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.

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 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 Cancer Proteomics: The State of the Art

2001 ◽  
Vol 17 (2) ◽  
pp. 49-57 ◽  
Author(s):  
Paul C. Herrmann ◽  
Lance A. Liotta ◽  
Emanuel F. Petricoin III
Keyword(s):  
Early Detection ◽  
Protein Interactions ◽  
State Of The Art ◽  
Careful Analysis ◽  
Cellular Protein ◽  
Protein Networks ◽  
New Paradigm ◽  
Detailed Understanding ◽  
Cellular Processes ◽  
Cancer Proteomics

Now that the human genome has been determined, the field of proteomics is ramping up to tackle the vast protein networks that both control and are controlled by the information encoded by the genome. The study of proteomics should yield an unparalleled understanding of cancer as well as an invaluable new target for therapeutic intervention and markers for early detection. This rapidly expanding field attempts to track the protein interactions responsible for all cellular processes. By careful analysis of these systems, a detailed understanding of the molecular causes and consequences of cancer should emerge. A brief overview of some of the cutting edge technologies employed by this rapidly expanding field is given, along with specific examples of how these technologies are employed. Soon cellular protein networks will be understood at a level that will permit a totally new paradigm of diagnosis and will allow therapy tailored to individual patients and situations.

scholarly journals easyCLIP analysis of RNA-protein interactions incorporating absolute quantification

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Douglas F. Porter ◽  
Weili Miao ◽  
Xue Yang ◽  
Grant A. Goda ◽  
Andrew L. Ji ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Absolute Quantification ◽  
Complex Frequency ◽  
Cross Link ◽  
Sequencing Library ◽  
Link Rate ◽  
Cancer Mutations ◽  
The Impact

AbstractQuantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.

scholarly journals easyCLIP Analysis of RNA-Protein Interactions Incorporating Absolute Quantification

2021 ◽  
Author(s):  
Douglas Porter ◽  
Paul Khavari
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Absolute Quantification ◽  
Specific Protein ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Cross Link ◽  
Cross Links ◽  
Methodological Problems ◽  
Target Rna

Abstract In general, an RNA-binding protein (RBP) may be considered a protein with an abnormally frequent interaction with RNA, and a "target RNA" for a specific protein may be considered an RNA with an abnormally frequent interaction with that protein. Traditional ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing analysis methods generally define interactions by methods that are indirectly assessing the latter. However, there has been limited direct assessment of these metrics by determining RNA cross-link rates or the RNA-binding profiles of non-RBPs.Here, we describe a method to determine RNA cross-link rates and create sequencing libraries for a protein of interest, either of which may be performed on their own. easyCLIP is a relatively short, reliable, and efficient CLIP method, with the capacity to directly visualize RNA libraries and diagnose methodological problems. By combining the sequencing data and cross-link rates, the absolute frequency of given cross-links may be compared between control and experimental proteins, to nominate potential RBPs and distinctive RNA-protein interactions.

scholarly journals In-cell identification and measurement of RNA-protein interactions

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Antoine Graindorge ◽  
Inês Pinheiro ◽  
Anna Nawrocka ◽  
Allison C. Mallory ◽  
Peter Tsvetkov ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cell Protein ◽  
Cellular Functions ◽  
Functional Regions ◽  
Rna Purification ◽  
High Throughput Method ◽  
Regulatory Rnas ◽  
Rna Interaction

AbstractRegulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.

scholarly journals Structural basis for the dimerization of Gemin5 and its role in protein recruitment and translation control

2019 ◽  
Vol 48 (2) ◽  
pp. 788-801 ◽  
Author(s):  
María Moreno-Morcillo ◽  
Rosario Francisco-Velilla ◽  
Azman Embarc-Buh ◽  
Javier Fernández-Chamorro ◽  
Santiago Ramón-Maiques ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Structural Basis ◽  
Middle Region ◽  
Protein Protein Interactions ◽  
Translation Control ◽  
Dimerization Domain ◽  
Multifunctional Protein ◽  
Cellular Processes ◽  
And Function

Abstract In all organisms, a selected type of proteins accomplishes critical roles in cellular processes that govern gene expression. The multifunctional protein Gemin5 cooperates in translation control and ribosome binding, besides acting as the RNA-binding protein of the survival of motor neuron (SMN) complex. While these functions reside on distinct domains located at each end of the protein, the structure and function of the middle region remained unknown. Here, we solved the crystal structure of an extended tetratricopeptide (TPR)-like domain in human Gemin5 that self-assembles into a previously unknown canoe-shaped dimer. We further show that the dimerization module is functional in living cells driving the interaction between the viral-induced cleavage fragment p85 and the full-length Gemin5, which anchors splicing and translation members. Disruption of the dimerization surface by a point mutation in the TPR-like domain prevents this interaction and also abrogates translation enhancement induced by p85. The characterization of this unanticipated dimerization domain provides the structural basis for a role of the middle region of Gemin5 as a central hub for protein-protein interactions.

scholarly journals A Live-Cell Assay for the Detection of pre-microRNA-Protein Interactions

2020 ◽  
Author(s):  
Sydney L. Rosenblum ◽  
Daniel A. Lorenz ◽  
Amanda L. Garner
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Live Cell ◽  
Cell Detection ◽  
Binding Domains ◽  
Genome Wide ◽  
Rna Interaction ◽  
And Function ◽  
Specific Sequences

AbstractRecent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of both coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry- and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA-protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-of-concept, we have utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs.

scholarly journals PARP1 Regulates the Biogenesis and Activity of Telomerase Complex Through Modification of H/ACA-Proteins

2021 ◽  
Vol 9 ◽  
Author(s):  
Nikita V. Savelyev ◽  
Nikita M. Shepelev ◽  
Olga I. Lavrik ◽  
Maria P. Rubtsova ◽  
Olga A. Dontsova
Keyword(s):  
Telomere Length ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Telomere Maintenance ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Rna Biogenesis ◽  
Cellular Dna ◽  
Rna Maturation

Poly(ADP-ribose) polymerase 1 (PARP1) is established as a key regulator of the cellular DNA damage response and apoptosis. In addition, PARP1 participates in the global regulation of DNA repair, transcription, telomere maintenance, and inflammation response by modulating various DNA-protein and protein-protein interactions. Recently, it was reported that PARP1 also influences splicing and ribosomal RNA biogenesis. The H/ACA ribonucleoprotein complex is involved in a variety of cellular processes such as RNA maturation. It contains non-coding RNAs with specific H/ACA domains and four proteins: dyskerin (DKC1), GAR1, NHP2, and NOP10. Two of these proteins, DKC1 and GAR1, are targets of poly(ADP-ribosyl)ation catalyzed by PARP1. The H/ACA RNA-binding proteins are involved in the regulation of maturation and activity of the telomerase complex, which maintains telomere length. In this study, we demonstrated that of poly(ADP-ribosyl)ation influences on RNA-binding properties of DKC1 and GAR1 and telomerase assembly and activity. Our data provide the evidence that poly(ADP-ribosyl)ation regulates telomerase complex assembly and activity, in turn regulating telomere length that may be useful for design and development of anticancer therapeutic approaches that are based on the inhibition of PARP1 and telomerase activities.

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