A Deep Boosting Based Approach for Capturing the Sequence Binding Preferences of RNA-Binding Proteins from High-Throughput CLIP-Seq Data

AbstractCharacterizing the binding behaviors of RNA-binding proteins (RBPs) is important for understanding their functional roles in gene expression regulation. However, current high-throughput experimental methods for identifying RBP targets, such as CLIP-seq and RNAcompete, usually suffer from the false positive and false negative issues. Here, we develop a deep boosting based machine learning approach, called DeBooster, to accurately model the binding sequence preferences and identify the corresponding binding targets of RBPs from CLIP-seq data. Comprehensive validation tests have shown that DeBooster can outperform other state-of-the-art approaches in predicting RBP targets and recover false negatives that are common in current CLIP-seq data. In addition, we have demonstrated several new potential applications of DeBooster in understanding the regulatory functions of RBPs, including the binding effects of the RNA helicase MOV10 on mRNA degradation, the influence of different binding behaviors of the ADAR proteins on RNA editing, as well as the antagonizing effect of RBP binding on miRNA repression. Moreover, DeBooster may provide an effective index to investigate the effect of pathogenic mutations in RBP binding sites, especially those related to splicing events. We expect that DeBooster will be widely applied to analyze large-scale CLIP-seq experimental data and can provide a practically useful tool for novel biological discoveries in understanding the regulatory mechanisms of RBPs.

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Large scale interaction analysis of RNA binding proteins/LncRNAs to identify lncRNA nuclear localization mechanisms

10.26226/morressier.5ebd45acffea6f735881b191 ◽

2020 ◽

Author(s):

Yile Huang

Keyword(s):

Nuclear Localization ◽

Large Scale ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Interaction Analysis ◽

Scale Interaction

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The Role of RNA-Binding Proteins in Vertebrate Neural Crest and Craniofacial Development

Journal of Developmental Biology ◽

10.3390/jdb9030034 ◽

2021 ◽

Vol 9 (3) ◽

pp. 34

Author(s):

Thomas E. Forman ◽

Brenna J. C. Dennison ◽

Katherine A. Fantauzzo

Keyword(s):

Gene Expression ◽

Neural Crest ◽

Binding Proteins ◽

Rna Binding ◽

Gene Expression Regulation ◽

Rna Binding Proteins ◽

Craniofacial Development ◽

Specific Sequence ◽

Altered Expression ◽

Binding Domains

Cranial neural crest (NC) cells delaminate from the neural folds in the forebrain to the hindbrain during mammalian embryogenesis and migrate into the frontonasal prominence and pharyngeal arches. These cells generate the bone and cartilage of the frontonasal skeleton, among other diverse derivatives. RNA-binding proteins (RBPs) have emerged as critical regulators of NC and craniofacial development in mammals. Conventional RBPs bind to specific sequence and/or structural motifs in a target RNA via one or more RNA-binding domains to regulate multiple aspects of RNA metabolism and ultimately affect gene expression. In this review, we discuss the roles of RBPs other than core spliceosome components during human and mouse NC and craniofacial development. Where applicable, we review data on these same RBPs from additional vertebrate species, including chicken, Xenopus and zebrafish models. Knockdown or ablation of several RBPs discussed here results in altered expression of transcripts encoding components of developmental signaling pathways, as well as reduced cell proliferation and/or increased cell death, indicating that these are common mechanisms contributing to the observed phenotypes. The study of these proteins offers a relatively untapped opportunity to provide significant insight into the mechanisms underlying gene expression regulation during craniofacial morphogenesis.

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Functional characterization of splicing regulatory elements

10.1101/2021.05.14.444228 ◽

2021 ◽

Author(s):

Scott I Adamson ◽

Lijun Zhan ◽

Brenton R Graveley

Keyword(s):

High Throughput ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Functional Characterization ◽

Regulatory Elements ◽

Small Subset ◽

General Sequence ◽

Splicing Regulators ◽

Splicing Regulatory Elements

Background: RNA binding protein-RNA interactions mediate a variety of processes including pre-mRNA splicing, translation, decay, polyadenylation and many others. Previous high-throughput studies have characterized general sequence features associated with increased and decreased splicing of certain exons, but these studies are limited by not knowing the mechanisms, and in particular, the mediating RNA binding proteins, underlying these associations. Results: Here we utilize ENCODE data from diverse data modalities to identify functional splicing regulatory elements and their associated RNA binding proteins. We identify features which make splicing events more sensitive to depletion of RNA binding proteins, as well as which RNA binding proteins act as splicing regulators sensitive to depletion. To analyze the sequence determinants underlying RBP-RNA interactions impacting splicing, we assay tens of thousands of sequence variants in a high-throughput splicing reporter called Vex-seq and confirm a small subset in their endogenous loci using CRISPR base editors. Finally, we leverage other large transcriptomic datasets to confirm the importance of RNA binding proteins which we designed experiments around and identify additional RBPs which may act as additional splicing regulators of the exons studied. Conclusions: This study identifies sequence and other features underlying splicing regulation mediated specific RNA binding proteins, as well as validates and identifies other potentially important regulators of splicing in other large transcriptomic datasets.

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Roles of Organellar RNA-Binding Proteins in Plant Growth, Development, and Abiotic Stress Responses

International Journal of Molecular Sciences ◽

10.3390/ijms21124548 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4548 ◽

Cited By ~ 1

Author(s):

Kwanuk Lee ◽

Hunseung Kang

Keyword(s):

Abiotic Stress ◽

Plant Growth ◽

Stress Responses ◽

Translational Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Metabolism ◽

Functional Roles ◽

Growth Development

Organellar gene expression (OGE) in chloroplasts and mitochondria is primarily modulated at post-transcriptional levels, including RNA processing, intron splicing, RNA stability, editing, and translational control. Nucleus-encoded Chloroplast or Mitochondrial RNA-Binding Proteins (nCMRBPs) are key regulatory factors that are crucial for the fine-tuned regulation of post-transcriptional RNA metabolism in organelles. Although the functional roles of nCMRBPs have been studied in plants, their cellular and physiological functions remain largely unknown. Nevertheless, existing studies that have characterized the functions of nCMRBP families, such as chloroplast ribosome maturation and splicing domain (CRM) proteins, pentatricopeptide repeat (PPR) proteins, DEAD-Box RNA helicase (DBRH) proteins, and S1-domain containing proteins (SDPs), have begun to shed light on the role of nCMRBPs in plant growth, development, and stress responses. Here, we review the latest research developments regarding the functional roles of organellar RBPs in RNA metabolism during growth, development, and abiotic stress responses in plants.

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First Identification of RNA-Binding Proteins That Regulate Alternative Exons in the Dystrophin Gene

International Journal of Molecular Sciences ◽

10.3390/ijms21207803 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7803

Author(s):

Julie Miro ◽

Anne-Laure Bougé ◽

Eva Murauer ◽

Emmanuelle Beyne ◽

Dylan Da Cunha ◽

...

Keyword(s):

Large Scale ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Dystrophin Gene ◽

Rna Seq ◽

Spatio Temporal ◽

Dmd Gene ◽

Shed Light

The Duchenne muscular dystrophy (DMD) gene has a complex expression pattern regulated by multiple tissue-specific promoters and by alternative splicing (AS) of the resulting transcripts. Here, we used an RNAi-based approach coupled with DMD-targeted RNA-seq to identify RNA-binding proteins (RBPs) that regulate splicing of its skeletal muscle isoform (Dp427m) in a human muscular cell line. A total of 16 RBPs comprising the major regulators of muscle-specific splicing events were tested. We show that distinct combinations of RBPs maintain the correct inclusion in the Dp427m of exons that undergo spatio-temporal AS in other dystrophin isoforms. In particular, our findings revealed the complex networks of RBPs contributing to the splicing of the two short DMD exons 71 and 78, the inclusion of exon 78 in the adult Dp427m isoform being crucial for muscle function. Among the RBPs tested, QKI and DDX5/DDX17 proteins are important determinants of DMD exon inclusion. This is the first large-scale study to determine which RBP proteins act on the physiological splicing of the DMD gene. Our data shed light on molecular mechanisms contributing to the expression of the different dystrophin isoforms, which could be influenced by a change in the function or expression level of the identified RBPs.

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High-throughput mutagenesis identifies mutations and RNA binding proteins controlling CD19 splicing and CART-19 therapy resistance

10.1101/2021.10.08.463671 ◽

2021 ◽

Author(s):

Mariela Cortés-López ◽

Laura Schulz ◽

Mihaela Enculescu ◽

Claudia Paret ◽

Bea Spiekermann ◽

...

Keyword(s):

High Throughput ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Single Point ◽

Point Mutations ◽

Therapy Resistance ◽

Regulatory Elements ◽

Splice Isoforms ◽

Exon 2

During CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to enhanced CD19 mis-splicing. Our dataset represents a comprehensive resource for potential prognostic factors predicting success of CART-19 therapy.

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Functional Roles of RNA-Binding Proteins in Plant Signaling

Life ◽

10.3390/life10110288 ◽

2020 ◽

Vol 10 (11) ◽

pp. 288

Author(s):

Victor Muleya ◽

Claudius Marondedze

Keyword(s):

Stress Responses ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Similarity ◽

Plant Signaling ◽

Affinity Capture ◽

Functional Roles ◽

Binding Domains ◽

Mrna Targets

RNA-binding proteins (RBPs) are typical proteins that bind RNA through single or multiple RNA-binding domains (RBDs). These proteins have a functional role in determining the fate or function of the bound RNAs. A few hundred RBPs were known through in silico prediction based on computational assignment informed by structural similarity and the presence of classical RBDs. However, RBPs lacking such conventional RBDs were omitted. Owing to the recent mRNA interactome capture technology based on UV-crosslinking and fixing proteins to their mRNA targets followed by affinity capture purification and identification of RBPs by tandem mass spectrometry, several hundreds of RBPs have recently been discovered. These proteome-wide studies have colossally increased the number of proteins implicated in RNA binding and unearthed hundreds of novel RBPs lacking classical RBDs, such as proteins involved in intermediary metabolism. These discoveries provide wide insights into the post-transcriptional gene regulation players and their role in plant signaling, such as environmental stress conditions. In this review, novel discoveries of RBPs are explored, particularly on the evolving knowledge of their role in stress responses. The molecular functions of these RBPs, particularly focusing on those that do not have classical RBDs, are also elucidated at the systems level.

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The increasing diversity and complexity of the RNA-binding protein repertoire in plants

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2020.1397 ◽

2020 ◽

Vol 287 (1935) ◽

pp. 20201397 ◽

Cited By ~ 1

Author(s):

C. Marondedze

Keyword(s):

Stress Responses ◽

Large Scale ◽

Rna Binding ◽

Rna Binding Proteins ◽

Functional Roles ◽

Binding Domains ◽

Quantitative Studies ◽

Current State ◽

Rna Interaction ◽

Post Transcriptional Regulation

Post-transcriptional regulation has far-reaching implications on the fate of RNAs. It is gaining increasing momentum as a critical component in adjusting global cellular transcript levels during development and in response to environmental stresses. In this process, RNA-binding proteins (RBPs) are indispensable chaperones that naturally bind RNA via one or multiple globular RNA-binding domains (RBDs) changing the function or fate of the bound RNAs. Despite the technical challenges faced in plants in large-scale studies, several hundreds of these RBPs have been discovered and elucidated globally over the past few years. Recent discoveries have more than doubled the number of proteins implicated in RNA interaction, including identification of RBPs lacking classical RBDs. This review will discuss these new emerging classes of RBPs, focusing on the current state of the RBP repertoire in Arabidopsis thaliana , including the diverse functional roles derived from quantitative studies implicating RBPs in abiotic stress responses. Notably, this review highlights that 836 RBPs are enriched as Arabidopsis RBPs while 1865 can be classified as candidate RBPs. The review will also outline outstanding areas within this field that require addressing to advance our understanding and potential biotechnological applications of RBPs.

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