Identification of RNA-binding Proteins in Macrophages by Interactome Capture

Liepelt, Anke; Vries, Isabel S. Naarmann-de; Simons, Nadine; Eichelbaum, Katrin; Föhr, Sophia; Archer, Stuart K.; Castelló, Alfredo; Usadel, Björn; Krijgsveld, Jeroen; Preiß, Thomas; Marx, Gernot; Hentze, Matthias W.; Ostareck, Dirk H.; Ostareck-Lederer, Antje

doi:10.1074/mcp.m115.056564

Cited by 74 publications

(74 citation statements)

References 93 publications

(93 reference statements)

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“…The majority of these proteins (88%; 438/496) had been identified in previous global analyses of RNA-and ribosome-binding proteins 1-12 (Supplemental Table 2), indicating that our list of polysome-interacting proteins is mostly in agreement with the literature.The curated 496 mRNA/Ribosome-bound proteins fell into several broad categories ( Figure 1F). This includes a wide array of metabolic enzymes ( Figure 1F), especially those involved in carbohydrate metabolism, similar to what had been uncovered by several genome-wide RNA/Ribosome binding protein surveys from a variety of species [1][2][3][4][5][6][7][8][9][10][11][12]18 . The most abundant by spectral counts were glycolytic enzymes, including both spliced isoforms of pyruvate kinase (PKM1 and 2).…”

supporting

confidence: 55%

Pyruvate Kinase M Links Glucose Availability to Protein Synthesis

Kejiou

Ilan

Aigner

et al. 2019

Preprint

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How human cells coordinate various metabolic processes, such as glycolysis and protein translation, remains unclear. One key insight is that various metabolic enzymes have been found to associate with mRNAs, however whether these enzymes regulate mRNA biology in response to changes in cellular metabolic state remains unknown. Here we report that the glycolytic enzyme, pyruvate kinase M (PKM), inhibits the translation of 7% of the transcriptome in response to elevated levels of glucose and pyruvate.Our data suggest that in the presence of glucose and pyruvate, PKM associates with ribosomes that are synthesizing stretches of polyacidic nascent polypeptides and stalls the elongation step of translation.PKM-regulated mRNAs encode proteins required for the cell cycle and may explain previous results linking PKM to cell cycle regulation. Our study uncovers an unappreciated link between glycolysis and the ribosome that likely coordinates the intake of glycolytic metabolites with the regulation of protein synthesis and the cell cycle. Results and Discussion Mass spectrometry analysis of ER and cytosolic polysomes and mRNPsOver the past decade, numerous proteins that lack RNA binding domains, such as metabolic enzymes, have been shown to exhibit mRNA and ribosome binding 1-12 . It is unclear whether these unconventional RNA binding proteins (RBP) associate with transcripts and ribosomes in a spatially defined manner, or if they link metabolic states to mRNA stability or translation. To determine the spatial distribution of RNA-, and ribosome-binding proteins, we isolated ER and cytosolic fractions from human osteosarcoma (U2OS) cells ( Figure 1A) and sedimented crude polysomes. The cytosol and ER represent the major division in cellular protein synthesis, each containing distinct pools of mRNAs and unique translational regulatory systems [13][14][15][16] . These isolated polysomes were then treated with RNase to liberate RNA-binding proteins ("RNA-bound fraction"), and resedimented to pellet ribosomes and associated proteins ("Ribosome-bound fraction"; Figure 1B). We then analyzed the composition of the RNA-bound and Ribosome-bound fractions ( Figure 1B-C) by mass spectrometry, as previously described 17 . In parallel we also isolated messenger ribonuclear protein (mRNP) complexes from the ER and cytosol using oligo-dT affinity chromatography ("mRNP-bound"; Figure 1B, D, "dT") and again analyzed these fractions by mass spectrometry. To control for non-specific binding to the oligo-dT resin we also performed the affinity chromatography with beads lacking any nucleic acid ( Figure 1B, "Mock Beads", 1D "B"). Our purification conditions, done in the absence of crosslinking, enabled recovery of proteins that are directly and indirectly bound to mRNAs and/or ribosomes.After statistical processing (see Methods), 370 proteins were present in the RNA-bound fraction, 414 were present in the ribosome-bound fraction, and 2690 proteins were enriched in the mRNPassociated fraction. Upon further manual curation (see methods), 496 protei...

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supporting

confidence: 55%

Pyruvate Kinase M Links Glucose Availability to Protein Synthesis

Kejiou

Ilan

Aigner

et al. 2019

Preprint

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“…Applied to HeLa and HEK293 cells, RNA interactome capture identified more than a thousand RBPs, hundreds of which were previously unknown to bind RNA. This method was successfully used to determine the poly(A)-RNA-binding proteome of different human cell types, including human (Baltz et al 2012;Castello et al 2012;Beckmann et al 2015) and murine (Kwon et al 2013;Liao et al 2016;Liepelt et al 2016) cells, as well as from organisms such as S. cerevisiae (Mitchell et al 2013;Beckmann et al 2015;Matia-González et al 2015), C. elegans (Matia-González et al 2015), D. melanogaster (Sysoev et al 2016;Wessels et al 2016), A. thaliana (Marondedze et al 2016;Reichel et al 2016), and the parasites plasmodium and trypanosoma (Bunnik et al 2016;Lueong et al 2016). However, the identification of the RBPs bound to specific RNAs remains challenging.…”

Section: Introductionmentioning

confidence: 99%

Specific RNP capture with antisense LNA/DNA mixmers

Rogell

Fischer

Rettel

et al. 2017

RNA

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RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

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“…During the last few years, RNA–protein interactome capture has become increasingly popular and expanded to commonly used model organisms and human parasites (Table ). It confirmed repeatedly that the spectrum of RBPs goes well beyond previous thoughts and includes a large fraction of unconventional RBPs that are devoid of classical RBDs, which further prompted the development novel approaches for defining noncanonical RNA‐binding regions on RBPs .…”

Section: List Of Poly(a) Rna–protein Interactome Studies Grouped To Tmentioning

confidence: 99%

Unconventional RNA‐binding proteins: an uncharted zone in RNA biology

Albihlal

Gerber

2018

FEBS Letters

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The RNA-binding proteins play essential roles in the post-transcriptional regulation of gene expression. While hundreds of RNA-binding proteins can be predicted computationally, the recent introduction of proteome-wide approaches has dramatically expanded the repertoire of proteins interacting with RNA. Besides canonical RNA-binding proteins that contain characteristic RNA-binding domains, many proteins that lack such domains but have other well-characterized cellular functions were identified; including metabolic enzymes, heat shock proteins, kinases, as well as transcription factors and chromatin-associated proteins. In the context of these recently published RNA-protein interactome datasets obtained from yeast, nematodes, flies, plants and mammalian cells, we discuss examples for seemingly evolutionary conserved 'unconventional' RNA-binding proteins that act in central carbon metabolism, stress response or regulation of transcription.

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Identification of RNA-binding Proteins in Macrophages by Interactome Capture

Cited by 74 publications

References 93 publications

Pyruvate Kinase M Links Glucose Availability to Protein Synthesis

Pyruvate Kinase M Links Glucose Availability to Protein Synthesis

Specific RNP capture with antisense LNA/DNA mixmers

Unconventional RNA‐binding proteins: an uncharted zone in RNA biology

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