Spatiotemporally-resolved mapping of RNA binding proteins via functional proximity labeling reveals a mitochondrial mRNA anchor promoting stress recovery

AbstractProximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, we show that it is possible to map specific protein subclasses within distinct compartments of living cells. In particular, we develop a method to enrich subcompartment-specific RNA binding proteins (RBPs) by combining peroxidase-catalyzed PL with organic-aqueous phase separation of crosslinked protein-RNA complexes (“APEX-PS”). We use APEX-PS to generate datasets of nuclear, nucleolar, and outer mitochondrial membrane (OMM) RBPs, which can be mined for novel functions. For example, we find that the OMM RBP SYNJ2BP retains specific nuclear-encoded mitochondrial mRNAs at the OMM during translation stress, facilitating their local translation and import of protein products into the mitochondrion during stress recovery. Functional PL in general, and APEX-PS in particular, represent versatile approaches for the discovery of proteins with novel function in specific subcellular compartments.

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Functional proximity mapping of RNA binding proteins uncovers a mitochondrial mRNA anchor that promotes stress recovery

10.1101/2020.11.17.387209 ◽

2020 ◽

Author(s):

Wei Qin ◽

Samuel A Myers ◽

Dominique K. Carey ◽

Steven A Carr ◽

Alice Y Ting

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Protein ◽

Stress Recovery ◽

Protein Enrichment ◽

Functional Protein ◽

Protein Mapping ◽

Versatile Tool ◽

Rna Complexes

AbstractProximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, it should be possible to map specific protein subclasses within distinct compartments of living cells. Here we demonstrate this capability for RNA binding proteins (RBPs), by combining peroxidase-based PL with organic-aqueous phase separation of crosslinked protein-RNA complexes (“APEX-PS”). We validated APEX-PS by mapping nuclear RBPs, then applied it to uncover the RBPomes of two unpurifiable subcompartments - the nucleolus and the outer mitochondrial membrane (OMM). At the OMM, we discovered the RBP SYNJ2BP, which retains specific nuclear-encoded mitochondrial mRNAs during translation stress, to promote their local translation and import of protein products into the mitochondrion during stress recovery. APEX-PS is a versatile tool for compartment-specific RBP discovery and expands the scope of PL to functional protein mapping.Graphic Abstract

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Functional proximity mapping of RNA binding proteins uncovers a mitochondrial mRNA anchor that promotes stress recovery

10.21203/rs.3.rs-103196/v1 ◽

2020 ◽

Author(s):

Wei Qin ◽

Samuel Myers ◽

Dominique Carey ◽

Steven Carr ◽

Alice Ting

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Protein ◽

Stress Recovery ◽

Protein Enrichment ◽

Functional Protein ◽

Protein Mapping ◽

Versatile Tool ◽

Rna Complexes

Abstract Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, it should be possible to map specific protein subclasses within distinct compartments of living cells. Here we demonstrate this capability for RNA binding proteins (RBPs), by combining peroxidase-based PL with organic-aqueous phase separation of crosslinked protein-RNA complexes (“APEX-PS”). We validated APEX-PS by mapping nuclear RBPs, then applied it to uncover the RBPomes of two unpurifiable subcompartments - the nucleolus and the outer mitochondrial membrane (OMM). At the OMM, we discovered the RBP SYNJ2BP, which retains specific nuclear-encoded mitochondrial mRNAs during translation stress, to promote their local translation and import of protein products into the mitochondrion during stress recovery. APEX-PS is a versatile tool for compartment-specific RBP discovery and expands the scope of PL to functional protein mapping.

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CLIP-Seq to Discover Transcriptome-Wide Imprinting of RNA Binding Proteins in Living Cells

Methods in Molecular Biology - Small Non-Coding RNAs ◽

10.1007/978-1-4939-2547-6_14 ◽

2015 ◽

pp. 151-160 ◽

Cited By ~ 3

Author(s):

Jérôme Saulière ◽

Hervé Le Hir

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Living Cells

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A protocol for PAIR: PNA-assisted identification of RNA binding proteins in living cells

Nature Protocols ◽

10.1038/nprot.2006.81 ◽

2006 ◽

Vol 1 (2) ◽

pp. 920-927 ◽

Cited By ~ 17

Author(s):

Fanyi Zeng ◽

Tiina Peritz ◽

Theresa J Kannanayakal ◽

Kalle Kilk ◽

Emelía Eiríksdóttir ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Living Cells

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Systematic Identification of Protein Phosphorylation-Mediated Interactions

10.1101/2020.09.18.304121 ◽

2020 ◽

Author(s):

Brendan M. Floyd ◽

Kevin Drew ◽

Edward M. Marcotte

Keyword(s):

Mass Spectrometry ◽

Protein Phosphorylation ◽

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Protein ◽

Mass Spectrometry Data ◽

Size Exclusion ◽

Functional Protein

ABSTRACTProtein phosphorylation is a key regulatory mechanism involved in nearly every eukaryotic cellular process. Increasingly sensitive mass spectrometry approaches have identified hundreds of thousands of phosphorylation sites but the functions of a vast majority of these sites remain unknown, with fewer than 5% of sites currently assigned a function. To increase our understanding of functional protein phosphorylation we developed an approach for identifying the phosphorylation-dependence of protein assemblies in a systematic manner. A combination of non-specific protein phosphatase treatment, size-exclusion chromatography, and mass spectrometry allowed us to identify changes in protein interactions after the removal of phosphate modifications. With this approach we were able to identify 316 proteins involved in phosphorylation-sensitive interactions. We recovered known phosphorylation-dependent interactors such as the FACT complex and spliceosome, as well as identified novel interactions such as the tripeptidyl peptidase TPP2 and the supraspliceosome component ZRANB2. More generally, we find phosphorylation-dependent interactors to be strongly enriched for RNA-binding proteins, providing new insight into the role of phosphorylation in RNA binding. By searching directly for phosphorylated amino acid residues in mass spectrometry data, we identified the likely regulatory phosphosites on ZRANB2 and FACT complex subunit SSRP1. This study provides both a method and resource for obtaining a better understanding of the role of phosphorylation in native macromolecular assemblies.

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Capturing RNA–protein interaction via CRUIS

Nucleic Acids Research ◽

10.1093/nar/gkaa143 ◽

2020 ◽

Vol 48 (9) ◽

pp. e52-e52 ◽

Cited By ~ 5

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.

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Current techniques for visualizing RNA in cells

F1000Research ◽

10.12688/f1000research.8151.1 ◽

2016 ◽

Vol 5 ◽

pp. 775 ◽

Cited By ~ 9

Author(s):

Lilith V.J.C. Mannack ◽

Sebastian Eising ◽

Andrea Rentmeister

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

State Of The Art ◽

Living Cells ◽

Enzymatic Modification ◽

Current State ◽

Rna Imaging ◽

In Cells ◽

Art Techniques

Labeling RNA is of utmost interest, particularly in living cells, and thus RNA imaging is an emerging field. There are numerous methods relying on different concepts ranging from hybridization-based probes, over RNA-binding proteins to chemo-enzymatic modification of RNA. These methods have different benefits and limitations. This review aims to outline the current state-of-the-art techniques and point out their benefits and limitations.

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Post-transcriptional control of gene expression following stress: the role of RNA-binding proteins

Biochemical Society Transactions ◽

10.1042/bst20160364 ◽

2017 ◽

Vol 45 (4) ◽

pp. 1007-1014 ◽

Cited By ~ 35

Author(s):

Robert Harvey ◽

Veronica Dezi ◽

Mariavittoria Pizzinga ◽

Anne E. Willis

Keyword(s):

Protein Synthesis ◽

Mammalian Cells ◽

Cellular Response ◽

Transcriptional Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Major Influence ◽

Functional Protein ◽

Control Of Gene Expression

The ability of mammalian cells to modulate global protein synthesis in response to cellular stress is essential for cell survival. While control of protein synthesis is mediated by the regulation of eukaryotic initiation and elongation factors, RNA-binding proteins (RBPs) provide a crucial additional layer to post-transcriptional regulation. RBPs bind specific RNA through conserved RNA-binding domains and ensure that the information contained within the genome and transcribed in the form of RNA is exported to the cytoplasm, chemically modified, and translated prior to folding into a functional protein. Thus, this group of proteins, through mediating translational reprogramming, spatial reorganisation, and chemical modification of RNA molecules, have a major influence on the robust cellular response to external stress and toxic injury.

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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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