scholarly journals Dynamic changes in RNA–protein interactions and RNA secondary structure in mammalian erythropoiesis

2021 ◽  
Vol 4 (9) ◽  
pp. e202000659
Author(s):  
Mengge Shan ◽  
Xinjun Ji ◽  
Kevin Janssen ◽  
Ian M Silverman ◽  
Jesse Humenik ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Interactions ◽  
Rna Secondary Structure ◽  
Rna Binding ◽  
Developmental Process ◽  
Global Analysis ◽  
Sequence Motifs ◽  
Rna Molecules ◽  
Interaction Sites ◽  
Rna Interaction

Two features of eukaryotic RNA molecules that regulate their post-transcriptional fates are RNA secondary structure and RNA-binding protein (RBP) interaction sites. However, a comprehensive global overview of the dynamic nature of these sequence features during erythropoiesis has never been obtained. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approach to reveal the global landscape of RNA secondary structure and RBP–RNA interaction sites and the dynamics of these features during this important developmental process. We identify dynamic patterns of RNA secondary structure and RBP binding throughout the process and determine a set of corresponding protein-bound sequence motifs along with their dynamic structural and RBP-binding contexts. Finally, using these dynamically bound sequences, we identify a number of RBPs that have known and putative key functions in post-transcriptional regulation during mammalian erythropoiesis. In total, this global analysis reveals new post-transcriptional regulators of mammalian blood cell development.

scholarly journals PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

2016 ◽  
Author(s):  
Alex M. Tamburino ◽  
Ebru Kaymak ◽  
Shaleen Shrestha ◽  
Amy D. Holdorf ◽  
Sean P. Ryder ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Small Rna ◽  
Rna Binding ◽  
Mrna Translation ◽  
Single Experiment ◽  
Novel Interactions ◽  
Transcriptional Gene Regulation ◽  
Rna Interaction ◽  
Eukaryotic Genomes

SUMMARYInteractions between RNA binding protein (RBP) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using ‘protein-centered’ (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest – in a ‘gene-centered’ manner, yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA ‘bait’ can be tested versus multiple RBP ‘preys’ in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3′UTRs. PRIMA faithfully recapitulates numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

scholarly journals RNA regulons and the RNA-protein interaction network

2012 ◽  
Vol 3 (5) ◽  
pp. 403-414 ◽  
Author(s):  
Jochen Imig ◽  
Alexander Kanitz ◽  
André P. Gerber
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Interaction Network ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Interaction Network ◽  
Coordinated Control ◽  
Non Coding Rnas ◽  
Rna Interaction

AbstractThe development of genome-wide analysis tools has prompted global investigation of the gene expression program, revealing highly coordinated control mechanisms that ensure proper spatiotemporal activity of a cell’s macromolecular components. With respect to the regulation of RNA transcripts, the concept of RNA regulons, which – by analogy with DNA regulons in bacteria – refers to the coordinated control of functionally related RNA molecules, has emerged as a unifying theory that describes the logic of regulatory RNA-protein interactions in eukaryotes. Hundreds of RNA-binding proteins and small non-coding RNAs, such as microRNAs, bind to distinct elements in target RNAs, thereby exerting specific and concerted control over posttranscriptional events. In this review, we discuss recent reports committed to systematically explore the RNA-protein interaction network and outline some of the principles and recurring features of RNA regulons: the coordination of functionally related mRNAs through RNA-binding proteins or non-coding RNAs, the modular structure of its components, and the dynamic rewiring of RNA-protein interactions upon exposure to internal or external stimuli. We also summarize evidence for robust combinatorial control of mRNAs, which could determine the ultimate fate of each mRNA molecule in a cell. Finally, the compilation and integration of global protein-RNA interaction data has yielded first insights into network structures and provided the hypothesis that RNA regulons may, in part, constitute noise ‘buffers’ to handle stochasticity in cellular transcription.

scholarly journals Biochemical and Structural Insights into RNA Binding by Ssh10b, a Member of the Highly Conserved Sac10b Protein Family in Archaea

2013 ◽  
Vol 289 (3) ◽  
pp. 1478-1490 ◽  
Author(s):  
Li Guo ◽  
Jingjin Ding ◽  
Rong Guo ◽  
Yanjie Hou ◽  
Da-Cheng Wang ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Secondary Structure ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Sulfolobus Shibatae ◽  
Β Sheet ◽  
Structural Insights ◽  
Dsdna Binding ◽  
Local Distortions

Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular β-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.

scholarly journals Structure and Sequence Determinants Governing the Interactions of RNAs with Influenza A Virus Non-Structural Protein NS1

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 947
Author(s):  
Alan Wacquiez ◽  
Franck Coste ◽  
Emmanuel Kut ◽  
Virginie Gaudon ◽  
Sascha Trapp ◽  
...  
Keyword(s):  
Influenza A ◽  
Rna Binding ◽  
3D Structure ◽  
Genetically Engineered ◽  
Phenotypic Characterization ◽  
Sequence Motifs ◽  
Structural Protein ◽  
Specific Sequence ◽  
Rna Interaction

The non-structural protein NS1 of influenza A viruses is an RNA-binding protein of which its activities in the infected cell contribute to the success of the viral cycle, notably through interferon antagonism. We have previously shown that NS1 strongly binds RNA aptamers harbouring virus-specific sequence motifs (Marc et al., Nucleic Acids Res. 41, 434–449). Here, we started out investigating the putative role of one particular virus-specific motif through the phenotypic characterization of mutant viruses that were genetically engineered from the parental strain WSN. Unexpectedly, our data did not evidence biological importance of the putative binding of NS1 to this specific motif (UGAUUGAAG) in the 3′-untranslated region of its own mRNA. Next, we sought to identify specificity determinants in the NS1-RNA interaction through interaction assays in vitro with several RNA ligands and through solving by X-ray diffraction the 3D structure of several complexes associating NS1′s RBD with RNAs of various affinities. Our data show that the RBD binds the GUAAC motif within double-stranded RNA helices with an apparent specificity that may rely on the sequence-encoded ability of the RNA to bend its axis. On the other hand, we showed that the RBD binds to the virus-specific AGCAAAAG motif when it is exposed in the apical loop of a high-affinity RNA aptamer, probably through a distinct mode of interaction that still requires structural characterization. Our data are consistent with more than one mode of interaction of NS1′s RBD with RNAs, recognizing both structure and sequence determinants.

scholarly journals Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein–RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing

2021 ◽  
Vol 22 (10) ◽  
pp. 5312
Author(s):  
Akio Masuda ◽  
Toshihiko Kawachi ◽  
Kinji Ohno
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Detection Efficiency ◽  
Fine Tuning ◽  
Interaction Sites ◽  
Rnap Ii ◽  
Rna Interaction ◽  
In Cells

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other’s activity, indicating an essential role of protein–RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein–RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein–RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein–RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein–RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.

scholarly journals Transcriptome-wide high-throughput mapping of protein–RNA occupancy profiles using POP-seq

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mansi Srivastava ◽  
Rajneesh Srivastava ◽  
Sarath Chandra Janga
Keyword(s):  
High Throughput ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Interaction Network ◽  
K562 Cells ◽  
Genomic Variation ◽  
Interaction Sites ◽  
Protein Occupancy ◽  
Rna Interaction

AbstractInteraction between proteins and RNA is critical for post-transcriptional regulatory processes. Existing high throughput methods based on crosslinking of the protein–RNA complexes and poly-A pull down are reported to contribute to biases and are not readily amenable for identifying interaction sites on non poly-A RNAs. We present Protein Occupancy Profile-Sequencing (POP-seq), a phase separation based method in three versions, one of which does not require crosslinking, thus providing unbiased protein occupancy profiles on whole cell transcriptome without the requirement of poly-A pulldown. Our study demonstrates that ~ 68% of the total POP-seq peaks exhibited an overlap with publicly available protein–RNA interaction profiles of 97 RNA binding proteins (RBPs) in K562 cells. We show that POP-seq variants consistently capture protein–RNA interaction sites across a broad range of genes including on transcripts encoding for transcription factors (TFs), RNA-Binding Proteins (RBPs) and long non-coding RNAs (lncRNAs). POP-seq identified peaks exhibited a significant enrichment (p value < 2.2e−16) for GWAS SNPs, phenotypic, clinically relevant germline as well as somatic variants reported in cancer genomes, suggesting the prevalence of uncharacterized genomic variation in protein occupied sites on RNA. We demonstrate that the abundance of POP-seq peaks increases with an increase in expression of lncRNAs, suggesting that highly expressed lncRNA are likely to act as sponges for RBPs, contributing to the rewiring of protein–RNA interaction network in cancer cells. Overall, our data supports POP-seq as a robust and cost-effective method that could be applied to primary tissues for mapping global protein occupancies.

scholarly journals easyCLIP Quantifies RNA-Protein Interactions and Characterizes Recurrent PCBP1 Mutations in Cancer

2019 ◽  
Author(s):  
Douglas F. Porter ◽  
Paul A. Khavari
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Cellular Protein ◽  
Good Efficiency ◽  
Cellular Processes ◽  
Simple Protocol ◽  
Link Rate ◽  
Cross Links ◽  
Rna Interaction ◽  
Insight Into

ABSTRACTRNA-protein interactions mediate a host of cellular processes, underscoring the need for methods to quantify their occurrence in living cells. RNA interaction frequencies for the average cellular protein are undefined, however, and there is no quantitative threshold to define a protein as an RNA-binding protein (RBP). Ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing, an effective and widely used means of characterizing RNA-protein interactions, would particularly benefit from the capacity to quantitate the number of RNA cross-links per protein per cell. In addition, CLIP-seq methods are difficult, have high experimental failure rates and many ambiguous analytical decisions. To address these issues, the easyCLIP method was developed and used to quantify RNA-protein interactions for a panel of known RBPs as well as a spectrum of random non-RBP proteins. easyCLIP provides the advantages of good efficiency compared to current standards, a simple protocol with a very low failure rate, troubleshooting information that includes direct visualization of prepared libraries without amplification, and a new form of analysis. easyCLIP, which uses sequential on-bead ligation of 5’ and 3’ adapters tagged with different infrared dyes, classified non-RBPs as those with a per protein RNA cross-link rate of <0.1%, with most RBPs substantially above this threshold, including Rbfox1 (18%), hnRNPC (22%), CELF1 (11%), FBL (2%), and STAU1 (1%). easyCLIP with the PCBP1L100 RBP mutant recurrently seen in cancer quantified increased RNA binding compared to wild-type PCBP1 and suggested a potential mechanism for this RBP mutant in cancer. easyCLIP provides a simple, efficient and robust method to both obtain both traditional CLIP-seq information and to define actual RNA interaction frequencies for a given protein, enabling quantitative cross-RBP comparisons as well as insight into RBP mechanisms.

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