT Fast Real-Time PCR System and data was collected using the Sequence Detection System 2.4 (SDS) software (Applied Biosystems). Expression of miRNAs was quantified using miRNA TaqMan probes (Applied Biosystems) and calculated using the Absolute Quantitation (AQ) standard curve method. RNU6B was used as an endogenous control as it showed expression levels that remained relatively constant with low variance and high abundance across the samples tested.P <0.05 was considered statistically significant.All numerical data are expressed as the mean ± s.e.m. Statistical differences among groups were analyzed by Student’s t–test, One–Way ANOVA with post–hoc correction, and Pearson’s correlation coefficients. Statistical significance was calculated using GraphPad Prism5 and SPSS 20. This study assessed several steps involved in the development of an ncRNA biomarker discovery pipeline, ranging from sample preparation to bioinformatic processing. At each stage, we evaluated the pros and cons of different techniques that may be suitable in some circumstances but not others, depending on experimental design. Evaluation methods included quality of data output in relation to hands-on laboratory time, cost, and efficiency of processing.There are several important parameters to test in order to establish a high-throughput biomarker discovery pipeline including quality of the sample, library preparation methods, input quantity, and sequencing coverage. However, prior to testing these parameters, we established a set of output measures to allow us to compare across methodologies and experimental conditions. These quality control measures are described in detail on Table First, we tested three commercially available library preparation methods for small RNA sequencing: (1) Novex TBE PAGE gel; (2) Pippin Prep automated gel system; and (3) AMPure XP beads Fig. . It is iBefore purification, adapter-ligated libraries for all samples showed a peak corresponding to miRNAs around 147 nt in length , PPS (N = 424), AMPure (N = 370) and control (N = 372).We obtained a total number of 109,956,847 raw reads, with a quality value of 37 in all libraries Table . The disThe Novex TBE PAGE gel proved to be the most specific for isolating the miRNA population in the samples. This is because we were able to manually and carefully cut the band between 145–160 nt corresponding to miRNAs from the gel and avoided any other smaller or larger RNA populations in the samples. However, we lost a significant amount of library product after purification from the gel, and in the end generated less reads after sequencing. In addition, this method requires a significant amount of hands-on time in the lab, which ultimately translates to very low throughput and significantly higher cost. We found purification by Novex gel to be a very good and specific method, particularly fit for small sample size projects where miRNAs are the main focus.PPS generated the highest number both of total reads and distinct miRNAs identified, as well as very high specificity to miRNAs. This can be attributed to several factors, for example: (1) the libraries purified with PPS contained more than 50 times more product after purification, as compared to the Novex gel methods. This is due to the fact that the PPS is an automated system that does not require extraction of the library products directly from the gel, which can lead to less library product; (2) the range of the automatically isolated bands can be optimized to a desired product size (we used 125–180 nt), due to size selection and specificity, PPS contained the least number of reads removed due to a size either smaller than 15 nt or larger than 40 nt; (3) PPS showed the lowest number of adapter-adapter ligated reads. However, because each PPS instrument limits a run to only 4 samples, we tested variations across instruments. We found a significant difference in the final number of miRNAs identified per machine with 50 more miRNAs identified with PPS2. The PPS showed limitations in terms of consistency, and while the protocol requires less hands-on time in the laboratory, it does not increase throughput (only 4 samples per run) or cost significantly. We believe this is a very good method for medium size projects.In contrast, purification by gel-free magnetic AMPure XP beads offers a high-throughput and consistent protocol. These libraries contained similar amounts of library product as compared to PPS (50X more than Novex). This method produced the highest quality of sequencing reads and the highest number of reads mapping back to other small RNA molecules. Nonetheless, because this method retained all products larger than 100 nt, AMPure beads produced the lowest specificity , number of surviving reads and overall number of miRNAs (50 less than PPS). However, AMPure beads offer a very consistent, time efficient, high-throughput protocol with a significant reduction in labor time and cost. We found that purification by AMPure beads is a very good method, particularly fit for large projects where not only miRNAs but all small ncRNAs are the main focus. The results from this section are summarized in Table Finally, control sample AC was not purified or size selected before sequencing in order to compare the results to the three methods tested. However, all libraries in the study (including control sample AC) were prepared using the Illumina TruSeq Small RNA protocol. This protocol is ideal for the investigation of small RNA species, as it takes advantage of the structure of most small RNA molecules by ligating specific adapters to the 5′-phosphate and 3′-hydroxyl group, which are molecular signatures of their biogenesis pathway. This means that if the adapter ligation works well, in theory, the libraries don’t require any further purification. However, the success of purification methods also depends on suppression of adaptor dimer products in order to keep their representation at acceptable levels, ideally <2.5 %. The AC control results were similar to AMPure XP beads because, as previously explained, AMPure XP beads do not contain a very specific size selection  as opposed to Novex (145–160 nt) or PPS (125 and 180 nt).Available amounts of starting RNA material are often a deciding factor when planning a study, as biological samples are often limited or hard to obtain. While planning an NGS project, there should be sufficient material set aside for an exploratory (profiling) experiment, as well as for technical validation and downstream experiments. We tested the feasibility of constructing good quality small RNA libraries from smaller amounts of RNA than the 1 μg suggested by Illumina Fig. . All libClinical samples can be prone to RNA degradation due to methodological issues with sample collection and long periods of storage. In addition, RNA degradation has a significant impact on the profiling of messenger RNA (mRNA), which translates as background noise in the data. In most cases, these samples are not used or thrown away. However, several studies have shown that miRNAs display robust stability and are less susceptible to degradation , 64–69. RNA sequencing coverage refers to the total number of reads to be sampled in a particular experiment and it is an important factor while planning NGS experiments. Coverage can have a significant effect on the quality of data, sensitivity of detection and overall cost of the project. Sequencing time and cost is considerably smaller when using a faster, lower-scale NGS platform. However, to our knowledge, there are no published reports that directly compare the levels and number of distinct miRNAs that can be measured from human blood using different scale sequencers, such as HiSeq and MiSeq. The purpose of this experiment was to determine thresholds for detectability of small ncRNAs and whether or not a fast-turnaround time sequencer like MiSeq can be used in biomarker discovery. Here, we sequenced 12 blood samples on both HiSeq2500 and MiSeq Illumina sequencers Fig. . We geneMicroRNA expression patterns can be tissue and cell specific. For example, miR-1 has been shown to be enriched in cardiomyocytes  while miP <0.0001, Pearson’s r = 0.98, r2 = 0.95) (Fig. We found tissue specific patterns of expression of miRNAs from these tissues. In whole-blood, the most abundant miRNAs were miR-486-5p, miR-486-3p which accounted for more than 90 % of all detectable miRNAs Fig.  Additio. We alsoWe also explored the expression of other small ncRNAs. In peripheral blood, we found an average of 11 % reads that mapped back to other species besides miRNAs. In other tissues we found more variation in the distribution of small RNAs. An average of 44.9 % of reads in brain tissue mapped back to other RNA species. Heart and liver showed 42.4 % and 65.4 % of reads mapping back to other small RNAs, respectively Fig. -b. The mThe goal of this study was to highlight some fundamental details of small ncRNA profiling, and provide the reader with general guidelines for quantification, data processing and analysis of sncRNAs from clinical samples using NGS. Our results show that good quality sequencing libraries can be prepared from small amounts of total RNA and that varying degradation levels in the samples do not have a significant effect on the overall quantification of sncRNAs via NGS. In addition, we discuss the strengths and limitations of three commercially available library preparation methods, describe our bioinformatics pipeline, provide recommendations for sequencing depth and coverage, and describe in detail the expression and distribution of all sncRNAs in four human tissues: whole-blood, brain, heart and liver. Ultimately, this study provides valuable information that will help researchers plan and execute future small RNA profiling studies that will contribute to the understanding of sncRNAs as potential biomarkers and mediators of biological functions and disease.


They repress constitutive decay elements containing mRNAs and play a critical role in RNA homeostasis and immunological self-tolerance. Here we present the crystal structures of the RNA-binding region of Roquin paralog RC3H2 in both apo- and RNA-bound forms. The RNA-binding region has a bipartite architecture composed of ROQ and HEPN domains, and can bind to stem-loop and double-stranded RNAs simultaneously. The two domains undergo a large orientation change to accommodate RNA duplex binding. We profiled E2 ubiquitin-conjugating enzymes that pair with Roquins and found that RC3H1 and RC3H2 interact with two sets of overlapping but not identical E2 enzymes to drive the assembly of polyubiquitin chains of different linkages. Crystal structures, small-angle X-ray scattering, and E2 profiling revealed that while the two paralogs are highly homologous, RC3H2 and RC3H1 are different in their structures and functions. We also demonstrated that RNA duplex binding to RC3H2 cross-talks with its E3 ubiquitin ligase function using an  Aberrant expression, mutation, or malfunction of RBPs are associated with many human diseases, including neurological disorders, cancers, and autoimmune diseases12tion Met19Arg in tRc3h1 RING-less and Rc3h1-null mice die prematurely, while Rc3h2 RING-less mice are viable and healthyAlthough RC3H1 and RC3H2 both repress the mRNAs of genes that control T follicular helper cell development, such as the inducible T-cell co-stimulator (ICOS), and the activation-induced tumor necrosis receptor superfamily 4 (TNFRS4)10Ubiquitination is the conjugation of a small regulatory protein, ubiquitin, to substrate proteins. This post-translational modification is catalyzed by a three-enzyme cascade  and plays an important role in almost all biological processes, such as proteasomal degradation, protein endocytosis and trafficking, DNA damage repair, and cell signaling12In vivo ubiquitination assays, in both HeLa and HEK293 cells, confirmed that the RING domain of RC3H2 promotes the ubiquitin-proteasome-mediated degradation of ASK1 and regulates the cellular response to oxidative stress. The C. elegans ortholog of Roquin, RLE-1, similarly ubiquitinates the ASK1 ortholog NSY-1 and regulates pathogen infection in the wormAn imaging-based siRNA screen identified human RC3H2, but not RC3H1, as the E3 ligase that regulates the turnover of apoptosis signal-regulating kinase 1 (ASK1)In vitro ubiquitination assay revealed that RC3H1 and RC3H2 can pair with two overlapping but dissimilar sets of E2 enzymes to drive auto-ubiquitination and form ubiquitin chains of different linkages. RNA binding influences RC3H2 ubiquitination activity, but has differential effects on polyubiquitination when RC3H2 is paired with various E2 enzymes. Thus, the cross-talk between RNA binding and ubiquitination by Roquins may be context dependent.Here, we report the crystal structures of the human RC3H2 RNA-binding region in both apo- and intermediate early response 3 (Ier3) CDE RNA-bound forms. We discovered that RC3H2 can bind to stem-loop and double-stranded (ds) RNAs simultaneously. A comparison with RC3H1 structures suggests that the RNA-binding region has a bipartite architecture and that the orientation of the two subdomains is flexible to accommodate dsRNA binding. E. coli protease that survived purification. We then performed in situ proteolysis by adding 1:1,000 (w/w) elastase to crystallization drops1992122Sulfolobus solfataricus. The alignment Z-score is 5.2 and the RMSD value is 2.45 Å over the Cα atoms of 106 residues  and RC3H2(a.a. 1–442) constructs containing the RING, HEPN, ROQ, and CCCH-type zinc finger (ZnF) domains using small-angle X-ray scattering (SAXS). Both proteins are monomeric in the solution and adopt an elongated conformation, with radii of gyration of 29.9 Å and 29.3 Å respectively, and a maximum dimension of 98.0 Å. The pair-distance distribution function of both proteins showed a tailing shape with two shoulder peaks , suggestWe next determined the crystal structure of RC3H2 in complex with a 15-nt constitutive decay element (CDE) of human intermediate early response 3 (Ier3) RNA at 2.5 Å resolution . Surpris2122The single-stranded Ier3 RNA forms a stem-loop structure containing five Watson-Crick base pairs and a UGU trinucleotide loop . The 5′ Unexpectedly, our RC3H2/RNA complex structure contains an anti-parallel dsRNA. The RNA duplex has 10 Watson-Crick base pairs at the stem regions of the RNA molecules . All the2 were observed in the ASU  and those solved by other groups 922 the ASU . A similWe measured the oligomeric status of the RC3H2 ROQ domain in the presence and absence of four CDE RNAsd of 356 ± 86 nM, and Tnf23 at 144 ± 50 nM  mRNA (XM_011251206), located 528 base downstream in the ICOS CDE sequence. The dsRNA was annealed from two complementary synthetic RNA chains, which do not form intra-chain base pairing. We discovered that the dsRNA can form a complex with RC3H2  that are involved in stem-loop RNA binding and measured their protein-RNA interactions using isothermal titration calorimetry (ITC). The wild-type RC3H2 binds to Ier3 RNA at a K ± 50 nM . No measth RC3H2 , which cd of 360 nM to 590 nM, as measured by ITC  and found that both Roquins can interact with most of the E2 conjugating enzymes tested and drive polyubiquitination (Ub(n) in Although an tination . HoweverSince the RING domain of Roquins contacts the HEPN but not the ROQ domain, we postulated that dsRNA but not stem-loop RNA binding may cross-talk with ubiquitination. Selecting a few E2 enzymes, we tested the effect of Tnf23 RNA binding on the E3 ubiquitin ligase activity of Roquins. Tnf23 RNA did not significantly alter the auto-ubiquitination activity of RC3H2 when paired with the UBE2N/UBE2V1 E2 complex (data not shown). However, Tnf23 binding seemed to promote auto-ubiquitination driven by UBE2K, while inhibiting UBE2D2-mediated polyubiquitination .When the complex structure of RC3H2 with Ier3 dsRNA is superimposed onto that of apo-RC3H1 (PDB:4TXA) in reference to the HEPN domain, we noticed a clash between the RING domain and the RNA duplex , suggestIn this report, we solved the crystal structures of the RNA-binding region of RC3H2 in apo-form and in complex with Ier3 15-nt RNA in two topological forms. The RNA-binding region can be separated into two domains, ROQ and HEPN, which move relatively independently. This is consistent with several recent structural models of the paralog RC3H12122α RMSDs of the ROQ domains are 0.39 Å and 0.28 Å, respectively, and that the key protein residues involved in the RNA interaction are identical for RC3H1 and RC3H2. All three RNAs use the 5′ arm backbone of a stem structure containing five Watson-Crick base pairs and a trinucleotide loop to interact with the ROQ domain. This suggests that stem-loop RNA recognition by the ROQ domain is sequence independent but conformation dependent, as has been noted previously21The winged helix-turn-helix motif of the ROQ domain21A Roquin/dsRNA complex structure was previously reported for RC3H1 and a 23-nt Tnf CDE RNAclamp” a dsRNA through domain movement. Indeed, when RC3H2 binds to the dsRNA, the ROQ domain rotates 42° along an axis intersecting the HEPN and ROQ domains to accommodate the interactions  with the purine ring of a guanosine. The side chains  may regulate the interaction of the HEPN/ROQ domains with dsRNA. The identity of the kinase that phosphorylates the S/T residue and how the phosphorylation affects RNA binding sanroque mutation Met199Arg. We hypothesize that the ZnF and ROQ domains form an auto-inhibited state, and that binding of stem-loop RNA to the ROQ domain may expose the ZnF domain. Since the ZnF domain could also potentially bind to RNA, the release of an auto-inhibited state may enhance the interaction between Roquin and RNAs. A crystal structure of the ROQ domain with a longer RNA transcript should provide further insight on this interaction.Our structure corroborates the conclusion of a previous study that the dimerization of Roquins is not required for binding RNAssanroque mice with the symptoms of human SLE, it is tempting to postulate that the RC3H1-UBE2L3 axis may act in the pathogenesis of the disease.Ubiquitin can use one of its seven lysines or the N-terminal methionine to form polyubiquitin chains of different linkages. These chains have various three-dimensional structures, dynamics, and surface properties and have vastly divergent physiological functions in the cell133435RNAs often exist in complex with proteins. The translational repression of mRNAs involves not only mRNA degradation, but also protein degradation3738in vivo may provide insights on how ubiquitination cross-talks with RNA homeostasis and reveal other functional roles of the Roquin family E3 ligases.In summary, our crystal structures of the RNA binding region of RC3H2 and ubiquitination assays confirmed that although the paralogs share many similarities, their functions are not identical. Future investigation of ubiquitination and RNA binding E. coli strain BL21 (DE3) harbouring the pRARE2 plasmid (Novagen) using Terrific Broth cultured in LEX Bioreactors (Harbinger Biotech). Protein production was induced using 0.5 mM IPTG overnight at 18 °C. All proteins were first purified using nickel-NTA agarose resin (Qiagen), and the His6-tag was then removed by TEV protease where applicable. Uncleaved protein and TEV protease were removed by another pass through the nickel-NTA resin. The proteins were further purified using cation-exchange chromatography , followed by size-exclusion chromatography . The purified proteins were concentrated to final concentrations of between 25–35 mg/mL using an Amicon ultracentrifugal filter , flash frozen in liquid nitrogen, and stored at −80 °C. RC3H1(a.a. 1–445)and RC3H2 (a.a. 1–442) were stored in a buffer containing 25 mM Bis-Tris, pH 6.5, 500 mM NaCl, 1 mM DTT and 20 μM ZnCl2, while the RNA-binding domain of RC3H2 was stored in a buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl and 1 mM DTT. For selenomethionine labelling, the target protein was over-expressed in E. coli using a prepacked M9 SeMet growth media kit (Medicilon) as per the manufacturer's instructions. All protein concentrations were determined by measuring UV absorbance at 280 nm using NanoDrop 2000 (Thermo Scientific).DNAs encoding the RING, HEPN, ROQ, and ZnF domains of human RC3H1 (a.a. 1–445) and RC3H2 (a.a. 1–442) and the RNA-binding region of RC3H2 (a.a. 87–404) were subcloned into either pET28-MHL  or pNIC-CH  vectors using the InFusion™ cloning kit (ClonTech). Site-directed mutagenesis was carried out using the PrimerSTAR HS DNA polymerase (Takara) and was verified by DNA sequencing. All proteins were over-expressed in a phage-resistant Four single-stranded RNA oligos, Ier3: AUGUUCUGUGAACAC, Tnf23: ACAUGUUUUCUGUGAAAACGGAG, Roquin: AUAACUUCUGUGAAGUUGC, and Roquin2: UUAAUAACUUCUGUGAAGUUGUUUAC, and one RNA duplex dsICOS: UCUUCAGUCUAGACAGUUCGCUU (and its complementary sequence) were chemically synthesized and purified by HPLC , and dissolved in a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, to a final concentration of 2 mM. All RNA concentrations were measured using UV absorbance at 260 nm. The RNA oligo stock solutions were heated at 98 °C for 5 minutes, then slowly cooled to room temperature, aliquoted, and stored at –80 °C. Repeated freezing and thawing were avoided.All crystals were grown at 20 °C using either the sitting or hanging drop vaporization methods. For RC3H1, the His6-tag-removed RC3H1 (a.a. 1–445) protein at a 25 mg/mL concentration was first mixed with elastase from porcine pancreas (Sigma-Aldrich) at a ratio of 1:1000 (w/w), then immediately mixed with a reservoir solution containing 25% PEG-8000, 0.2 M NaCl, 0.1 M HEPES, pH 7.5, 5% ethyl glycol at a ratio of 1:1. Crystals of the apo-form RC3H2 RNA-binding region were grown in a sitting drop mixed from 2 μL protein solution  with 1 μL well solution containing 3.2 M NaCl, 0.1 M sodium acetate, pH 4.6. For the RC3H2/Ier3 RNA complex, N-terminal His6-tag-removed RC3H2 protein was first diluted to 6.7 mg/ml, then mixed with Ier3 RNA solution (2 mM) at a molar ratio of 1:1.5 before being set in a hanging drop. The drop was mixed from 1.5 μL protein/RNA complex and 1.5 μL well solution consisting of 17% PEG-10K, 0.1 M Bis-Tris, pH 6.5, and 5% ethyl glycol. All crystals for data collection were cryo-protected via an initial immersion in the corresponding well solution plus 10–15% ethylene glycol, then an immersion in Paratone-N (Hampton Research), before being flash-frozen in liquid nitrogen.X-ray diffraction data for apo-form RC3H2 was collected at 100K on CMCF beamline 08ID-1 at Canadian Light Source. The RC3H1 and RC3H2/RNA data were collected on beamline 19ID at Advanced Photon Source (APS), Argonne National Laboratory. All datasets were processed using the HKL-3000 suited, entropy, enthalpy, and Gibbs free energy were also calculated during curve fitting.All ITC measurements were carried out at 25 °C on a MicroCal ITC200 . All protein and RNA samples were diluted from stock solutions into the same buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1mM TCEP before measurement. For each titration, 15–18 injections were performed by injecting 2 μl 200–500 μM RNA into a sample well containing 0.25 mL of 15–30 μM proteins. The concentrations of the proteins and RNAs were determined by measuring UV absorbance at 280 nm and 260 nm, respectively. Binding isotherms were plotted, analyzed, and fitted to a one-site binding model using Origin software . The dissociation constant K2. All the enzymes, and RNA where applicable, were premixed, and ubiquitination was initiated by adding ATP to a final concentration of 2 mM. An E2 Screening Kit (UBPbio) was used for E2 profiling, which contained 27 His6-tagged E2 enzymes or complexes and one GST-tagged UBE2K. After incubation at 37 °C for 2 hours, SDS-PAGE loading buffer was added to stop the reaction. The proteins were electrophoretically separated on NuPAGE Novex 4–12% Bis-Tris gels (ThermoFisher) in MOPS SDS running buffer. Ubiquitination products were probed using a P4D1 monoclonal ubiquitin antibody (BioLegend). E2 enzymes were probed using a DyLight 680 conjugated anti-His antibody (Life Technologies). IRDye® secondary antibodies (LI-COR) were used for indirect detection and the results were visualized on an Odyssey CLx infrared imaging system (LI-COR). For chain linkage specificity test, a ubiquitin linkage screen kit I (UBPBio) was used.All auto-ubiquitination was carried out in a 20 μL reaction system containing 100 nM E1 enzyme UBE1, 2 μM individual E2 enzymes, 2 μM RC3H1 (a.a. 1–445) or RC3H2 (a.a. 1–442) protein and 50 μM ubiquitin. The reaction buffer contained 20 mM Tris-HCl, pH 7.6 (4 °C), 50 mM NaCl, 1 mM β-mercaptoethanol and 5 mM MgClFor SEC experiments, samples of the wild-type and mutant RC3H2 ROQ domains were mixed with different RNAs at a 1:3 molarity ratio and passed through an analytical column  pre-equilibrated with 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM TCEP. The column was calibrated using gel filtration standard (Bio-Rad).ab initio shape envelopes were determined using the program DAMMIN from the ATSAS packageSAXS measurements were performed at room temperature at the 12ID-B beamline of the Advanced Photon Source (APS), Argonne National Laboratory. A 14-KeV X-ray beam was used as the photon source with a sample-to-detector distance of 3 m for SAXS setup. Simultaneous wide-angle X-ray scattering (WAXS) data were recorded as well. Thirty two-dimensional (2D) images were recorded for each buffer and sample solution using a flow cell, with an exposure time of 0.5–1 seconds to minimize radiation damage. Concentration series measurements for the same sample were carried out to remove the scattering contribution due to inter-particle interactions and to extrapolate the data to infinite dilution. The concentrations were 0.75, 1 and 5 mg/ml for RC3H1(a.a. 1–445) and 1, 3, 5 and 10 mg/ml for RC3H2 (a.a. 1–442) in a final buffer of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM TCEP. The 2D images were reduced to 1D scattering profiles using the MATLAB software package at the beamline. The 1D SAXS profiles were grouped by sample and averaged, after which the buffer background was subtracted. The procedures for data collection, processing, and analysis are similar to those previously describedHow to cite this article: Zhang, Q. et al. New Insights into the RNA Binding and E3 Ubiquitin Ligase Activities of Roquins. Sci. Rep.5, 15660; doi: 10.1038/srep15660 (2015).


Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a ‘seed’ region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at a 3.48 Å resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein–RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target.Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of DOI:http://dx.doi.org/10.7554/eLife.05375.001 A crucial step in the production of proteins is the translation of messenger RNA molecules. Other RNA molecules called small RNAs are also involved in this process: these small RNAs bind to the messenger RNA molecules to either increase or decrease the production of proteins.Bacteria and other microorganisms use small RNA molecules to help them respond to stress conditions and to changes in their environment, such as fluctuations in temperature or the availability of nutrients. The ability to rapidly adapt to these changes enables bacteria to withstand harmful conditions and to make efficient use of resources available to them.Many small RNA molecules use a protein called Hfq to help them interact with their target messenger RNAs. In some cases this protein protects the small RNA molecules when they are not bound to their targets. Hfq also helps the small RNA to bind to the messenger RNA, and then recruits other enzymes that eventually degrade the complex formed by the different RNA molecules.Previous research has shown that six Hfq subunits combine to form a ring-shaped structure and has also provided some clues about the way in which Hfq can recognise a short stretch of a small RNA molecule, but the precise details of the interaction between them are not fully understood. Now Dimastrogiovanni et al. have used a technique called X-ray crystallography to visualize the interaction between Hfq and a small RNA molecule called RydC.These experiments reveal that a particular region of RydC adopts a structure known as a pseudoknot and that this structure is critical for the interactions between the RydC molecules and the Hfq ring. Dimastrogiovanni et al. find that one RydC molecule interacts with one Hfq ring, and they identify the contact points between the RydC molecule and different regions of the Hfq ring.Based on this information, Dimastrogiovanni et al. propose a model for how the RydC:Hfq complex is likely to interact with a messenger RNA molecule. The next step will be to test this model in experiments.DOI:http://dx.doi.org/10.7554/eLife.05375.002 The expression of genetic information is controlled and synchronised through intricate regulatory networks. In bacteria, the control of gene expression post-transcription is mediated in part by small RNAs (sRNAs), and their contributions enrich the computational complexity and repertoire of regulatory circuits . BacteriEscherichia coli and other eubacteria forms a compact hexameric toroid that presents two structurally non-equivalent concave surfaces for molecular recognition; these faces are referred to as the proximal and distal faces, with the former exposing an N-terminal α-helix , transcription was stopped by the addition of rifampicin, and RNA levels were monitored at various time-points after treatment. As previously shown (1/2) >32 min), while RydC-S1 decayed rapidly (t1/2 ∼2 min). In contrast, the half-life of RydC is already markedly reduced in hfq mutant cells (t1/2 ∼4 min) and there is some further acceleration of the decay rate for RydC-S1, matching that of RydC in the absence of Hfq. This observation led us to assume that RydC stability depends on both the integrity of the pseudoknot fold and the ability to associate with the Hfq protein. Both the wild-type RydC and RydC-S1 mutant bind Hfq in electrophoretic mobility shift assays with in vitro synthesized RNA although the affinity of the mutant is lower than for the wild-type RydC . Unbiased, interpretable density for the RNA was apparent in the early maps calculated from the positioned protein hexamer, and a model for most of RydC could be fitted into the electron density .The strong association of RydC with Hfq both in vivo and in vitro as well as its highly compact structure made it an ideal candidate for co-crystallizations. Co-crystals of full length  density . The fol density . The RNA density . The 3′-The organisation of the RNA in the crystal would suggest that a single RydC molecule could form a closed complex with two sandwiching Hfq hexamers. However, solution studies using analytical ultracentrifugation (AUC) and multi-angle laser light scattering (SEC-MALS) indicate that the RydC can form a stable 1:1 complex with the Hfq hexamer under various buffer conditions when the components are present in unitary stoichiometry . When HfSalmonella Typhimurium Hfq/polyU  and the 3′-end of the sRNA MicA is as efficient in target gene regulation as the wild-type RydC , mutant RydC-LII had a reduced half-life (t1/2 ∼11 min). Both variants RydC-LI and the double mutant RydC-LI/LII were even less stable (t1/2 ∼2 min) and especially the RydC-LI and RydC-LI/LII variants were less potent than the wild-type to regulate the reporter target Cfa–GFP  was expressed and purified, and its binding rates and affinities were estimated from interferometry (ForteBio Octet Red96). For this experiment, the 5′ region of cfa  and the Hfq rim mutant (HfqRim) have similar affinities and association rate constants for the immobilised cfa.To explore the contribution of the potential rim contacts to mediate the interaction between RydC and cfa changes. The binding affinity of HfqWT for cfa decreases by a factor of roughly 5, suggesting that the formation of the Hfq–sRNA complex affects the Hfq affinity for the mRNA fragment. Moreover, the presence of RydC decreases by an order of magnitude the association rate constant of Hfq for cfa. This could result from cfa being bound in a slightly different way in the Hfq/cfa complex as compared to the Hfq/RydC/cfa ternary complex. Strikingly, in the presence of RydC, the HfqRim mutant no longer has detectable binding to cfa. This finding suggests that the RydC interacts differently with HfqWT and the HfqRim lateral surface mutant in a way that perturbs cfa binding. Thus, the rim interactions shown in In the presence of RydC, the association of Hfq with The C-terminal regions of Hfq, beyond residue 70, are generally disordered, but density could be observed for some of the protomers into which the model could be partially extended. Although poorly ordered, the C-terminal tails appear to be making distributive contacts over the surface of RydC. The N-terminal regions of three Hfq protomers, exposed on the proximal face of the protein, also interact with RydC, mainly in the vicinity of nucleotides 23–25, 51–52, and 59.1/2 ∼5 min) and was comparable with the half-life observed for an hfq null mutant (t1/2 ∼4 min). However, upon initial decay of a subfraction of RydC, the remaining population appears to be more stable and is only degraded at a rate comparable to RNA in the wild-type background. In vitro binding experiments show that the affinity of purified Hfq truncated at residue 66 (Hfq66) for binding cfa is similar to that of the wild-type protein; however, the presence of RydC decreases substantially the binding of Hfq66  are not reduced in their cellular abundance, a second class of sRNAs is strongly affected by the absence of the C-terminal end. This latter group includes RydC and also several sRNAs highly abundant in the wild-type , and it is tempting to speculate that this subpopulation of sRNAs is associated with Hfq through supporting interactions with the C-terminal tail. The differing roles of the tails for different sRNAs may account for the apparently conflicting results reported in the literature on the effects of the C-terminus on sRNA binding and activity  to avoid hetero-hexamer formation. The mutant proteins were purified with the same protocol used for the wild-type Hfq, with a HiTrap Heparin purification step added . The presence of mutations or truncations was confirmed by mass spectrometry analyses.Wild-type Salmonella RydC RNA in vitro transcription (IVT), a DNA template was amplified by PCR from pKF42-1 using primers RydC_T7_fwd and RydC_T7_rev  for 4 hr at 37°C, and then Turbo DNase (Ambion) was added to digest the template. The IVT product was separated on a 10% polyacrylamide gel containing 7 M urea. The band corresponding to the transcribed RydC was visualized by UV-shadowing, excised from the gel, and the RNA was electro-eluted overnight using an EluTrap system . The RNA sample was concentrated by ultrafiltration through a Vivaspin 500 concentrator (5 kDa cut-off).Transcription mixtures containing DNA-template, ribonucleotides (rNTPs), T7 RNA polymerase, DTT (dithiothreitol), and RNase Out  were incubated in transcription buffer (400 mM Tris (pH 7.9/8.0), 250 mM MgCl6 and RydC in a ratio of 2:1, the Hfq hexamer concentration being 15 mg/ml. Crystals of the Hfq–RydC complex were grown by sitting drop vapour diffusion after adding an equal volume of crystallization buffer , 15% vol/vol isopropanol) to the protein–RNA mixture. Crystals were harvested using 25% PEG400 as cryoprotectant and flash frozen in liquid nitrogen. X-ray data were collected on station I24 at Diamond Light source. Data were collected at 100 K at a wavelength of 0.9778 Å.The RNA–protein complex was prepared by mixing purified HfqImages obtained from the two best crystals having the same space group and cell dimensions were merged and scaled together, before proceeding with the structure determination.E. coli Hfq as the search model. Electron density for RNA was apparent in the early maps. Maps were improved with density modification with PARROT using histogram matching with tRNA/aminoacyl-tRNA synthase experimental structure factors as the reference distribution. This structure has a similar solvent content and RNA/protein mass ratio as the RydC/Hfq crystals, and density modification with this method improved the map and visualisation of the RNA. The structure was refined using BUSTER , which was also used to prepare all dilutions, for dissociation and neutralisation. A fragment of cfa mRNA  labelled with biotin. The binding of wild-type and mutant Hfq were assayed at 0, 5, 10, 25, 75, 150, 250, and 500 nM protein over 400 s, in the absence or presence of 1 μM chemically synthesized RydC . The dissociation was monitored over 300 s and was followed by regeneration of the sensors using 1 M MgCl2. Another set of tips was saturated with MBP-biotin and the measurements were then repeated for all Hfq and Hfq–RNA concentration series. The data were fitted with Data Analysis software (ForteBio) with a 1:1 binding model. The plots were prepared with Profit  using the following equation for response fit:Kinetic measurements with Bio-Layer Interferometry were performed using an Octet RED96 equipped with Streptavidin sensors  on 96-well plates. The experiment was performed in the binding buffer ,Y = Rm × Xwhere Y is the observed binding, X is the molar concentration of the ligand, Rm is the maximum specific binding, and n is the Hill’s coefficient.Sequences of all oligonucleotides employed in this study are listed in L-promoter, plasmid pKF42-1 served as template for PCR amplification with primer pairs JVO-10915/JVO-10916 , JVO-10913/JVO-10914 , and the linear fragments were purified and self-ligated. Similarly, pPL-RydC-LI/LII (pKF225) was constructed by self-ligation of a PCR product of JVO-10915/10,916 using pKF223 as template. Competent E. coli TOP10 were used for all cloning purposes.For plasmids expressing loop mutants of RydC from the PFor the in vivo analyses, bacteria were grown aerobically in L-Broth (LB) medium at 37°C. Where appropriate, liquid and solid media were supplemented with antibiotics at the following concentrations: 100 µg/ml ampicillin, 50 µg/ml kanamycin, and 20 µg/ml chloramphenicol.Salmonella enterica serovar Typhimurium strain SL1344 (JVS-0007) is referred to as wild-type strain and was used for mutant construction. Single mutant derivatives were constructed by the λRed recombinase one-step inactivation method. To obtain JVS-10701 (SL1344 hfq70::STOP), Salmonella cells carrying the pKD46 helper plasmid were transformed with a DNA fragment amplified from pKD4 using JVO-10909/JVO-10910. Subsequently, the KanR cassette of λRed-derived mutants was eliminated by transformation with the FLP recombinase expression plasmid pCP20 , and pellets were resuspended in 1X protein loading buffer  to a final concentration of 0.01 OD/µl. Samples corresponding to 0.1 OD were loaded per lane and resolved by SDS-PAGE, after which proteins were transferred to PVDF membranes as described in To prepare whole-cell samples for Western blot analyses, bacteria were collected by centrifugation  to abrogate transcription. RNA samples were withdrawn at indicated time-points, and RNA decay was determined by Northern blot analysis as previously described  and reverse transcribed and 5′-end-labelled as described previously . Labelle review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “Recognition of the small regulatory RNA RydC by the bacterial Hfq protein” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision. All three thought the structure of a full small regulatory RNA bound to Hfq was a major advance. Their comments listed below are related to the need for clarification of some data.1) 2) 3) cfa in vivo? Is this mutant derivative present at wild type levels in the cell? Does the lateral rim mutant show wild type binding to cfa in the absence of RydC and reduced binding in the presence of RydC in vivo?4) cfa RNA in a similar way what accounts for this difference? The authors could support their conclusions from the SPR data by including electrophoretic gel shift data on HfqWT and Hfqrim binding to the cfa RNA and RydC.Also, no data is shown on response vs time to obtain the kinetic rates for the SPR experiments in 5) 6) Discussion: The authors should comment on the fact that some Hfq homologs do not have this extension as well as name the proteins that have unstructured regions important to other nucleic acid/protein complexes. 1): Is it clear that the wild-type RydC and the RydC-S1 mutant, while binding Hfq with similar affinities (cited as results not shown), bind to the same face and in the same way to Hfq? One question is how much the findings with this pseudoknot RNA can be extrapolated to other Hfq-binding sRNAs.shows that RydC-S1 expressed in cells in the presence of Hfq is at a very low level at the time rifampicin is added (t=0). Hfq may bind to RydC-S1, but it apparently is unable to protect it from degradation. The data clearly shows the presence of Hfq inhibits degradation of RydC. This could be an indirect effect, but it could be due to Hfq binding/stabilizing the RydC pseudoknot fold and inhibiting degradation from the 5' end. The conclusions and comments here should be clarified.To address the question of whether RydC and RydC-S1 bind the same face of Hfq, we performed EMSA competition experiments. Our results demonstrate that the affinity for Hfq of the S1 mutant is lower than that of wild type RydC  panel A.Regarding the generality of the RydC/Hfq interaction and its bearing on other sRNA/Hfq complexes, we note that the interactions with the 3’ polyU tail and the UU steps are likely to also occur in the recognition of many other sRNAs (1/2 > 32’), and its half-life cannot further increase in a temperature sensitive RNase E mutant (rne-ts) compared to the control strain in the course of a 32’ experiment. We do observe an even flatter decay rate though. This suggests that RydC is mostly immune to the action of RNase E when bound to Hfq. In contrast, the half-life of the S1 pseudoknot mutant was increased in the absence of RNase E (from ∼3 min to ∼6.5 min), suggesting that the S1 mutant is more susceptible to degradation. However, the S1 mutant is not as stable as wild-type RydC and might be preferentially attacked by other cellular nucleases. Whether this susceptibility is due to an alternative structure or reduced binding capability to Hfq is unclear. We have clarified in the text that both the formation of the pseudoknot and the binding of Hfq may contribute to the in vivo stabilization of RydC.The reviewer also raises a question about the protection of RydC by Hfq from decay. We assume that the lower stability of RydC-S1 is most likely arising from the susceptibility of the RNA to RNase attack when not bound by Hfq. Wild-type RydC is very stable in vivo (t2): The major issue from the crystallography is that in the crystal, RydC is interacting with two adjacent Hfq hexamers. The authors suggest that the contacts seen on neighboring hexamers can also form on a single one, and that such single interaction is likely to be the functional complex. A clearer discussion of what interacts with each hexamer would at least clarify whether there are mutational tests that might demonstrate the importance of each type of interaction. Related to this point,is the only figure that provides a picture of the whole structure and therefore is important for readers to understand the major conclusions, as well as the nature of the complex with two Hfq hexamers. It might be easier to understand this if the second RydC (at upper right) in 2B were in a different shade, for instance (lighter), and possibly if a schematic were included to make it clear what is going on . Highlighting in some fashion (maybe box and expand to the side) the rim interaction region is important in trying to understand the implications of this sandwiched structure for the rim interaction. It is hard to read the label in some cases. Is the U-rich tail on one hexamer and the 5' end on the other Hfq hexamer? How important is the 5' end for function?Concerning the discussion of what interacts with each hexamer in the crystal lattice, the 3’ poly U tail contacts the proximal face of one hexamer, while the 5’ end interacts with the lateral face of a neighbouring Hfq hexamer in the crystal lattice. Also, the UU steps make pseudoequivalent contacts, so that U23/U24 make interactions with the rim portion in one hexamer while the U46/U47 make nearly the same interactions with the same residues in the second hexamer : The data in this figure is difficult to understand. By stability measurements alone, LI and LI/LII should be similarly defective; however, for activity, LI is much more active than LI/LII, and in terms of accumulation of RydC, LI looks better than LII (or the double). Is there an explanation for this?cfa activation. Such effects are difficult to distinguish in vivo and will need the development of a suitable in vitro assay that can separate sRNA decay and activity in a more quantitative fashion. We suggest that LI and LII are both interacting with Hfq, but there might be two alternative binding patterns involving LI or LII. One of the binding options (the one using site LI) is more beneficial for RydC with regard to stability. If interacting via LI RydC is capable of target binding, but also protection by Hfq. If interacting via LII, RydC is still able to interact with cfa but is more susceptible to degradation. The LI/LII double mutant does not bind Hfq, and is degraded without ever interacting with the target. We have changed the text to clarify the limitations in interpreting these data.Regarding the differences in LI and LII, we suggest the causes are complex in vivo. Synthesis rate may not be a major factor since all the variants are expressed from the same constitutive promoter. Instead, the differences might be due to rates of assembly onto Hfq, rates of target binding, and of decay. Our tentative explanation for this is that the assembly rate of LI is sufficiently high to make enough active regulator whereas the LI/II double mutant may be compromised for both assembly and stability, resulting in an intracellular copy number too low for 4): Why was an Hfq mutant with 6 changes used for this experiment? It is difficult to know which rim interactions are in fact important, given this drastic change. Is this active for RydC regulation of cfa in vivo? Is this mutant derivative present at wild type levels in the cell? Does the lateral rim mutant show wild type binding to cfa in the absence of RydC and reduced binding in the presence of RydC in vivo?cfa binding using EMSA. We have not explored if this mutant is present at wild-type levels in cells, or if it is active for RydC regulation of cfa in vivo. The binding experiments in vivo are difficult to evaluate in a quantitative manner. Both wild type Hfq and the mutant seem to bind cfa in a similar way but with different affinity .We did try individual point mutations, but we couldn’t see any effects on RydC or Also, no data is shown on response vs time to obtain the kinetic rates for the SPR experiments in. This data and the fit should be added to supplementary information. The response values shown inare low and raise a concern on the accuracy of the evaluated parameters. Additionally, the asymptotic maximum response value for the Hfqrim (1.8) is less than half than that for HfqWT (5.0). If both Hfq's bind cfa RNA in a similar way what accounts for this difference? The authors could support their conclusions from the SPR data by including electrophoretic gel shift data on HfqWT and Hfqrim binding to the cfa RNA and RydC.We have included below representative kinetic profiles from the octet measurements . There aWT and Hfqrim, but we should point out that the measurements are not from an SPR device (such as a biacore), but from interferometry using an Octet instrument, and the response units are proportional to the layer thickness. We have found that these signals differ for different constructs, likely due to differences in the relationship of the various Hfq species and the surface of the sensor. The electrophoretic gel shift mobility data show two bands with titrations for the HfqWT while only one band is seen for the Hfqrim mutant, and these may account for the different layer thicknesses of the two samples : The authors need to comment on these decay curves. While they emphasize here the very early points , it is striking that only a fraction of the RNA is in fact degraded (40%?) in this mutant. Does that suggest that a subpopulation becomes sensitive, but an almost equal part is stably bound to Hfq? Is this truncated Hfq active for RydC regulation in vivo?cfa regulation by RydC in the presence of Hfq70, and found that cfa activity was only slightly compromised  Discussion: The authors should comment on the fact that some Hfq homologs do not have this extension as well as name the proteins that have unstructured regions important to other nucleic acid/protein complexes.We have added these points to the Discussion.


RNA sequencing has increasingly become an indispensable tool for biological research. While sequencing costs have fallen dramatically in recent years, the current cost of RNA sequencing, nonetheless, remains a barrier to even more widespread adoption. Here, we present a simple RNA sequencing protocol with substantially reduced costs. This protocol uses as little as 10 ng of total RNA, allows multiplex sequencing of up to 96 samples per lane, and is strand specific. Extensive validation using human embryonic stem cells showed high consistency between technical replicates at various multiplexing levels. RNA sequencing measures gene expression by sequencing cDNA libraries converted from mRNA and counting reads that map to each gene. Since its introduction six years ago1The most critical step in RNA sequencing is the construction of a cDNA library suitable for sequencing. Several protocols for this purpose have been developed5678910111213141517581315Ligation Mediated RNA sequencing) that dramatically reduces the cost of sample preparation. Reagents used in this protocol are fully disclosed and widely available. The whole protocol is highly streamlined and a single researcher can process up to 192 samples in two days by hand. We also reduced sequencing costs by designing indexes to allow multiplex sequencing up to 96 samples per lane. Using this protocol, we sequenced up to 95 technical replicates of mRNAs from human embryonic stem cells and found this protocol to produce highly consistent results between technical replicates at various multiplexing levels.One obstacle that prevents the wider use of RNA sequencing is the high cost of cDNA library preparation using commercially available kits. In this report, we developed a strand-specific RNA sequencing library construction protocol (LM-Seq: 2 = 0.998), indicating the protocol is very consistent. The Spearman's rank correlation between the same hES cell sample sequenced with LM-Seq and Illumina's TruSeq is 0.96   . Librarictively) . We did ctively) , and thaWith the current HiSeq2500 from Illumina, we routinely get >150 M reads per lane of sequencing, which is much more than needed for standard gene expression analysis. So next we evaluated the performance of LM-Seq with highly multiplexed samples. To accomplish that, we designed a set of 10 nt DNA indexes with 5 nt editing distance between any two indexes . Two majWe developed LM-Seq as a cost-effective strand-specific RNA sequencing protocol to enable large-scale comparative gene expression analysis. This protocol is highly streamlined, with minimal hands on time. It is fully compatible with multi-channel pipetting to allow parallel processing of a large number of samples. A single person can process up to 192 samples within two days. The most time consuming step in this protocol is the ligation of the partial Illumina's 5′ adaptor. If preferred, this ligation time can be shortened to as little as one hour with minimal affect on gene quantification . HoweverData generated by LM-Seq showed high correlation with those generated by Illumina's TruSeq . HoweverLM-Seq selectively sequences poly-A-tailed mRNA transcripts. Information about RNA transcripts that are not poly-A-tailed, such as some forms of non-coding RNAs, will be lost. If information for non-poly-A-tailed transcripts is desired, the mRNA selection step in LM-Seq can be replaced with a ribosomal RNA reduction step using commercially available kits, such as Ribo-Zero rRNA removal kit from Epicentre.Read coverage using LM-Seq has a slight bias towards the 5′ end of the transcript , which iWith the ever-increasing data output per lane of a flowcell on the Illumina platform, the ability to multiplex becomes critical for any RNA sequencing protocol. We designed a set of 96 indexes for LM-Seq for this purpose. These indexes have a 5 nt editing distance between any two indexes to allow unambiguous differentiation between samples with different indexes. With an increased number of samples per lane, the sequencing depth per sample becomes a concern, especially for complex transcriptomes like that of human. Our data suggested that genes with low expression values suffer more than genes with high expression values when sequencing depth is limited. For pilot screen experiments where large changes in gene expression are expected and/or tracking expression patterns of high or moderate expressers is the goal, multiplexing with up to 96 samples per lane for human is likely adequate. Also, for species such as yeast that have a less complex transcriptome, one can probably multiplex even more without significant loss of sensitivity.Human embryonic stem cell line H1 was cultured on Matrigel coated plates in E8 medium as previously describedA detailed step-by-step protocol can be found in the Indexed cDNA libraries are pooled and sequenced on an Illumina HiSeq2500 with a single 51 bp read and a 10 bp index read. FASTQ files were generated by CASAVA (v1.8.2). Reads were mapped to the human transcriptome (RefGene v1.1.17) using BowtieZ.H., S.S., R.S. and J.A.T. designed research; Z.H., A.E., B.K.N. and J.B. performed research; Z.H., P.J., R.S. and J.A.T. analyzed data; and Z.H. and J.A.T. wrote the paper.Supplementary Information


RNA viruses have genomes with a distinct nucleotide composition and codon usage. We present the global characteristics of the RNA genome of Zika virus (ZIKV), an emerging pathogen within the Flavivirus genus. ZIKV was first isolated in 1947 in Uganda, caused a widespread epidemic in South and Central America and the Caribbean in 2015 and has recently been associated with microcephaly in newborns.The nearly 11 kb positive-stranded RNA genome of ZIKV was analyzed for its nucleotide composition, also in the context of the folded RNA molecule. Nucleotide trends were investigated along the genome length by skew analyses and we analyzed the codons used for translation of the ZIKV proteins.ZIKV RNA has a biased nucleotide composition in being purine-rich and pyrimidine-poor. This preference for purines is a general characteristic of the mosquito-borne and tick-borne flaviviruses. The virus-specific nucleotide bias is further enriched in the unpaired, single-stranded regions of the structured ZIKV RNA genome, thus further imposing this ZIKV-specific signature. The codons used for translation of the ZIKV proteins is also unusual, but we show that it is the underlying bias in nucleotide composition of the viral RNA that largely dictates these codon preferences.The ZIKV RNA genome has a biased nucleotide composition that dictates the codon usage of this flavivirus. We discuss the evolutionary scenarios and molecular mechanisms that may be responsible for these distinctive ZIKV RNA genome features. Flaviviridae that was first isolated in 1947 from a sentinel rhesus monkey placed in the Zika Forest near Lake Victoria in Uganda [Aedes africanus, but the virus was also isolated from other Aedes species . ZIKV iiewed in , 7. A raiewed in –10. Hereiewed in . Neural iewed in .Given the clinical relevance, we performed a detailed analysis of several features of the ZIKV RNA genome, including the nucleotide (nt) composition, also in the context of the structured RNA genome, and the viral codon usage. This insight can be central to the understanding of factors that govern virus evolution. Mutation pressure has been shown to be the dominant factor shaping the nucleotide composition and codon usage in mammalian genomes –15. The bona fide open reading frames.Viral RNA genome sequences were obtained from GenBank. The MR-766 prototype ZIKV strain originates from the index case: a monkey infected in 1947 in Uganda (Genbank entry NC_012532). Other ZIKV isolates used: KU497555 (Brazil), KU509998 (Haiti), KU501215 (Puerto Rico), KU312312 (Suriname), KU647676 (Martinique), KJ776791 (French Polynesia), KU701217 , KU681082 (Philippines) and KF268950 . The full genome sequences were manually curated into Phylogenetic and molecular evolutionary analyses were conducted with MEGA v6 . The opeRNA secondary structure prediction was performed by the MFold v3.6 algorithm with default settings . The MFoBase composition along the complete RNA genome length and the accompanying ss and ds fasta files was analyzed by cumulative skew diagrams using overlapping windows , 20. Overd codon position (GC3) [Escherichia coli [Aedes genes were derived from codon usage tables [The single ZIKV reading frame was analyzed using the “Nc-plot”, which plots the effective number of codons  versus the GC-content at the 3on (GC3) . A contion (GC3)  and Eschhia coli . The rathia coli . ENC ande tables . All calWe first analyzed the complete RNA genome of the ZIKV MR-766 prototype strain , which oThese basic characteristics do not deviate significantly for other ZIKV isolates. We analyzed two early isolates  and 8 recent isolates. Phylogeny of these 10 strains is presented in Fig. This nt bias is likely to influence the codon usage in the single ORF that encodes the ZIKV polyprotein, but in fact the same nt trends are apparent for segments of the RNA genome that are not translated into protein: the short 5’-untranslated region (5’UTR) and 3’UTR of the ZIKV genome Table . These rAedes [We previously reported that the unequal nucleotide composition of a viral RNA genome may become even more biased in unpaired domains of the folded RNA molecule. This initial study was performed for HIV-1 RNA, using both MFold-generated structure prediction and the experimentally probed RNA structure , 29. TheAedes –32. A moA powerful way to visualize trends in nt usage is a nucleotide skew analysis along the viral genome Fig. . The genWe subsequently analyzed the ds and ss positions separately . The continuous grey line indicates the ENC values expected (ENCexp) for random codon usage at a particular GC3 value. Deviation from this line in the direction of lower ENC-values  points to the selection of a preferred set of codons. The ratio ENCobs/ENCexp provides an easy measure for the extent of deviation: the value of 1 (ENCobs = ENCexp) indicates the absence of any codon bias, values below 0.8 may suggest weak codon bias, and even lower values can advocate stronger codon bias. We included two early and several recent ZIKV isolates, but all cluster in a very tiny area of the Nc-plot, confirming their very close genetic relationship. Most importantly, the ENCobs/ENCexp ratio of all ZIKV isolates is above 0.88, which is close to the unbiased value. We also plotted ENC/GC3 values derived from average codon usage in a set of human and mosquito mRNAs  compared with the values for ZKIV and human genes (0.53). Between virus and hosts we did not observe different codon preferences, which are related to codon bias by translational control and indicated by low ENCobs/ENCexp values. Overall, this analysis suggests that the virus-specific codon usage trends reflect the biased nt composition of these viral RNA genomes.Codon usage in the single ZIKV ORF was also analyzed by means of an “Nc-plot” Fig. , with thitions GC. The conFlaviviridae, including the major pathogens dengue virus [This survey of the ZIKV RNA genome indicates a preference for purines over pyrimidines that is enhanced in the unpaired domains of the viral RNA and that influences the codons used for translation of the viral proteins. No significant differences were found among the highly related ZIKV strains concerning these basic properties, ranging from the prototype Uganda strain isolated in 1947 to recent south American isolates. The strong correlation between nucleotide composition and codon usage bias suggests that mutation pressure in ZIKV is an important determinant of the codon bias observed. This finding is consistent with previous findings for other viruses, which demonstrate a wide variation in codon usage that usually correlates with the viral RNA-specific nucleotide composition . The ZIKue virus , yellow ue virus  and hepaue virus . Althougue virus –50, as fAedes hosts.We previously presented two possible causes for the presence of viral RNA genomes with a biased nt-composition . One freWe previously reported that virus-specific compositional signatures are commonly enhanced in the unpaired domains of a structured RNA genome. This is true for HIV-1, with an average A-count of 36.2 % that increases to 47.5 % in the unpaired genome segments, and also for other retroviruses with a distinct nt bias , 34. We Many reports dwell on the exotic codon usage employed by viruses. For instance, a recent report documented codon usage adaptation in pandemic ZIKV virus strains . However


The human antigen R (HuR) stabilizes many mRNAs of proto-oncogene, transcription factors, cytokines and growth factors by recognizing AU-rich elements (AREs) presented in their 3’ or 5’ untranslated region (UTR). Multiple lines of experimental evidence suggest that this process plays a key role in cancer development. Thus, destabilizing HuR/RNA interaction by small molecules presents an opportunity for cancer treatment/prevention. Here we present an integrated approach to identify inhibitors of HuR/RNA interaction using a combination of fluorescence-based and NMR-based high throughput screening (HTS). The HTS assay with fluorescence polarization readout and Z’-score of 0.8 was used to perform a screen of the NCI diversity set V library in a 384 well plate format. An NMR-based assay with saturation transfer difference (STD) detection was used for hits validation. Protein NMR spectroscopy was used to demonstrate that some hit compounds disrupt formation of HuR oligomer, whereas others block RNA binding. Thus, our integrated high throughput approach provides a new avenue for identification of small molecules targeting HuR/RNA interaction. RNA bin2β4 fold ,20, the 2β4 fold ,22, wher2β4 fold ,23. RRM32β4 fold .1H-15N HSQC is unparalleled in its ability to identify the binding site, even though its utility is limited to smaller proteins that yield good NMR spectra [1H-15N protein HSQC spectra revealed that one of the compounds (C10) disrupts HuR/RNA interaction, whereas another compound (C11) impedes HuR oligomerization. Our results suggest that this integrated approach can be a valuable strategy for screening compound libraries for inhibitors of the HuR function.Elevated expression of HuR is linked to carcinogenesis in many human tumors and correlates with poor outcome –29. For  spectra . 12 comp5’-FAM labeled RNA oligonucleotide (AUUUUUAUUUU) derived from the 3’ UTR of the c-Fos proto-oncogene was purchased from MWG operon (HPLC purified and RNase free). Unlabeled c-Fos RNA oligo was purchased from IDT (HPLC purified and RNase free).E.coli. BL21 Rosetta cells. The culture was grown to OD260~0.6 at 37°C, and the protein expression was induced by addition of 1mM IPTG. The incubator temperature was reduced to 18°C immediately following IPTG addition and the cells cultured for additional 16–18hrs. The cells were harvested by centrifugation for 20 min at 6000rpm and sonicated in the presence of protease inhibitors and 15% Glycerol in 50mM Tris pH8.0. Cell lysate was spun down at 18000rpm and the supernatant was collected. The protein was purified using metal chelation chromatography, followed by size exclusion chromatography on the G75  or G200 (for full-length construct) resin. Size exclusion chromatography buffer contained 50mM phosphate buffer, 100mM NaCl, 5mM TCEP at pH7.0. 13C/15N or 15N labeled proteins were expressed in 13C/15N or 15N-enriched M9 minimal media respectively and purified using the same protocol.Full length (residue 1–326), RRM1-2 (residue 1–190), RRM1 (residue 1–98) and RRM2 (residue 102–186) constructs of HuR was PCR amplified from pMal-c2x plasmid containing full length HuR (gift from Dr. Gorospe) and cloned into pET14b vector. They were both expressed in Fluorescence polarization measurements were performed in the 384-well plate format on a Biotek Synergy2 plate reader using 480/20nm excitation and 520/20nm emission filters. The NCI diversity set V library containing 1597 compounds was screened in this study. All samples were dissolved in phosphate buffer . The assay mixture contained 50nM HuR and 10nM RNA to yield FP values approximately 80% of the maximum value observed for fully bound RNA. NCI diversity set V library in 96-well format (10mM stock in DMSO) was transferred to 384-well plate, and then diluted into 50uM in phosphate buffer. 5ul of 50uM compound stock was added to 20 ul assay mixture to yield 10uM final compound concentration.2O or H2O. Saturation transfer difference (STD) NMR spectra were acquired at 298K on Avance III Bruker 500 MHz spectrometer equipped with a 1.7mm TCI cryogenic probe and an automated SampleJet sample changer. Compound concentration in the samples was 0.5 mM and the HuR concentration was 10uM. Protein saturation was achieved using the STDDIFFESGP.3 pulse. The on resonance was set for -500Hz (-1ppm) and off resonance was set for 25000Hz (50ppm), the saturation time is 3s. 16 scans were acquired with 1.5s relaxation delay between the scans. The data was processed using nmrPipe and plotted using Xmgrace. 2D 1H-15N HSQC was acquired with 2048 and 128 complex points in the direct and indirect dimensions, respectively. Protein concentration was 0.1~0.2mM. Backbone assignment of RRM1-2 was derived from the analysis of 3D HNCACB, CBCACONH.Protein samples were prepared in 50mM phosphate buffer at pH 7.0 containing 100mM NaCl, 5mM TCEP in DSedimentation velocity analytical ultracentrifugation was performed on a Beckman XLA analytical ultracentrifuge using an AN50 Ti rotor with standard Epon 2-channel centerpieces. The samples were spun at 25000rpm for~12hrs at 20 C. 25 scans measuring absorbance at 280nm were collected. The data were analyzed using Ultrascan III software.We first characterized the binding of the full-length HuR with 11-mer polyribonucleotide derived from the 3’ UTR of the mRNA of the C-fos oncogene . The binTo evaluate the assay readiness, we performed competition assay with unlabeled RNA. For FP based competitive HTS, Z’ score is the quality factor for the evaluation of an assay: the higher of Z’ value, the more robust of the assay is . We calcWe then performed initial screening using NCI diversity set V, containing 1597 compounds as described in the Material and Methods section. The standard deviation for FP measurement in the screen was 18.2 in unfiltered raw data . CompounNMR-based assays are good secondary assays for HTS, because they provide evidence for direct interaction of the hit compounds with the target protein, thus removing the possibility of the interaction with the RNA or another false-positive effect arising from the direct influence of the hit compound on the fluorescent tag. STD-NMR is a powerful way of monitoring small-molecule/protein interaction because of the ease to implement, the need for low concentration of protein (less than 10uM protein), and the no-limitation for molecular weight. 4 out of 12 compounds from the primary screens displayed STD difference effects  and thos1H-15N HSQC spectrum of the 15N-labeled full length protein reveals that very few signals are visible and they display poor spectral dispersion (1H-15NHSQC spectra of a shorter HuR construct containing only domains RRM1-2. Only compound C10 was observed to have an effect on the spectrum of RRM1-2 (13C/15N labeled RRM1-2 (maximum concentration we can get is about 0.15mM due to the precipitation above 0.15mM), the assignment of RRM1-2 becomes more challenging task. At the end, we have managed to obtain 85% backbone assignment  is one of most robust, reliable and reproducible binding assays used today : CSP is spersion . This inspersion . In the spersion . This isspersion , althougspersion . We thenf RRM1-2 . Upon adf RRM1-2 . The crysignment . The chesignment , which wsignment , indicat15N HSQC  Figs. ThRNA binding proteins (RBPs) that associate with specific mRNA and function as mRNA turnover and translation regulatory RNA binding protein (TTR-RBP) have emerged as pivotal post-transcriptional regulators of gene expression in mammalian cells –4. The aHuR contains three distinct modules known as RNA recognition motifs (RRMs): the N-terminal tandem RRM1-2 and the C-terminal RRM3 . RRM1 anE. coli expression system and a fluorescently-labeled RNA oligo. We also show that NMR-based assays can be used as a secondary filter to remove false positives arising in the primary screening assay by selecting compounds that directly interact with the target protein. During the time of preparing manuscripts, a paper using FP to screen HuR inhibitor was published [1H-15N HSQC are commonly used for studying the protein-ligand binding, which provide the information for residue specific interaction as illustrated in Previous efforts to discover inhibitors of HuR/RNA interaction yielded a low molecular weight inhibitor using confocal fluctuation spectroscopic assay , in whicublished . SPR wasublished  and is nublished ,42. NMR ublished –39. Thus1H-15N HSQC experiments can be used to evaluate effects of the compounds on protein oligomerization and protein/RNA interaction as is demonstrated in this study. HSQC spectra revealed that compound C11 restores signals that are not observed in the spectra of the full-length HuR protein. This striking effect most likely arises from the inhibition of HuR oligomerization caused by C11 binding to the protein, since there is no domain-domain interaction among three RRMs 1H-15N HSIn summary, our preliminary studies reveal that small molecules can inhibit HuR/RNA interaction either by directly competing for the RNA binding site on the RRM1-2 segment  or indirS1 Fig(A) The standard deviation is 18.2 in unfiltered raw data. The average FP value is 171.22 (red solid line). Compounds producing FP readings significantly higher than the average  were ignored due to protein precipitation and/or fluorescence quenching caused by compounds. The hits having 5-standard deviation  below the average were also removed due to their intrinsic fluorescence. (B) The standard deviation was 12.4 with the average of fluorescence reading at 171.40 (red dotted line) after the removal of 18 compounds from (A). The hits having 3-standard deviation  below the average were selected for further verification. (C) The selected hits from (B) were subjected retesting using original condition. The cut-off is same as initial hits pick-up.(TIF)Click here for additional data file.S2 FigCompounds at 250uM in final concentration in the assay volume of 25uL were added to the mixture of 10nM RNA and 50nM HuR in the first well in 384 format plate followed by a serial dilution into next well where the concentration of RNA and HuR was kept constant.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig15N-labeled RRM1. The end-point spectrum (RRM2/RRM1 = 9:1) (red) was overlaid with that of free RRM1 (blue). Two spectra overlaid very well, indicating that RRM1 did not interact with RRM2.Unlabeled RRM2 was titrated into 0.1mM (TIF)Click here for additional data file.S5 Fig13  using Ultrascan III software, while our AUC data indicated that only ~10% monomer existed in solution, the majority of species was dimer/oligomer. We tested 4 compounds from secondary screening. 30% and 50% fraction of boundary were in orange. 10uM HuR and 20uM compounds were used in AUC experiments in the total volume of 500ul. We found that both C10 and C11 shifted the boundary of species toward monomer, while C2 and C8 did not, indicating that C10 and C11 partially disrupted the formation of HuR dimer/oligomer.Sedimentation velocity studies are informative of the overall shape of molecule. The predicted moomer of full length HuR (36 kDa) is ~2.5x10(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigRRM1 and RRM2 are colored green. RNA is in orange. The residues interact with RNA was shown in sticks and colored red.(TIF)Click here for additional data file.S8 FigThe panel A-F represents compounds C1-6 respectively.(TIF)Click here for additional data file.S9 FigThe panel A-F represents compounds C7-12 respectively.(TIF)Click here for additional data file.S1 Table0-X)*p)).IC50s were obtained by fitting to the equation of ΔP = ΔPmax/(1+10^((Logx(DOC)Click here for additional data file.


In Situ Hybridisation–Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos.Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing  Although there is increasing interest in post-transcriptional gene regulation and RNA modulation ,2, the tIn-situ hybridisation Proximity Ligation Assay (ISH-PLA) (in situ hybridisation (ISH) with the proximity ligation assay (PLA) and one of its first achievements show that di-methylation of lysine 4 of histone H3 (H3K4me2) at the MYH11 locus is restricted to the smooth muscle cell (SMC) lineage in human and mouse tissue sections. Further, it showed that this mark persisted even in phenotypically modulated SMC present in atherosclerotic lesions that show no detectable expression of SMC marker genes [ISH-PLA)  is a relIn order to study cells of a specific type in their normal environment there is a need to use techniques to purify them, which often involves generating transgenic lines of animals . AlternaInitially in the ISH-PLA assay a nucleic acid probe targets a specific genomic sequence close to the binding site of the cognate binding protein. Two primary antibodies raised in different species are added, one (raised in rabbit) targets the cognate protein and a second antibody (raised in mouse) targets the biotinylated nucleic acid probe. Subsequently two secondary species-specific PLA antibodies with conjugated PLA oligonucleotides are added. Since the probe and the cognate binding protein are in close proximity the conjugated oligonucleotides are also in close proximity and are circularised upon introduction of a linear connector oligonucleotide, allowing rolling circle replication to commence. After the amplification reaction, far-red fluorescently labelled complementary oligonucleotide probes are added to highlight the product. Normally in ISH-PLA the nucleic acid probe binding to the target gene is around 500 bp in length, however since mature messenger RNA transcripts are generally in the several kilobase range this length probe would lack resolution in terms of determining the site bound by the RNA binding protein. Further, RNA may adopt a wide range of secondary and tertiary structures which can interfere with probe binding ,12. TogeXenopus staufen (Stau1) with Vg1 mRNA, the complex of which localises to the vegetal pole in oocytes . Staufen1 is a double stranded RNA binding protein (dsRBP) [To develop the RNA based ISH-PLA we have exploited the well-established interaction of  (dsRBP)  which bi (dsRBP) . The Vg1 (dsRBP) –17. Stau (dsRBP) . Both mo (dsRBP) . An advaThere is a clear need for a sensitive assay for RNA-protein interactions that permits subcellular and single cell resolution analysis in a variety of samples including tissue sections and whole-mounts. Here we describe the modifications necessary to adapt the existing DNA based ISH-PLA technique to mRNA.2, 1 mM DTT, 1 mM EDTA, protease inhibitor cocktail tablet (Roche)) and one embryo equivalent resolved by SDS-PAGE gel electrophoresis and electro-blotted onto a nitrocellulose blotting membrane (Life Sciences). The membrane was blocked and probed following standard protocols [Embryo protein lysate was extracted in embryo extraction buffer (10 mM HEPES (pH 8.5), 2 mM MgClrotocols , with thXenopus laevis were obtained from the European Xenopus Resource Centre (EXRC) at the University of Portsmouth and maintained at 18.5C in recirculating water with 10% daily changes. They were fed 3–5 times daily on a high protein pelleted diet. All animal care and experimentation was approved by the University of Portsmouth AWERB and performed under ASPA (1986) licences. This work, licensed under project number 70/7661, complies with Home Office regulations regarding the use of animals in scientific research. All surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering.Adult female Xenopus laevis females and oocytes separated by collagenase treatment and staged [Ovarian lobes were removed from d staged . OocytesXenopus laevis oocytes [TM 2 Primerdesign). Forward (AATATGTGTGCTACAGATCC) and reverse (TGACCTGTTAGATCTATCACT) primers were used to PCR amplify a 480-bp of the Vg1 3’UTR from the library  and the product subcloned into the pCR®2.1-TOPO TA vector , putative clones were confirmed by Sanger sequencing (Source Bioscience). The confirmed clone was then subcloned into pBluescript II between SacI and NotI restriction sites 5’ and 3’ respectively. RNA was in vitro transcribed from the construct using MEGAascript (Ambion).RNA was extracted from stage III/IV  oocytes  and revein vitro transcribed 480-bp 3’UTR of the Vg1 mRNA was labelled and analysed by EMSA which was conducted as described previously [TM Precast Gel from BIO-RAD. The samples were run at 20 mA for 75 min at 4°C.Either the DNA analogue of the proposed LNA probe (Invitrogen) or the eviously . The nucin-situ hybridisation was performed on stage III-IV Xenopus laevis oocytes following the protocol described previously [in situ hybridisation buffer for 30 minutes at 70°C and 5.5 hours at 60°C. Finally for the probe visualisation, a fluorescent antibody  was used at a 1:100 dilution instead of an anti-digoxygenin antibody.Whole-mount eviously  with theA full protocol is provided in Xenopus staufen (Stau1) with Vg1 mRNA, the complex of which localises to the vegetal pole in oocytes. Initially a rabbit anti Xenopus Stau1 antibody , was assayed for its specificity and applicability to in-situ hybridisation experiments. This was first tested by Western blot analysis, Xenopus laevis total protein extract at a range of developmental stages with the anti-XStau1 antibody. The antibody is specific to a single protein corresponding to a molecular weight of 80 kDa, the expected size of Xenopus Staufen. Given the propensity of antibodies to recognize their epitopes when in the exposed state of a Western blot but not when in a complete in vivo complex we also tested the Stau1 antibody as the primary antibody in a whole-mount Immunohistochemistry assay  probe rather than a DNA or RNA probe due to the improved specificity and sensitivity of LNAs ,12,26, wu assays –29 and cXenopus laevis Vg1 mRNA, accession number: NM-001095591.1) was initially predicted by Mfold . This information was then fed into RNAcomposer, an automated RNA structure 3D Modeling Server (http://rnacomposer.cs.put.poznan.pl/), to provide a tertiary structure (Staufen does not bind to the entire Vg1 mRNA sequence but rather to two repetitive motifs (VM1 and E2) found in the 3’ UTR of the Vg1 mRNA that has extensive secondary structure. Primary considerations of the probe design are; (1) The LNA probe must be in close proximity (30–40 nm) to the binding site of XStau1 to Vg1 mRNA; it is important to note that in a highly structured RNA molecule sequences that are far apart in the primary sequence may be close in three dimensional space. (2) That the probe is targeting an exposed region of the RNA; if the RNA target sequence is already directly in intra-molecular or inter-molecular interactions then probe binding will be inhibited. Both of these considerations require an understanding of the predicted tertiary structure of the RNA. To this end the secondary structure of the 3’ UTR region  and an electrophoretic mobility shift assay (EMSA) was performed . Initialhe probe ; a smallTo check for the ability of the probe to bind in the presence of the protein complex a second EMSA was performed on a native gradient gel (4–20%). The interaction between the labeled DNA analogue of the LNA probe (referred to as “probe” hereafter) and the Stau1 3’ UTR was confirmed ; this muin-situ hybridisation assay (WISH) conducted to optimize the protocol prior to extending to the rISH-PLA methodology. Xenopus laevis oocytes in 10 out of 10 of the stage IV and V oocytes analysed. Our results are in agreement with previously published data [Having confirmed that the DNA analogue of the proposed LNA probe was able to interact with Vg1 encoding mRNA in the presence of the Staufen protein the probe sequence was synthesised as a biotinylated LNA oligonucleotide. This probe was next tested in preliminary whole-mount hed data , which aAfter the successful testing and optimization of the whole-mount immunohistochemistry and WISH assay, these two assays were merged along with the proximity ligation assay (PLA) to assess and optimise the ISH-PLA assay for RNA analysis. Two separate experiments were performed, each using 10 oocytes. In 70% 14/20) of the tested oocytes the observed signal pattern generated by rISH-PLA  was stro/20 of thWe have shown that an RNA-protein complex can be detected with good spatial resolution using a variation of the ISH-PLA technique: rISH-PLA. There are a number of advantages of the rISH-PLA technique over traditional RNA immunoprecipitation assays used for studying RNA-protein interactions such as as RIP and CLIP. Principally these relate to its application to mixed cell populations and ease of use. ISH-PLA can provide information on where RNA-protein complexes are localised and allows the identification of a given RNA-protein interaction at subcellular and single cell resolution, thus avoiding issues associated with heterogenous cell populations from which conventional RNA-protein assays suffer. The detailed examination of RBP interactions with a specific RNA has always been a technically demanding series of experiments. Thus the potential to identify interactions at a cellular level in a relatively straightforward process, that can be adopted by any laboratory which routinely uses either immunohistochemistry or in situ hybridisation promises significant advantages for those investigating the role of such complexes in gene regulation, a rapidly growing area of research.However, the rISH-PLA technique does appear to have at least some of the false positive issues associated with immunoprecipitation techniques. The results showed that 10% of the oocytes (3 out of 30) had a faint but visible localised signal at the vegetal pole in the control antibody samples. The reason for this is unclear but one possibility is that the anti-mouse PLA antibodies are anchored to the mouse anti-biotin antibody and that the anti-rabbit PLA antibody is not completely washed away due to permeability issues associated with the high lipid content of the oocytes in the vegetal region. Should this occur, then at the ligation step there is stochastic chance of the ligation of the two PLA antibodies interacting  causing a signal at the vegetal pole. It will be interesting to discover whether this is an oocyte-specific problem caused by the high lipid content. Also it was important to have detailed information about the RNA-protein complex in order to design the probe efficiently. A lack of such information would not preclude the assay being applied but which probe to use would have to be determined empirically.There are many RNA-protein interactions occurring that are of interest both for basic biology and biomedical research that may be advanced by rISH-PLA. Recently it has been found that more than 800 RBPs, which are often highly conserved across organisms, have been implicated as having crucial roles in several types of cancer . RNA binDrosophila, Zebrafish and Xenopus) that have good sequence data.The development of the rISH-PLA assay for RNA analysis provides a powerful tool to advance our understanding of the role of RNA binding proteins in gene regulation at the post-transcriptional level and their role in wider cellular activity. This method can potentially be used in other organisms, however crucial to the technique is the design of the LNA probe. This requires knowledge of the primary sequence of the target mRNA for secondary and tertiary structure prediction. Therefore its utility will be limited to organisms (such as S1 TextThe full in detail step-by-step method for rISH-PLA is provided in the protocol shown in supplementary information.(DOCX)Click here for additional data file.