scholarly journals Binding specificities of human RNA binding proteins towards structured and linear RNA sequences

2018 ◽  
Author(s):  
Arttu Jolma ◽  
Jilin Zhang ◽  
Estefania Mondragón ◽  
Ekaterina Morgunova ◽  
Teemu Kivioja ◽  
...  
Keyword(s):  
Active Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Metabolism ◽  
Rna Sequences ◽  
Binding Domains ◽  
A Genome ◽  
Rna Binding Domains ◽  
Rna Motif

ABSTRACTSequence specific RNA-binding proteins (RBPs) control many important processes affecting gene expression. They regulate RNA metabolism at multiple levels, by affecting splicing of nascent transcripts, RNA folding, base modification, transport, localization, translation and stability. Despite their central role in most aspects of RNA metabolism and function, most RBP binding specificities remain unknown or incompletely defined. To address this, we have assembled a genome-scale collection of RBPs and their RNA binding domains (RBDs), and assessed their specificities using high throughput RNA-SELEX (HTR-SELEX). Approximately 70% of RBPs for which we obtained a motif bound to short linear sequences, whereas ~30% preferred structured motifs folding into stem-loops. We also found that many RBPs can bind to multiple distinctly different motifs. Analysis of the matches of the motifs in human genomic sequences suggested novel roles for many RBPs. We found that three cytoplasmic proteins, ZC3H12A, ZC3H12B and ZC3H12C bound to motifs resembling the splice donor sequence, suggesting that these proteins are involved in degradation of cytoplasmic viral and/or unspliced transcripts. Surprisingly, structural analysis revealed that the RNA motif was not bound by the conventional C3H1 RNA-binding domain of ZC3H12B. Instead, the RNA motif was bound by the ZC3H12B’s PilT N-terminus (PIN) RNase domain, revealing a potential mechanism by which unconventional RNA binding domains containing active sites or molecule-binding pockets could interact with short, structured RNA molecules. Our collection containing 145 high resolution binding specificity models for 86 RBPs is the largest systematic resource for the analysis of human RBPs, and will greatly facilitate future analysis of the various biological roles of this important class of proteins.

scholarly journals Thermodynamic modeling reveals widespread multivalent binding by RNA-binding proteins

2021 ◽  
Author(s):  
Salma Sohrabi-Jahromi ◽  
Johannes Söding
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Sequence Specificity ◽  
Rna Sequences ◽  
Link Type ◽  
Binding Domains ◽  
Regulatory Processes ◽  
Multivalent Binding

AbstractMotivationUnderstanding how proteins recognize their RNA targets is essential to elucidate regulatory processes in the cell. Many RNA-binding proteins (RBPs) form complexes or have multiple domains that allow them to bind to RNA in a multivalent, cooperative manner. They can thereby achieve higher specificity and affinity than proteins with a single RNA-binding domain. However, current approaches to de-novo discovery of RNA binding motifs do not take multivalent binding into account.ResultsWe present Bipartite Motif Finder (BMF), which is based on a thermodynamic model of RBPs with two cooperatively binding RNA-binding domains. We show that bivalent binding is a common strategy among RBPs, yielding higher affinity and sequence specificity. We furthermore illustrate that the spatial geometry between the binding sites can be learned from bound RNA sequences. These discovered bipartite motifs are consistent with previously known motifs and binding behaviors. Our results demonstrate the importance of multivalent binding for RNA-binding proteins and highlight the value of bipartite motif models in representing the multivalency of protein-RNA interactions.AvailabilityBMF source code is available at https://github.com/soedinglab/bipartite_motif_finder under a GPL license. The BMF web server is accessible at https://bmf.soedinglab.org.

scholarly journals Validation and classification of RNA binding proteins identified by mRNA interactome capture

2021 ◽  
Author(s):  
Vaishali ◽  
Lyudmila Dimitrova-Paternoga ◽  
Kevin Haubrich ◽  
Mai Sun ◽  
Anne Ephrussi ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Modular Organization ◽  
Binding Domains ◽  
Mammalian Cell Lines ◽  
Rna Immunoprecipitation ◽  
Rna Binding Domains ◽  
Rna Binders

AbstractRNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we employed a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RNA binding proteins. Half of the analysed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster End binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1’s RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather non-specific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

scholarly journals Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap

2017 ◽  
Vol 12 (12) ◽  
pp. 2447-2464 ◽  
Author(s):  
Alfredo Castello ◽  
Christian K. Frese ◽  
Bernd Fischer ◽  
Aino I Järvelin ◽  
Rastislav Horos ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Cultured Cells ◽  
Rna Binding Proteins ◽  
Binding Domains ◽  
Wide Scale ◽  
Rna Binding Domains

scholarly journals RNA interactome capture in Brachypodium reveals a flowering plant core RBPome

2020 ◽  
Author(s):  
Meixia Li ◽  
Zhicheng Zhang ◽  
Sam Balzarini ◽  
Bhavesh Parmar ◽  
Boonen Kurt ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Carbon Fixation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Brachypodium Distachyon ◽  
Functional Characterization ◽  
Flowering Plant ◽  
Transcriptional Level ◽  
Binding Domains ◽  
Rna Binding Domains

Abstract BackgroundRNA binding proteins regulate gene expression at the post-transcriptional level by controlling the fate of RNA, in processes such as mRNA localization, translation, splicing and stability. The annotation of RNA binding proteins is mainly based on the well-known RNA binding domains and motifs. However, novel RNA binding proteins without such conventional domains have been identified in different species using in vivo RNA interactome capture. To find support for novel conserved RNA binding proteins in plants, we applied an optimized RNA interactome capture to the monocot model Brachypodium distachyon.ResultsWe provide experimental evidence for 203 RNA binding proteins isolated from Brachypodium shoot tissue and leaf mesophyll protoplasts, and grouped these into classic RNA binding proteins with recognizable RNA binding domains and motifs, and candidate RNA binding proteins without such domains. Compared to RNA binding proteins captured in Arabidopsis thaliana, candidate RNA binding proteins involved in carbon fixation and carbon metabolic pathways are highly conserved. We tried to validate the RNA binding proteins captured in this research through a silica-based method, but this method appears not efficient for plants. This may indicate that optimized methods to validate high throughout RNA binding proteome are required for plants.ConclusionsOur results provide classic and candidate RNA binding proteins in Brachypodium distachyon and conserved RNA binding proteins in flowering plants. Future functional characterization should point out what the significance of RNA binding is for the function of these proteins.

scholarly journals Cooperativity boosts affinity and specificity of proteins with multiple RNA-binding domains

2021 ◽  
Author(s):  
Simon H. Stitzinger ◽  
Salma Sohrabi-Jahromi ◽  
Johannes Söding
Keyword(s):  
High Throughput ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Binding Domains

AbstractNumerous cellular processes rely on the binding of proteins with high affinity to specific sets of RNAs. Yet most RNA binding domains display low specificity and affinity, to the extent that for most RNA-binding domains, the enrichment of the best binding motif measured by high-throughput RNA SELEX or RNA bind-n-seq is usually below 10-fold, dramatically lower than that of DNA-binding domains. Here, we develop a thermodynamic model to predict the binding affinity for proteins with any number of RNA-binding domains given the affinities of their isolated domains. For the four proteins in which affinities for individual domains have been measured the model predictions are in good agreement with experimental values. The model gives insight into how proteins with multiple RNA-binding domains can reach affinities and specificities orders of magnitude higher than their individual domains. Our results contribute towards resolving the conundrum of missing specificity and affinity of RNA binding proteins and underscore the need for bioinformatic methods that can learn models for multi-domain RNA binding proteins from high-throughput in-vitro and in-vivo experiments.

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

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.

scholarly journals RNA Is a Double-Edged Sword in ALS Pathogenesis

2021 ◽  
Vol 15 ◽  
Author(s):  
Benjamin L. Zaepfel ◽  
Jeffrey D. Rothstein
Keyword(s):  
Motor Neurons ◽  
Cortical Neurons ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Metabolism ◽  
Small Subset ◽  
Toxic Rna ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that affects upper and lower motor neurons. Familial ALS accounts for a small subset of cases (<10–15%) and is caused by dominant mutations in one of more than 10 known genes. Multiple genes have been causally or pathologically linked to both ALS and frontotemporal dementia (FTD). Many of these genes encode RNA-binding proteins, so the role of dysregulated RNA metabolism in neurodegeneration is being actively investigated. In addition to defects in RNA metabolism, recent studies provide emerging evidence into how RNA itself can contribute to the degeneration of both motor and cortical neurons. In this review, we discuss the roles of altered RNA metabolism and RNA-mediated toxicity in the context of TARDBP, FUS, and C9ORF72 mutations. Specifically, we focus on recent studies that describe toxic RNA as the potential initiator of disease, disease-associated defects in specific RNA metabolism pathways, as well as how RNA-based approaches can be used as potential therapies. Altogether, we highlight the importance of RNA-based investigations into the molecular progression of ALS, as well as the need for RNA-dependent structural studies of disease-linked RNA-binding proteins to identify clear therapeutic targets.

PUB1: a major yeast poly(A)+ RNA-binding protein

1993 ◽  
Vol 13 (10) ◽  
pp. 6114-6123
Author(s):  
M J Matunis ◽  
E L Matunis ◽  
G Dreyfuss
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Uv Light ◽  
Binding Protein ◽  
Gene Replacement ◽  
Yeast Saccharomyces Cerevisiae ◽  
Binding Domains ◽  
Development And Differentiation

The expression of RNA polymerase II transcripts can be regulated at the posttranscriptional level by RNA-binding proteins. Although extensively characterized in metazoans, relatively few RNA-binding proteins have been characterized in the yeast Saccharomyces cerevisiae. Three major proteins are cross-linked by UV light to poly(A)+ RNA in living S. cerevisiae cells. These are the 72-kDa poly(A)-binding protein and proteins of 60 and 50 kDa (S.A. Adam, T.Y. Nakagawa, M.S. Swanson, T. Woodruff, and G. Dreyfuss, Mol. Cell. Biol. 6:2932-2943, 1986). Here, we describe the 60-kDa protein, one of the major poly(A)+ RNA-binding proteins in S. cerevisiae. This protein, PUB1 [for poly(U)-binding protein 1], was purified by affinity chromatography on immobilized poly(rU), and specific monoclonal antibodies to it were produced. UV cross-linking demonstrated that PUB1 is bound to poly(A)+ RNA (mRNA or pre-mRNA) in living cells, and it was detected primarily in the cytoplasm by indirect immunofluorescence. The gene for PUB1 was cloned and sequenced, and the sequence was found to predict a 51-kDa protein with three ribonucleoprotein consensus RNA-binding domains and three glutamine- and asparagine-rich auxiliary domains. This overall structure is remarkably similar to the structures of the Drosophila melanogaster elav gene product, the human neuronal antigen HuD, and the cytolytic lymphocyte protein TIA-1. Each of these proteins has an important role in development and differentiation, potentially by affecting RNA processing. PUB1 was found to be nonessential in S. cerevisiae by gene replacement; however, further genetic analysis should reveal important features of this class of RNA-binding proteins.

scholarly journals The structural basis for RNA selectivity by the IMP family of RNA-binding proteins

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jeetayu Biswas ◽  
Vivek L. Patel ◽  
Varun Bhaskar ◽  
Jeffrey A. Chao ◽  
Robert H. Singer ◽  
...  
Keyword(s):  
Neural Development ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Stability ◽  
Underlying Mechanism ◽  
Structural Basis ◽  
General Mechanism ◽  
Binding Domains ◽  
Variable Loops

Abstract The IGF2 mRNA-binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine systematic evolution of ligands by exponential enrichment (SELEX) and NMR spectroscopy to demonstrate that the major RNA-binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.

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

2020 ◽  
Vol 21 (12) ◽  
pp. 4548 ◽  
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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