RNA Mapping Research Articles

Evolution of RNA-Based Networks.

RNA molecules have served for decades as a paradigmatic example of molecular evolution that is tractable both in in vitro experiments and in detailed computer simulation. The adaptation of RNA sequences to external selection pressures is well studied and well understood. The de novo innovation or optimization of RNA aptamers and riboswitches in SELEX experiments serves as a case in point. Likewise, fitness landscapes building upon the efficiently computable RNA secondary structures have been a key toward understanding realistic fitness landscapes. Much less is known, however, on models in which multiple RNAs interact with each other, thus actively influencing the selection pressures acting on them. From a computational perspective, RNA-RNA interactions can be dealt with by same basic methods as the folding of a single RNA molecule, although many details become more complicated. RNA-RNA interactions are frequently employed in cellular regulation networks, e.g., as miRNA bases mRNA silencing or in the modulation of bacterial mRNAs by small, often highly structured sRNAs. In this chapter, we summarize the key features of networks of replicators. We highlight the differences between quasispecies-like models describing templates copied by an external replicase and hypercycle similar to autocatalytic replicators. Two aspects are of importance: the dynamics of selection within a population, usually described by conventional dynamical systems, and the evolution of replicating species in the space of chemical types. Product inhibition plays a key role in modulating selection dynamics from survival of the fittest to extinction of unfittest. The sequence evolution of replicators is rather well understood as approximate optimization in a fitness landscape for templates that is shaped by the sequence-structure map of RNA. Some of the properties of this map, in particular shape space covering and extensive neutral networks, give rise to evolutionary patterns such as drift-like motion in sequence space, akin to the behavior of RNA quasispecies. In contrast, very little is known about the influence of sequence-structure maps on autocatalytic replication systems.

Genome-Wide Mapping and Subtype Classification of Long Non-Coding RNA in Acute Myeloid Leukemia

Background: Most efforts to characterize acute myeloid leukemia (AML) have focused so far on genetic and epigenetic aberrations, which can ultimately lead to altered protein-coding gene function. The roles of long non-coding RNAs (lncRNAs), which orchestrate cell physiology and act as key regulators of the AML oncogenic state, remain uncharacterized globally.Material and Methods: We performed a genomic analysis of GENCODE lncRNAs in 179 clinically annotated cases of de novo AML, using The Cancer Genome Atlas (TCGA) RNA-Seq data. In addition, we described global correlations between lncRNAs and the expression of cis- and trans-acting genes. We also established lncRNA-based subtype classification based on distinct signatures and then correlated that classification with fusion transcripts and genetic alterations.Results: Using stringent criteria (RPKM ≥1 in at least 10% of AML), we identified 2,913 expressed lncRNAs and used an integrative analysis to predict those that are potential regulators of AML oncogenic state. The expression of 1,935 (66.4%) lncRNAs showed positive correlations with the mRNA expression of their neighboring genes, while only 14 (0.4%) of the lncRNAs showed negative correlations. Gene ontology analysis using GREAT revealed enrichment of cis-neighboring genes in the PML body gene set (p=8.2x10-7). Unsupervised clustering of lncRNA-based expression showed five robust molecular clusters (C1 to C5), which were highly correlated with the mRNA-based classification. Of those, three clusters (C1, C2 and C5) were tightly associated with recurrent fusion transcripts; cluster C1 (n=16) was composed exclusively of promyelocytic leukemias, while cluster C5 (n=45) was enriched for MLL-rearranged cases (24.4%), and cluster C2 (n=31) was enriched for MYH11-CBFB or RUNX1-RUNX1T1 (55.2%) rearranged cases. Importantly, cluster C4 (n=30), which includes cytogenetically normal leukemias, was highly enriched for NPM1 (p=2.3x10-11) and FLT3 (p=1.6x10-4) mutations; conversely, cluster C3 (n=53) was highly enriched for recurrent copy-number alterations as well as RUNX1 (p=0.001) and TP53 somatic mutations (p=0.004). We further discovered a core of 37 lncRNAs significantly associated with a MLL-signature and 16 lncRNAs with a NPM1-mutated signature.Conclusion: This study describes the first genome-wide mapping and characterization of lncRNAs in AML and proposes a robust lncRNA-based classification. This classification may serve in defining core lncRNAs that orchestrate key oncogenic states in the different clinical subtypes. DisclosuresNo relevant conflicts of interest to declare.

A genome-wide map of hyper-edited RNA reveals numerous new sites.

Adenosine-to-inosine editing is one of the most frequent post-transcriptional modifications, manifested as A-to-G mismatches when comparing RNA sequences with their source DNA. Recently, a number of RNA-seq data sets have been screened for the presence of A-to-G editing, and hundreds of thousands of editing sites identified. Here we show that existing screens missed the majority of sites by ignoring reads with excessive (‘hyper’) editing that do not easily align to the genome. We show that careful alignment and examination of the unmapped reads in RNA-seq studies reveal numerous new sites, usually many more than originally discovered, and in precisely those regions that are most heavily edited. Specifically, we discover 327,096 new editing sites in the heavily studied Illumina Human BodyMap data and more than double the number of detected sites in several published screens. We also identify thousands of new sites in mouse, rat, opossum and fly. Our results establish that hyper-editing events account for the majority of editing sites.

Genome-wide Mapping and Characterization of Notch-Regulated Long Noncoding RNAs in Acute Leukemia

Notch signaling is a key developmental pathway that is subject to frequent genetic and epigenetic perturbations in many different human tumors. Here we investigate whether long noncoding RNA (lncRNA) genes, in addition to mRNAs, are key downstream targets of oncogenic Notch1 in human Tcell acute lymphoblastic leukemia (T-ALL). By integrating transcriptome profiles with chromatin state maps, we have uncovered many previously unreported T-ALL-specific lncRNA genes, a fraction of which are directly controlled by the Notch1/Rpbjκ activator complex. Finally we have shown that one specific Notch-regulated lncRNA, LUNAR1, is required for efficient T-ALL growth invitro and invivo due to its ability to enhance IGF1R mRNA expression and sustain IGF1 signaling. These results confirm that lncRNAs are important downstream targets of the Notch signaling pathway, and additionally they are key regulators of the oncogenic state in T-ALL.

Standardization of RNA Chemical Mapping Experiments

Chemical mapping experiments offer powerful information about RNA structure but currently involve ad hoc assumptions in data processing. We show that simple dilutions, referencing standards (GAGUA hairpins), and HiTRACE/MAPseeker analysis allow rigorous overmodification correction, background subtraction, and normalization for electrophoretic data and a ligation bias correction needed for accurate deep sequencing data. Comparisons across six noncoding RNAs stringently test the proposed standardization of dimethyl sulfate (DMS), 2′-OH acylation (SHAPE), and carbodiimide measurements. Identification of new signatures for extrahelical bulges and DMS “hot spot” pockets (including tRNA A58, methylated in vivo) illustrates the utility and necessity of standardization for quantitative RNA mapping.

Comparative Chromosome Mapping of U2 snRNA and 5S rRNA Genes in Gymnotus Species (Gymnotiformes, Gymnotidae): Evolutionary Dynamics and Sex Chromosome Linkage in G. pantanal

A comparative mapping of U2 small nuclear RNA (snRNA) and 5S ribosomal RNA (rRNA) genes was performed in 6 Gymnotus species. All species analyzed presented the U2 snDNA organized in conspicuous blocks and not co-located with rRNA genes. In addition, 5 species showed the U2 snDNA located in a single pair of chromosomes, which seems to be a conserved trait in this genus. Conversely, G. pantanal was the only species displaying several terminal signals in different chromosome pairs, including the X₁ sex chromosome but not the Y chromosome. This is the first report of U2 snRNA genes in sex chromosomes of fishes. The absence of sites in the Y chromosome of G. pantanal indicates a possible loss of terminal segments of the chromosomes involved in the Y formation.

Single nucleotide mapping of RNA 5' and 3' ends.

Nuclease protection assay is a sensitive method for detection, quantitation, and mapping of a specific RNA in an extremely heterogeneous mixture of RNAs, such as total cellular RNA. The assay is based on a small volume solution hybridization of a single-stranded synthetic antisense and labeled RNA probe to a RNA sample. Thus, it is much more efficient than the common immobilized hybridization on a membrane, such as in northern-blot analysis. After solution hybridization, different nucleases are used to remove any remaining single-stranded nucleotides within the probe and sample RNA by digestion. Then, the remaining probe-target hybrids are purified and separated on a denaturing polyacrylamide gel. Using a radioactive labeled probe, the protected probe can be visualized by direct autoradiography and the copy number can be calculated based on the specific radioactivity of the RNA probe and the length of protected fragment. Because of its high sensitivity and resolution, nuclease protection assay is the most effective procedure for mapping internal and external boundaries in mRNA compared to other RNA detection methods such as RT-PCR.

Rapid mapping of RNA 3' and 5' ends.

In recent years, an enormous progress has been made in applied genomics leading to identification and isolation of novel cDNAs. However, most attempts result in the acquisition of transcribed sequences that represent only a part of the mRNA's complete sequence. Rapid Amplification of cDNA Ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. Since the first report of this technique, many significant improvements have been made on the basic approach. This chapter describes the most recent update of the relatively simple and versatile classic RACE protocol.

MISIS: A bioinformatics tool to view and analyze maps of small RNAs derived from viruses and genomic loci generating multiple small RNAs

In eukaryotes, diverse small RNA (sRNA) populations including miRNAs, siRNAs and piRNAs regulate gene expression and repress transposons, transgenes and viruses. Functional sRNAs are associated with effector proteins based on their size and nucleotide composition. The sRNA populations are currently analyzed by deep sequencing that generates millions of reads which are then mapped to a reference sequence or database. Here we developed a tool called MISIS to view and analyze sRNA maps of genomic loci and viruses which spawn multiple sRNAs. MISIS displays sRNA reads as a histogram where the x-axis indicates positions of the 5′- or 3′-terminal nucleotide of sense and antisense sRNAs, respectively, along a given reference sequence or its selected region and the y-axis the number of reads starting (for sense sRNA) or ending (for antisense sRNA) at each position. Size-classes of sRNAs can be visualized and compared separately or in combination. Thus, MISIS gives an overview of sRNA distribution along the reference sequence as well as detailed information on single sRNA species of different size-classes and abundances. MISIS reads standard BAM/SAM files outputted by mapping tools and generates table files containing counts of sRNA reads at each position of the reference sequence forward and reverse strand and for each of the chosen size-classes of sRNAs. These table files can be used by other tools such as Excel for further quantitative analysis and visualization. MISIS is a Java standalone program. It is freely available along with the source code at the following website: http://www.fasteris.com/apps.

RAMPAGE: Promoter Activity Profiling by Paired‐End Sequencing of 5′‐Complete cDNAs

RNA annotation and mapping of promoters for analysis of gene expression (RAMPAGE) is a method that harnesses highly specific sequencing of 5'-complete complementary DNAs to identify transcription start sites (TSSs) genome-wide. Although TSS mapping has historically relied on detection of 5'-complete cDNAs, current genome-wide approaches typically have limited specificity and provide only scarce information regarding transcript structure. RAMPAGE allows for highly stringent selection of 5'-complete molecules, thus allowing base-resolution TSS identification with a high signal-to-noise ratio. Paired-end sequencing of medium-length cDNAs yields transcript structure information that is essential to interpreting the relationship of TSSs to annotated genes and transcripts. As opposed to standard RNA-seq, RAMPAGE explicitly yields accurate and highly reproducible expression level estimates for individual promoters. Moreover, this approach offers a streamlined 2- to 3-day protocol that is optimized for extensive sample multiplexing, and is therefore adapted for large-scale projects. This method has been applied successfully to human and Drosophila samples, and in principle should be applicable to any eukaryotic system.

Allelic DNA Methylation Analysis in <italic>Arabidopsis</italic> Hybrids Associated with siRNA

Recently, accumulating evidence suggests that epigenetic factors including DNA methylation, small RNA and histone modifications may play important roles in hybrid vigor in plants. However, knowledge about the mechanism of these epigenetic factors in hybrids is still limited. Based on genome-wide DNA methylation and small RNA maps of <italic>Arabidopsis thaliana</italic> Ler and C24 ecotypes and their reciprocal F₁ hybrids in previous work, we further analyzed allelic specific DNA methylation. Our study indicated that both <italic>trans</italic>- and<italic> cis</italic>-factors play roles in DNA methylation regulations in hybrids. In addition, we found that siRNAs correlate with DNA methylation <italic>trans</italic>-regulations. High degree <italic>trans</italic>-regulation DNA methylation is significantly concurrent with enriched siRNA regions. Our study analyzed DNA methylation regulations associated with siRNAs in hybrids, and shed light on understanding the mechanism of hybrid vigor.

In-gel probing of individual RNA conformers within a mixed population reveals a dimerization structural switch in the HIV-1 leader

Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2′OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR–17–5p, and miR–20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

HiTRACE-Web: an online tool for robust analysis of high-throughput capillary electrophoresis.

To facilitate the analysis of large-scale high-throughput capillary electrophoresis data, we previously proposed a suite of efficient analysis software named HiTRACE (High Throughput Robust Analysis of Capillary Electrophoresis). HiTRACE has been used extensively for quantitating data from RNA and DNA structure mapping experiments, including mutate-and-map contact inference, chromatin footprinting, the Eterna RNA design project and other high-throughput applications. However, HiTRACE is based on a suite of command-line MATLAB scripts that requires nontrivial efforts to learn, use and extend. Here, we present HiTRACE-Web, an online version of HiTRACE that includes standard features previously available in the command-line version and additional features such as automated band annotation and flexible adjustment of annotations, all via a user-friendly environment. By making use of parallelization, the on-line workflow is also faster than software implementations available to most users on their local computers. Free access: http://hitrace.org.

Visiting “Noncodarnia”

Visiting “Noncodarnia”

Small RNA sequencing of Potato leafroll virus-infected plants reveals an additional subgenomic RNA encoding a sequence-specific RNA-binding protein

Potato leafroll virus (PLRV) is a positive-strand RNA virus that generates subgenomic RNAs (sgRNA) for expression of 3′ proximal genes. Small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs revealed coverage of the entire viral genome with the exception of four distinctive gaps. Remarkably, these gaps mapped to areas of PLRV genome with extensive secondary structures, such as the internal ribosome entry site and 5′ transcriptional start site of sgRNA1 and sgRNA2. The last gap mapped to ∼500nt from the 3′ terminus of PLRV genome and suggested the possible presence of an additional sgRNA for PLRV. Quantitative real-time PCR and northern blot analysis confirmed the expression of sgRNA3 and subsequent analyses placed its 5′ transcriptional start site at position 5347 of PLRV genome. A regulatory role is proposed for the PLRV sgRNA3 as it encodes for an RNA-binding protein with specificity to the 5′ of PLRV genomic RNA.

Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars

Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.

Fast-Forward Genetics Identifies Plant CPL Phosphatases as Regulators of miRNA Processing Factor HYL1

MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.

From specific to global analysis of posttranscriptional regulation in eukaryotes: posttranscriptional regulatory networks

Regulation of gene expression occurs at several levels in eukaryotic organisms and is a highly controlled process. Although RNAs have been traditionally viewed as passive molecules in the pathway from transcription to translation, there is mounting evidence that their metabolism is controlled by a class of proteins called RNA-binding proteins (RBPs), as well as a number of small RNAs. In this review, I provide an overview of the recent developments in our understanding of the repertoire of RBPs across diverse model systems, and discuss the computational and experimental approaches currently available for the construction of posttranscriptional networks governed by them. I also present an overview of the different roles played by RBPs in the cellular context, based on their cis-regulatory modules identified in the literature and discuss how their interplay can result in the dynamic, spatial and tissue-specific expression maps of RNAs. I finally present the concept of posttranscriptional network of RBPs and their cognate RNA targets and discuss their cross-talk with other important posttranscriptional regulatory molecules such as microRNAs s, resulting in diverse functional network motifs. I argue that with rapid developments in the genome-wide elucidation of posttranscriptional networks it would not only be possible to gain a deeper understanding of regulation at a level that has been under-appreciated in the past, but would also allow us to use the newly developed high-throughput approaches to interrogate the prevalence of these phenomena in different states, and thereby study their relevance to physiology and disease across organisms.

An RNA Mapping DataBase for curating RNA structure mapping experiments

We have established an RNA mapping database (RMDB) to enable structural, thermodynamic and kinetic comparisons across single-nucleotide-resolution RNA structure mapping experiments. The volume of structure mapping data has greatly increased since the development of high-throughput sequencing techniques, accelerated software pipelines and large-scale mutagenesis. For scientists wishing to infer relationships between RNA sequence/structure and these mapping data, there is a need for a database that is curated, tagged with error estimates and interfaced with tools for sharing, visualization, search and meta-analysis. Through its on-line front-end, the RMDB allows users to explore single-nucleotide-resolution mapping data in heat-map, bar-graph and colored secondary structure graphics; to leverage these data to generate secondary structure hypotheses; and to download the data in standardized and computer-friendly files, including the RDAT and community-consensus SNRNASM formats. At the time of writing, the database houses 53 entries, describing more than 2848 experiments of 1098 RNA constructs in several solution conditions and is growing rapidly. Freely available on the web at http://rmdb.stanford.edu. rhiju@stanford.edu. Supplementary data are available at Bioinformatics Online.