Identification and Characterization of RNA-binding Proteins through Three-hybrid Analysis

ABSTRACTRNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs), and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches.We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using Position Specific Scoring Matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome of Escherichia coli. APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available at https://pypi.python.org/pypi/bio-apricot

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Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies

Nucleic Acids Research ◽

10.1093/nar/gkm016 ◽

2007 ◽

Vol 35 (4) ◽

pp. 1289-1300 ◽

Cited By ~ 74

Author(s):

K. Buchet-Poyau ◽

J. Courchet ◽

H. L. Hir ◽

B. Seraphin ◽

J.-Y. Scoazec ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Processing Bodies ◽

Evolutionarily Conserved ◽

Identification And Characterization

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Characterization of Viral Double-Stranded RNA-Binding Proteins

Methods ◽

10.1006/meth.1998.0626 ◽

1998 ◽

Vol 15 (3) ◽

pp. 225-232 ◽

Cited By ~ 11

Author(s):

Bertram L. Jacobs ◽

Jeffrey O. Langland ◽

Teresa Brandt

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Double Stranded Rna

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Discovery of RNA-binding proteins and characterization of their dynamic responses by enhanced RNA interactome capture

Nature Communications ◽

10.1038/s41467-018-06557-8 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 40

Author(s):

Joel I. Perez-Perri ◽

Birgit Rogell ◽

Thomas Schwarzl ◽

Frank Stein ◽

Yang Zhou ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Jurkat Cells ◽

Dynamic Responses ◽

Capture Probe ◽

Signal To Noise ◽

Low Background ◽

Detailed Composition

AbstractFollowing the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal-to-noise ratios compared to existing methods. In Jurkat cells, eRIC reduces the rRNA and DNA contamination by >10-fold compared to RIC and increases the detection of RNA-binding proteins. Due to its low background, eRIC also empowers comparative analyses of changes of RNA-bound proteomes missed by RIC. For example, in cells treated with dimethyloxalylglycine, which inhibits RNA demethylases, eRIC identifies m6A-responsive RNA-binding proteins that escape RIC. eRIC will facilitate the unbiased characterization of RBP dynamics in response to biological and pharmacological cues.

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