Comparative and integrative analysis of RNA structural profiling data: current practices and emerging questions

Choudhary, Krishna; Deng, Feiqi; Aviran, Sharon

doi:10.1007/s40484-017-0093-6

Cited by 44 publications

(76 citation statements)

References 232 publications

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“…To decouple the effect of the modification from the underlying reverse transcriptase background for every construct assayed, we employed a signal-to-noise analysis per position on the SHAPE transcript that is normally referred to as "reactivity" (12, 34-39) (see Supplementary Fig. 3 and associated discussion for other definitions of the reactivity (38,40,41)). In our version of this observable, we first compute the ratio of the modified to unmodified read counts at each nucleotide, and subsequently subtract one from the result (in similar manner to (41); this and other methods for reactivity calculation have been reviewed by (38)).…”

Section: In Vitro Shape-seq Reveal An Extended Protected Region By Pcpmentioning

confidence: 99%

Anin vivobinding assay for RNA-binding proteins based on repression of a reporter gene

Katz

Kaufmann

Cohen

et al. 2017

Preprint

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We employ a reporter assay and Selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Qβ, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wideswath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation.Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo. thought to be highly dynamic, and is dependent on many factors such as temperature, cellular RNAbinding protein (RBP) content, presence or absence of translating ribosomes, and interaction with other RNA molecules (3). Thus, a typical RBP-RNA interaction is likely to depend not only on the presence of a specific binding sequence, but also on many other factors.In bacteria, post-transcriptional regulation has been studied extensively in recent decades.There are well-documented examples of RBPs that either inhibit or directly compete with ribosome binding via a variety of mechanisms. These include direct competition with the 30S ribosomal subunit for binding via single stranded recognition (4), entrapment of the 30S subunit in an inactive complex via a nested pseudoknot structure (5) and ribosome assembly inhibition when the RBP is bound to a structured RBP binding site, or hairpin (6-9). These hairpins have been studied in three distinct positions: either immediately downstream and including the AUG (7), upstream of the Shine-Dalgarno sequence (8), or as structures that entrap Shine-Dalgarno motifs, as is the case for the PP7 and MS2 phage coat-protein binding sites. There is also a well-characterized example of translation stimulation:binding of the phage Com RBP was shown to destabilize a sequestered ribosome binding site (RBS) of the Mu phage mom gene, thereby facilitating translation (10, 11). While these studies indicate a richness of RBP-RNA-based regulatory mechanisms, a systematic understanding of the relationship between RBP binding, sequence specificity, the underlying secondary and tertiary RNA structure, and the resultant regulatory output is still lacking.In recent years, advances in next generation ...

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Section: In Vitro Shape-seq Reveal An Extended Protected Region By Pcpmentioning

confidence: 99%

Anin vivobinding assay for RNA-binding proteins based on repression of a reporter gene

Katz

Kaufmann

Cohen

et al. 2017

Preprint

View full text Add to dashboard Cite

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“…We used Bowtie-2 [53] with the settings recommended by [54] (options -trim5=3, -N=1). Transcripts with poor read coverage tend to bias towards zero-valued reactivities [55]. To mediate this bias, low information content profiles with less than one percent non-zero reactivities were excluded.…”

Section: Datamentioning

confidence: 99%

Empowering the annotation and discovery of structured RNAs with scalable and accessible integrative clustering

Miladi

Sokhoyan

Houwaart

et al. 2019

Preprint

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RNA plays essential roles in all known forms of life. Clustering RNA sequences with common sequence and structure is an essential step towards studying RNA function. With the advent of high-throughput sequencing (HTS) techniques, experimental and genomic data are expanding to complement the predictive methods. However, the existing methods do not effectively utilize and cope with the immense amount of data becoming available. Hundreds of thousands of non-coding RNAs (ncRNAs) have been detected, however, the annotation of these ncRNAs is lacking behind. Here we present GraphClust2, a comprehensive approach for scalable clustering of RNAs based on sequence and structural similarities. GraphClust2 bridges the gap between HTS and structural RNA analysis, and provides an integrative solution by incorporating diverse experimental and genomic data in an accessible manner via the Galaxy framework. GraphClust2 can efficiently cluster and annotate large datasets of RNAs and supports structure probing data. We demonstrate that the annotation performance of clustering functional RNAs can be considerably improved. Furthermore, an off-the-shelf procedure is introduced for identifying locally conserved structure candidates in long RNAs. We suggest the presence and the sparsity of phylogenetically conserved local structures for a collection of long non-coding RNAs. By clustering data from two CLIP experiments, we demonstrate the benefits of GraphClust2 for motif discovery under the presence of biological and

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“…Reverse transcription is simultaneously applied to an 116 untreated sample of the RNA. One way to determine a reactivity value per nucleotide is 117 to compute the difference between the modification rates per-site on the reagent-treated 118 and control samples [39,40]. The reactivity resulting from this background-subtraction 119 is a measure of the nucleotide's sensitivity towards the reagent and correlates with the 120 local backbone flexibility [41].…”

Section: Overview Of Shape Experiments and Reactivity Reconstruction 108mentioning

confidence: 99%

Extracting information from RNA SHAPE data: Kalman filtering approach

Vaziri

Koehl

Aviran

2018

Preprint

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RNA SHAPE experiments have become important and successful sources of information for RNA structure prediction. In such experiments, chemical reagents are used to probe RNA backbone flexibility at the nucleotide level, which in turn provides information on base pairing and therefore secondary structure. Little is known, however, about the statistics of such SHAPE data. In this work, we explore different representations of noise in SHAPE data and propose a statistically sound framework for extracting reliable reactivity information from multiple SHAPE replicates. Our analyses of RNA SHAPE experiments underscore that a normal noise model is not adequate to represent their data. We propose instead a log-normal representation of noise and discuss its relevance.Under this assumption, we observe that processing simulated SHAPE data by directly averaging different replicates leads to bias. Such bias can be reduced by analyzing the data following a log transformation, either by log-averaging or Kalman filtering. Application of Kalman filtering has the additional advantage that a prior on the nucleotide reactivities can be introduced. We show that the performance of Kalman filtering is then directly dependent on the quality of that prior. We conclude the paper with guidelines on signal processing of RNA SHAPE data. September 4, 2018 1/31 Introduction 1 Beyond its role in protein synthesis and the transfer of genetic information, RNA exists 2 as a dynamic cellular component at the core of gene regulation [1]. From microRNAs 3 involved in regulating gene expression [2] and long noncoding RNAs similarly regulating 4 gene expression [3] to ribozymes acting as chemical catalysts [4], RNA plays a central 5 role in a multitude of cellular activities. The diverse repertoire of biological functions 6 that RNAs adopt is deeply rooted in their abilities to form complex three-dimensional 7 structures [1]. This interplay between structure and function underscores the need for 8 robust structural analysis as a prerequisite to a full understanding of the physiological 9 role of RNA [5]. Despite its importance, determining the complex 3D structures of RNA 10 remains a challenging problem, particularly for longer RNAs [6, 7]. 11 Considering the hierarchical nature of RNA folding [8], much of the efforts in 12 structure determination have been devoted to its two-dimensional base-pairing pattern, 13 also known as its secondary structure. This secondary structure is generally considered 14 to be more stable than and independent of the final 3D conformation [8]. Though 15 experimental methods such as nuclear magnetic resonance (NMR) [9] and 16 crystallography [10] can be used to accurately resolve 3D RNA structures, they are 17 time-consuming, expensive, and often preclude the analysis of long or flexible 18 molecules [11]. Comparative sequence analysis, the process of inferring base-pairing 19from co-variations observed in the alignment of homologous sequences, is a robust 20 method for defining the secondary structure of RNA [11,12]. Howeve...

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Comparative and integrative analysis of RNA structural profiling data: current practices and emerging questions

Cited by 44 publications

References 232 publications

Anin vivobinding assay for RNA-binding proteins based on repression of a reporter gene

Anin vivobinding assay for RNA-binding proteins based on repression of a reporter gene

Empowering the annotation and discovery of structured RNAs with scalable and accessible integrative clustering

Extracting information from RNA SHAPE data: Kalman filtering approach

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