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Most transcriptome studies involve sequencing and quantification of steady-state mRNA by isolating and sequencing poly (A) RNA. Although this type of sequencing data is informative to determine steady-state mRNA levels, it does not provide information on transcriptional output and thus may not always reflect changes in transcriptional regulation of gene expression . Furthermore, sequencing poly (A) RNA may miss transcribed regions of the genome not usually modified by polyadenylation which includes many long non-coding RNAs including enhancer RNA (eRNA). Here, we describe nuclear RNA sequencing (nucRNA-seq) which investigates the transcriptional landscape through sequencing and quantification of nuclear RNAs which are both unspliced and spliced transcripts for protein-coding genes and nuclear-retained long non-coding RNAs.

Background Osteosarcoma (OS) is the most common malignant bone tumor and has a poor prognosis. The potential involvement of circular RNAs (circRNAs) in OS progression remains unexplored. Here, we report that CircECE1, a circular RNA derived from human ECE1, plays a critical role in energy metabolism in OS. Methods The RIP chip sequence assay was performed to confirm CircECE1, through overexpression or knockdown of CircECE1 to verify its function in 143B and U2OS. RNA immunoprecipitation and immunoprecipitation were used to verify CircECE1’s regulation of protein c-Myc and co- immunoprecipitation was used to verified the competitive binding relationship between CircECE1 and SPOP. The influence of CircECE1 on energy metabolism was evaluated by seahorse experiment, western blot, and immunohistochemistry. Results We found that CircECE1 is highly expressed in OS tissues and cells and that CircECE1 knockdown suppresses tumor proliferation and metastasis both in vitro and in vivo. Further, CircECE1 significantly promotes glucose metabolism in OS cells in vitro and in vivo. Mechanistically, CircECE1 interacts with c-Myc to prevent speckle-type POZ-mediated c-Myc ubiquitination and degradation. C-Myc inhibits thioredoxin binding protein (TXNIP) transcription and subsequently activates the Warburg effect. Conclusions CircECE1 regulates the Warburg effect through the c-Myc/TXNIP axis. CircECE1 mediated signal transduction plays a important role in OS process and energy metabolism. These findings may identify novel targets for OS molecular therapy.

Cancer cells can develop a strong addiction to discrete molecular regulators, which control the aberrant gene expression programs that drive and maintain the cancer phenotype. Here, we report the identification of the RNA-binding protein HuR/ELAVL1 as a central oncogenic driver for malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive sarcomas that originate from cells of the Schwann cell lineage. HuR was found to be highly elevated and bound to a multitude of cancer-associated transcripts in human MPNST samples. Accordingly, genetic and pharmacological inhibition of HuR had potent cytostatic and cytotoxic effects on tumor growth, and strongly suppressed metastatic capacity in vivo. Importantly, we linked the profound tumorigenic function of HuR to its ability to simultaneously regulate multiple essential oncogenic pathways in MPNST cells, including the Wnt/beta-catenin, YAP/TAZ, RB/E2F, and BET pathways, which converge on key transcriptional networks. Given the exceptional dependency of MPNST cells on HuR for survival, proliferation, and dissemination, we propose that HuR represents a promising therapeutic target for MPNST treatment. This work was funded by grants to AW from the Spanish Association Against Cancer (AECC; JP Vizcaya); Spanish Ministry of Science, Innovation and Universities (MCIU)/Agencia Estatal de Investigacion (AEI)/European Regional Development Fund (FEDER), European Union (EU) (Subprograma Ramon y Cajal RYC2010-06901; Proyectos Retos Investigacion RTI2018-097503-B-I00, SAF2015-65360-R; Proyectos Explora Ciencia SAF2015-72416-EXP; Proyectos Europa Excelencia SAF2015-62588-ERC); the BBVA Foundation; Basque Department of Industry, Tourism and Trade (Elkartek) and Education (PI2013-46); Fundacion Vasca de Innovacion e Investigacion Sanitarias EiTB Maratoia (BIO13/CI/015); the Neurofibromatosis Therapeutic Acceleration Program; and the European Research Council (consolidator grant under the EU's Horizon 2020 research and innovation programme; grant agreement 865157). MVR is grateful for the support of a 2017 Leonardo Grant for Researchers and Cultural Creators (BBVA Foundation), Accion Estrategica Ciber Emergentes 2018 (Ciberehd-ISCIII), and Gilead Sciences International Research Scholars Program in Liver Disease. The work of AC is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and Education (IKERTALDE IT1106-16); the BBVA Foundation; the MCIU/AEI (SAF2016-79381-R [FEDER/EU]; Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks SAF20168-1975-REDT); the European Training Networks Project (H2020-MSCA-ITN-308 2016 721532); the AECC (IDEAS175CARR, GCTRA18006CARR); La Caixa Foundation (HR17-00094); and the European Research Council (Starting Grant 336343, PoC 754627). MG was supported by the Intramural Research Program of the National Institute on Aging, NIH. MPI is grateful for the support of the Basque Government of Education fellowship. MTC is grateful for the support of "Ayudas para contratos predoctorales para la formacion de doctores" (MCIU/AEI/FEDER, EU). COH acknowledges funding from Brain Tumour Research. RB acknowledges MCIU/AEI/FEDER, EU (BFU2014-52282-P and BFU2017-84653-P). ES and CL are supported by the Carlos III National Health Institute funded by FEDER funds - a way to build Europe (CIBERONC) and the Government of Catalonia (2017SGR496). MLM-C acknowledges support from MCIU/AEI/FEDER, EU (SAF2017-87301-R); Asociacion Espanola Contra El Cancer (AECC); AECC Canceres Raros; BBVA Biomedicina (UMBRELLA); and La CAIXA Foundation (LCF/PR/HP17/52190004). AP was supported by Associazione Italiana Ricerca sul Cancro (AIRC; IG 2018 Id.21548). CIBERehd is funded by the Instituto de Salud Carlos III. We thank MCIU for the Severo Ochoa Excellence Accreditation (SEV-2016-0644).

CD4 T cell activation is a central component of the mammalian adaptive immune response and is underscored by a dramatic change in the gene expression profile in these cells. The changes in gene expression that occur during T cell activation are regulated in multiple ways including post-transcriptionally by complexes of RNA-binding proteins. Recently, our study explored the role of the RNA-binding protein U2AF2 and its interacting proteins in mediating posttranscriptional changes in constitutive and alternative splicing during T cell activation. First, we used RNA-seq to identify the global changes in gene expression and splicing that occur with T cell activation. Next, we used RIP-seq to identify the specific genes bound to U2AF2 during T cell activation. After identification of the protein interacting partners of U2AF2, we used splicing sensitive microarrays to measure the effects on global gene expression of using siRNAs to knock down a sampling of these proteins. Finally, we used RIP-chip to measure the effects of the same siRNA knockdown on the transcripts specifically bound to U2AF2. Here we provide the experimental details and analysis of the gene expression data for each of these techniques, which have been deposited into Gene Expression Omnibus (GEO) with the Superseries ID: GSE62923.

Background MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. Methods We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on miRNA binding sites, both in the 3’UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the enriched or underrepresented genes in the immunoprecipitated fractions, independently for AGO2 and GW182 related samples. Results For each of the two proteins, we trained and tested several support vector machine algorithms capable of distinguishing the enriched from the underrepresented genes that were experimentally detected. The most efficient algorithm for distinguishing the enriched genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3’UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for distinguishing the enriched genes in the GW182 immunoprecipitated samples was the length of the coding region. Conclusions Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC recruits genes based on miRNA binding sites in the 3’UTR and coding region, but only the longer mRNAs probably remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs. Electronic supplementary material The online version of this article (10.1186/s12859-019-2683-y) contains supplementary material, which is available to authorized users.

Plastiden enthalten ihr eigenes Genom, das u.a. für Untereinheiten des photosynthetischen Apparates kodiert. Die Expression dieses Apparates wird hauptsächlich posttranskriptionell reguliert. Dafür notwendige Faktoren sind vor allem RNA-Bindeproteine, welche fast ausschließlich kernkodiert und posttranslational in die Plastiden importiert werden. Dazu gehören auch die äußerst abundanten chloroplastidären Ribonukleoproteine (cpRNPs). Die bisher näher untersuchten Mitglieder der cpRNP-Familie aus Arabidopsis thaliana sind an der Prozessierung und Stabilisierung von plastidären Transkripten beteiligt und phylogenetisch eng miteinander verwandt. In dieser Studie wurden zwei noch unbekannte Mitglieder der cpRNP Familie, CP33A und CP33B, näher untersucht. CP33A ist ein essentielles Protein der Chloroplastenbiogenese. Mutanten von CP33A keimen nur in der Gegenwart einer externen Kohlenstoffquelle. Die Blätter sind albinotisch, in ihrer Struktur anomal und das gesamte Wachstum ist stark eingeschränkt. Untersuchungen der RNA-Interaktionspartner von CP33A durch RIP-Chip-Analysen (RNA-Immunopräzipitation und Chip-Hybridisierung) zeigen, dass CP33A mit allen mRNAs assoziiert. Des Weiteren führt der Verlust von CP33A zu einer starken Reduktion vieler Transkripte, vor allem RNAs, die durch die plastidär kodierte RNA Polymerase (PEP) transkribiert werden und unprozessierte Vorläufer-Transkripte. CP33B interagiert ebenfalls mit multiplen plastidären RNAs. Dabei zeigt CP33B eine Präferenz für psbA. Feinkartierung der CP33B-Bindung innerhalb des psbA Leserahmens verdeutlichten, dass CP33B vor allem mit dem 3´Ende des Transkriptes interagiert. Phänotypische und genetische Untersuchungen der cp33b-Nullmutante ließen keinen vom Wildtyp abweichenden Phänotyp identifizieren und zeigten dass CP33B keinen essentiellen Einfluss auf die Proteinakkumulation photosynthetischer Untereinheiten, die Expression plastidärer Transkripte, das Spleißen und die Edierung seiner Ziel-RNAs hat., Plastids harbour their own genome, which encodes for essential subunits of the photosynthetic apparatus. The expression of these subunits is mainly regulated on the posttranscriptional level. The important factors for posttranscriptional processing are RNA-binding proteins (RBPs), which are almost exclusively nuclear-encoded and imported posttranslational into the plastids. Among them are the chloroplast ribonucleoproteins (cpRNPs). The cpRNPs are a family of highly abundant RNA-binding proteins found in the chloroplast of land plants. Members of the Arabidopsis thaliana cpRNP family, that have been investigated in more detail, are involved in processing and stabilization of plastid transcripts and are phylogenetically closely related. In this study two unknown members of the cpRNPs, CP33A and CP33B, which cluster outside of this phylogenetic group, are investigated. CP33A is essential for chloroplast biogenesis. Null alleles of CP33A only germinate in the presence of an external carbon source. cp33a seedlings are albino, show strong growth inhibition and an abnormal leaf structure. Investigating RNA-ligands of CP33A using RIP-Chip (coimmunoprecipitation coupled to microarray analysis) shows an association with all chloroplast mRNAs. The loss of CP33A leads to a reduction of almost all transcripts, predominantly affecting RNAs transcribed by the plastid-encoded RNA polymerase (PEP) and unspliced and unprocessed precursor mRNAs. CP33B also interacts with multiple plastid RNAs. The main target is the mRNA of psbA. More than 90% of the stromal psbA mRNA is associated with CP33B. Fine mapping efforts suggest that CP33B preferentially interacts with the 3’-end of the psbA reading frame. Phenotypic and genetic analyses of cp33b-null mutants do not show any differences compared to wild-type plants. CP33B has no essential impact on: Protein accumulation of photosynthetic subunits, expression of plastid transcripts, RNA-splicing or RNA-editing of its target RNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the translational repression and degradation of target mRNAs in the cell [1]. The RNA-induced silencing complexes (RISCs) include mature miRNAs used as templates to recognize the complementary mRNA targets. Several prediction tools are available to predict miRNA targets, however, only a small part of the interaction pairs has been validated by experimental approaches. In addition, none of these tools does take into account the network structure of miRNA-mRNA interactions, which involves collaborative and competition [2] effects that are crucial to efficiently predict the miRNA regulatory effects in a specific cellular context. A first solution to consider collaborative effects has been given by the web tool ComiR [3], which predicts the targets of a weighted set of miRNAs, provided the miRNA expression profile of the samples/tissues of interest. The analysis of the expression profile of the RNA fraction immunoprecipitated (IP) with the RISC proteins, namely Rip-Chip or Rip-Seq, has been widely used to detect which genes are regulated by the RISC machinery. Recently, we found that AGO2 and GW182 are associated with two distinct sets of mRNA characterized by different features [4]. One of the relevant features is the presence of putative miRNA binding sites in the coding region of the mRNA, in addition to the already widely explored involvement of the binding sites in the 3'UTR. Next, we tested whether ComiR would be improved by the introduction of such features

We introduce RNA G-quadruplex sequencing (rG4-seq), a transcriptome-wide RNA G-quadruplex (rG4) profiling method that couples rG4-mediated reverse transcriptase stalling with next-generation sequencing. Using rG4-seq on polyadenylated-enriched HeLa RNA, we generated a global in vitro map of thousands of canonical and noncanonical rG4 structures. We characterize rG4 formation relative to cytosine content and alternative RNA structure stability, uncover rG4-dependent differences in RNA folding and show evolutionarily conserved enrichment in transcripts mediating RNA processing and stability.

RNA secondary structure and RNA-protein interactions are necessary for maintaining biological functionality and regulatory mechanisms within eukaryotic transcriptomes. Determining the structural characteristics and protein-bound sites of RNA molecules has therefore become a major research objective and requires the development of global methods for probing intra- and intermolecular RNA interaction sites. Sequencing RNAs treated with single-strand- and double-strand-specific ribonucleases in the absence of proteins allows the inference of RNA secondary structure. These samples can be compared to samples treated with nucleases in the presence of interacting proteins to identify protein-bound sequences. Thus, these four libraries reveal a comprehensive, transcriptome-wide view of RNA secondary structure and RNA protein interaction sites in a single experiment for any plant species of interest. © 2016 by John Wiley & Sons, Inc.

Many features of how gene transcription occurs in human cells remain unclear, mainly because of a lack of quantitative approaches to follow genome transcription with nucleotide precision in vivo. Here we present a robust genome-wide approach for studying RNA polymerase II (Pol II)-mediated transcription in human cells at single-nucleotide resolution by native elongating transcript sequencing (NET-seq). Elongating RNA polymerase and the associated nascent RNA are prepared by cell fractionation, avoiding immunoprecipitation or RNA labeling. The 3' ends of nascent RNAs are captured through barcode linker ligation and converted into a DNA sequencing library. The identity and abundance of the 3' ends are determined by high-throughput sequencing, which reveals the exact genomic locations of Pol II. Human NET-seq can be applied to the study of the full spectrum of Pol II transcriptional activities, including the production of unstable RNAs and transcriptional pausing. By using the protocol described here, a NET-seq library can be obtained from human cells in 5 d.

icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3–modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ~5 d, and the sequencing and bioinformatics data analysis take an additional 4–5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.

The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem–loop (SL) structure. This SL structure interacts with the stem–loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3′hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3′ end of RD-histone mRNAs together with SLBP.

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection–based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

We describe a method called META RNA profiling (for “modular early-tagged amplification”) that can quantify a broad panel of microRNAs or mRNAs simultaneously across many samples – and requires far less sequence depth than existing digital profiling technologies. The method assigns quantitative tags during reverse-transcription to permit up-front sample pooling before competitive amplification and deep sequencing. This simple, scalable, and inexpensive approach brings large-scale gene expression studies within more practical reach.

RNA-binding proteins (RBPs) play an important role in regulating gene expression at the posttranscriptional level, including the steps of pre-mRNA splicing, polyadenylation, mRNA stabilization, mRNA export from the nucleus to the cytoplasm, mRNA localization, and translation. RBPs regulate these processes primarily by binding to specific sequence elements in newly synthesized or mature transcripts. While many RPBs are known to recognize certain nucleotide sequences in RNA, information is insufficient for others. In particular, RBPs often compete for RNA binding or interact with RNA cooperatively. Hence, it is of importance to study the RNA-protein interactions in vivo. Numerous methods have been developed to identify the target nucleotide sequences of RBPs. The methods include the electrophoretic mobility shift assay (EMSA), systematic evolution of ligands by exponential enrichment (SELEX), RNA pull-down assay, RNA footprinting, RNA immunoprecipitation (RIP), UV-induced crosslinking immunoprecipitation (CLIP) and its variants, and measurement of the level for newly synthesized transcripts. Each of the methods has its limitation, and several methods supplementing each other should be employed in order to detect the RNA sequence to which a protein binds.

Long noncoding RNAs (lncRNAs) have important roles in shaping chromatin by targeting chromatin-modifying enzymes to distinct genomic sites. This section covers two methods to analyze lncRNA-protein interactions. The RNA-protein pull-down assays use either bead-bound proteins to capture in vitro transcripts, or immobilized synthetic RNAs to bind proteins from cell lysates. In the RNA immunoprecipitation (RIP) assay, endogenous RNAs are co-immunoprecipitated with a protein of interest. Both the methods can be applied to material from proliferating and quiescent cells, thus providing insights into how lncRNA-protein interactions are altered between these two cellular states.

Researchers have recently had a growing interest in understanding the functional role of long noncoding RNAs (lncRNAs) in chromatin organization. Accumulated evidence suggests lncRNAs could act as interphase molecules between chromatin and chromatin remodelers to define the epigenetic code. However, it is not clear how lncRNAs target chromatin remodelers to specific chromosomal regions in order to establish a functionally distinct epigenetic state of chromatin. We developed and optimized chromatin RNA immunoprecipitation (ChRIP) technology to characterize the lncRNAs associated with active and inactive chromatin compartments. Use of ChRIP to identify chromatin-bound lncRNA will further improve our knowledge regarding the functional role of lncRNAs in establishing epigenetic modifications of chromatin.

Chromatin immunoprecipitation (ChIP) has become the most effective and widely used tool to study the interactions between specific proteins or modified forms of proteins and a genomic DNA region. Combined with genome-wide profiling technologies, such as microarray hybridization (ChIP-on-chip) or massively parallel sequencing (ChIP-seq), ChIP could provide a genome-wide mapping of in vivo protein-DNA interactions in various organisms. Here, we describe a protocol of ChIP-on-chip that uses tiling microarray to obtain a genome-wide profiling of ChIPed DNA.

RNA-binding proteins (RBPs) mediate important co- and post-transcriptional gene regulation by binding pre-mRNA in a sequence- and/or structure-specific manner. For a comprehensive understanding of RBP function, transcriptome-wide mapping of the RNA-binding sites is essential, and CLIP-seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP-seq combines protein/RNA UV-crosslinking with immunoprecipitation (CLIP) followed by high-throughput sequencing of crosslinked RNA fragments. To overcome the limitations of low RNA-protein crosslinking efficiency in standard CLIP-seq, photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) has been developed. Here, living cells or whole organisms are fed photo-activatable nucleoside analogs that are incorporated into nascent RNA transcripts before UV treatment. This allows greater crosslinking efficiency at comparable radiation doses for enhanced RNA recovery and separation of crosslinked target RNA fragments from background RNA degradation products. Moreover, it facilitates the generation of specific UV-induced mutations that mark the crosslinking nucleotide and allow transcriptome-wide identification of RBP binding sites at single-nucleotide resolution. © by 2017 John Wiley & Sons, Inc.

High-throughput sequencing is subject to sequence dependent bias, which must be accounted for if researchers are to make precise measurements and draw accurate conclusions from their data. A widely studied source of bias in sequencing is the GC content bias, in which levels of GC content in a genomic region effect the number of reads produced during sequencing. Although some research has been performed on methods to correct for GC bias, there has been little effort to understand the underlying mechanism. The availability of sequencing protocols that target the specific location of structure in nucleic acid molecules enables us to investigate the underlying molecular origin of observed GC bias in sequencing. By applying a parallel analysis of RNA structure (PARS) protocol to bacterial genomes of varying GC content, we are able to observe the relationship between local RNA secondary structure and sequencing outcome, and to establish RNA secondary structure as the significant contributing factor to observed GC bias.

Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.

ARGONAUTE (AGO) proteins function in small RNA (sRNA)-based RNA silencing pathways to regulate gene expression and control invading nucleic acids. In posttranscriptional RNA silencing pathways, plant AGOs associate with sRNAs to interact with highly sequence-complementary target RNAs. Once the AGO–sRNA-target RNA ternary complex is formed, target RNA is typically repressed through AGO-mediated cleavage or through other cleavage-independent mechanisms. The universe of sRNAs associating with diverse plant AGOs has been determined though AGO immunoprecipitation (IP) and high-throughput sequencing of co-immunoprecipitated sRNAs. To better understand the biological functions of AGO–sRNA complexes, it is crucial to identify the repertoire of target RNAs they regulate. Here I present a detailed AGO–RNA IP followed by high-throughput sequencing (AGO RIP-Seq) methodology for the isolation of AGO ternary complexes from plant tissues and the high-throughput sequencing of AGO-bound target RNAs. In particular, the protocol describes the IP of slicer-deficient hemagglutinin (HA)-tagged AGO proteins expressed in plant tissues, the isolation of AGO-bound RNAs, and the generation of target RNA libraries for high-throughput sequencing., This work was supported by an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie grant agreement No. 655841 to A.C.

Salivary biomarkers for disease detection, diagnostic and prognostic assessments have become increasingly well established in recent years. Despite salivary transcriptomic analyses have created a new paradigm in the emerging field for non-invasive molecular diagnosis, other types of RNA, especially non-coding RNAs (ncRNAs), have been the main interest in the scientific community, due to their better stability in a cell-free context than mRNAs. In this chapter, we will overview the development of sensitive and robust technique for the discovery of salivary ncRNAs, such as RNA-sequencing (RNA-seq), technique applicable not only to find disease biomarkers in human saliva, but also to fully describe the landscape of extracellular RNAs (exRNAs) in saliva. RNA is isolated from cell-free saliva (CFS) and quality controls (QCs) have been done to evaluate exRNAs present in saliva from healthy donors. In this chapter we explain the current leading technology that has been used to characterize ncRNAs: HiSeq from Illumina platform for RNA sequencing. Therefore, the chapter is divided into two main sections regarding the type of the library (small and long ncRNA libraries) constructed, from RNA extraction and quantification to cDNA generation and corresponding QCs. Using these invaluable technical tools, one can identify thousands of ncRNA species in saliva. These methods indicate that salivary exRNA provides an efficient medium for biomarker discovery of oral and systemic diseases.

N 1-methyladenosine (m1A), N 3-methylcytidine (m3C), and N 1-methylguanosine (m1G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m1A, m3C, or m1G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

The generation of diverse cell types in multicellular organisms often requires the activity of cell-type-specific transcription factors. Understanding where these transcription factors bind in controlling specific cellular programs is critical. However, probing these cell-type-specific factors in vivo with standard chromatin immunoprecipitation (ChIP) assays remains a challenge. We have developed an optimized ChIP assay termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which improves ChIP sensitivity and allows the detection of cell-type-specific signals at a genome-wide scale. Here, I describe the procedure for implementing this method for the study of plant transcription factors. Besides being useful for cell-type-specific studies, MOBE-ChIP can also be employed as a general strategy for enhancing ChIP signals.

Brassinosteroids (BRs) play important roles in many growth and developmental processes. BRs signal to regulate BR-INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1) transcription factors (TFs), which, in turn, regulate several hundreds of transcription factors (termed BES1/BZR1-targeted TFs or BTFs) and thousands of genes to mediate various BR responses. Chromatin Immunoprecipitation followed by high-throughput sequencing (ChIP-seq) with BES1/BZR1 and BTFs is an important approach to identify BR target genes. In combination with RNA-sequencing experiments, these genomic methods have become powerful tools to detect BR target genes and reveal transcriptional networks underlying BR-regulated processes.

Recent work has shown that RNA polymerase II mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of non-coding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of non-coding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetics marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of non-coding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets.

RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of a RBP to specific regions of its target RNAs in adherent human cell lines.

Today, whole genomes are analyzed by Next-Generation Sequencing systems, or displayed by DNA microarray in situ synthesis. However, genomic data are mainly static and do not exactly reveal the complexity of protein interaction networks of any organism or cell. To investigate protein interactions, the generation of protein microarrays, displaying whole proteomes is a prerequisite. But traditional protein microarray generation is time consuming, costly, and is restricted by a wide range of technical difficulties concerning cell culturing, protein purification, and transfer onto microarray. Some of these obstacles can be bypassed by application of cell-free expression systems, enabling fast in situ synthesis of protein microarrays without need for cell culturing. This review provides a historical timeline of the different methods to generate protein microarrays by cell-free expression, highlights differences and similarities, and reports the current state of the different approaches.

Technological advances in the sequencing field support in-depth characterization of the transcriptome. Here, we review genome-wide RNA sequencing methods used to investigate specific aspects of gene expression and its regulation, from transcription to RNA processing and translation. We discuss tag-based methods for studying transcription, alternative initiation and polyadenylation events, shotgun methods for detection of alternative splicing, full-length RNA sequencing for the determination of complete transcript structures, and targeted methods for studying the process of transcription and translation. With the ensemble of technologies available, it is now possible to obtain a comprehensive view on transcriptome complexity and the regulation of transcript diversity.

We recently developed a novel transcriptome analysis method, termed 5'-bromo-uridine (BrU) immunoprecipitation chase-deep sequencing analysis (BRIC-seq). BRIC-seq enables the determination of genome-wide RNA stability by chasing chronological decreases of BrU-labeled RNAs under physiologically undisturbed conditions. The RNA half-life of each transcript is calculated from the decreasing number of BrU-labeled RNA sequence tags measured by deep sequencing of BrU-labeled RNAs. Here, we describe a detailed protocol and provide tips for BRIC-seq, followed by computational analysis.

High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA (small RNA-seq or sRNA-seq), such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias.

An emergent strategy for the transcriptome-wide study of protein-RNA interactions is CLIP-seq (crosslinking and immunoprecipitation followed by high-throughput sequencing). We combined CLIP-seq and mRNA-seq to identify direct RNA binding sites of eIF4AIII in human cells. This RNA helicase is a core constituant of the Exon Junction Complex (EJC), a multifunctional protein complex associated with spliced mRNAs in metazoans. Here, we describe the successive steps of the CLIP protocol and the computational tools and strategies we employed to map the physiological targets of eIF4AIII on human RNAs.

Eukaryotic gene expression is regulated in a combinatorial manner and on several interconnected layers, ranging from epigenetic mechanisms, transcription, RNA processing to protein stages. mRNA processing plays a major role in tissue- and development-dependent regulation, in particular alternative pre-mRNA splicing, which greatly enhances the capacity and composition of the proteome. Within the last decade, novel methods have been developed to systematically study the complex networks between regulatory alternative splicing factors and their RNA targets. This minireview focuses on cross-linking and immunoprecipitation methods, which – in combination with deep RNA sequencing – have made an important contribution in unraveling these networks.

The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 57 (ORF57)-encoded protein (Mta) is a multifunctional regulator of viral gene expression. ORF57 is essential for viral replication, so elucidation of its molecular mechanisms is important for understanding KSHV infection. ORF57 has been implicated in nearly every aspect of viral gene expression, including transcription, RNA stability, splicing, export, and translation. Here we demonstrate that ORF57 interacts with the KSHV K-bZIP protein in vitro and in cell extracts from lytically reactivated infected cells. To further test the biological relevance of the interaction, we performed a chromatin immunoprecipitation and microarray (ChIP-chip) analysis using anti-ORF57 antibodies and a KSHV tiling array. The results revealed four specific areas of enrichment, including the ORF4 and K8 (K-bZIP) promoters, as well as oriLyt, all of which interact with K-bZIP. In addition, ORF57 associated with DNA corresponding to the PAN RNA transcribed region, a known posttranscriptional target of ORF57. All of the peaks were RNase insensitive, demonstrating that ORF57 association with the viral genome is unlikely to be mediated exclusively by an RNA tether. Our data demonstrate that ORF57 associates with the viral genome by using at least two modes of recruitment, and they suggest that ORF57 and K-bZIP coregulate viral gene expression during lytic infection.

The relevance of microRNAs (miRNAs) in mammalian species was highlighted in the past decade. Inherent to the rapid advancements in the miRNA field was the growing need for applicable and validated experimental tools that enabled rapid progress from discovery to development of diagnostic and therapeutic applications. In the last few years, different technologies have been developed to measure miRNA expression, and one important challenge is making sense of miRNA profiles. In fact, technical and analytical aspects have substantial impact on subsequent biological interpretations and their translational applications. In the context of the workflow of the entire process of miRNA analysis, we will herein (i) consider some of the more relevant pre-analytical aspects such as study design, sample processing, and miRNA extraction, (ii) describe the available analytical tools for miRNA profiling (reverse transcription quantitative PCR, microarray hybridization, next-generation sequencing) and discuss their strengths and weaknesses, (iii) highlight the available postanalytical strategies for quality control, normalization, and statistical and bioinformatic analyses of miRNA profiling data, and (iv) summarize cross-platform comparison studies.

The proteins interaction with DNA is one of the key regulatory elements of many biological processes; including gene transcription, epigenetic modification or cell differentiation. Immunoprecipitation of chromatin; ChIP; is a method used to assess the interaction of the protein with a DNA sequence, and determines the localization of specific locus in the genome. The main steps of this method are fixation, sonication, immunoprecipitation and analysis of DNA. Although the immunoprecipitation assay is a multipurpose tool applied in biochemistry and biotechnology, it requires optimization. This paper describes several critical parameters that should be taken into account when immunoprecipitation assay is applied.

Identification and validation of microRNA (miRNA) target genes is essential for gaining a better understanding of the many different functions miRNAs have in healthy and diseased cells. From a practical standpoint, validated target genes are also useful for monitoring pharmacological activity of developmental therapeutics that modulate miRNAs, such as anti-miRNA oligonucleotides (anti-miR). Here, we describe a method that uses changes in Argonaute 2-RNA immunoprecipitation in response to competition by anti-miR, titrated ex vivo, as physical evidence for target validation.

High-throughput sequencing of RNA is used to analyze the transcriptomes of viruses and cells, providing information about transcript structure and abundance. A wide array of programs and pipelines has been created to manage and interpret the abundance of data generated from high-throughput RNA sequencing experiments. This protocol details the use of free and open-source programs to align RNA-Seq reads to a reference genome, visualize read coverage and splice junctions, estimate transcript abundance, and evaluate differential expression of transcripts in different conditions. Particular concerns related to EBV and viral transcriptomics are addressed and access to EBV reference files is provided.

In mammalian cells, DNA methylation at the 5-position of cytosine leads to recruitment of proteins that selectively recognize and bind 5-methylcytosine (5mC). Taking advantage of the structural identity of 5mC, various affinity purification-based protocols have been developed to enrich for either DNA that is modified by 5mC or proteins that recognize 5mC. In this protocol, an antibody against 5mC is used to immunoprecipitate the methylated DNA. The method can be scaled up to perform genome-wide DNA methylation analysis. Because immunoprecipitation is a straightforward procedure that does not require any prior modification of genomic DNA, we also describe several commercial kits available to perform the immunoprecipitation-based detection of DNA methylation.

Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure. PIP-seq utilizes single- and double-stranded RNA-specific nucleases in the absence of proteins to infer RNA secondary structure. These libraries are also compared to samples that undergo nuclease digestion in the presence of proteins in order to find enriched protein-bound sequences. Combined, these four libraries provide a comprehensive, transcriptome-wide view of RNA secondary structure and RNA protein interaction sites from a single experimental technique. © 2016 by John Wiley & Sons, Inc.

With the many advances in genome-wide sequencing, it has been discovered that much more of the genome is transcribed into RNA than previously appreciated. These nonprotein-coding RNAs (ncRNAs) come in many different forms, and they have been shown to have a variety of functions within the cell, influencing processes such as gene expression, mRNA splicing, and transport, just as a few examples. As we delve deeper into studying their mechanisms of action, it becomes important to understand how they play these roles, in particular by understanding what proteins these ncRNAs interact with. This protocol describes one technique that can be used to study this, ultra-violet light cross-linking RNA immunoprecipitation (UV-RIP), which uses an antibody to pull down a specific protein of interest and then detects RNA that is bound to it. This technique utilizes UV light to cross-link the cells, which takes advantage of the fact that UV light will only cross-link proteins and nucleic acids that are directly interacting. This approach can provide key mechanistic insight into the function of these newly identified ncRNAs.

The relevance of RNA-protein interactions in modulating mRNA and noncoding RNA function is increasingly appreciated and several methods have been recently developed to map them. The RNA immunoprecipitation (RIP) is a powerful method to study the physical association between individual proteins and RNA molecules in vivo. The basic principles of RIP are very similar to those of chromatin immunoprecipitation (ChIP), a largely used tool in the epigenetic field, but with some important caveats. The approach is based on the use of a specific antibody raised against the protein of interest to pull down the RNA-binding protein (RBP) and target-RNA complexes. Any RNA that is associated with this protein complex will also be isolated and can be further analyzed by polymerase chain reaction-based methods, hybridization, or sequencing.Several variants of this technique exist and can be divided into two main classes: native and cross-linked RNA immunoprecipitation. The native RIP allows to reveal the identity of RNAs directly bound by the protein and their abundance in the immunoprecipitated sample, while cross-linked RIP leads to precisely map the direct and indirect binding site of the RBP of interest to the RNA molecule.In this chapter both the protocols applied to mammalian cells are described taking into account the caveats and considerations required for designing, performing, and interpreting the results of these experiments.

We present a detailed protocol for the experimental identification of miRNA-target RNA interaction sites using cross-linking, ligation, and sequencing of hybrids (CLASH). The basis of the technique is the purification of UV-stabilized Argonaute (AGO)-RNA complexes assembled in living cells, with subsequent ligation of AGO-associated RNA-RNA duplexes to form chimeric RNAs. Following cDNA synthesis, DNA library preparation and high-throughput sequencing, interacting RNA molecules are unambiguously identified as chimeric reads in bioinformatic analysis of sequencing data. CLASH potentially recovers any RNA duplex that is bound by RNA-binding protein, so modified approaches would be suitable for the identification of many other inter- and intramolecular RNA-RNA interactions. Since CLASH analysis is independent of bioinformatic predictions it allows the identification and analysis of RNA targeting rules in an unbiased way.

The physical and biological activity of any organisms is mainly depended on the genetic information which stored in DNA. A process at which a gene gives rise to a phenotype is called as gene expression. Analysis of gene expression can be used to interpret the changes that occur at biological level of a stressed cell or tissue. Hybridization technology helps to study the gene expression of multiple cell at a same time. Among them microarray technology is a high- throughput technology to study the gene expression at transcription level (DNA) or translation level (Protein). Analysis the protein only can predict the accurate changes that happens in a tissue, when they are infected by a disease causing organisms. Protein microarray mainly used to identify the interactions and activities of proteins with other molecules, and to determine their function for a system at normal state and stressed state. The scope of this chapter is to outline a detail description on the fabrication, types, data analysis, and application of protein microarray technology towards gene expression profiling.

Recent genome-wide studies have yielded new insights into the biological function of long noncoding RNAs (lncRNAs), predominantly through analysis of their genomic addresses. These studies have revealed that a large number of lncRNAs map to regulatory elements in eukaryotic genome regions known as promoter and enhancer elements. Here, we review the principles of current methodologies for analyzing lncRNAs with high-throughput sequencing approaches. These include (1) direct RNA sequencing, (2) sequencing coupled with transcription, and (3) isolation of protein complexes associated with lncRNAs followed by high-throughput sequencing. Within these categories, we also describe detailed protocols for chromatin-associated RNA sequencing, nascent transcript Global run-on sequencing, and photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation.

Motivation: RIP-chip is a high-throughput method to identify mRNAs that are targeted by RNA-binding proteins. The protein of interest is immunoprecipitated, and the identity and relative amount of mRNA associated with it is measured on microarrays. Even if a variety of methods is available to analyse microarray data, e.g. to detect differentially regulated genes, the additional experimental steps in RIP-chip require specialized methods. Here, we focus on two aspects of RIP-chip data: First, the efficiency of the immunoprecipitation step performed in the RIP-chip protocol varies in between different experiments introducing bias not existing in standard microarray experiments. This requires an additional normalization step to compare different samples and even technical replicates. Second, in contrast to standard differential gene expression experiments, the distribution of measurements is not normal. We exploit this fact to define a set of biologically relevant genes in a statistically meaningful way. Results: Here, we propose two methods to analyse RIP-chip data: We model the measurement distribution as a gaussian mixture distribution, which allows us to compute false discovery rates (FDRs) for any cut-off. Thus, cut-offs can be chosen for any desired FDR. Furthermore, we use principal component analysis to determine the normalization factors necessary to remove immunoprecipitation bias. Both methods are evaluated on a large RIP-chip dataset measuring targets of Ago2, the major component of the microRNA guided RNA-induced silencing complex (RISC). Using published HITS-CLIP experiments performed with the same cell line as used for RIP-chip, we show that the mixture modelling approach is a necessary step to remove background, which computed FDRs are valid, and that the additional normalization is a necessary step to make experiments comparable. Availability: An R implementation of REA is available on the project website (http://www.bio.ifi.lmu.de/REA) and as supplementary data file. Contact: florian.erhard@bio.ifi.lmu.de Supplementary information: Supplementary data are available at Bioinformatics online.

Functional protein microarray is an important tool for high-throughput and large-scale systems biology studies. Besides the progresses that have been made for protein microarray fabrication, significant advancements have also been achieved for applying protein microarrays on determining a variety of protein biochemical activities. Among these applications, detection of protein binding properties, such as protein-protein interactions (PPIs), protein-DNA interactions (PDIs), protein-RNA interactions, and antigen-antibody interactions, are straightforward and have substantial impacts on many research fields. In this review, we will focus on the recent progresses in protein-protein, protein-DNA, protein-RNA, protein-small molecule, protein-lipid, protein-glycan, and antigen-antibody interactions. We will also discuss the challenges and future directions of protein microarray technologies. We strongly believe that protein microarrays will soon become an indispensible tool for both basic research and clinical applications.

Analysis of mature microRNA (miRNA) expression is important to understand its physiological functions and pathological implications. Microarray is a powerful technology to profile global miRNA expression. In this study, we developed a rapid miRNA labeling method by which miRNA was directly labeled in total RNA for microarray detection. This method consists of RNA tailing by poly(A) polymerase, reverse transcription, and template-switching by moloney murine leukemia virus (MMLV) reverse transcriptase. After these reactions, the small RNA cDNA was ready for labeling for microarray detection. The whole process of prearray sample preparation was dramatically shortened to 2 h. Furthermore, this method allows very limited starting total RNA (100 ng) for microarray analysis. Our data showed that the results from our method were highly consistent with that of real-time polymerase chain reaction (PCR).

Long noncoding RNAs (lncRNAs) represent a new frontier in molecular genetics and molecular biology. They have a tremendous potential for advancing our comprehensive understanding of biological processes in huma n health and disease. The transcripts of lncRNAs are easy to find, but sorting out what they do remains the biggest challenge in lncRNAs' research field. In the paper, we highlight recent progress regarding the methods to explore the roles of lncRNAs.