RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

Lambert, Nicole; Robertson, Alex D.; Jangi, Mohini; McGeary, Sean E.; Sharp, Phillip A.; Burge, Christopher B.

doi:10.1016/j.molcel.2014.04.016

Cited by 374 publications

(481 citation statements)

References 46 publications

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“…The RBFOX motifs downstream of the CELF2-repressed exons are also more highly conserved than those around CELF2-unresponsive exons (Fig. 1F), which is an additional hallmark of functional relevance (Lambert et al 2014;Taliaferro et al 2016).…”

Section: Rbfox Motifs and Binding Are Enriched Downstream Of Celf2-rementioning

confidence: 97%

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

Gazzara¹,

Mallory

Roytenberg

et al. 2017

Genome Res.

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Over 95% of human multi-exon genes undergo alternative splicing, a process important in normal development and often dysregulated in disease. We sought to analyze the global splicing regulatory network of CELF2 in human T cells, a well-studied splicing regulator critical to T cell development and function. By integrating high-throughput sequencing data for binding and splicing quantification with sequence features and probabilistic splicing code models, we find evidence of splicing antagonism between CELF2 and the RBFOX family of splicing factors. We validate this functional antagonism through knockdown and overexpression experiments in human cells and find CELF2 represses RBFOX2 mRNA and protein levels. Because both families of proteins have been implicated in the development and maintenance of neuronal, muscle, and heart tissues, we analyzed publicly available data in these systems. Our analysis suggests global, antagonistic coregulation of splicing by the CELF and RBFOX proteins in mouse muscle and heart in several physiologically relevant targets, including proteins involved in calcium signaling and members of the MEF2 family of transcription factors. Importantly, a number of these coregulated events are aberrantly spliced in mouse models and human patients with diseases that affect these tissues, including heart failure, diabetes, or myotonic dystrophy. Finally, analysis of exons regulated by ancient CELF family homologs in chicken, Drosophila, and Caenorhabditis elegans suggests this antagonism is conserved throughout evolution.

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Section: Rbfox Motifs and Binding Are Enriched Downstream Of Celf2-rementioning

confidence: 97%

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

Gazzara¹,

Mallory

Roytenberg

et al. 2017

Genome Res.

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“…To further explore the potential of cPPRs to predictably target RNAs, we designed a cPPR that specifically bound the RNA recognition sequence of the human MBNL1 protein 30 ( Supplementary Fig. 4a,b).…”

Section: A Consensus Ppr Protein Scaffoldmentioning

confidence: 99%

An artificial PPR scaffold for programmable RNA recognition

et al. 2014

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Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism in eukaryotic cells. Although recent computational and structural studies have provided insights into RNA recognition by PPR proteins, their highly insoluble nature and inconsistencies between predicted and observed modes of RNA binding have restricted our understanding of their biological functions and their use as tools. Here we use a consensus design strategy to create artificial PPR domains that are structurally robust and can be programmed for sequence-specific RNA binding. The atomic structures of these artificial PPR domains elucidate the structural basis for their stability and modelling of RNA-protein interactions provides mechanistic insights into the importance of RNA-binding residues and suggests modes of PPR-RNA association. The modular mode of RNA binding by PPR proteins holds great promise for the engineering of new tools to target RNA and to understand the mechanisms of gene regulation by natural PPR proteins.

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“…For example, the dynamics of alternative splicing during postnatal heart development correlate with expression changes of many RBPs, including CUG-BP, Elavlike family member 1 (CELF1), Muscleblind-like 1 (MBNL1), and FOX proteins (8). Detailed biochemical studies have elucidated the mechanisms by which these splicing factors regulate splicing in a position-and context-dependent manner (9,10). The function of other RBPs during heart development has also been studied.…”

mentioning

confidence: 99%

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function

Beetz

O’Keeffe

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

106

118

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We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.T he expression of more than 95% of human genes is affected by alternative pre-mRNA splicing (AS) (1, 2). Differentially spliced isoforms play distinct roles in a temporally and spatially specific manner (3), and mutations that lead to aberrant splicing are the cause of many human genetic diseases (4). RNA-binding proteins (RBPs) play a central role in the regulation of alternative splicing during development and disease. They function primarily by positively or negatively regulating splice-site recognition by the spliceosome (1). Many RBPs are expressed in specific tissues, and AS is regulated by the combinatorial activities of these factors on specific pre-mRNAs through their interactions with distinct regulatory sequences in premRNA that function as splicing enhancers or silencers (5).The developing heart is one of the best studied systems where splicing changes occur during normal development, and mutations affecting specific splicing outcomes contribute to cardiomyopathy (6, 7). Although these mutations can either disrupt splicing elements or affect the expression of specific splicing factors, the latter mechanism is clearly responsible for the distinct splicing profiles at different developmental stages. For example, the dynamics of alternative splicing during postnatal heart development correlate with expression changes of many RBPs, including CUG-BP, Elavlike family member 1 (CELF1), Muscleblind-like 1 (MBNL1), and FOX proteins (8). Detailed biochemical studies have elucidated the mechanisms by which these splicing factors regulate splicing in a position-and context-dependent manner (9, 10). The function of other RBPs during heart development has also been studied. For example, two of the muscle-specific splicing factors, RBM20 and RBM24, play distinct roles in splicing regulation. RBM20 mainly acts as a splicing repressor, as its absence leads to multiple exon inclusion events in the heart. For example, the Titin gene is one of...

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RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

Cited by 374 publications

References 46 publications

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

An artificial PPR scaffold for programmable RNA recognition

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function

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