Transcriptome-wide discovery of coding and noncoding RNA-binding proteins

Huang, Rongbing; Han, Meihua; Meng, Liying; Chen, Xing

doi:10.1073/pnas.1718406115

Cited by 145 publications

(140 citation statements)

References 76 publications

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“…This technique was an important advance but, like RIP, identifies the species involved but not the site of interaction, and is limited to mature mRNAs. To identify the total RNA‐bound proteome, the approach of 5‐ethynyluridine (EU) labelling of RNAs followed by biotin ligation using the click reaction (RICK) was recently developed (Bao et al , ; Huang et al , ) as well as approaches based on phase separation, in which RNA–protein conjugates are recovered from an aqueous/phenol interface (Queiroz et al , ; Trendel et al , ). In addition, MS analyses have been developed to identify the precise amino acid at the site of RNA–protein crosslinking (Kramer et al , ).…”

Section: Introductionmentioning

confidence: 99%

Defining the RNA interactome by total RNA ‐associated protein purification

Shchepachev

Bresson

Spanos

et al. 2019

Molecular Systems Biology

123

139

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The RNA binding proteome ( RBP ome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA ‐associated protein purification ( TRAPP ) based on 254 nm UV crosslinking and purification of all RNA –protein complexes using silica beads. In a variant approach ( PAR ‐ TRAPP ), RNA s were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR ‐ TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBP s. In comparison, TRAPP identified many more proteins not expected to bind RNA , and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNA s. Exploiting PAR ‐ TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA –peptide conjugates, using iTRAPP , providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBP ome characterization.

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Section: Introductionmentioning

confidence: 99%

Defining the RNA interactome by total RNA ‐associated protein purification

Shchepachev

Bresson

Spanos

et al. 2019

Molecular Systems Biology

123

139

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“…High throughput RNA association annotations were primarily collected from Hentze et al 2018supplemental table S2 (Hentze et al, 2018. In addition, we gathered more recent high throughput datasets from (Bao et al, 2018;Huang et al, 2018;Queiroz et al, 2019;Trendel et al, 2019).…”

Section: Rna-associated Annotations Overlap Comparisons and Score Pementioning

confidence: 99%

“…Further, RNPs are strongly implicated in human diseases including amyotrophic lateral sclerosis (ALS) (Scotter et al, 2015), spinocerebellar ataxia (Yue et al, 2001), and autism (Voineagu et al, 2011). Accordingly, substantial recent effort has been focused on systematic identification of RNA-associated proteins (Baltz et al, 2012;Bao et al, 2018;Brannan et al, 2016;Castello et al, 2012Castello et al, , 2016He et al, 2016;Huang et al, 2018;Queiroz et al, 2019;Treiber et al, 2017;Trendel et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Systematic discovery of endogenous human ribonucleoprotein complexes

Mallam

Sae-Lee

Schaub

et al. 2018

Preprint

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RNA-binding proteins (RBPs) play essential roles in biology and are frequently associated with human disease. While recent studies have systematically identified individual RBPs, their higher order assembly into Ribonucleoprotein (RNP) complexes has not been systematically investigated. Here, we describe a proteomics method for systematic identification of RNP complexes in human cells. We identify 1,428 protein complexes that associate with RNA, indicating that over 20% of known human protein complexes contain RNA. To explore the role of RNA in the assembly of each complex, we identify complexes that dissociate, change composition, or form stable protein-only complexes in the absence of RNA. Importantly, these data also provide specific novel insights into the function of well-studied protein complexes not previously known to associate with RNA, including replication factor C (RFC) and cytokinetic centralspindlin complex. Finally, we use our method to systematically identify cell-type specific RNA-associated proteins in mouse embryonic stem cells. We distribute these data as a resource, rna.MAP (rna.proteincomplexes.org) which provides a comprehensive dataset for the study of RNA-associated protein complexes. Our system thus provides a novel methodology for further explorations across human tissues and disease states, as well as throughout all domains of life. ! 3! ! 4! upon CF-MS by comparing chromatographic separations of cellular lysate under control and RNA degrading conditions ( Figure 1A). A statistical framework is then applied to discover RNP complexes by identifying concurrent shifts of known protein complex subunits upon RNA degradation ( Figure 1A).Analysis of DIF-FRAC data answers important questions as to the role of RNA plays in macromolecular complexes. Specifically, we identify RNP complexes that 1) dissociate, 2) form stable protein-only complexes, and 3) change composition in the absence of RNA suggesting specific roles for RNA in each of these cases. Because DIF-FRAC is independent of UV crosslinking, nucleotide incorporation, genetic manipulation or poly(A) RNA capture efficiency, it can therefore be used to investigate a wide variety of cell types, tissues and species. To demonstrate this versatility, we apply DIF-FRAC to mouse embryonic stem cells (mESCs), identifying 1,165 RNA-associated proteins, to show the method is highly adaptable and can be extended to discover RNP complexes in diverse samples.Finally, we created a system-wide resource of 1,428 RNP complexes, many of which are previously unreported as having an RNA component, representing 20% of known human protein complexes. We provide our resource, rna.MAP, to the community as a fully searchable web database at rna.proteincomplexes.org. Results and Discussion Differential fractionation (DIF-FRAC) identifies RNP complexesThe DIF-FRAC strategy builds upon our previous strategy of Co-Fractionation Massspectroscopy (CF-MS) for identifying protein complexes in cellular lysate (Havugimana et al., 2012;Wan et al., 2015). CF-MS chromatograp...

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“…What has been missing is a methodology to purify RNPs in an unbiased fashion. Methods based on RNA in vivo labeling with modified nucleotides as RBR-ID [9], RICK [10] and CARIC [11] have been introduced. These approaches utilise modified RNA bases to either identify RBPs or to directly purify RNPs from cells.…”

Section: Introductionmentioning

confidence: 99%

Fast and unbiased purification of RNA-protein complexes after UV cross-linking

Urdaneta

Beckmann

2019

Preprint

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Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 hrs) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.

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Transcriptome-wide discovery of coding and noncoding RNA-binding proteins

Cited by 145 publications

References 76 publications

Defining the RNA interactome by total RNA ‐associated protein purification

Defining the RNA interactome by total RNA ‐associated protein purification

Systematic discovery of endogenous human ribonucleoprotein complexes

Fast and unbiased purification of RNA-protein complexes after UV cross-linking

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