Experimental and Computational Considerations in the Study of RNA-Binding Protein-RNA Interactions

Nostrand, Eric L. Van; Huelga, Stephanie C.; Yeo, G

doi:10.1007/978-3-319-29073-7_1

Cited by 16 publications

(17 citation statements)

References 82 publications

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“…(3) How do we define, measure, or experimentally validate functional binding sites? Orthogonal assays to measure RBP function can address these questions . For example, a splicing map can be generated from an analysis of alternative splicing to compare binding location relative to exons that are alternatively spliced upon knockdown of a protein of interest .…”

Section: The Next Step: Identification Of Functional Binding Sitesmentioning

confidence: 99%

“…Orthogonal assays to measure RBP function can address these questions. 48 For example, a splicing map can be generated from an analysis of alternative splicing to compare binding location relative to exons that are alternatively spliced upon knockdown of a protein of interest. 49 Emerging highthroughput screening techniques using CRISPR genome editing, tethering, 50 and other methods to assay RNA processing allows for the manipulation of specific binding sites to evaluate function.…”

Section: The Next Step: Identification Of Functional Binding Sitesmentioning

confidence: 99%

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Advances and challenges in the detection of transcriptome‐wide protein–RNA interactions

2017

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RNA binding proteins (RBPs) play key roles in determining cellular behavior by manipulating the processing of target RNAs. Robust methods are required to detect the numerous binding sites of RBPs across the transcriptome. RNA‐immunoprecipitation followed by sequencing (RIP‐seq) and crosslinking followed by immunoprecipitation and sequencing (CLIP‐seq) are state‐of‐the‐art methods used to identify the RNA targets and specific binding sites of RBPs. Historically, CLIP methods have been confounded with challenges such as the requirement for tens of millions of cells per experiment, low RNA yields resulting in libraries that contain a high number of polymerase chain reaction duplicated reads, and technical inconveniences such as radioactive labeling of RNAs. However, recent improvements in the recovery of bound RNAs and the efficiency of converting isolated RNAs into a library for sequencing have enhanced our ability to perform the experiment at scale, from less starting material than has previously been possible, and resulting in high quality datasets for the confident identification of protein binding sites. These, along with additional improvements to protein capture, removal of nonspecific signals, and methods to isolate noncanonical RBP targets have revolutionized the study of RNA processing regulation, and reveal a promising future for mapping the human protein‐RNA regulatory network. WIREs RNA 2018, 9:e1436. doi: 10.1002/wrna.1436This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA RecognitionRNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional ImplicationsRNA Methods > RNA Analyses in Cells

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Section: The Next Step: Identification Of Functional Binding Sitesmentioning

confidence: 99%

Section: The Next Step: Identification Of Functional Binding Sitesmentioning

confidence: 99%

Advances and challenges in the detection of transcriptome‐wide protein–RNA interactions

2017

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“…Since then, dozens of long-range RNA structures that modulate pre-mRNA splicing have been confirmed experimentally [27,28]. The use of highthroughput assays for large-scale profiling of intronic pre-mRNA structure is still limited because the intronic signal is often reduced or missing, which is also a common problem in other methods [30]. Computational identification of long-range RNA structure therefore remains a great challenge in RNA biology.Comparative genomics provides a powerful alternative to de novo RNA folding by detecting signatures of evolutionary covariation [31,32].…”

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confidence: 99%

Conserved long-range base pairings are associated with pre-mRNA processing of human genes

Kalmykova

Kalinina

Denisov

et al. 2020

Preprint

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The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. While DNA employs it for genome replication, RNA molecules fold into complicated secondary and tertiary structures. Current knowledge on functional RNA structures in human protein-coding genes is focused on locally-occurring base pairs. However, chemical crosslinking and proximity ligation experiments have demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved long-range RNA structures in the human transcriptome, which consists of 1.1 million pairs of conserved complementary regions (PCCRs). PCCRs tend to occur within introns proximally to splice sites, suppress intervening exons, circumscribe circular RNAs, and exert an obstructive effect on cryptic and inactive splice sites. The double-stranded structure of PCCRs is supported by a significant decrease of icSHAPE nucleotide accessibility, high abundance of A-to-I RNA editing sites, and frequent nearby occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNA Pol II slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. Additionally, transcript starts and ends are strongly enriched in regions between complementary parts of PCCRs, leading to an intriguing hypothesis that RNA folding coupled with splicing could mediate co-transcriptional suppression of premature cleavage and polyadenylation events. PCCR detection procedure is highly sensitive with respect to bona fide validated RNA structures at the expense of having a high false positive rate, which cannot be reduced without loss of sensitivity.The catalog of PCCRs is visualized through a UCSC Genome Browser track hub to facilitate further genome research on long-range RNA structures.Double-stranded structure is a key feature of nucleic acids that enables replicating the genomic information and underlies fundamental cellular processes [1,2]. Many RNAs adopt functional secondary structures, and mRNAs are no exception although their main role is to encode proteins [3,4,5,6]. In eukaryotes, RNA structure affects gene expression through modulating all steps of pre-mRNA processing including splicing [7], cleavage and polyadenylation [8], and RNA editing [9]. The loss of functional RNA structure has been increasingly reported as implicated in hereditary disease and cancer [10,11,12,13].Recent progress in high-throughput sequencing techniques enabled several experimental strategies to determine RNA structure in vivo [14,15,16]. Chemical RNA structure probing can reveal which bases are single-or double-stranded, but it cannot determine which nucleotides form base pairs [17,18,19,20,21].In order to identify the interacting partners, RNA structure probing has to be combined with de novo RNA structure prediction [22], but due to a number of technical limitations such methods cannot account for base pairings that are far apart [23]. Photo-inducible RNA crosslinking a...

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“…Since there are no commercial antibodies available for fly mettl4, we generated a Drosophila KC cell line with a FLAG-tagged mettl4 for the eCLIP-seq experiment [7]. In total, we generated two biological replicates for immunoprecipitation (IP ) samples, and their respective input samples, together with one IP-control and Input-control sample for the quality control and enrichment analysis [8]. The two replicates showed a strong correlation with a Spearman correlation coefficient of 0.97, indicating great consistency between the replicates (Fig.S3).…”

Section: Identification Of Potential Substrates By Eclip-seqmentioning

confidence: 99%

CG14906 (mettl4) mediates m6A methylation of U2 snRNA inDrosophila

Wang

Chen

et al. 2020

Preprint

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Recent studies reported that METTL4 regulates DNA 6mA in vivo and therefore is a candidate DNA m6A methyltransfease. However, the enzymatic activity of METTL4 in vitro has not been demonstrated in part due to t he difficulties of obtaining well-folded proteins. Here we show that mettl4 is a major methyltransfase responsible for m6A methylation of U2 snRNA both in vitro and in vivo in fly, and identify adenosine at 29th position as the site of m6A methylation. This study answered a long-standing question regarding the enzymatic activity of METTL4, and thus paved the way for further investigating the functions of METTL4 in different biological settings.

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Experimental and Computational Considerations in the Study of RNA-Binding Protein-RNA Interactions

Cited by 16 publications

References 82 publications

Advances and challenges in the detection of transcriptome‐wide protein–RNA interactions

Advances and challenges in the detection of transcriptome‐wide protein–RNA interactions

Conserved long-range base pairings are associated with pre-mRNA processing of human genes

CG14906 (mettl4) mediates m6A methylation of U2 snRNA inDrosophila

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