A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5′-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.

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Suppression of C9orf72 RNA repeat-induced neurotoxicity by the ALS-associated RNA-binding protein Zfp106

eLife ◽

10.7554/elife.19032 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 22

Author(s):

Barbara Celona ◽

John von Dollen ◽

Sarat C Vatsavayai ◽

Risa Kashima ◽

Jeffrey R Johnson ◽

...

Keyword(s):

Motor Neurons ◽

Binding Proteins ◽

Rna Binding ◽

Zinc Finger Protein ◽

Rna Binding Proteins ◽

Binding Protein ◽

Affinity Purification ◽

Rna Binding Protein ◽

Severe Motor ◽

Affinity Purification Mass Spectrometry

Expanded GGGGCC repeats in the first intron of the C9orf72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS), but the mechanisms underlying repeat-induced disease remain incompletely resolved. One proposed gain-of-function mechanism is that repeat-containing RNA forms aggregates that sequester RNA binding proteins, leading to altered RNA metabolism in motor neurons. Here, we identify the zinc finger protein Zfp106 as a specific GGGGCC RNA repeat-binding protein, and using affinity purification-mass spectrometry, we show that Zfp106 interacts with multiple other RNA binding proteins, including the ALS-associated factors TDP-43 and FUS. We also show that Zfp106 knockout mice develop severe motor neuron degeneration, which can be suppressed by transgenic restoration of Zfp106 specifically in motor neurons. Finally, we show that Zfp106 potently suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Thus, these studies identify Zfp106 as an RNA binding protein with important implications for ALS.

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SPACE exploration of chromatin proteome to reveal associated RNA-binding proteins

10.1101/2020.07.13.200212 ◽

2020 ◽

Author(s):

Mahmoud-Reza Rafiee ◽

Julian A Zagalak ◽

Giulia Tyzack ◽

Rickie Patani ◽

Jernej Ule ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Genome Maintenance ◽

Purification Method ◽

Comprehensive Knowledge ◽

Associated Proteins ◽

Versatile Technique ◽

Dna Binders ◽

Lateral Sclerosis

AbstractChromatin is composed of many proteins that mediate intermolecular transactions with the genome. Comprehensive knowledge of these components and their interactions is necessary for insights into gene regulation and other activities; however, reliable identification of chromatin-associated proteins remains technically challenging. Here, we present SPACE (Silica Particle Assisted Chromatin Enrichment), a stringent and straightforward chromatin-purification method that helps identify direct DNA-binders separately from chromatin-associated proteins. We demonstrate SPACE’s unique strengths in three experimental set-ups: the sensitivity to detect novel chromatin-associated proteins, the quantitative nature to measure dynamic protein use across distinct cellular conditions, and the ability to handle 10-25 times less starting material than competing methods. In doing so, we reveal an unforeseen scale of association between over 500 nuclear RNA-binding proteins (RBPs) with chromatin and DNA, providing new insights into their roles as important regulators of genome maintenance and chromatin composition. Applied to iPSC-derived neural precursors, we discover a new role for the amyotrophic lateral sclerosis (ALS)-causing Valosin Containing Protein (VCP) in recruiting DNA-damage components to chromatin, thus paving the way for molecular mechanistic insights into the disease. SPACE is a fast and versatile technique with many applications.

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Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

Bioscience Reports ◽

10.1007/bf01124794 ◽

1987 ◽

Vol 7 (3) ◽

pp. 231-238 ◽

Cited By ~ 94

Author(s):

P. Manjunath ◽

M. R. Sairam ◽

J. Uma

Keyword(s):

Seminal Plasma ◽

Binding Proteins ◽

Affinity Purification ◽

Polyacrylamide Gel Electrophoresis ◽

Apparent Molecular Weight ◽

Single Step ◽

High Yield ◽

Property A ◽

Purification Method ◽

Elution Pattern

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, −A2 and −A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.

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Using RNA Affinity Purification Followed by Mass Spectrometry to Identify RNA-Binding Proteins (RBPs)

Methods in Molecular Biology - RNA Tagging ◽

10.1007/978-1-0716-0712-1_14 ◽

2020 ◽

pp. 241-253

Author(s):

Mengge Shan ◽

Brian D. Gregory

Keyword(s):

Mass Spectrometry ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Affinity Purification

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Differential Binding of Mitochondrial Transcripts by MRB8170 and MRB4160 Regulates Distinct Editing Fates of Mitochondrial mRNA in Trypanosomes

mBio ◽

10.1128/mbio.02288-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 10

Author(s):

Sameer Dixit ◽

Michaela Müller-McNicoll ◽

Vojtěch David ◽

Kathi Zarnack ◽

Jernej Ule ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Affinity Purification ◽

Mitochondrial Rna ◽

Nucleotide Resolution ◽

Additional Role

ABSTRACT A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei . Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome. IMPORTANCE Trypanosoma brucei mitochondrial mRNAs undergo maturation by RNA editing, a unique process involving decrypting open reading frames by the precise deletion and/or insertion of uridine (U) residues at specific positions on an mRNA. This process is catalyzed by multiprotein complexes, such as the RNA editing core complex, which provides the enzymatic activities needed for U insertion/deletion at a single editing site. Less well understood is how RNA editing occurs throughout an mRNA bearing multiple sites. To address this question, we mapped at single-nucleotide resolution the RNA interactions of two unique RNA-binding proteins (RBPs). These RBPs are part of the mitochondrial RNA-binding complex 1, hypothesized to mediate multiple rounds of RNA editing. Both RBPs were shown to mark mRNAs for the process in correlation with the number of editing sites on the transcript. Surprisingly, one also binds mRNAs that bypass RNA editing, indicating that it may have an additional role outside RNA editing.

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FLASH: ultra-fast protocol to identify RNA–protein interactions in cells

Nucleic Acids Research ◽

10.1093/nar/gkz1141 ◽

2019 ◽

Vol 48 (3) ◽

pp. e15-e15 ◽

Cited By ~ 1

Author(s):

Ibrahim Avsar Ilik ◽

Tugce Aktas ◽

Daniel Maticzka ◽

Rolf Backofen ◽

Asifa Akhtar

Keyword(s):

High Throughput ◽

Protein Interactions ◽

Binding Sites ◽

Binding Proteins ◽

High Throughput Sequencing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Affinity Purification ◽

Sequencing Library

Abstract Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding sites has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein–RNA interactions in living cells. The entire FLASH protocol, starting from cells on plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo.

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Identifying mRNAs bound by RNA-binding proteins using affinity purification and differential display

Methods ◽

10.1016/s1046-2023(02)00014-2 ◽

2002 ◽

Vol 26 (2) ◽

pp. 115-122 ◽

Cited By ~ 4

Author(s):

N Rodgers

Keyword(s):

Differential Display ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Affinity Purification

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uvCLAP: a fast, non-radioactive method to identify in vivo targets of RNA-binding proteins

10.1101/158410 ◽

2017 ◽

Cited By ~ 1

Author(s):

Daniel Maticzka ◽

Ibrahim Avsar Ilik ◽

Tugce Aktas ◽

Rolf Backofen ◽

Asifa Akhtar

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Neurological Diseases ◽

Affinity Purification ◽

Target Selection ◽

Rna Motif

AbstractRNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expression regulating splicing, localization, translation and stability of mRNAs. Understanding the exact contribution of RBPs to gene regulation is crucial as many RBPs are frequently mis-regulated in several neurological diseases and certain cancers. While recently developed techniques provide binding sites of RBPs, they are labor-intensive and generally rely on radioactive labeling of RNA. With more than 1,000 RBPs in a human cell, it is imperative to develop easy, robust, reproducible and high-throughput methods to determine in vivo targets of RBPs. To address these issues we developed uvCLAP (UV crosslinking and affinity purification) as a robust, reproducible method to measure RNA-protein interactions in vivo. To test its performance and applicability we investigated binding of 15 RBPs from fly, mouse and human cells. We show that uvCLAP generates reliable and comparable data to other methods. Unexpectedly, our results show that despite their different subcellular localizations, STAR proteins (KHDRBS1-3, QKI) bind to a similar RNA motif in vivo. Consistently a point mutation (KHDRBS1Y440F) or a natural splice isoform (QKI-6) that changes the respective RBP subcellular localization, dramatically alters target selection without changing the targeted RNA motif. Combined with the knowledge that RBPs can compete and cooperate for binding sites, our data shows that compartmentalization of RBPs can be used as an elegant means to generate RNA target specificity.

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Influence of hypoxia on the RNA binding proteins complexes and translation in leukemia cells

10.26226/morressier.5ebd45acffea6f735881af3d ◽

2020 ◽

Author(s):

Paulina Podszywalow-Bartnicka

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Leukemia Cells

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