Allele-specific binding of RNA-binding proteins reveals functional genetic variants in the RNA

Yang, Ei-Wen; Bahn, Jae Hoon; Hsiao, Esther Yun-Hua; Tan, Boon Xin; Sun, Yiwei; Fu, Ting; Zhou, Bo; Nostrand, Eric L. Van; Pratt, Gabriel A.; Freese, Peter; Wei, Xintao; Quinones-Valdez, Giovanni; Urban, Alexander; Graveley, Brenton R.; Burge, Christopher B.; Yeo, G; Xiao, Xinshu

doi:10.1038/s41467-019-09292-w

Cited by 42 publications

(51 citation statements)

References 54 publications

(71 reference statements)

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“…The ENCODE RNA binding protein resource contains 1,223 replicated datasets for 356 RBPs, including in vivo targets by eCLIP, in vitro binding motifs by RNA Bind-N-Seq, subcellular localization by immunofluorescence, factor-responsive expression and splicing changes by knockdown/RNA-seq, and DNA associations by ChIP-seq [20]. This unique resource has already proven useful in characterizing allele-specific RBP interactions [51,52], identify candidate regulators of miRNA processing [53], predicting whether RNAs are protein-coding or non-coding [54], and identifying novel factors which act to suppress improper RNA processing caused by retrotransposable elements [36], and will continue to enable researchers to ask broad questions about basic RNA processing mechanisms, deeply consider the functional roles of an individual RBP, or even query an RNA of interest in order to gain insight into potential regulators. Here we describe examples how integrated analyses of binding profiles obtained from eCLIP can yield novel insights into both processing of standard mRNAs as well as other RNA families, including identifying new characteristics of ribosomal RNA processing and the role of RBP interactions with retrotransposable elements.…”

Section: Discussionmentioning

confidence: 99%

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

Nostrand

Pratt

Yee

et al. 2019

Preprint

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AbstractA critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enabled mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale integrated analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the large-scale mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types. Here we describe our analysis of 223 enhanced (eCLIP) datasets characterizing 150 RBPs in K562 and HepG2 cell lines, revealing a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multi-copy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation (enabling identification of branch points with single-nucleotide resolution) and provides genome-wide validation for a branch point-based scanning model for 3’ splice site recognition. Further, we show that eCLIP peak co-occurrences across RBPs enables the discovery of novel co-interacting RBPs. Finally, we present a protocol for visualization of RBP:RNA complexes in the eCLIP workflow using biotin and standard chemiluminescent visualization reagents, enabling simplified confirmation of ribonucleoprotein enrichment without radioactivity. This work illustrates the value of integrated analysis across eCLIP profiling of RBPs with widely distinct functions to reveal novel RNA biology. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

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Section: Discussionmentioning

confidence: 99%

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

Nostrand

Pratt

Yee

et al. 2019

Preprint

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“…225 We further investigated whether allele-specific RBP binding (ASB) was occurring specifically at sQTLs. 226 We obtained a set of ASB variants identified in the ENCODE eCLIP dataset using BEAPR (Binding Estima-227 tion of Allele-specific Protein-RNA interaction) 38 39 . Indeed, the SNP rs9876026 ( Fig.…”

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confidence: 99%

“…Selected covariates were regressed out from the relative abundances of each gene's transcript 35 isoforms by sQTLseekeR2 before testing for association with the genotype. 36 Software 37 For sQTL mapping we employed sQTLseekeR2 v1.0.0, an enhanced version (see also Supplementary 38 Note 1) of the sQTLseekeR R package 20 , which identifies genetic variants that are associated with multi- 39 variate changes in the relative abundances of a gene's transcript isoforms (i.e. splicing ratios).…”

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confidence: 99%

“…Of these, for 12 RBPs with significantly different |deltaSVM| values 235 between sQTLs and non-sQTLs (Wilcoxon Rank-Sum test, FDR < 0.1), we obtained the 100 highest-236 21 scoring 10-mers, aligned them using mafft v7.407 (high accuracy mode L-INS-1) 82 , removed the columns 237 of the alignment with more than 50% of gaps and built sequence logos using WebLogo standalone v3.6.0 238 (http://weblogo.threeplusone.com/). 239 To evaluate allele-specific RBP binding (ASB), we obtained the ASB variants identified in the same 240 eCLIP dataset using BEAPR (Binding Estimation of Allele-specific Protein-RNA interaction), available from 241 Yang et al 38 . In short, BEAPR is a method to identify ASB events in protein-RNA interactions from eCLIP 242 data.…”

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confidence: 99%

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Identification and analysis of splicing quantitative trait loci across multiple tissues in the human genome

Garrido-Martín

Borsari

Calvo

et al. 2020

Preprint

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We have developed an efficient and reproducible pipeline for the discovery of genetic variants affecting splicing (sQTLs), based on an approach that captures the intrinsically multivariate nature of this phenomenon. We employed it to analyze the multi-tissue transcriptome GTEx dataset, generating a comprehensive catalogue of sQTLs in the human genome. A core set of these sQTLs is shared across multiple tissues. Downstream analyses of this catalogue contribute to the understanding of the mechanisms underlying splicing regulation. We found that sQTLs often target the global splicing pattern of genes, rather than individual splicing events. Many of them also affect gene expression, but not always of the same gene, potentially uncovering regulatory loci that act on different genes through different mechanisms. sQTLs tend to be preferentially located in introns that are post-transcriptionally spliced, which would act as hotspots for splicing regulation. While many variants affect splicing patterns by directly altering the sequence of splice sites, many more modify the binding of RNA-binding proteins (RBPs) to target sequences within the transcripts. Genetic variants affecting splicing can have a phenotypic impact comparable or even stronger than variants affecting expression, with those that alter RBP binding playing a prominent role in disease. 33 splice sites, many more modify the binding of RNA-binding proteins (RBPs) to target sequences within 34 the transcripts. We have observed that sQTLs often impact GWAS traits and diseases more than variants 35 affecting only gene expression, confirming earlier reports which suggest that splicing mutations underlie 36 many hereditary diseases 15,19 . For many conditions, GWAS associations are particularly strong for sQTLs 37 3 altering RBP binding sites. 40 For sQTL mapping, we developed sQTLseekeR2, a software based on sQTLseekeR 20 , which identi-41 fies genetic variants associated with changes in the relative abundances of the transcript isoforms of a 42 given gene. sQTLseekeR uses the Hellinger distance to estimate the variability of isoform abundances 43 across observations, and Anderson's method 21,22 , a non-parametric analogue to multivariate analysis 44 of variance, to assess the significance of the associations (see Methods and Supplementary Note 1). 45 Among other enhancements, sQTLseekeR2 improves the accuracy and speed of the p value calcula-46 tion, and allows to account for additional covariates before testing for association with the genotype, 47 while maintaining the multivariate statistical test in sQTLseekeR. It also implements a multiple testing 48 correction scheme that empirically characterizes, for each gene, the distribution of p values expected un-49 der the null hypothesis of no association (see Methods and Supplementary Note 1). To ensure highly 50 parallel, portable and reproducible sQTL mapping, we embedded sQTLseekeR2 in a Nextflow 23 (plus 51 Docker, https://www.docker.com/) computational workflow named sqtlseeker2-nf, available at 52 https://gi...

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“…Furthermore, 31 putative functional SNPs were testable for allele-specific binding (ASB) using the ENCODE eCLIP data in our previous study 40 , 18 (58%) of which had significant ASB supporting their functional roles. To experimentally confirm the ASB patterns, we carried out electrophoretic mobility shift assays (EMSA, or gel shift) for BUD13, the protein with the largest number of eCLIP peaks overlapping putative functional SNPs ( Supplementary Fig.…”

Section: Experimental Support Of Functional Snps For Gmasmentioning

confidence: 99%

Allele-specific alternative splicing in human tissues

Amoah¹,

Hsiao²,

Bahn

et al. 2020

Preprint

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Alternative splicing is an RNA processing mechanism that affects most genes in human, contributing to disease mechanisms and phenotypic diversity. The regulation of splicing involves an intricate network of cis-regulatory elements and trans-acting factors. Due to their high sequence specificity, cis-regulation of splicing can be altered by genetic variants, significantly affecting splicing outcomes. Recently, multiple methods have been applied to understanding the regulatory effects of genetic variants on splicing. However, it is still challenging to go beyond apparent association to pinpoint functional variants. To fill in this gap, we utilized large-scale datasets of the Genotype-Tissue Expression (GTEx) project to study genetically-modulated alternative splicing (GMAS) via identification of allele-specific splicing events. We demonstrate that GMAS events are shared across tissues and individuals more often than expected by chance, consistent with their genetically driven nature. Moreover, although the allelic bias of GMAS exons varies across samples, the degree of variation is similar across tissues vs. individuals. Thus, genetic background drives the GMAS pattern to a similar degree as tissue-specific splicing mechanisms. Leveraging the genetically driven nature of GMAS, we developed a new method to predict functional splicing-altering variants, built upon a genotype-phenotype concordance model across samples. Complemented by experimental validations, this method predicted >1000 functional variants, many of which may alter RNA-protein interactions. Lastly, 72% of GMASassociated SNPs were in linkage disequilibrium with GWAS-reported SNPs, and such association was enriched in tissues of relevance for specific traits/diseases. Our study enables a comprehensive view of genetically driven splicing variations in human tissues.

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Allele-specific binding of RNA-binding proteins reveals functional genetic variants in the RNA

Cited by 42 publications

References 54 publications

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

Identification and analysis of splicing quantitative trait loci across multiple tissues in the human genome

Allele-specific alternative splicing in human tissues

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