Xenopus egg extracts treated with RNase, and its activity is stimulated in vitro by RNA binding. Spindle assembly defects following RNase-treatment are partially rescued by inhibiting MCAK, a microtubule depolymerase that is inactivated by AurB-dependent phosphorylation. These findings implicate AurB as an important RNA-dependent spindle assembly factor, and demonstrate a translation-independent role for RNA in stimulating AurB.Accurate chromosome segregation is essential for cell viability. The mitotic spindle is crucial for chromosome segregation, but much remains unknown about factors that regulate spindle assembly. Recent work implicates RNA in promoting proper spindle assembly independently of mRNA translation; however, the mechanism by which RNA performs this function is currently unknown. Here, we show that RNA regulates both the localization and catalytic activity of the mitotic kinase, Aurora-B (AurB), which is present in a ribonucleoprotein (RNP) complex with many mRNAs. Interestingly, AurB kinase activity is reduced in  The spindle is a macromolecular assembly of microtubules and associated proteins that controls the segregation of chromosomes during each eukaryotic cell division. During mitosis, centrosomes nucleate microtubules that search for and capture chromosomes, resulting in chromosome alignment at the metaphase plate Xenopus), and Survivin–localizes to chromatin, centromeres, and microtubules during mitosis to promote spindle assembly . Ran-GTP is produced near chromosomes and releases spindle assembly factors–such as NuMA Xenopus egg extracts Although many proteins regulating spindle assembly have already been characterized, recent work has demonstrated that RNA also plays a critical role in promoting proper spindle assembly. Many mRNAs encoding mitotic regulators and components of the translation machinery localize to mitotic and meiotic spindles Xenopus and human cells Xenopus egg extracts to explore the hypothesis that the CPC is another spindle assembly factor that is regulated by RNA binding. We show that AurB activity is reduced in RNase-treated extracts, and that AurB is present in a complex with RNAs. We also show that the CPC binds to RNA in vitro, leading to AurB activation. Thus, we propose that AurB is an additional spindle regulatory enzyme governed by RNA-binding.Our previous study indicated that a Rae1-containing RNP regulated spindle assembly in Xenopus egg extracts, we assayed AurB activity by monitoring Op18 hyperphosphorylation in vitro in the presence or absence of RNA  and active (mitotic) – and assve RNase –J. The pve RNase , but hadve RNase . Total Rve RNase , albeit X. laevis genome. Gene Ontology analysis −16), demonstrating that AurB associates with mRNAs that are enriched on mitotic spindles. The binding of three transcripts enriched in mitotic AurB immunoprecipitations was confirmed by qPCR , which is reminiscent of the mode of interaction of PRC2 with RNA in vitroOur experiments in extracts did not define whether RNA binds directly to the CPC, or binds indirectly via other proteins that may associate with the complex. To determine whether the CPC binds directly to RNA, we examined the interaction of recombinant CPC with RNA in vitro , B usingin vitro –that disin vitro . The CPCl. 19006 , Table 1in vivo. Taken together, our results demonstrate that the CPC is a bona fide RNA binding-complex with at least 2 RNA-binding domains: one in AurB and another in Survivin/DasraA (likely in DasraA).To further define the RNA-binding subunits within the CPC, we tested RNA binding of AurB alone or in complex with Incenp, and Survivin alone or in complex with DasraA. Full-length AurB alone displayed appreciable affinity for RNA , S3A, whin vitro .To determine if RNA-binding by the CPC activates AurB we adapted a protocol in vitro . Two difin vitro . Fixed images were acquired with the spinning disk confocal setting.Mouse αphospho-H3 (ser10) (Millipore) was used at 1/5000 for western blot. Rabbit αCenpE was a gift of Don Cleveland Nuclei were cycled through interphase in the presence or absence of Cy5-dUTP as described above. Unlabeled and Cy5-labeled interphase reactions were re-arrested in mitosis with control or RNase-treated extract, respectively, each containing rhodamine-labeled tubulin and 10 µM nocodazole, as described above. After 1 hr., reactions were fixed with paraformaldehyde as described above, combined, and spun onto a single coverslip. The coverslip was then processed for αMCAK immunofluorescence using Alexa488-labeled secondary antibody. Pairs of control and RNase-treated nuclei  lying in the same field of view were selected for analysis. Kinetochores were selected by manual thresholding based on the Alexa488 signal on control nuclei, and the integrated intensity of the remaining foci on each nucleus was computed using Metamorph.in vitro. Capping reactions were conducted with the ScriptCap m7G capping system as described (CellScript). IRES-containing RNA was not capped. RNA was incubated at 10 nM in extracts at 20°C for 1 hr., and luciferase activity measured using the Dual-Glo Lucifearse Assay System (Promega).Renilla Luciferase DNA (Promega) without (pMB 341) or with  an IRES sequence was transcribed 2 pH7.7) plus 300 mM NaCl and 0.1% TritonX-100, and collected on a magnet. Beads were subsequently washed in 1 ml PBS with or without RNaseA at 0.01 mg/ml and rotated at room temperature for 10 min. A second wash with RNaseA was conducted without incubation, then beads were washed 3 more times with XB+ NaCl+ TritonX-100 as before. Beads were washed once with kinase buffer . Kinase reactions with MCAK substrate contained 0.1 mM ATP, 5 µM GFP-MCAK-NT, 0.5 µCi g32P-ATP, and AurB bound to 0.1 µg αAurB antibody on beads in 20 µl kinase buffer. For kinase assay rescues, RNA was prepared from separate extracts and corresponding AurB immunoprecipitations using the RNeasy kit (Qiagen) as described below. Reactions were incubated for 15 min at room temperature. Beads were collected on a magnet and the supernatant was saved in final 1x SDS loading dye. Samples were run on 10% PAGE, the gel was then dried and analyzed for 32P incorporation using the PhosphorImager system. Reactions with histone H3 substrate included 2 mg/ml histones (Type VIII-S) instead of GFP-MCAK-NT, and were detected by western blot.Kinase assays were performed as described 32P-ATP, 0.5x kinase buffer (see above), and 1 mM sodium vanadate to inhibit the phosphatase. 500 nM of RNA or double-stranded DNA was added as indicated. Reactions were incubated at room temperature for 15 minutes, then stopped by the addition of SDS loading dye. Samples were analyzed as described above.Kinase assays were performed as described Xenopus laevis Unigene transcript database using Bowtie AurB was immunoprecipitated from 100 µl of extract treated with puromycin for 30 min. Immunoprecipitation was conducted on ice for 1 hr with 80 µg αAurB antibody. Beads were washed five times with 1 ml PBS+1% TritonX-100. RNA was isolated according to the RNeasy kit “RNA clean up” protocol (Qiagen). RNA libraries were prepared for sequencing using the TruSeq RNA Sample Preparation v2 kit (Illumina) according to manufacturer’s instructions. Four libraries were barcoded and sequenced per lane on an Illumina HiSeq machine. Comparison of enrichment of AurB associated RNAs was performed by sequencing three biological replicates of RNA from AurB immunoprecipitations and from matched total extracts. Comparison of changes in AurB- bound RNAs upon changes in cell cycle state (mitotic vs interphase extracts) was performed from a single extract by comparing the enrichment of RNA in AurB immunoprecipitations vs total extract in each phase. Sequencing reads were aligned to the X. laevis Unigene database as described for AurB-associated RNAs. To perform correlation analysis between AurB-bound and spindle-bound RNAs, we examined only transcripts that had a total of 100 reads in each sample.Spindle-associated RNAs were obtained by diluting cycled spindles in 1x BrB80+30% glycerol +0.5% TritonX-100 and pelleting through a 10 ml 1x BrB80+60% glycerol cushion as described RNA sequences associated with this study have been deposited into the NIH SRA under accession numbers (Bioproject: PRJNA191571 and PRJNA247381).http://meme.sdsc.edu/meme/cgi-bin/meme.cgi) using the discriminatory motif discovery mode.Motif analysis was performed by obtaining the sequences for all RNAs enriched in AurB IPs greater than two standard deviations from the mean, and filtered against a negative-control set of sequences derived from transcripts that were under-represented in AurB immunoprecipitations. The negative control set was four times larger than the set of AurB-enriched RNAs in terms of total nucleotide length. Prior to motif or base composition analysis, poly-A tails were removed from sequences. These sequences were used as input for the MEME motif discovery software . Individual transcripts were quantified using the iQ SYBR Green system (Bio-Rad).Xenopus laevis cDNA with primers oMB 1668 (CAGGACTTGGTTGAGACAAAATG) and oMB 1669 (GAGCATCCAATTCTTCATCACC), and a 320 bp fragment of Xl. 19006 was amplified similarly using primers oMB 1644 (CAAATTGATAAAGAGACAGATG) and oMB 1645 (TGCTCTCCAAGATGCTTGTCC). Both fragments were cloned into pCR2.1 TOPO (Invitrogen) . A 41 nt region identified by MEME motif analysis in pMB 732, consisting of bases GGCAAAATCAGATCACAAATCTGAAAGAGAAAAAGTAGAAA was mutated to ATGGGGCTGAGCTGTGGGCTCAGGGAGAGGGGGACGAGGG (pMB 733). DNA templates were amplified with T7 and M13 Reverse primers and used for in vitro transcription with T7 RNA polymerase. The resulting RNAs were capped as described for luciferase assays, and added to extracts together at a final concentration of 1 nM each. In vitro transcribed RNAs were incubated in extracts for 30 min., followed by treatment with puromycin for 30 min. RNA bound to AurB was isolated as described for RNA sequencing, reverse transcribed with random hexamer primers, and quantified by qPCR as described above using a plasmid-specific forward primer oMB 681 (GGGCCCTCTAGATGCATGCTC) and the following reverse primers: oMB 1669 (GAGCATCCAATTCTTCATCACC) for Xl. 84202, oMB 1810 (CTCTTTCAGATTTGTGATCTG) for Xl. 19006 WT, or oMB 1811 (TCTCCCTGAGCCCACAGCTCAG) for Xl. 19006 mutant.A 318 bp fragment of Xl. 84202 was amplified from GFP-3xHA-AurB was made by cloning full-length AurB DNA with an N-terminal 3xHA tag into a GFP-pET30a vector (pMB 599). GFP-MCAK-NT was made by cloning DNA encoding amino acids 2–149 of MCAK into a GFP-pET30a vector (pMB 730). Plasmids encoding AurB/Incenp and Survivin/DasraA have been previously described in vitro transcription using PCR products as a template and T7 RNA polymerase. In vitro transcripts were labeled with Cy5-CTP (ratio of incorporation: 1∶20). RNAs were purified by LiCl precipitation, diluted to 100 nM and stored at −80°C.RNAs were produced by Final EMSA reactions contained: 20 mM Hepes (pH 7.8), 100 mM NaCl, 2 mM MgCl2, 4% glycerol, 1 mM beta-mercaptoethanol, 0.1 mg/ml yeast tRNA, 1 nM Cy5-labeled transcript and varying amounts of recombinant protein. 20 µL reactions were incubated at room temperature for 30 min and separated on 4% native acrylamide gels (prepared with 19∶1 acrylamide: bis-acrylamide). Gels were electrophoresed for 1 hr at 110 Volts and scanned using a Typhoon Imager (GE).Percentage of RNA bound by each protein was calculated using ImageJ. Apparent dissociation constants were calculated from each lane using the equation:d  =  (([Protein]−[complex])*([RNA]−[complex]))/[complex]. Each titration was performed a minimum of two independent times and the reported Kd is an average of all lanes from two trials. Kd plots were produced using R.KAll animal work was performed according to standards of animals care and approved by MGH IACUC (OLAW Assurance #: A3596-01). All animal work performed in this study was approved by the Massachusetts General Hospital Subcommittee on Research Animal Care. Frogs were housed in Aquatic Habitats recirculating water housing systems. Water was maintained at a conductivity of ∼1800 µS and a pH between 7.5–8. Animals were fed frog brittle (Nasco). Frogs were provided with PVC tubes and plastic lily pads as enrichment. Frogs are handled minimally and all injections are performed using the best possible practices to minimize distress during handling.Figure S1AurB kinase activity requires RNA in a translation-independent manner.A) Hyperphosphorylation of Op18 in control or RNase-treated extracts containing phosphatase inhibitors, and sperm nuclei at a concentration of 5000/µl. Arrow indicates AurB-dependent hyperphosphorylated form of Op18. Tubulin is shown as a loading control. B) Indicated volumes of total egg extract were probed by western blot using custom αAurB antibody. Arrow indicates AurB. C) Phosphorylation of GFP-PP7 (non-specific protein) or GFP-MCAK-NT by AurB in vitro. D) Phosphorylation of histone H3 in vitro by AurB isolated from control or RNase-treated extracts incubated with sperm nuclei, and washed in the presence or absence of RNase. AurB is shown as a loading control. E) Same as (D), using recombinant AurB added to extracts and immunoprecipitated with anti-GFP antibody. D-E) Data are representative of experiments performed at least in triplicate. F) Inhibition of cap-dependent and IRES-dependent translation by puromycin. Luciferase RNA containing either a 5′ cap or the IRES sequence from HIV-1 was translated in extracts, and protein levels were monitored after 1 hour of incubation. n = 3 extracts. Error bars represent SEM. Luciferase RNA levels were comparable in control and puromycin-treated extracts during the course of the assay as assessed by RT-PCR. Total RNA is shown as a loading control. The decrease in IRES RNA during the experiment results from degradation due to an absence of a 5′ cap; note that degradation is unaffected by puromycin. G) Phosphorylation of MCAK in vitro by AurB isolated from mitotic extracts in the presence or absence of the translation inhibitor puromycin. Activity of AurB from interphase extract is also shown. All extracts were incubated with sperm nuclei prior to AurB isolation. MCAK substrate and AurB amounts are shown as loading controls. Data are representative of experiments performed at least in triplicate. H) Mitotic extract was incubated with sperm nuclei for 1 hr., and subjected to immunoprecipitation with αAurB antibodies. RNA was isolated from total extract prior to immunoprecipitation (input), or from the immunoprecipitation (AurB IP). The resulting RNA pools were added to reactions shown in I) Detection of RNaseA by western blot in input and AurB immunoprecipitations. 0.5 µl of control extract, or 0.05, 0.1, 0.2, 0.5 µl of RNase-treated extract  were run in parallel with control or RNase-treated AurB immunoprecipitations. J) RNA added to kinase reactions pre-treated with RNase is stable. Total RNA was incubated with RNasin (0.8 U/µl) and with or without AurB beads treated with RNase as indicated. Data are representative of experiments performed at least in duplicate. K) Phosphorylation of MCAK in vitro by AurB isolated from control extract containing sperm nuclei. Each indicated RNA type was added at a concentration of 1.25 µg/ml. Data are representative of experiments performed at least in duplicate.(TIF)Click here for additional data file.Figure S2Binding of CPC complex members to RNA in vitro.A) Purified proteins used in gel shift assays. B) Purified CPC was blotted using myc antibody, which recognizes the myc epitope present on the C-terminus of Incenp. Western blot demonstrates the presence of many Incenp degradation products that co-purify with the CPC. Gel shift assays to assess binding of C) GFP-3xHA-AurB, D) Survivin, E) Survivin/DasraA, or F) AurB/Incenp to RNA fragments derived from Xl. 19006, Xl. 19006 lacking the poly-A motif defined in (TIF)Click here for additional data file.Figure S3Binding of CPC complexes to RNA in vitro (see). Quantificaiton of RNA binding of A) GFP-AurB, B) Survivin, C) Survivin/DasraA, D) AurB/Incenp, or E) full CPC to Xl. 19006, Xl. 19006 mutant, or Xl. 84202 RNAs, or Xl. 19006 DNA. Data from 2–3 independent EMSA experiments from each protein-nucleic acid combination are shown. Best-fit curve for each combination is plotted in blue. Independent experiments are indicated by circle and triangle plotting symbols.(TIF)Click here for additional data file.Figure S4RNA stimulates AurB in vitro and is required for proper spindle morphology in extracts.A) Phosphorylation of AurB and Incenp in vitro by purified, full CPC in the presence or absence of Xl. 84202 or Xl. 19006 transcripts. B) Quantitation of spindle lengths from (TIF)Click here for additional data file.Table S1Gene ontology analysis of AurB and spindle –enriched transcripts. Transcripts enriched in AurB IP (Tab1), purified spindles (Tab2), and both (Tab3) were used as input for the DAVID Gene Ontology browser. Prior to analysis all X. laevis Unigene transcripts were converted into human Uniprot names using BlastX. Significantly enriched categories are presented.(XLSX)Click here for additional data file.Table S2Correlations between two different, representative sequencing libraries. Pearson correlation coefficients were calculated using RPKM per transcript from sequencing libraries derived from total RNA in 2 separate extracts, or from RNA co-immunoprecipitated with AurB from the corresponding extracts. In addition, the correlation of transcript enrichment in the AurB immunoprecipitation (Aurora-B IP(rpkm)/Total extract(rpkm)) was calculated with respect to the relative enrichment of each transcript on purified spindles, and with the base composition of each transcript (% of each base).(DOCX)Click here for additional data file.Table S3X. laeivs Unigene sequences from two AurB IP and Total extract pairs, and from two purified spindle and Total extract pairs. Data are presented as raw read counts and normalized RPKM values for each library.Sequencing reads aligned to (TXT)Click here for additional data file.


Metastasis is the major cause of death among cancer patients, yet early detection and intervention of metastasis could significantly improve their clinical outcomes. We have sequenced and analyzed RNA (Expression) and DNA (Mutations) from the primary tumor (PT), tumor extension (TE) and lymphatic metastatic (LM) sites of patients with clear cell renal cell carcinoma (CCRCC) before treatment. Here, we report a three-nucleotide deletion near the C-region of Plk5 that is specifically associated with the lymphatic metastasis. This mutation is un-detectable in the PT, becomes detectable in the TE and dominates the LM tissue. So while only a few primary cancer cells carry this mutation, the majority of metastatic cells have this mutation. The increasing frequency of this mutation in metastatic tissue suggests that this Plk5 deletion could be used as an early indicator of CCRCC metastasis, and be identified by low cost PCR assay. A large scale clinical trial could reveal whether a simple PCR assay for this mutation at the time of nephrectomy could identify and stratify high-risk CCRCC patients for treatments. Renal cell carcinoma (RCC) accounts for about 2-3% of all cancers and worldwide more than 250,000 new cases each year . The mosHere, we employed a novel strategy with next generation sequencing (NGS) to identify specific mutations as biomarkers for metastatic CCRCC. We compare the genetic profiles from different tumor tissues of the same patient (primary tumor vs tumor extension vs lymphatic metastasis) to identify metastasis-specific mutations. Genomic instability in tumors generates large-scale cellular heterogeneity within a tumor population, from which rare variants evolve through a Darwinian selection process leading to successful metastasis , 7. ConsD. Melanogaster, Polo-Like Kinases (Plk) are a family of evolutionary conserved proteins characterized by their expression of one or more polo box protein binding domain (PBD) near the C terminal and a Ser/Thr kinase near the N-terminal [2/M transition [Initially identified in terminal . Plk's aansition . Among aansition . Human Pansition , 12. Theansition  as an inansition , 12.Traditionally, metastasis-related genes are identified by comparing differential gene expression profiles between tumor and metastatic cells. Here, we report a novel approach with next generation sequencing (NGS). To identify the mutations that are unique to the metastatic RCC cells, we harvested both DNA and RNA from tissue slides of primary tumor (PT), tumor extension (TE) and lymph node metastasis (LM) from the same patient for NGS on HiSeq 2000 . Only tissue slides with >80% tumor cells (examined by a pathologist) were used for DNA and RNA extraction. To avoid sampling variations and improve the reliability of NGS, both DNA and RNA of a sample were extracted from the same tube with TRIzol . As expected, the majority of genetic alternations  are common for all three sites because they are from the same patient Figure . However® Pathway Analysis (IPA®) indicated that cyclins and the cyclin dependent kinases (Cdk) were significantly enriched among differentially expressed genes. These differentially expressing genes involve multiple pathways including the Plk cell division pathway  deletion at position 1535177 of Chromosome 19  was detected with dominant frequency in LM  was reported with unknown function in the “1000 Genomes” project. Previously, the anti-proliferative effect of murine and human Plk5 protein has been reported  and DNA The expression of Plk5 protein in HEK295, a human embryonic kidney cell line has been reported previously . TherefoTo rule out the patient specific possibility, we further performed target sequencing of Plk5 with DNA from other stage-4 CCRCC patients. The sequencing data confirmed that the Plk5 mutation (rs58035688) is predominating in metastatic CCRCC, but not detectable or barely detectable in the primary tumor  in cancer have been reported , and tarAccumulating literature associates Plk function abnormalities with malignancy in various types of cancer . Unlike ® reagent .Fresh tissues from primary tumors, tumor extension, and metastatic lymph nodes were obtained at surgery from all cases with IRB approval and patient consent. Surgical specimen isolation and handling was done in compliance to standards of clinical care. Each specimen was cut into two parts. One part was used for routine pathological diagnosis, and the other half was frozen in liquid nitrogen for molecular analysis (DNA and RNA extraction). The diagnosis of CCRCC and stage of diseases was confirmed by independent pathologists. After identifying tumor cells by a pathologist, genomic DNA and mRNA were isolated from slides with 80% or more tumor cells by using TRIzolFor each sample, 1μg of gDNA and 5μg of RNA was prepared and submitted for sequencing. The DNA and RNA quality was evaluated and libraries were constructed with a library construction kit . Libraries were sequenced on the Illumina HiSeq 2000 platform . The raw reads generated were filtered according to sequencing quality and with regard to adaptor contamination and duplicated reads. Thus, only high-quality reads were remained and used in the genome assembly. Both RNA-seq and Exome-seq data were analyzed with Partek Flow version 4 . Bases with Phred score less than 20 were trimmed from both ends of the raw sequencing reads, and trimmed reads shorter than 25 nt were excluded from downstream analyses. Both pre- and post-alignment QA/QC was carried out with default settings as part of Flow workflow.www.gencodegenes.org) was used to quantify aligned reads to genes/transcripts using Partek E/M method. [For RNA-seq samples, trimmed reads were mapped onto human genome hg38 using Tophat 2.0.8 as implemented in Flow with default settings, and using Gencode 20 annotation as guidance. Gencode 20 annotation , COSMIC v71, GWAS (February 17 2015) EVS for known exon variants (cESP6500) ClinVar (2015-02), the pharmaco Genomic Mutation Database 2015.1(beta), Allele frequency from 1000 Genomes (dbSNP141) and dbNSFP for non-synonymous functional prediction (v2.9). Before constructing the analysis set, we removed dubious short reading frames and obviously unrelated genes resulting from the filtering parameters.For Exome-seq samples, trimmed reads were mapped onto human genome hg38 using BWA-MEM 0.7.9a  as impleFrozen tumor samples from patients were fixed in 4% PFA for 24 hours, followed by paraffin block embedding and sectioning. Formalin fixed sections are cut at 4 uM and baked at 60 degree for at least 1 hour before deparaffinization in xylene. Then they were washed with 100% ethanol, followed by rehydration in 95% ethanol for 5-10 minute each. The slides are put in 3% hydrogen peroxide in absolute methanol in order to quench endogenous peroxidase. After washing the slides in distilled water, antigen retrieval is performed using citrate buffer (pH 6) and microwaving for 30 minutes. After cooling off at room temperature for 20 minutes, the slides are then blocked with normal serum for 20 minutes to block the non-specific binding. Primary antibody against the Plk5 N terminal region  were added and left on the slides for 30 minutes. The slides are washed with PBS for 10 minutes. Chromogen of 0.03% diaminobenzidine is applied for 10 minutes and Hematoxylin is used as the counter stain. The slides are de-hydrated and cover slipped before imaging analysis. Relative intensity of Plk5 protein expression is quantified by Imagej .®, USA) and the quality and concentration of the resulting DNA was tested on a nanodrop before sequencing by GENEWIZ.Primers for exon 14 of Plk5 were designed using NCBI primer designed tools. A 307 nucleotide sequence was PCR amplified with a forward primer (CTTTGCAGGTGAGCTTCAGT), and a reverse primer(GGTTGAACAGTCATGCCACA). The resulting PCR products were confirmed with a 1.4% agarose gel for fragment size. DNA was then column purified (zymo


From transcription, processing, storage, and translation, all the stages in the life cycle of an RNA depend on interactions with proteins. Although technologies are making remarkable progress in unraveling the landscape of protein–RNA interactions, many key issues are unclear. We still have to identify how many proteins have RNA-binding ability, what are their targets and functional pathways. Moreover, while we know the number of protein-coding genes in the human genome, functional non-coding RNAs are still poorly defined. What is the function of the non-coding part of the eukaryotic transcriptome? A clear understanding of the biological functions of coding and non-coding transcripts would provide novel insights in molecular biology. What are the protein components binding to an RNA while it is being produced? Our lack of understanding of how ribonucleoprotein complexes assemble is a major rate-limiting factor to future progress in the field. We need to generate an in-depth characterization of protein–RNA complexes that form in cells during development and in response to external Life on earth might descend from an RNA world  particles. The assembly of functional RNPs and delivery to their destinations often involves progression through a series of intermediate complexes and subcellular compartments. For instance, ns Gall, . Similarns Gall, . What arnucleoli. Processing of the ribosomal RNAs initiates in the dense fibrillar part of the nucleolus and continues in the granular component, where the RNAs self-assemble with ribosomal proteins to form nearly completed subunits. Other examples of protein–RNA assemblies found in the nucleoplasm include speckles and promyelocytic leukemia bodies, among others  have started to unveil the targets of a number of RNA-binding proteins in the cell at defined conditions  and RNAs  as well as The field of protein–RNA interactions is moving fast and a number of fascinating hypotheses have been recently formulated on ribonucleoprotein complexes. A PubMed search using the terms “protein” and “RNA” shows that the number of indexed publications increased progressively in the last decades: 736 (period 1985–1995), 1377 (1995–2005), and 1768 (2005–2016) manuscripts. The overall increase of about 250% indicates the strong interest in the field and that in the future we might witness a revolution in the study of protein–RNA networks!Indeed, we do not have yet a complete understanding of how protein–RNA binding specificity is achieved and how the regulatory function of individual proteins is influenced by synergy and competition with other molecules. Novel approaches based on biochemical and functional studies, such as for instance SHAPE , through the European Research Council, under grant agreement RIBOMYLOME_309545 (GT), and from the Spanish Ministry of Economy and Competitiveness (BFU2014-55054-P). I also acknowledge support from AGAUR (2014 SGR 00685), the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017” (SEV-2012-0208).The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments.High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for  IDH1/2 hotspot mutations have been observed in the vast majority of astrocytomas and oligodendrogliomas [BRAFV600E mutation is frequently seen in pleomorphic xanthoastrocytomas, gangliogliomas, extra-cerebellar pilocytic astrocytoma, and a subset of diffuse gliomas [IDH1/2 and BRAFV600E are highly anticipated, not only for molecular diagnostics, but also for decision-making regarding molecular-targeted therapies.Gliomas account for over 30% of all primary brain tumors, and the majority require adjuvant treatments, including chemotherapy and/or radiotherapy . Recent ogliomas –7, and t gliomas –10. ClinImmunohistochemistry (IHC) and Sanger sequencing (SS) are conventionally performed to detect glioma-related mutations; however, clinical applications of these analyses are still limited. One of the primary issues that limit the clinical applications is the intrinsic heterogeneity of gliomas; highly sensitive platforms are required to detect genetic alterations . ToleranIDH1/2 and BRAFV600E mutations are heterozygous, HRM is a suitable method for their detection in clinical environments [High resolution melting (HRM) analysis is an alternative technique developed to screen for variation in DNA sequences. The development of novel saturation dyes has enabled the creation of the HRM platform, which is based on the principle that variation in DNA sequences produces alterations in the melting characteristics of DNA amplicons –14. Clinronments . Howeverronments . Accordironments . Conventronments . UnequivIDH1/2 and BRAF mutations in clinical glioma samples. We further developed a numeric expression for an objective mutation indicator, the HRM-mutation index (MI).In the present study, a differential calculus analysis was established in order to develop an unequivocal method for mutation scanning using HRM. The effectiveness of this method was explored using IDH1/2 analysis, surgically obtained tissue samples were collected from 192 patients, including 91 glioblastomas, 5 gliosarcomas, 23 anaplastic astrocytomas, 29 anaplastic oligodendrogliomas, 5 anaplastic oligoastrocytomas, 20 diffuse astrocytomas, 12 oligodendrogliomas, 1 oligoastrocytoma, 1 gliomatosis cerebri, 1 astroblastoma, 1 anaplastic pilocytic astrocytoma, 1 pleomorphic xanthoastrocytoma, 1 dysembryoplastic neuroepithelial tumor, and 1 histologically normal tissue sample. As a validation set for the IDH1 analysis, we collected 46 additional glioma tissue samples whose corresponding pathological specimen for IHC analysis were available, including 31 glioblastomas, 2 anaplastic astrocytomas, 12 diffuse astrocytomas, and 1 oligodendroglioma. Tumor tissue samples collected from 52 patients were used for the BRAFV600E mutation analysis; these included 24 pilocytic astrocytomas, 4 pleomorphic xanthoastrocytomas, 6 gangliogliomas, 14 adamantinomatous craniopharyngiomas, and 4 papillary craniopharyngiomas. Tumor DNA was also isolated from formalin-fixed paraffin-embedded (FFPE) specimens, using a RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE . Corresponding nontumor DNAs were isolated from a blood sample from the same patient using the QIAamp DNA Blood Mini Kit (Qiagen). The present investigation was approved by the Ethics Committee of Kyushu University and complied with the current laws of the country in which it was performed. Written informed consent was obtained from all participants.Brain tumor samples were obtained from patients during surgery at Kyushu University Hospital. A section of tumor tissue was snap-frozen in liquid nitrogen and stored at -80°C. Tumor DNA was isolated from the frozen blocks, using a QIAamp DNA Mini Kit . Histological diagnoses were made by senior neuropathologists according to World Health Organization guidelines. For the initial Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/). In silico PCR applications were used to verify the theoretical specificity of the primers (http://genome.ucsc.edu/cgi-bin/hgPcr). Amplicon size was optimized experimentally  in a total volume of 20 μL, according to the manufacturer’s protocol. A 7500 Fast Real-Time PCR System (Applied Biosystems) was used for amplification, and the cycling conditions were as follows: an initial denaturation of 10 min at 95°C; 40 cycles of 95°C for 15 s, 60°C for 1 min; and a dissociation cycle consisting of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. Auto-call genotyping data determined by differentiation of the normalized and temperature-adjusted melting curves for analyzed samples were acquired using the HRM v2.0 software (Applied Biosystems).® Terminator v1.1 (for IDH1/2) or v3.1 (for BRAF) Cycle Sequencing Kits (Applied Biosystems). Following purification, the electrophoresis and analyses were conducted using a PRISM® 3100 Genetic Analyzer (Applied Biosystems). Raw data were analyzed using the phred/phrap/consed package (http://www.phrap.org/consed/consed.html#howToGet) for sequence determination.PCR products were purified using ExoSap-IT . Thereafter, cycle sequencing was carried out using BigDyeR132H was performed on 6-μm-thick, formalin-fixed, paraffin-embedded tumor sections using an antibody specific for the mutant IDH1R132H protein . The tissue sections were deparaffinized and dehydrated, followed by endogenous peroxide quenching in 3% H2O2 for 30 min. Heat-induced epitope retrieval was performed at 120°C for 10 min in citrate buffer. The sections were then incubated with blocking serum followed by incubation with mutant IDH1R132H protein  overnight at 4°C. The staining procedure was performed using a Vectastain Elite ABC Kit  and DAB according to the manufacturer’s instructions.IHC for IDH1IDH1 codon R132, and were classified as either seq-mut  or seq-wt (n = 113). Duplicate HRM reactions  for the corresponding DNA samples were performed, and the mutation status of each sample was interpreted independently. The concordance rate of SS and HRM was 94.5% (363/384), and sporadic discrepancies between duplicate HRM reactions were observed . We surveyed the difference plots and derivative curves of discordant results and speculated that the reproducibility of the analysis using HRM software is limited when the change in the melting curve shape is equivocal  by plotting these calculated values throughout the melting phase. The -d2 curve of the mutant DNA sample contained an easily interpretable extra peak derived from the heteroduplex, in marked contrast to that of the wild-type DNA sample, which showed a simple curve. Thus, this analysis enabled an unequivocal visual discrimination of mutation status  experiments. The temperatures of the high melting transition and low melting transition within the extra heteroduplex peak in the -d2 curve were also recorded for each sample . As shown in d2 curve.Based on the hypothesis that the reliability of HRM depends on the precise discrimination ability of the melting curve, we developed a differential calculus approach in order to establish a robust analytical method. Negative first derivative values of fluorescence intensities, which are automatically provided in increments of 0.04 degrees by the default HRM software, were outputted. Using these values, the forward differences in increments of 0.04 degrees were then calculated. We edited approximated the negative second derivative curves (n status . The toln status . To estih sample . We defit (°C) as −d2f(t) We hypothesized that the difference in −d2f(t) between LTMT and HTMT would provide a numerical estimation of the mutation status. Accordingly, we defined the LTMT and HTMT zones within each 4SD range, which was calculated by the Shapiro-Wilk W test mentioned above, as ZLTMT and ZHTMT, respectively.Our approach improved the discrimination ability for mutation HRM curves; nonetheless, inter-observer variability cannot be completely excluded as long as the determination is dependent upon visual inspection or a comparison to wild-type curves. To establish an objective indicator of mutation status, we aimed to develop a numerical expression to identify the heteroduplex-derived peak. First, we defined the negative second derivative value of Hence,d2f(t) of ZHTMT from the supremum −d2f(t) of ZLTMT and defined the resulting value as the HRM-MI as follows:Subsequently, we subtracted the infimum −.d2 curve data of 113 seq-wt DNA samples. All of the HRM-MI values of the seq-mut DNA samples were distributed in the positive range , which had the rare IDH1R132S genotype. While discrepancies between the results of conventional HRM and SS were seen as frequently as those in the initial sample set , the results of HRM-MI and SS were completely concordant, validating the high reliability of our new method.To validate the reliability of our new analytical method, we analyzed To further estimate the sensitivity of our method, we performed reconstitution experiments in which a pair of DNA samples from a tumor and healthy tissue (blood) of the same individual were mixed at various ratios and subjected to HRM analyses ). Using IDH2R172 mutations were analyzed for the 192 glioma samples by HRM and SS, and mutation status was interpreted independently. IDH2 mutations were detected in only 4 samples by SS, so a statistical analysis was not performed; however, the -d2 curves of mutational DNA samples presented demarcated heteroduplex peaks, which can be precisely interpreted by visual inspection , and HRM, are available to analyze genetic mutation profiles in clinical tissue samples. Among them, IHC is the most popular approach and can be performed using clinical pathology techniques and specimens. Its low cost, lack of a DNA purification step, and high resolution  support the potential of IHC for clinical applications; however, IHC can detect only a targeted mutation, and inter-laboratory and inter-observer variabilities are not negligible . SS is tIDH1R132G and IDH1R132S; however, other mutant genotypes or variant sequences, such as SNPs, were not evaluated in the present study. The hotspot mutations detected in neoplasms, such as IDH or BRAF mutations, are located at specific genetic loci leading to gains-of-function [DNA melt curve profiles generated by HRM distinguish small differences between sequences, which enables reliable mutation scanning by most quantitative PCR machines . HRM is function . These sIDH mutations produce 2-hydroxyglutarate and are related to gliomagenesis by inducing a hypermethylator phenotype [IDH1 inhibitor, which is anticipated to be utilized for future molecular-targeted therapy [BRAF, p.V600E is the most frequent mutation; it constitutively activates RTK signaling pathways and is prevalent in various cancers [BRAF-targeted therapy has already been shown clinical efficacy for metastatic melanoma [BRAF inhibitor in preclinical models of BRAFV600E-mutant tumors, including gliomas [henotype –35. A pr therapy . Within  cancers . BRAF-tamelanoma . Moreove gliomas . Simple S1 FileTm was outputted from the HRM software for 108 (36 × 3) experiments. The temperatures of the high melting transition and low melting transition within the extra heteroduplex peak in the -d2 curve were also recorded for each sample.Triplicate HRM reactions from the same mutant DNA samples were performed in 36 variable well positions. Each (XLSX)Click here for additional data file.S2 FileIDH1-mutant and 113 wildtype DNA samples.The 384 HRM-MIs were calculated by the duplicate HRM results of the 79 (XLSX)Click here for additional data file.S3 FileTm was outputted from the HRM software and the temperatures of the high melting transition and low melting transition were recorded for the calculation of HRM-MIs.Decuplicate HRM reactions from mixed DNA samples were performed. Each (XLSX)Click here for additional data file.S4 FileThe 104 HRM-MIs were calculated by the duplicate HRM results of the 15 BRAF-mutant and 37 wildtype DNA samples.(XLSX)Click here for additional data file.


The Kaposi’s sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman’s disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exon-intron junction of the pre-mRNA. We selected four candidates  and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation. During viral replication, the oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) modulates both host and viral gene expression. KSHV ORF57 is a multifunctional posttranscriptional regulator that is essential for viral replication and stabilizes viral RNAs. Previous studies demonstrated that ORF57 RNA-binding is essential for its activity, but the full spectrum of ORF57 targets are unknown. Here we employed a high-throughput analysis to identify RNA fragments bound by ORF57 during lytic reactivation. As expected, we found targets that mapped to the viral genome, and we further uncovered novel host targets, a subset of which had ORF57 bound near their 5´ ends. Further examination of this subset demonstrated that ORF57 bound preferentially at the 5´-most exon-intron boundary. ORF57 affected the pre-mRNA abundance from these genes, most likely by stabilizing otherwise unstable inefficiently spliced pre-mRNAs. In at least one case, this stabilization led to increases in mRNA expression of the host gene. We suggest that KSHV employs the same mechanism to stabilize intronless viral RNAs and cellular unspliced pre-mRNAs to modulate viral and host gene expression during lytic reactivation. Kaposi’s sarcoma-associated herpesvirus  is a human gammaherpesvirus and the etiological agent for Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), KSHV-inflammatory cytokine syndrome (KICS), and some cases of multicentric Castleman’s disease (MCD)–4. The Ktranscription and export (TREX) complex, a multisubunit protein complex including the REF/Aly or UIF proteins that promotes cellular mRNA export [One KSHV factor critical for viral gene expression is the ORF57 protein (Mta) –12. WhilA export ,20. TREXA export , but thiA export . TherefoA export ,23–34, bA export ,29,33. IA export ,31,35. IA export .polyadenylated nuclear (PAN) RNA is up-regulated by co-expression of ORF57 in transient transfections[ORF57 stabilizes viral RNAs in the cell nucleus, independent of its reported ability to export intronless RNAs. This function was first suggested by the observation that the levels of the sfections,33. Diresfections,37. Moresfections,39. In asfections,25,31,35sfections,41. Thersfections, regulatsfections, genome sfections, translasfections, and maysfections,46,47.ORF57 binds directly to RNA, but whether binding is driven by specific RNA sequences, cellular factors, and/or if ORF57 binding is coupled to RNA synthesis or processing remains unclear. In the case of PAN RNA, two groups reported the presence of specific sequences in the 5´ end of PAN RNA, dubbed the ORF57-responsive element (ORE)  or Mta-rBecause of the importance of ORF57 RNA-binding for function, a comprehensive understanding of the RNAs bound by ORF57 during lytic infection is required to understand ORF57’s essential activities in viral replication. Indeed, an unbiased screen for ORF57-bound RNAs revealed novel targets, including one host RNA (IL6 mRNA), and suggested a new ORF57 mechanism . HoweverIn this study, we have adapted high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) for identification of ORF57 targets during lytic reactivation . As predTo identify RNAs bound by ORF57 during lytic reactivation, we performed HITS-CLIP ,52 in lyDue to ORF57’s propensity to be lost in insoluble nuclear fractions and to precipitate in lysate, we had to perform extensive optimization of the cross-linking and extract preparation steps. Under these conditions, immunoprecipitation of RNA was undetectable when we immunoprecipitated with non-specific antibodies  with those from cells at 12 hpi. For the 0 hpi samples, we added 4SU and harvested RNA from uninduced cells 2 hrs later; for 12 hpi samples we added 4SU at 10 hpi and we collected RNA at 12 hpi . For GAPBecause steady-state levels of the selected candidates were monitored in the context of viral infection, it is possible that changes in their RNA levels were due to the action of viral proteins other than ORF57. Clearly, the contributions of host shut-off to the steady-state and newly made RNA levels Figs.  and 7 caSo far, we have shown that ORF57 binds Figs.  and 5 anWe therefore examined the effects of ORF57 on BTG1, ZFP36 and EGR1 mRNA levels in transfected HEK293 cells . While ZThis model proposes that under normal cell culture conditions EGR1 pre-mRNAs are inefficiently spliced and degraded. Importantly, cells tightly regulate EGR1 expression by rapidly inducing EGR1 upon re-addition of serum to serum-starved cells . We reasIn this study, we performed HITS-CLIP of the KSHV ORF57 protein in a lytically reactivated PEL cell line to globally identify ORF57-bound RNA fragments. We recovered enriched clusters that map to both human and viral genomes Figs. –5. For tThe identification of known ORF57 targets and the identification of novel host RNAs affected by ORF57 validate our approach. Admittedly, our analysis is not without its limitations. HITS-CLIP data presents bioinformatic challenges and various methods have been applied to filter bona fide bound fragments from background (discussed in reference ). The coWe identified a subset of enriched clusters that showed enhanced ORF57 binding at the 5´ ends of host transcripts, near the first exon-intron boundary . ExaminaIf ORF57 protects both viral and cellular RNAs from host nuclear RNA decay factors, then it is of interest to identify the cellular RNA decay pathway involved. We have recently identified a nuclear RNA decay pathway that is dependent on the nuclear poly(A)-binding protein, PABPN1, and the poly(A) polymerases PAPα and PAPγ ,28,41,84In an alternative model, increases in pre-mRNA levels result from inhibition of splicing by ORF57. Consistent with this idea, the herpes simplex virus ORF57 homolog ICP27 inhibits splicing –79. In aWhile we favor the stability model, we are cautious in our interpretations of the mechanisms driving the changes in steady-state levels of pre-mRNA and mRNA. The pre-mRNA-to-mRNA ratio is often taken as a measurement of splicing efficiency. However, when one considers nuclear pre-mRNA decay as a contributing factor, interpretation of this ratio becomes muddled. For example, in our preferred model , the incIn principle, our data are also consistent with a role for ORF57 in inducing transcription of these targets, particularly in the case of EGR1. The EGR1 response to ORF57 in HEK293 cells  does notde novo motif discovery algorithm [ORF57 is sufficient to increase the abundance of intronless RNAs including viral mRNAs, PAN RNA, and intronless reporters. In the case of PAN RNA and vIL6, this up-regulation is enhanced by the presence of the specific cis-acting sequences that appear to serve as specific ORF57 binding sites ,28,43. Hlgorithm , we werelgorithm  supportiHow could ORF57 achieve apparent specificity in binding and function if it has little or no sequence specificity? ORF57 has been reported to interact with a number of host RNA-binding proteins, so it is possible that these proteins dictate the apparent specificity of binding ,25,37,55Because of its critical role in the viral life cycle, a mechanistic understanding of ORF57 functions is essential to the understanding of KSHV replication and pathogenesis. Our high throughput screening of ORF57-bound RNAs begins to address interactions of ORF57 with viral and host RNAs. This work extends existing data supporting a general role for ORF57 in the stabilization of a wide variety of viral RNAs. In addition, these data suggest that ORF57 nuclear RNA stabilization function is not restricted to viral RNAs, but further modulates the processing and decay of host transcripts during lytic reactivation. Ongoing studies seek to identify the precise molecular mechanisms of ORF57 interactions with the host cell RNA decay machinery that promote the stabilization of viral and host transcripts in the nucleus. In addition, it is of great interest to determine how changes in gene expression induced by ORF57 binding of host RNAs affect viral replication and/or pathogenesis.TREx BCBL1-Rta cells  were car7 cells were reactivated at 5x105 cells/ml and then collected at 20 hpi. Cells were washed in phosphate buffered saline , and resuspended in 3 mL of ice-cold PBS. Crosslinking was performed on ice in a Spectrolinker (Spectronics Corporation) at 125mJ/cm2 ~2 cm from the 254nm UV bulb. Five separate immunoprecipitations were performed for each biological replicate for a total of 108 cells per pellet sample. Cells were pelleted, frozen on dry ice and stored at -80°C.HITS-CLIP was performed essentially as previously described ,52, but 2 was added to 5 mM with 30 U of RQ1 DNase (Promega) and incubated for 15 min at 25°C. For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract. No dilution was performed for the “high MNase” samples . Input RNAs were treated with MNase, end-labeled, and size selected prior to library preparation to mirror the pellet samples. Libraries were made according to Illumina’s TruSeq Stranded mRNA Sample Preparation Guide, but the fragmentation step was omitted. Sequencing was performed on an Illumina HiSeq 2500 sequencer at the Eugene McDermott Center for Human Genetics. A detailed step-by-step lab protocol for HITS-CLIP with ORF57 can be obtained by contacting the corresponding author (NKC). Raw sequencing reads for all biological samples in this study are available, along with processed data files, under GEO accession number GSE64413.Gel purification of complexes was performed essentially as described in ,52. BrieAdaptors marked as N at the 3’ends of the paired-end sequencing data were first trimmed. Then Gsnap was used to align the sequencing data with parameters “-A sam—maxsearch 1-N 1-t 4-n 1” ,101. ThePaired-end read pairs were merged into single-end format if they overlap by at least 1 bp. Merged reads that have the same chromosome, strand, left-most mapping coordinate and right-most mapping coordinate were defined as PCR amplification duplicates and collapsed to unique tags, keeping only the tag with the highest sequencing quality. Then tags from all three pellet and three input experiments were simultaneously overlapped to identify CLIP clusters with at least 10 tags from any one or more conditions. CLIP clusters were continuous regions with non-zero binding tag count on each base within the clusters. Then the CLIP clusters were binned by 20bp and the tag counts and mutation counts on each base were summed for each bin within each experimental condition separately.Deletions and T→C mutations were chosen as characteristic RT-induced mutations of ORF57 binding according to comparative analysis of all the 12 types of substitutions, deletions and insertions (see below). The summed total tag count and summed characteristic mutation count within each bin were multiplied by 0.8 and multiplied by 0.2, respectively, and further summed to yield a value we termed the overall binding intensity for that bin. As a result, each bin will have 6 overall binding intensity numbers corresponding to the three pellet and three input samples.We used DESeq to analyze the binding intensity data of the three pellet vs. three input samples . For DESIn order to identify reverse transcription mutations that are characteristic of ORF57 crosslinking sites [Enriched clusters mapping to the human genome were annotated by genomic features including coding sequence (CDS), 3´ UTR, 5´ UTR, intron, 2kb upstream region, 2kb downstream region, miRNA/snoRNA and intergenic region. 2kb upstream region represents 2kb sequences before all genes’ transcription start sites. 2kb downstream region represents 2kb sequences after all genes’ transcription stop sites. Intergenic regions were defined as those parts of the genome that do not belong to any of the other categories listed here. If a cluster or a tag overlaps more than one feature, its count were divided proportionally and added towards each of the corresponding feature categories.Metabolic labeling and isolation of newly made RNAs were modified from previous protocols ,76 TwentS1 Fig(A) The 37-kDa, 50-kDa and larger (combined 100- and 150-kDa) protein-RNA complexes were cut from the nitrocellulose membrane, proteinase K treated and the purified RNAs were run on a denaturing urea gel. RNAs isolated from the condition treated with high MNase concentrations are loaded on the left, while the fragments digested with low MNase concentrations are on the right. Mobility of a DNA ladder (nt) is shown on the right. (B) RNAs digested with a low MNase concentration were extracted from the 37-kDa, 50-kDa and combined 100- and 150-kDa protein-RNA complex bands. These RNAs were used for northern blot analysis and probed for PAN RNA (B) or 5.8S rRNA (C). Positions of bands from a DNA ladder are included on the left of each membrane.(EPS)Click here for additional data file.S2 Fige(raw tag count + 1). Pearson’s correlations represent the correlation between raw tag counts of any 2 conditions.Scatter plots are shown on the upper right and Pearson’s correlations are shown on the lower left. For scatter plots, the CLIP tags in the human and KSHV genomes were binned (20 bp/bin) and the raw tag count data was log transformed as log(EPS)Click here for additional data file.S3 Fig10 (black) while the minus strand clusters are displayed as log10 (orange).The x-axis represents position on the KSHV genome (U75698) and the midpoint of each cluster was used as the x-coordinate. In this dataset, the KSHV enriched clusters ranged from 19–4339 nt; the mean viral enriched cluster length was 209 nt and the median was 99 nt. A statistical cutoff of 0.05 was used to define enriched clusters (dashed lines). For display, the RNA fragments mapping to the KSHV plus strand were assigned -log(EPS)Click here for additional data file.S4 Fig(A) Pie chart of the gene feature annotations of human enriched clusters from the low stringency dataset. If a cluster spanned multiple different annotations, the cluster annotation was split proportionally among the annotations. Upstream and downstream 2 kb refer to clusters mapping within 2 kb immediately flanking the nearest annotated gene. (B) The bar graph compares the number human enriched clusters (ECs) relative to their position on the closest annotated transcript. 0.0 and 1.0 correspond to the transcription start site (TSS) and the poly(A) site, respectively. Importantly, the length in this graph is relative to the annotated gene and is not a measure of the distance in base pairs. The brackets denote the 5´-enriched clusters examined in C-E. (C) Pie graph classifies the low stringency 5´-enriched clusters based on their gene feature annotations. (D) Bar graph comparing the number (y-axis) and position (x-axis) of 5´ enriched clusters relative to the transcription start site (TSS). The distance is measured in base pairs from the TSS (TSS = 0). (E) Bar graph comparing the number (y-axis) and position (x-axis) of 5´ enriched clusters relative to the 5´-most exon-intron junction. The distance is measured in base pairs from the first exon-intron boundary.(EPS)Click here for additional data file.S5 Fig(A) The number of genes (y axis) plotted according to the specific number of enriched clusters (EC) per gene for both the high and low stringency datasets. Additional enriched cluster statistics are given in the legend. (B) The number of exons (x-axis) was analyzed for the genes identified as containing enriched clusters. The enriched clusters were further divided into several different categories. For reference, the average number of exons in the entire human genome is shown (red). The “genes in all the enriched clusters” category includes the annotated genes of all the enriched clusters identified by our analysis (yellow). Genes with clusters at 5´ end represents the annotated genes from clusters concentrated at the 5´ ends of transcripts Click here for additional data file.S6 FigRNA was harvested at the indicated time points subsequent to lytic reactivation of TREx BCBL1-Rta cells with dox only (instead of butyrate and dox). (A) mRNA, (B) pre-mRNA, or (C) control RNA levels were monitored by RT-qPCR; positions of primers are shown in (EPS)Click here for additional data file.S7 Fig(A) The ratio of the total number of each type of mutation in the pellet tags divided by the number of mutations found in the input samples. All three biological replicates were combined into a single value for both the input and pellet mutation counts. (B) The total mutation count for each type of mutation for each sample in the sequencing data. The input samples (I1-I3) are displayed in shades of orange whereas the pellet samples (P1-P3) are shown in gray or black. The numbers refer to the three independent biological replicates. (C) The total mutation count in each of the three input samples was divided by the number of sequencing tags in the same samples to determine a mutation rate (orange). The same calculation was performed for the pellet samples (black). The numbers above the brackets are the fold differences between the pellet rates relative to the input rates for that mutation type. (D) Pearson correlations were determined comparing the total tag and mutant tag count from all bins in all clusters for each mutation type. A correlation for each of the six samples was determined as indicated.(EPS)Click here for additional data file.S1 TableThe coordinates for all enriched clusters in the human and viral genomes are listed for both the high and low stringency datasets. The first sheet provides a description of the data found in each of the columns. The additional spreadsheet contains the data.(XLSX)Click here for additional data file.S2 TableThis spreadsheet includes GO analysis of genes from enriched clusters from both the high and low-stringency datasets. The first sheet provides a description of the nomenclature and data found on each sheet.(XLSX)Click here for additional data file.S3 TableThis table provides the names and sequences for all primers used in this study.(DOCX)Click here for additional data file.S4 TableThe number of reads sequenced and mapped for these studies are given for three independent biological replicates of the input and pellet samples. The distribution of mapped reads to the human (hg19 reference genome) or KSHV genomes (U75698.1) is shown.(DOCX)Click here for additional data file.S5 TableThe chromosomal location of each of the T→C or deletion mutations is shown as is the p-value that corresponds to the statistical test applied to determine whether the mutation was overrepresented in the pellets compared to the inputs see .(XLSX)Click here for additional data file.


RNA-protein interactions govern many viral and host cell processes. Conventional ‘footprinting’ to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2′ hydroxyl reactivity (‘SHAPE’) that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. ‘XL-SHAPE’ was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome. RNA molecules, like proteins, can adopt intricate three-dimensional structures3gag gene, is known as the packaging signal (psi)9psi12psi RNA14psi, together with a cross linked region in the poly(A) stem loop whose 3D structure has not yet been modeled.We used this technique to gain new insight into HIV-1 genome packaging. HIV-1 is a global pathogen, infecting 35 million people worldwide and causing 7,000 new infections a day7The pipeline for conducting XL-SHAPE is depicted in Capillary sequencing using fluorophore-labeled primers is used to make the technique high-throughputUV irradiation at 254 nm induces cross-links between RNA and protein as nucleobase excitation at this wavelength leads to the formation of a covalent bond with a nearby amino acid side-chain1718The HIV TAR RNA and its ligand the Tat protein have been studied by many techniques including NMR. A Tat peptide RKKRRQRRR23242526 232425The strongest cross-links should occur where protein and RNA interact stably, however transient nonspecific interactions might generate cross-links. The Tat peptide was therefore compared to a control peptide with a high proportion of basic residues that was known to interact nonspecifically with TAR . To ascertain SHAPE reactivity before addition of Tat peptide, RNA or RNA-Tat peptide were probed with NMIA in DMSO or with DMSO only. Reproducible SHAPE reactivity changes were considered to be a difference of more than 0.2 units that was consistently seen in the majority (>66%) of experiments. These are marked colorimetrically on We chose the bacteriophage MS2 coat-binding protein (CBP)—stem-loop (SL) interaction to ensure that our system was sensitive enough to be generally applicable to RNA-protein interactions including those that do not have 1:1 stoichiometryDimerization of the HIV-1 genomic RNA is an important step in the viral lifecycle12All 20 amino acid side chains have been shown to cross-link, as has each of the bases171718psi region, which are distinct and separated in space (the poly(A) stem loop has not been modeled in 3D to date). All but a few cross-link sites are on exposed faces of the structure. Those which are not exposed are likely revealed during the conformational change occurring during Gag binding, a process which has most clearly been demonstrated for SL3The nucleotide regions cross-linking Gag were mapped on to nucleotides 104–344 in the 3D structure of this RNA  and compAs expected, Gag cross-links with SL3, which is recognized as a major packaging signal, at A319 and G320 in the GGAG loop. 5′ of SL3 is a short AU rich sequence in which U307, 308 and 309 and G310 also cross-link. In the published 3D structure these two loops are closely adjacent and provide a binding face for Gag interaction . SHAPE dIn SL1 bases U250, C252 and U253 and C267 on opposite sides of the terminal helix present a cross-linking motif ; SHAPE iNot previously recorded is a Gag binding site in the U5:AUG duplex on the 3′ face, G338 and 340 . SHAPE rA variety of cross-linking sites and SHAPE reactivity changes are noted in the PBS (primer binding site) region. Interestingly the four bases identified by crosslinking are adjacent in 3D space  whereas There are several central ‘embedded’ bases A133, G221, A222 and A225 and G298 whose SHAPE reactivity decreases but at which no cross-linking is seen. Indeed comparing the overall reactivity of many structural motifs in the Gag bound model suggests that certain motifs, such as SL3, are destabilized by Gag binding, whereas others, including the 3-helix junction above the U5:AUG duplex and the apical loop of the poly(A) stem are stabilized by Gag binding. These bidirectional changes are consistent with the structural switch described below.psi structure and also the poly(A) stem loop. Amongst these are sites in common with a previous footprinting study using Gag-GSTet al. detected structural change/binding to loop B of SL1, as do wein vitro at least, is not affected by the p6 domain.Previous studies that featured cross-linking of unmodified RNA to proteins have identified a minimal number of binding sites1621A previous NMR study of the interaction of SL3 RNA with NC showed interaction of NC with the 3′GAG in the terminal GGAG tetraloopXL-SHAPE detected previously unidentified Gag binding sites: the poly(A) loop and upper stem, the 3′ side of the U5:AUG helix, the GU-rich region around nt 120 and loop A of SL1. These may all reflect binding sites unique to the dimer that the virus may use to facilitate its selection over the monomer: the conformations of the U5:AUG helix and the GU-rich region change greatly during the structural switch1219in vitro transcription, using templates with T7 promoters. Templates were prepared by PCR using 1 × Biomix red (Bioline) and 0.5 μg template DNA. Templates used were pSVC21In vitro transcriptions were performed with the Ambion T7 Megascript kit and purified with Ambion MegaClear columns. Briefly, each transcription reaction contained 7.5 mM deoxyribonucleotide triphosphates (dNTPs), 1 × reaction buffer, 2 μg of template DNA and 2 μl of T7 RNA polymerase and was incubated for 3 h at 37 °C. DNA was degraded with 4 U of DNase  for 1 hr at 37 °C. RNA was eluted from MEGAclear columns (Applied Biosystems) in water and stored at −20 °C. The structural integrity of each RNA was assessed on denaturing polyacrylamide gels .RNA was prepared by BamHI and XhoI sites, and sequenced. Gag Δp6E.coli Bl21(DE3) cells. Cells were induced at an OD of 0.6, with 0.5 mM IPTG for 90 min at 30 °C. Cells were harvested and resuspended in a minimal volume of resuspension buffer , protease inhibitor (Roche Complete mini EDTA-free) 1 tablet per 10 mL) before treatment with 0.5 mg/mL lysozyme for 5 min at 4 °C. Cells were sonicated for 3 × 1 min at 50% power output in a cup-horn sonicator, with cooling, and treated with 0.5% triton X-100 for 5 min. The suspension was clarified at 12,000 × g for 20 mins; supernatant was applied to a GSTrap FF column  that had been previously equilibrated in binding buffer , 1 mM DTT) using an AKTApurifier (GE). The column was washed with binding buffer until the UV trace reached baseline and equilibrated with Prescission cleavage buffer . Prescission protease (660 U/5 mL column) was applied to the column and incubated for 3 h at 4 °C. A further GSTrap FF column was equilibrated in Prescission cleavage buffer and attached below the first column, to remove cleaved GST. Protein was eluted in a mixture of PBS-DTT and Prescission cleavage buffer  concentrated , and assessed by polyacrylamide gel electrophoresis, Western blot and Bradford assay.Tat and HSV peptides were purchased from Thermo Scientific and resuspended in water. The pMS2-GFP construct was obtained from Addgene. MS2-GFP was amplified using primers: 5′CCCGGGATCCATGGCTTCTAACTTTACTCAG3′ and 5′GCCGCTCGAGTTATTTGTATAGTTCATCCATGCC3′, cloned into pGEX-6P-1 using 20, heated at 95 °C for 2 min and snap-cooled on ice. The sample was then diluted to 1 μM in 10 mM Tris-HCl, 140 mM KCl, 10 M NaCl, 1 mM MgCl2 and incubated for 1 hour at 37 °C. RNA was placed at room temperature and the volume was increased to 150 μl per sample with 30 μl binding buffer , along with 15 μl RNasin and 100× excess (w/w) yeast tRNA. Each sample was divided into two: 75 μl aliquots were added into each of two wells of a round-bottom 96-well plate. For RNA-only samples, 75 μl of Protein elution buffer was added; for RNA-protein samples, 75 μl of protein in protein elution buffer was added. Plates were incubated at room temperature for 15 min. Samples were cross-linked on ice for 2 min at default power in an XL-1500 Spectrolinker UV cross-linker (Spectronics Corporations). Non-cross-linked control samples were incubated on ice for 2 min with no cross-linking. SDS was added to 0.5% final concentration, alongside 200 μg/mL Proteinase K. Plates were incubated on heat blocks set to 55 °C for 60 min. RNA was recovered by transferring sample to 1.5 mL microfuge tubes, phenol-chloroform extraction and ethanol precipitation.The cross-linking protocol was developed from a previous studyRNA was renatured and RNA only or RNA-protein complexes were prepared and incubated on 96-well plates as described above. 10 mM  NMIA in DMSO or an equal volume of DMSO only was added. Plates were incubated at 37 °C for 45 min and RNA was recovered with SDS and Proteinase K treatment and phenol-chloroform extraction as described above.in vitro transcription, using the Jena DNA cycle sequencing kit and 1.3 nmol primers labeled with either NED or PET. Each sequencing reaction contained 50 ng template, 1× sequencing kit buffer, enzyme and extension mix as per the manufacturer’s instructions. Cycle sequencing was for 5 min at 95 °C, then 30 cycles at 95 °C for 30 s, 37 °C for 30 s and 70 °C for 1 min (Labnet Multigene thermocycler). The amount of ladders and RT products combined to be sequenced was titrated to ensure that signals from the fluorophores did not bleed into one another. Combined cDNA samples and ladders were then purified by ethanol precipitation, resuspended in 10 μl and dried onto sequencing plates before being resuspended in 30 μl formamide and fractionated by capillary electrophoresis .RNA samples from either NMIA modification or cross-linking experiments were resuspended in 12 μl 2.1 mM Tris pH 8.0, 42 μM EDTA, with 5 nmol primer (designed to bind to the 3′end of the RNA and labeled with either 6FAM or VIC). For SHAPE probing experiments, this was always 6FAM for NMIA probed RNA and VIC for the DMSO only control. For cross-linking experiments, samples for the fluorophore controls were labeled with either 6FAM or VIC. For cross-linked and non cross-linked experiments on RNA-protein complexes, the RNA-protein sample was reverse transcribed with 6FAM and the RNA only control with VIC. Primer annealing took place at 85 °C for 1 min, 60 °C for 5 min and 35 °C for 5 min. 8 μl extension mix , 12.5 mM DTT, 1.25 mM dCTP, dATP, dUTP and 7-deaza dGTP was added and reverse transcription was allowed to proceed at 55 °C for 1 h. Each pair of samples to be analyzed together was combined and RNA was hydrolyzed at 95 °C for 2 min with 200 mM NaOH, followed by 5 min at 4 °C and the addition of an equal amount of HCl and the sequencing ladders. These were prepared from the same DNA templates used for This system, analyzed with SHAPEfinder softwarerd quartile plus 1.5 times the interquartile range. For NMIA experiments, each sample was normalized in this manner. For cross-linking experiments, Fx control samples were normalized in this manner and their average value used to normalize the rest of the experimental samples. In order to generate a value of cross-linking magnitude, the value of the 6FAM minus VIC peak at each nucleotide position for the fluorophore control cross-linked sample was subtracted from its counterpart in the experimental cross-linked sample, and the value of the fluorophore control for the non cross-linked sample was subtracted from its counterpart in the non cross linked control sample. The non-cross-linked data were then subtracted from the cross-linked data at each nucleotide position, to give the amount of signal attributable to RNA-protein cross-links.Traces were aligned with sequencing ladders and integrated using SHAPEfinder software. Mobility shift controls were first performed for each primer as describedHow to cite this article: Kenyon, J. C. et al. A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader. Sci. Rep.5, 14369; doi: 10.1038/srep14369 (2015).


Animals are often required to adequately respond to novel stimuli on the basis of their previous sensory experiences. To investigate the neural mechanism underlying this sensory-guided decision making, we conducted multiunit recordings from the rats performing a two-alternative choice task. The rats were trained to make a LEFT or a RIGHT choice in response to two auditory cues, and then their behavior was tested for these familiar cues and other novel cues. Their choice probabilities generally varied in a graded manner such that the probability of choosing an option changed gradually according to frequency differences between familiar and novel cues. However, we also observed large variability in choice behavior across rats: choice probabilities for novel cues were near the chance level in some rats, while it systematically changed with tone frequency in other rats. We elucidated the mechanism underlying such decision behavior and the possible origin of individual behavioral differences in a neural network model Fig. . Our modOur model successfully replicated the gradual choice behavior observed in our experiment. We further analyzed how neural dynamics in the reservoir network determines the choice probability for familiar and novel cues. We found that a familiar stimulus sequentially activates a relatively small portion of reservoir neurons, and reinforcement learning trained output connections from these neurons such that only an adequate output neuron is activated by the neural trajectory evoked in the reservoir. We further revealed that choice responses to novel cues become graded due to trial-by-trial overlaps between the familiar trajectories and novel-cue-evoked trajectories.Interestingly, our model also exhibited similar variability in choice responses to that observed in individual rats. We found that if input neurons are highly sensitive to external stimuli, that is, if the width of their frequency tuning curves is broad, the model network likely shows gradual choice behavior for novel stimuli. In contrast, the model with more sensitive input neurons (with broader tuning curves) tends to generate near-random choice behavior, displaying a flat dependence of choice probability on novel stimuli. We compared choice behavior between the models and the rats by introducing quantitative measures and found that the behavioral tendency of our model is consistent with that of the rats Fig. .These results may suggest that some individual differences in decision making behavior emerge from neural population dynamics rather than differences in higher-level behavioral strategies.


RNA silencing pathways were first discovered in plants. Through genetic analysis, it has been established that the key silencing components called Dicer-like (DCL) genes have been shown to cooperatively process RNA substrates of multiple origin into distinct 21, 22 and 24 nt small RNAs. However, only few detailed biochemical analysis of the corresponding complexes has been carried out in plants, mainly due to the large unstable complexes that are hard to obtain or reconstitute in heterologous systems. Reconstitution of activity needs thorough understanding of all protein partners in the complex, something that is still an ongoing process in plant systems. Here, we use biochemical analysis to uncover properties of two previously identified native dicer-like activities from wheat germ. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs of 21 and 24 nt in size. The 21 nt dicing activity, likely an siRNA producing complex known as DCL4, is 950 kDa-1.2 mDa in size and is highly unstable during purification processes but has a rather vast range for activity. On the contrary, the 24 nt dicing complex, likely the DCL3 activity, is relatively stable and comparatively smaller in size, but has stricter conditions for effective processing of dsRNA substrates. While both activities could process completely complementary dsRNA albeit with varying abilities, we show that DCL3-like 24 nt producing activity is equally good in processing incompletely complementary RNAs. Eukaryotes possess an efficient system of gene regulation through the production of small RNA (sRNA) called RNA silencing. The 21–24 nt sRNAs are either generated from partial complimentary precursor RNAs or a completely complimentary double-stranded RNAs (dsRNA) of multiple and often exogenous origin. The small RNAs which are produced from partial complimentary precursors are either involved in mRNA degradation or in inhibition of mRNA translation are termed as micro (mi) RNAs with important roles in development and disease. The small RNAs of 21–24 nt produced from perfect complimentary RNA molecules have much wider roles, such as resistance against pathogens, controlling transposons, development and heterochromatin formation [Small RNAs are processed from RNA substrates through the action of Dicer-like (DCL) proteins in plants. They can process RNAs based on their origin and structure into 21–24 nts. Once small RNAs are formed, they have the opportunity to make complexes with Argonautes (AGO) and this ribo-nucleoprotein complex is responsible for targeting RNAs that have sequence complementarity with small RNAs. Such a targeting can result in mRNA degradation or translational inhibition. This suppression of RNAs acts as a natural defense mechanism evolved to protect eukaryotic genomes against invasive nucleic acids such as viruses, transposable elements and transgenes. RNA silencing has emerged as a powerful tool with a wide range of applications in functional genomics and genetic engineering.Drosophila and four or more Dicer-like (DCL) proteins in Arabidopsis and other plants [Dicers and DCLs are large (∼200 kDa), multidomain proteins that contain a putative RNA helicase domain, PAZ (Piwi/Argonaute/Zwille) domain, two tandem ribonuclease III (RNase III) domains and one or two dsRNA-binding domains . In humar plants ,9.Arabidopsis [All the four Arabidopsis DCLs have specialized functions ,11. DCL1bidopsis –28.Arabidopsis, few dsRNA-binding proteins are involved in RNA silencing and are known to act synergistically [Hyl1) gene encodes a dsRNA-binding protein with two dsRNA-binding motifs, a putative nuclear localisation signal and a C-terminal repeat structure that may be involved in protein-protein interaction [Arabidopsis, functionally equivalent to dsRNA binding proteins in animals [Plant DCLs are not known to act on dsRNA substrates alone. Few additional factors have been associated with DCL action on RNAs. In plants, such as stically . HYPONASeraction . HYL1 fueraction –38. SERReraction –41 and heraction . A set o animals ,44.Arabidopsis. AGO4 functions in different small RNA pathways related to DNA methylation and epigenetic regulation. AGO7/ZIPPY function in developmental timing and AGO10 (Zwille) resembles AGO1 in its function. Other plant AGOs have similar key roles during plant development and genome regulation (reviewed in [One strand of siRNA is incorporated in an RNA-induced silencing complex (RISC) which guides ATP-dependent cleavage of homologous transcripts at a position corresponding to the nucleotides 10 and 11 of the siRNA. An important component of RISC complex is the AGO protein. AGOs are highly basic ∼100 kDa proteins and have been identified to be involved in RNA silencing through mutant screens. They have the characteristic N-terminal PAZ domain and a C-terminal PIWI domain. AGO1 functions as ‘slicer’ in iewed in ).Examining the poplar and rice genomes revealed that they contain five and six Dicer-like genes, respectively . AnalysiArabidopsis using DCL activities derived from flowers or immature floral buds were not successful mainly due to the unstable nature of protein complexes (unpublished results from the lab). However, Arabidopsis cell culture derived cells were good enough to isolate native DCL1 and DCL3 complexes [Arabidopsis seedling-derived crude extracts exhibited DCL4 activities and importance of DRB4 for their action [Arabidopsis crude extracts were also used by the same group to show difference in the specificity for substrates by DCL3 (that prefers short substrate) and DCL 4 (prefers long substrate) [In vitro reconstituted pri-miRNA processing reactions using recombinant DCL1 indicated that DCL1 alone is able to release miRNA:miRNA* duplex, but the processing was inaccurate [Although indirect effects of temperature, bivalent cations and substrates for DCL activity has been established –54, theromplexes , althougr action . Howeverbstrate) . At the accurate . An exteaccurate .in vitro system, they showed that in plants ATP-dependent, DCL enzymes cleave dsRNA into small RNAs that have the structure of siRNAs. Tang et al. [Extremely valuable information using native DCL complexes comes from limited studies in plants using wheat germ as the source of native protein complexes. Tang et al.  showed tg et al.  also shoin vitro system wherein the individual complexes of wheat germ are further purified to have either a 21 nt processing activity  and a 24 nt processing activity (likely DCL3 as this is the only DCL that is capable of generating 24 nt siRNAs efficiently). Each of these native complexes were subjected to in vitro assays to determine their biochemical requirements to process dsRNAs into small RNAs. Our results largely correlate with the genetic analysis done using A. thaliana model system, for example, temperature sensitivity of DCLs as well as inhibition of their action due to certain bivalent cations. This is one of the few biochemical attempts to understand cleaving abilities of few individual plant DCLs and the method holds promise for further characterization to understand mechanism of DCL action.Here we have developed an Wheat germs were prepared according to protocol described in Madin et al. . Brieflyg for 30 min. The resulting supernatant was subjected to gel-filtration on a G-25 (fine) column, equilibrated with two volumes of buffer A. The void volume was collected and centrifuged at 30,000 × g for 10 min. Extracts were frozen until use.The method used is a slight modification of the procedure described by Madin et al. . Washed Following buffers were used for column chromatography. Sephacryl S300 gel filtration buffer: 20 mM Tris-Acetate (pH 7.6), 90 mM KAc, 2 mM MgAc, 10 mM MET, 5% glycerol. Q-sepharose column buffer: 20mM Tris-HCl (pH7.5), 50 mM KCl, 4 mM MgCl2, 8 mM MET, 5% glycerol, gradient of KCl from 50 to 500mM. DCL4 was eluted at 220 mM of KCl and DCL3 at 260 mM.Ammonium sulphate (Sigma-Aldrich) was used at various concentrations to precipitate proteins. The precipitated proteins are desalted using desalting columns , and used for functional assays.HindIII restriction site is in caps). The second PCR product was amplified using primers:5’-TAATACGACTCACTATAGGGttacttgtacagctcgtccatgccga (contains T7 promoter directing production of anti-sense GFP RNA) and 5’- AAGCTTatggtgagcaagggcgaggagctg (containing HindIII restriction site). From the resulting clones, sense and antisense RNAs were transcribed using T7 polymerase in the presence of P32 UTP after digesting with HindIII. Sense and antisense RNAs were annealed to produce 700 bp dsRNA. This labelled dsRNA substrate was used for all dsRNA processing assays except the ones presented for substrate specificities.To produce 700 bp long dsRNA, two PCR products made on template of GFP sequence (pEGFP-1) were cloned into the base plasmid. The first PCR product was produced using primer pairs: 5’-TAATACGACTCACTATAGGGatggtgagcaagggcgaggagctg (contains T7 promoter directing production of sense GFP RNA is in caps) and 5’-AAGCTTttacttgtacagctcgtccatgccga (The following oligonucleotides were used to make dsRNA substrates of 60 bp versions: SENSE1 FOR (5’-TAATACGACTCACTATAGGGACTTACAACAGTACGAATGTTACAATCAGATTCATAGTTAACTGAGGCCCGCGCCC) and SENSE1:REV (5’-GGGCGCGGGC CTCAGTTAACTATGAATCTGATTGTAACATTCGTACTGTTGTAAGTCCCTATAGTGAGTCGTATTA) oligos were annealed together and then transcribed using T7 polymerase (Fermentas) in the presence of alpha P32 UTP to produce a 60 nt sense RNA.Similarly, ANTISENSE1(cc)FOR (5’-TAATACGACTCACTATAGGGCGCGGGCCTCAGTTA ACTATGAATCTGATTGTAACATTCGTACTGTTGTAAGTCCC and ANTISENSE1(cc)REV (5’-GGGACTTACAACAGTACGAATGTTACAATCAGATTCATAGTTAACTGA GGCCCGCGCCCTATAGTGAGTCGTATTA) were annealed together and transcribed to make a substrate that is complementary to sense RNA (60 nt antisense RNA). These two RNAs were annealed together to make a 60 bp dsRNA (60-P). Oligos ANTISENSE2(LOOP)FOR (5’-TAATACGACTCACTATAGGGCGCGGGCCTCACTTAACTATGATTCTGATTGTAA GTTTCGTACTGTAGTAAGTCCC) and ANTISENSE2(LOOP)REV (5’-GGGACTTACTAC AGTACGAAACTTACAATCAGAATCATAGTTAAGTGAGGCCCGCGCCCTATAGTGAGTCGTATTAT) were annealed and then transcribed to make a 60 nt antisense RNA that when annealed with 60 nt sense RNA can make imperfectly complementary 60 bp dsRNA (60-IMP).For analysis of dsRNA processing, 5 nM internally -32P-UTP labeled dsRNA was incubated in a 10 μL reaction containing 5μL of wheat germ extract, 100 μM GTP, 500 μM ATP, 10 mM creatine phosphate, 10 μg/mL creatine phosphokinase, 5 mM DTT, and 0.1 U/μL RNasin (Promega) at 25°C for 3 h. Reactions were stopped by the addition of 2× proteinase K buffer  sodium dodecyl sulfate) and deproteinized with ∼2 mg/mL proteinase K at 65°C for 15 min. Products were precipitated with 3 volumes cold ethanol and analyzed by electrophoresis in a 15% polyacrylamide gel. Radiolabelled size marker was prepared as discussed previously . The bloArabidopsis [Arabidopsis, or the difference in source tissue (cultured cells or inflorescence for Arabidopsis and germ for wheat). Interestingly, the ∼24 nt sRNA generating complex is much smaller at around 450 kDa . This prompted us to perform additional steps of purification before subjecting the crude extract S23 to gel filtration.Arabidopsis extracts with such activities, wheat germ derived extracts were stable through freeze-thaw cycles and were amenable to study in vitro requirements for DCL activities.A series of saturations with ammonium sulphate that is ideal in precipitating proteins was performed using S23 extract . The pelIt was shown previously that DCL activities require salt for their activity with an optimal range of 50–100 mM NaCl as reported for human Dicer  and planE. coli RNase III, Mg2+ and Ca2+ act to stabilize complex formation of the enzyme with the bacteriophage T7 R1.1 RNA, which has a hairpin structure [In tructure . Bindingtructure . Howevertructure . Other dtructure  but actiE. coli expressed DCL1 to cleave a miRNA precursor [Similar requirement of divalent cations for plant DCLs has not been explored for native complexes, however, Mg2+ is absolutely required for recursor . In orderecursor . In ordeArabidopsis also required ATP for activity but GTP also promoted DCL activity albeit at lower levels when compared to ATP [Human Dicer requires ATP for its activity . Recombid to ATP . Wheat gd to ATP . In orded to ATP  who showMost enzymatic reactions require specific pH and temperature and DCLs must have a specific requirement. It has been widely shown that plant PTGS is inhibited at low temperatures . HoweverSimilarly, at varying pH ranging between 4 and 9, crude extract as well as individual activities were incubated with radiolabelled substrate . The DCLA highly structured RNA is the source of miRNAs among plants. Sources of siRNAs are thought to be completely complementary RNAs of substantial length. In order to understand the substrate processing abilities of native DCL4 and DCL3 complexes, we incubated both perfectly complementary and imperfectly complementary RNAs with native complexes . A labelLength of the substrate may also affect processivity of Dicers and DCLs . IncubatArabidopsis have met with little success, especially because the plant DCL complexes are huge, low abundant, have many partners and their extracts are unstable [DCL proteins are RNAse III type nucleases that cleave structured RNA species to produce small RNAs. These small RNAs are incorporated into effectors called Argonautes to target RNAs that have complementarity with the small RNAs. Genetic data and observations made using virus infected plants showed that DCLs have specificity for their substrates. Genetic data also showed, based on some of the signatures of DCL activities in virus infected or stressed plants, that certain environmental factors influence activities of DCLs. Despite RNA silencing being discovered in plants, biochemical analyses of silencing players in plants such as unstable . Thus, munstable . Howeverin vivo conditions, in contrast to the behavior of recombinant proteins. Indeed, small RNA profiles observed in our assays match well with those found in plants under normal conditions as well as during stresses, such as temperature and cadmium stress. Complexes reconstituted from individual components may behave differently than the natural complete ones due to incomplete reconstitution or incomplete modification of the components and thus may provide erroneous results. For example, plant DCL1 expressed in E. coli can cleave a long RNA but the small RNAs generated from this activity did not have signatures of typical miRNAs indicating that isolated DCLs possess the ability to process various RNA substrates but are unlikely to match the precision of the complete DCL1 complex. However, when HYL1 (DRB1), identified as a partner required for accurate processing of long RNA, was provided, the activity became specific and matched in vivo results. We believe that our purified complexes match in vivo conditions perfectly. Our method using purified complexes is slightly better using crude extracts because crude extracts contain various additional proteins, unknown co-factors that may associate with DCLs to influence the biochemical requirements and substrate specificities. It has been shown for example that individual DRBs that are essential for distinctive DCL activities may also partner with other DCLs. Such a scenario is easily possible in crude extracts since there are opportunities to have unlimited number and amounts of proteins that may be natural or conditional partners.We used wheat germ derived purified native complexes of DCLs unlike most reported studies on Dicer activities and requirements. Our observations are likely applicable to other monocots and some dicots as well. DCL complexes used here are likely to have all co-factors and accessory factors necessary for their activity. This system is highly advantageous when compared to recombinant proteins expressed in artificial heterologous systems for at least two reasons. The protein behavior of wheat germ derived complexes is likely similar to Arabidopsis plants from 22°C to 30°C, transgene-induced PTGS becomes severely inhibited [trans-acting siRNAs produced by DCL4 also get inhibited at 30°C [In our assays we found some properties of DCL complexes correlating well with genetic and whole-genome analysis. These include restriction of optimal temperature for efficient silencing of viruses and production of endogenous siRNAs. In our assays, the activity of DCL4, a major antiviral protein, as well as a participant of transgene PTGS, is restricted to optimal conditions. It has negligible activity at 30°C and above. Indeed it has been shown that by moving nhibited . Concurr at 30°C . Our dat at 30°C . In our Substrate requirements for DCL3 in our assays have been surprising. DCL3 processes dsRNA substrates that have been thought to be completely complementary . HoweverFurther understanding of activities and structures of DCLs should elucidate how plant DCLs work in a more precise manner. Wheat germ system in the context of such details and identifying partner proteins need backing from genome sequence data, that is now available .S1 Fig(TIF)Click here for additional data file.S2 Figa) Gel filtration of 40% saturated ammonium sulfate pellet to remove unwanted proteins. Assay was carried out to purify DCL3 from DCL4 background. See b) Q-Sepharose chromatography to purify DCL3 complex from DCL4 background. Concentration of NaCl used for elution is mentioned.(TIF)Click here for additional data file.