scholarly journals Unbiased identification of trans regulators of ADAR and A-to-I RNA editing

2019 ◽  
Author(s):  
Emily C. Freund ◽  
Anne L. Sapiro ◽  
Qin Li ◽  
Sandra Linder ◽  
James J. Moresco ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rna Editing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Neuroblastoma Cells ◽  
Negative Regulator ◽  
Interacting Proteins ◽  
The Novel ◽  
Site Specific

AbstractAdenosine-to-Inosine RNA editing is catalyzed by ADAR enzymes that deaminate adenosine to inosine. While many RNA editing sites are known, few trans regulators have been identified. We perform BioID followed by mass-spectrometry to identify trans regulators of ADAR1 and ADAR2 in HeLa and M17 neuroblastoma cells. We identify known and novel ADAR-interacting proteins. Using ENCODE data we validate and characterize a subset of the novel interactors as global or site-specific RNA editing regulators. Our set of novel trans regulators includes all four members of the DZF-domain-containing family of proteins: ILF3, ILF2, STRBP, and ZFR. We show that these proteins interact with each ADAR and modulate RNA editing levels. We find ILF3 is a global negative regulator of editing. This work demonstrates the broad roles RNA binding proteins play in regulating editing levels and establishes DZF-domain containing proteins as a group of highly influential RNA editing regulators.

scholarly journals TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins

Cell ◽  
2016 ◽  
Vol 165 (3) ◽  
pp. 742-753 ◽  
Author(s):  
Aoife C. McMahon ◽  
Reazur Rahman ◽  
Hua Jin ◽  
James L. Shen ◽  
Allegra Fieldsend ◽  
...  
Keyword(s):  
Rna Editing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Editing Enzyme

scholarly journals Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins

2014 ◽  
Vol 11 (10) ◽  
pp. 1064-1070 ◽  
Author(s):  
Katharina Kramer ◽  
Timo Sachsenberg ◽  
Benedikt M Beckmann ◽  
Saadia Qamar ◽  
Kum-Loong Boon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
High Resolution Mass Spectrometry ◽  
Cross Linking ◽  
High Resolution Mass ◽  
Resolution Mass

scholarly journals Screening for New Interaction Partners for a Group IIB Intron Ribozyme with Surface Plasmon Resonance Imaging Coupled to MALDI Mass Spectrometry

2020 ◽  
Author(s):  
Ulrike Anders ◽  
Maya Gulotti-Georgieva ◽  
Susann Zelger-Paulus ◽  
Fatima-Ezzahra Hibti ◽  
Chiraz Frydman ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Living Organism ◽  
Maldi Mass Spectrometry ◽  
Mitochondrial Fraction ◽  
Ionization Mass ◽  
Rna Maturation

ABSTRACTRNA maturation is a highly regulated process whose precision is indispensable for the correct transfer of genetic information and, thus, the survival of any living organism. While in higher eukaryotes, this process is known to be assisted by the spliceosome, a very complex system assembled from numerous proteins, in bacteria splicing is catalyzed by ribozymes only. In lower eukaryotes however, e.g., yeast, RNA maturation is also expected to be less complex or simplified. Here, we focus on the mitochondrial group IIB intron RNA Sc.ai5γ from Saccharomyces cerevisiae (Sc.) and Mss116, a protein of the DEAD-box helicase family, known to play a crucial role in the Sc.ai5γ maturation pathway, acting as a co-factor in vivo. Although to date, only Mss116 has been described to be involved in the maturation of Sc.ai5γ, we hypothesize that the folding and splicing of Sc.ai5γ is regulated by more than one protein co-factor, i.e., that a complex or series of several proteins participate in folding and splicing the immature RNA correctly. For the identification of new potential Sc.ai5γ binders we coupled SPR imaging with matrix-assisted laser desorption/ionization mass spectrometry. This combination results in a powerful method to screen for specific RNA-binding proteins from complex mixtures, specifically lysate of the coarse mitochondrial fraction from yeast. Our results indicate that several proteins other than the well-known co-factor Mss116 interact with Sc.ai5γ, namely Dbp8, Prp8, Mrp13, and Cullin-3. With this novel approach, we report the identification of RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach.

scholarly journals Quantitative Proteomics to Identify Nuclear RNA-Binding Proteins of Malat1

2020 ◽  
Vol 21 (3) ◽  
pp. 1166 ◽  
Author(s):  
Marian Scherer ◽  
Michal Levin ◽  
Falk Butter ◽  
Marion Scheibe
Keyword(s):  
Quantitative Proteomics ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Full Length ◽  
Interacting Proteins ◽  
Non Coding Rna ◽  
Nuclear Rna ◽  
Long Non Coding Rna ◽  
Shed Light

The long non-coding RNA Malat1 has been implicated in several human cancers, while the mechanism of action is not completely understood. As RNAs in cells function together with RNA-binding proteins (RBPs), the composition of their RBP complex can shed light on their functionality. We here performed quantitative interactomics of 14 non-overlapping fragments covering the full length of Malat1 to identify possible nuclear interacting proteins. Overall, we identified 35 candidates including 14 already known binders, which are able to interact with Malat1 in the nucleus. Furthermore, the use of fragments along the full-length RNA allowed us to reveal two hotspots for protein binding, one in the 5′-region and one in the 3′-region of Malat1. Our results provide confirmation on previous RNA-protein interaction studies and suggest new candidates for functional investigations.

scholarly journals Capturing RNA–protein interaction via CRUIS

2020 ◽  
Vol 48 (9) ◽  
pp. e52-e52 ◽  
Author(s):  
Ziheng Zhang ◽  
Weiping Sun ◽  
Tiezhu Shi ◽  
Pengfei Lu ◽  
Min Zhuang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dna Damage ◽  
Protein Interactions ◽  
Noncoding Rna ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Living Cells ◽  
Innovative Methods ◽  
Rna Biology

Abstract No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA–protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA–protein interactions in their natural environment, and provides new insights into RNA biology.

scholarly journals Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Stacia L. Phillips ◽  
Erik J. Soderblom ◽  
Shelton S. Bradrick ◽  
Mariano A. Garcia-Blanco
Keyword(s):  
Mass Spectrometry ◽  
Dengue Virus ◽  
Virus Replication ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Viral Rna ◽  
Host Proteins ◽  
Infected Cells

ABSTRACT Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches. IMPORTANCE Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Viral RNA molecules physically interact with cellular RNA-binding proteins (RBPs) throughout the course of infection; the identification of such interactions will lead to the elucidation of the molecular mechanisms of virus replication. Until now, the identification of host proteins bound to dengue viral RNA has been accomplished using in vitro strategies. Here, we used a method for the specific purification of dengue viral ribonucleoprotein (RNP) complexes from infected cells and subsequently identified the associated proteins by mass spectrometry. We then validated a functional role for the majority of these proteins in mediating efficient virus replication. This approach has broad relevance to virology and RNA biology, as it could theoretically be used to purify any viral RNP complex of interest.

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