scholarly journals Individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to determine protein-RNA interactions

2017 ◽  
Author(s):  
Christopher R. Sibley
Keyword(s):  
High Throughput ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cdna Libraries ◽  
Cross Linking ◽  
Rna Transcripts ◽  
Rna Targets ◽  
Disease Aetiology ◽  
Nucleotide Resolution ◽  
Rna Complexes

AbstractRNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, whilst their perturbed activity can contribute to disease aetiology. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. iCLIP achieves this by exploiting UV-induced covalent crosslinks formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct crosslink sites in the high throughput sequenced cDNA libraries.

scholarly journals hnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons

2018 ◽  
Vol 115 (12) ◽  
pp. E2859-E2868 ◽  
Author(s):  
Michael Briese ◽  
Lena Saal-Bauernschubert ◽  
Changhe Ji ◽  
Mehri Moradi ◽  
Hanaa Ghanawi ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Muscular Atrophy ◽  
Axon Growth ◽  
Axon Elongation ◽  
Rna Targets ◽  
Motoneuron Diseases ◽  
Nucleotide Resolution ◽  
Nuclear Ribonucleoprotein

Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance.

scholarly journals Implementation of PAR-CLIP to characterize RNA-protein interactions in prokaryotes at nucleotide resolution

2020 ◽  
Author(s):  
Sandeep Ojha ◽  
Chaitanya Jain
Keyword(s):  
Protein Interactions ◽  
Messenger Rna ◽  
High Efficiency ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleoside Analogs ◽  
E Coli ◽  
Rna Targets ◽  
Genome Wide ◽  
Nucleotide Resolution

AbstractThe identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as “Crosslinking and Immunoprecipitation” (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs incorporated into cellular RNA has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP). Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA, and for the isolation of crosslinked RNA. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes.

scholarly journals Architecture of RNA influences plant biology

2021 ◽  
Author(s):  
Jiaying Zhu ◽  
Changhao Li ◽  
Xu Peng ◽  
Xiuren Zhang
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mirna Biogenesis ◽  
Plant Responses ◽  
Tertiary Structures ◽  
Rna Transcripts ◽  
Environmental Variations ◽  
Living Organisms ◽  
Processing Stability

Abstract The majority of the genome is transcribed to RNA in living organisms. RNA transcripts can form astonishing arrays of secondary and tertiary structures via Watson-Crick, Hoogsteen or wobble base pairing. In vivo, RNA folding is not a simple thermodynamics event of minimizing free energy. Instead, the process is constrained by transcription, RNA binding proteins (RBPs), steric factors and micro-environment. RNA secondary structure (RSS) plays myriad roles in numerous biological processes, such as RNA processing, stability, transportation and translation in prokaryotes and eukaryotes. Emerging evidence has also implicated RSS in RNA trafficking, liquid-liquid phase separation and plant responses to environmental variations such as temperature and salinity. At the molecular level, RSS is correlated with regulating splicing, polyadenylation, protein systhsis, and miRNA biogenesis and functions. In this review, we summarized newly reported methods for probing RSS in vivo and functions and mechanisms of RSS in plant physiology.

scholarly journals SSMART: Sequence-structure motif identification for RNA-binding proteins

2018 ◽  
Author(s):  
Alina Munteanu ◽  
Neelanjan Mukherjee ◽  
Uwe Ohler
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Motif ◽  
Primary Sequence ◽  
Sequence Structure ◽  
Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

scholarly journals The SRSF4-GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy

2021 ◽  
Author(s):  
Javier Larrasa-Alonso ◽  
María Villalba ◽  
Carlos Martí-Gómez ◽  
Paula Ortiz-Sánchez ◽  
Marina López-Olañeta ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Cardiac Hypertrophy ◽  
Diastolic Dysfunction ◽  
Ventricular Hypertrophy ◽  
Transcriptional Activity ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Left Ventricular ◽  
Therapeutic Tools ◽  
Nucleotide Resolution

Rationale: RNA-binding proteins (RBPs) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP serine/arginine-rich splicing factor 4 (SRSF4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. Objective: To investigate the role of SRSF4 in the heart. Methods and Results: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long non-coding RNAs (lncRNAs), including GAS5 (growth arrest specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide-resolution cross-linking and immuno-precipitation (iCLIP). GAS5 is a repressor of the glucocorticoid receptor (GR) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. Conclusions: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in Cushing's syndrome patients.

scholarly journals Functional characterization of splicing regulatory elements

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.

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

2016 ◽  
Author(s):  
Shuya Li ◽  
Fanghong Dong ◽  
Yuexin Wu ◽  
Sai Zhang ◽  
Chen Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Binding Proteins ◽  
Rna Binding ◽  
Gene Expression Regulation ◽  
Rna Binding Proteins ◽  
False Negative ◽  
Functional Roles ◽  
Potential Applications ◽  
Validation Tests

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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