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

2019 ◽  
Author(s):  
Eric L Van Nostrand ◽  
Gabriel A Pratt ◽  
Brian A Yee ◽  
Emily Wheeler ◽  
Steven M Blue ◽  
...  
Keyword(s):  
Rna Processing ◽  
Regulatory Networks ◽  
Large Scale ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cell Types ◽  
Integrated Analysis ◽  
Branch Points

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.

scholarly journals Calreticulin Interacts with C/EBPα and C/EBPβ mRNAs and Represses Translation of C/EBP Proteins

2002 ◽  
Vol 22 (20) ◽  
pp. 7242-7257 ◽  
Author(s):  
Lubov T. Timchenko ◽  
Polina Iakova ◽  
Alana L. Welm ◽  
Z.-J. Cai ◽  
Nikolai A. Timchenko
Keyword(s):  
Rna Processing ◽  
Posttranscriptional Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Posttranscriptional Control ◽  
Multifunctional Protein ◽  
Virus Rna

ABSTRACT We previously identified an RNA binding protein, CUGBP1, which binds to GCN repeats located within the 5′ region of C/EBPβ mRNAs and regulates translation of C/EBPβ isoforms. To further investigate the role of RNA binding proteins in the posttranscriptional control of C/EBP proteins, we purified additional RNA binding proteins that interact with GC-rich RNAs and that may regulate RNA processing. In HeLa cells, the majority of GC-rich RNA binding proteins are associated with endogenous RNA transcripts. The separation of these proteins from endogenous RNA identified several proteins in addition to CUGBP1 that specifically interact with the GC-rich 5′ region of C/EBPβ mRNA. One of these proteins was purified to homogeneity and was identified as calreticulin (CRT). CRT is a multifunctional protein involved in several biological processes, including interaction with and regulation of rubella virus RNA processing. Our data demonstrate that both CUGBP1 and CRT interact with GCU repeats within myotonin protein kinase and with GCN repeats within C/EBPα and C/EBPβ mRNAs. GCN repeats within these mRNAs form stable SL structures. The interaction of CRT with SL structures of C/EBPβ and C/EBPα mRNAs leads to inhibition of translation of C/EBP proteins in vitro and in vivo. Deletions or mutations abolishing the formation of SL structures within C/EBPα and C/EBPβ mRNAs lead to a failure of CRT to inhibit translation of C/EBP proteins. CRT-dependent inhibition of C/EBPα is sufficient to block the growth-inhibitory activity of C/EBPα. This finding further defines the molecular mechanism for posttranscriptional regulation of the C/EBPα and C/EBPβ proteins.

scholarly journals A new method to identify global targets of RNA-binding proteins in plants

2021 ◽  
Author(s):  
You-Liang Cheng ◽  
Hsin-Yu Hsieh ◽  
Shih-Long Tu
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Developmental Stages ◽  
Catalytic Domain ◽  
Rna Binding Proteins ◽  
Cell Types ◽  
Protein Coding ◽  
Polypyrimidine Tract Binding Protein ◽  
Model Protein

Background: RNA-binding proteins (RBPs) play crucial roles in various aspects of post-transcriptional gene expression; their functions can vary between tissues, cell types, developmental stages, and environmental conditions. Identifying RBP target RNAs and investigating whether they are differentially bound by RBPs in different cell types, stages, or conditions could shed light on RBP functions. Although several strategies have been designed to identify RBP targets, they involve complicated biochemical steps and require large quantities of material, and only a few studies using these techniques have been performed in plants. The TRIBE (targets of RNA binding proteins identified by editing) method was recently developed to identify RBP targets using a RBP coupled to the catalytic domain of a Drosophila RNA editing enzyme and expressing this fusion protein in vivo. The resulting novel editing events can be identified by sequencing. This technique uses little material and does not require complex biochemical steps, however it is not yet adapted for use in plants. Results: We successfully applied an optimized genome-wide TRIBE method in plants. We selected the splicing regulator polypyrimidine tract-binding protein (PTB) as a model protein for testing the TRIBE system in the moss Physcomitrium patens. We demonstrated that 13.81% of protein-coding gene transcripts in P. patens are targets of PTB. Most potential PTB binding sites are located in coding sequences and 3 prime untranslated regions, suggesting that PTB performs multiple functions besides pre-mRNA splicing in this moss. In addition, TRIBE showed reproducible results compared to other methods. Conclusions: We have developed an alternative method based on the TRIBE system to identify RBP targets in plants globally, and we provide guidance here for its application in plants.

scholarly journals RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

2021 ◽  
Vol 7 (1) ◽  
pp. 11 ◽  
Author(s):  
André P. Gerber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Cell Protein ◽  
Transcriptional Regulatory Networks ◽  
Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

scholarly journals Identification of mRNAs Associated with αCP2-Containing RNP Complexes

2003 ◽  
Vol 23 (19) ◽  
pp. 7055-7067 ◽  
Author(s):  
Shelly A. Waggoner ◽  
Stephen A. Liebhaber
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Cell Function ◽  
Rna Binding Proteins ◽  
Translation Control ◽  
Viral Mrnas ◽  
Posttranscriptional Gene Regulation ◽  
Direct Binding

ABSTRACT Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The α-globin poly(C) binding proteins (αCPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. αCPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of αCPs to cell function, we have identified a set of mRNAs that associate in vivo with the major αCP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of αCP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for αCP2 in this infantile neurodegenerative disease. Surprisingly, αCP2 mRNA itself was represented in αCP2-RNP complexes, suggesting autoregulatory control of αCP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of αCP2 within their 3′ untranslated regions. These data expand the list of mRNAs that associate with αCP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.

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 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.

The expanding world of metabolic enzymes moonlighting as RNA binding proteins

2021 ◽  
Author(s):  
Nicole J. Curtis ◽  
Constance J. Jeffery
Keyword(s):  
Gene Expression ◽  
Regulatory Networks ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Molecular Structures ◽  
Intermediary Metabolism ◽  
The Many ◽  
And Function

RNA binding proteins play key roles in many aspects of RNA metabolism and function, including splicing, transport, translation, localization, stability and degradation. Within the past few years, proteomics studies have identified dozens of enzymes in intermediary metabolism that bind to RNA. The wide occurrence and conservation of RNA binding ability across distant branches of the evolutionary tree suggest that these moonlighting enzymes are involved in connections between intermediary metabolism and gene expression that comprise far more extensive regulatory networks than previously thought. There are many outstanding questions about the molecular structures and mechanisms involved, the effects of these interactions on enzyme and RNA functions, and the factors that regulate the interactions. The effects on RNA function are likely to be wider than regulation of translation, and some enzyme–RNA interactions have been found to regulate the enzyme's catalytic activity. Several enzyme–RNA interactions have been shown to be affected by cellular factors that change under different intracellular and environmental conditions, including concentrations of substrates and cofactors. Understanding the molecular mechanisms involved in the interactions between the enzymes and RNA, the factors involved in regulation, and the effects of the enzyme–RNA interactions on both the enzyme and RNA functions will lead to a better understanding of the role of the many newly identified enzyme–RNA interactions in connecting intermediary metabolism and gene expression.

scholarly journals A unique ribonucleoprotein complex assembles preferentially on ecdysone-responsive sites in Drosophila melanogaster.

1993 ◽  
Vol 13 (9) ◽  
pp. 5323-5330 ◽  
Author(s):  
S A Amero ◽  
M J Matunis ◽  
E L Matunis ◽  
J W Hockensmith ◽  
G Raychaudhuri ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Polytene Chromosomes ◽  
Cell Extract ◽  
Sequence Motifs ◽  
Drosophila Cell ◽  
Rnase Digestion

The protein on ecdysone puffs (PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains sequence motifs characteristic of transcription factors and RNA-binding proteins (S. A. Amero, S. C. R. Elgin, and A. L. Beyer, Genes Dev. 5:188-200, 1991). PEP is associated with RNA in vivo, as demonstrated here by the sensitivity of PEP-specific chromosomal immunostaining in situ to RNase digestion and by the immunopurification of PEP in Drosophila cell extract with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As revealed by sequential immunostaining, PEP is found on a subset of chromosomal sites bound by the HRB (heterogeneous nuclear RNA-binding) proteins, which are basic Drosophila hnRNPs. These observations lead us to suggest that a unique, PEP-containing hnRNP complex assembles preferentially on the transcripts of ecdysone-regulated genes in Drosophila melanogaster presumably to expedite the transcription and/or processing of these transcripts.

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