Analysis of the Non-Specific Binding Proteins in the RNA Pull-Down Experiment

Background: To investigate the interactions between RNA and proteins is essential to understand how these macromolecule complexes exert their functions. RNA pull-down is a classic technique to enrich RNA binding proteins, however, a large number of non-specific binding proteins may be enriched during sample preparation, interfering with the downstream mass spectrometric analyses and also causing false positives. Objective: In this study we examined the background contaminates in RNA pull-down experiment using mass spectrometric analysis. Method Antisense MALAT1 was first synthesized using in vitro transcription and incubated with cellular proteins extracted from HepG2 cells. The non-specific binding proteins were isolated using streptavidin conjugated magnetic beads and separated on SDS-PAGE. Each gel lane was divided into nine bands and digested with trypsin for the downstream LC-MS/MS analyses. Results: 191 protein groups were identified as non-specific binding proteins in RNA pull-down samples. In addition, comparison between different sample preparation conditions showed that the level of background contaminates were mostly induced by the solid phase support and not affected by the studied RNA. In addition, using more stringent detergent and streptavidin magnetic beads with smaller size could reduce the amount of background interfering proteins. Conclusion: This study provides a reference to distinguish bona fide RNA interacting proteins from the background contaminants. The results also demonstrate that different sample preparation conditions have great impacts on the level of enriched background contaminates, shedding new light on the optimization of RNA pull-down experiment.

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Predicting localized affinity of RNA binding proteins to transcripts with convolutional neural networks

10.1101/2021.06.02.446817 ◽

2021 ◽

Author(s):

Alexander Kitaygorodsky ◽

Emily Jin ◽

Yufeng Shen

Keyword(s):

Binding Affinity ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Binding ◽

Computational Prediction ◽

Data Sets ◽

Coding Region ◽

Protein Coding ◽

Specific Affinity

RNA binding proteins (RBPs) are important regulators of transcriptional and post-transcriptional processes. Computational prediction of localized RBP binding affinity with transcripts is important for interpretation of genetic variation, especially variants outside of protein coding region. Here we describe POLARIS (Prediction Of Localized Affinity for RBPs In Sequence), a new deep-learning method for achieving fast, site-specific binding affinity predictions of RNA-binding proteins (RBPs) to the transcribed genome. POLARIS has two modules: 1. a convolutional neural network (CNN) to predict overall RBP binding within a region based on transcript sequence content and expression level; 2. a Gradient-weighted Class Activation Mapping (GradCAM) implementation for efficient signal backpropagation to individual sequence positions. We trained the model using enhanced crosslinking and immunoprecipitation (eCLIP) data from ENCODE. POLARIS has good performance with a median AUC ~ 0.96 for 160 RBPs across three different cell lines, substantially higher than selected popular published methods trained and tested on the same data sets. When tested on data from a different cell line with the same RBPs, the overall performance is maintained, supporting the ability of cell-type specific affinity prediction. Finally, the GradCAM module allows the model to identify the informative sites in a region that drive prediction. The localized prediction facilitates interpretation of the results and provides basis for inference of functional impact of noncoding variants.

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Identification of hemicatenane-specific binding proteins by fractionation of HeLa nuclei extracts

Biochemical Journal ◽

10.1042/bcj20190855 ◽

2020 ◽

Vol 477 (2) ◽

pp. 509-524

Author(s):

Oumayma Rouis ◽

Cédric Broussard ◽

François Guillonneau ◽

Jean-Baptiste Boulé ◽

Emmanuelle Delagoutte

Keyword(s):

Spatial Organization ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Binding ◽

Regulation Of Gene Expression ◽

Double Stranded Dna ◽

Nuclear Extracts ◽

Interesting Possibility

DNA hemicatenanes (HCs) are four-way junctions in which one strand of a double-stranded helix is catenated with one strand of another double-stranded DNA. Frequently mentioned as DNA replication, recombination and repair intermediates, they have been proposed to participate in the spatial organization of chromosomes and in the regulation of gene expression. To explore potential roles of HCs in genome metabolism, we sought to purify proteins capable of binding specifically HCs by fractionating nuclear extracts from HeLa cells. This approach identified three RNA-binding proteins: the Tudor-staphylococcal nuclease domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing family, the paraspeckle protein component 1 and the splicing factor proline- and glutamine-rich protein. Since these proteins were partially pure after fractionation, truncated forms of these proteins were expressed in Escherichia coli and purified to near homogeneity. The specificity of their interaction with HCs was re-examined in vitro. The two truncated purified SND1 proteins exhibited specificity for HCs, opening the interesting possibility of a link between the basic transcription machinery and HC structures via SND1.

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Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis

Journal of Mass Spectrometry ◽

10.1002/jms.606 ◽

2004 ◽

Vol 39 (3) ◽

pp. 233-254 ◽

Cited By ~ 349

Author(s):

György Vas ◽

Károly Vékey

Keyword(s):

Sample Preparation ◽

Solid Phase Microextraction ◽

Solid Phase ◽

Mass Spectrometric ◽

Mass Spectrometric Analysis ◽

Spectrometric Analysis

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The design and structural characterization of a synthetic pentatricopeptide repeat protein

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714024869 ◽

2015 ◽

Vol 71 (2) ◽

pp. 196-208 ◽

Cited By ~ 23

Author(s):

Benjamin S. Gully ◽

Kunal R. Shah ◽

Mihwa Lee ◽

Kate Shearston ◽

Nicole M. Smith ◽

...

Keyword(s):

Crystal Structure ◽

Binding Proteins ◽

Rna Binding ◽

De Novo ◽

Rna Binding Proteins ◽

Specific Binding ◽

Consensus Sequence ◽

Pentatricopeptide Repeat ◽

Ppr Protein ◽

Ppr Proteins

Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to thede novodesign of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2 Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.

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Identification of the Junctional Plaque Protein Plakophilin 3 in Cytoplasmic Particles Containing RNA-binding Proteins and the Recruitment of Plakophilins 1 and 3 to Stress Granules

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0708 ◽

2006 ◽

Vol 17 (3) ◽

pp. 1388-1398 ◽

Cited By ~ 72

Author(s):

Ilse Hofmann ◽

Marialuisa Casella ◽

Martina Schnölzer ◽

Tanja Schlechter ◽

Herbert Spring ◽

...

Keyword(s):

Binding Proteins ◽

Stress Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Gradient Centrifugation ◽

Intercellular Junctions ◽

Stress Granules ◽

Spectrometric Analysis ◽

Adhesive Interactions

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell–cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25–35 S and 45–55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in “stress granules” known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.

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Allele-specific binding of RNA-binding proteins reveals functional genetic variants in the RNA

10.1101/396275 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ei-Wen Yang ◽

Jae Hoon Bahn ◽

Esther Yun-Hua Hsiao ◽

Boon Xin Tan ◽

Yiwei Sun ◽

...

Keyword(s):

Genetic Variants ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Binding ◽

Specific Protein ◽

Genome Wide Association Studies ◽

Genome Wide ◽

Allele Specific ◽

Rna Interaction

AbstractAllele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants influencing RNA processing and gene expression phenotypes. Recently, genome-wide detection of in vivo binding sites of RNA binding proteins (RBPs) is greatly facilitated by the enhanced UV crosslinking and immunoprecipitation (eCLIP) protocol. Hundreds of eCLIP-Seq data sets were generated from HepG2 and K562 cells during the ENCODE3 phase. These data afford a valuable opportunity to examine allele-specific binding (ASB) of RBPs. To this end, we developed a new computational algorithm, called BEAPR (Binding Estimation of Allele-specific Protein-RNA interaction). In identifying statistically significant ASB sites, BEAPR takes into account UV cross-linking induced sequence propensity and technical variations between replicated experiments. Using simulated data and actual eCLIP-Seq data, we show that BEAPR largely outperforms often-used methods Chi-Squared test and Fisher’s Exact test. Importantly, BEAPR overcomes the inherent over-dispersion problem of the other methods. Complemented by experimental validations, we demonstrate that ASB events are significantly associated with genetic regulation of splicing and mRNA abundance, supporting the usage of this method to pinpoint functional genetic variants in post-transcriptional gene regulation. Many variants with ASB patterns of RBPs were found as genetic variants with cancer or other disease relevance. About 38% of ASB variants were in linkage disequilibrium with single nucleotide polymorphisms from genome-wide association studies. Overall, our results suggest that BEAPR is an effective method to reveal ASB patterns in eCLIP and can inform functional interpretation of disease-related genetic variants.

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Assembly of the α-Globin mRNA Stability Complex Reflects Binary Interaction between the Pyrimidine-Rich 3′ Untranslated Region Determinant and Poly(C) Binding Protein αCP

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.4572 ◽

1999 ◽

Vol 19 (7) ◽

pp. 4572-4581 ◽

Cited By ~ 82

Author(s):

Alexander N. Chkheidze ◽

Dmitry L. Lyakhov ◽

Alexander V. Makeyev ◽

Julia Morales ◽

Jian Kong ◽

...

Keyword(s):

Mrna Stability ◽

Untranslated Region ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Binding ◽

Direct Interaction ◽

Binary Interaction ◽

Globin Mrna ◽

Hnrnp K

ABSTRACT Globin mRNAs accumulate to 95% of total cellular mRNA during terminal erythroid differentiation, reflecting their extraordinary stability. The stability of human α-globin mRNA is paralleled by formation of a sequence-specific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3′ untranslated region (3′UTR), the α-complex. The proteins of the α-complex are widely expressed. The α-complex or a closely related complex also assembles at pyrimidine-rich 3′UTR segments of other stable mRNAs. These data suggest that the α-complex may constitute a general determinant of mRNA stability. One or more αCPs, members of a family of hnRNP K-homology domain poly(C) binding proteins, are essential constituents of the α-complex. The ability of αCPs to homodimerize and their reported association with additional RNA binding proteins such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that the α-complex is a multisubunit structure. In the present study, we have addressed the composition of the α-complex. An RNA titration recruitment assay revealed that αCPs were quantitatively incorporated into the α-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between each of the three major αCP isoforms and the α-globin 3′UTR was detected, suggesting that each of these proteins might be sufficient for α-complex assembly. This sufficiency was further supported by the sequence-specific binding of recombinant αCPs to a spectrum of RNA targets. Finally, density sedimentation analysis demonstrated that the α-complex could accommodate only a single αCP. These data established that a single αCP molecule binds directly to the α-globin 3′UTR, resulting in a simple binary structure for the α-complex.

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