The mRNA Binding Proteome of Proliferating and Differentiated Muscle Cells

Hiller, Monika; Geissler, M; Janssen, George M.C.; Veelen, Peter A. van; Aartsma‐Rus, Annemieke; Spitali, Pietro

doi:10.1016/j.gpb.2020.06.004

Cited by 5 publications

(7 citation statements)

References 38 publications

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“…Consistent with the considerations above, we observed that several classical core RBPs (splicing factors, ribosomal proteins, and hnRNPs) show decreased binding in four comparative RIC studies employing different perturbations in distinct mammalian cell lines ( Figure 2 ; Milek et al, 2017 ; Perez-Perri et al, 2018 ; Garcia-Moreno et al, 2019 ; Hiller et al, 2020 ). Conversely, proteins with moonlighting RNA-binding activity such as metabolic enzymes tend to show increased binding.…”

Section: Introductionsupporting

confidence: 87%

“…Quantitative interpretation of CLIP-like experiments is difficult ( Ramanathan et al, 2019 ), and only performed in exceptional cases ( Schueler et al, 2014 ) or indirectly ( Gregersen et al, 2014 ; Milek et al, 2017 ; Smith et al, 2021 ). In contrast, RIC-like experiments are often used to assess changes in RNA-binding for RBPs across conditions ( Hentze et al, 2018 ), and several groups have identified context-specific regulatory RBPs in mammalian tissue culture cells ( Boucas et al, 2015 ; Liepelt et al, 2016 ; Milek et al, 2017 ; Perez-Perri et al, 2018 ; Garcia-Moreno et al, 2019 ; Ignarski et al, 2019 ; Queiroz et al, 2019 ; Trendel et al, 2019 ; Hiller et al, 2020 ; Smith et al, 2021 ), zebrafish and fly embryos ( Sysoev et al, 2016 ; Despic et al, 2017 ), yeast ( Shchepachev et al, 2019 ; Bresson et al, 2020 ), and plant cells ( Marondedze et al, 2019 ). While it is tempting to interpret differences in RIC-like experiments as changes in RBP-RNA interaction (RNA-bound protein fraction), this is not to be taken for granted.…”

Section: Introductionmentioning

confidence: 99%

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Opportunities and Challenges in Global Quantification of RNA-Protein Interaction via UV Cross-Linking

Vieira-Vieira

Selbach

2021

Front. Mol. Biosci.

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RNA-binding proteins (RBPs) are key mediators of posttranscriptional gene expression control. However, the links between cell signaling on the one hand and RBP function on the other are understudied. While thousands of posttranslational modification (PTM) sites on RBPs have been identified, their functional roles are only poorly characterized. RNA-interactome capture (RIC) and cross-linking and immunoprecipitation (CLIP) are attractive methods that provide information about RBP-RNA interactions on a genome-wide scale. Both approaches rely on the in situ UV cross-linking of RBPs and RNAs, biochemical enrichment and analysis by RNA-sequencing (CLIP) or mass spectrometry (RIC). In principle, RIC- and CLIP-like methods could be used to globally quantify RBP-RNA interactions in response to perturbations. However, several biases have to be taken into account to avoid misinterpretation of the results obtained. Here, we focus on RIC-like methods and discuss four key aspects relevant for quantitative interpretation: (1) the RNA isolation efficiency, (2) the inefficient and highly variable UV cross-linking, (3) the baseline RNA occupancy of RBPs, and (4) indirect factors affecting RBP-RNA interaction. We highlight these points by presenting selected examples of PTMs that might induce differential quantification in RIC-like experiments without necessarily affecting RNA-binding. We conclude that quantifying RBP-RNA interactions via RIC or CLIP-like methods should not be regarded as an end in itself but rather as starting points for deeper analysis.

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Section: Introductionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Opportunities and Challenges in Global Quantification of RNA-Protein Interaction via UV Cross-Linking

Vieira-Vieira

Selbach

2021

Front. Mol. Biosci.

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“…Thus, post-transcriptional regulation of gene expression orchestrated by RBPs but also other factors, such as non-coding RNAs and even MRFs ( Panda et al, 2016 ; Luo et al, 2021 ), plays crucial roles in muscle development and regeneration. Many RBPs are differentially associated with target mRNAs in proliferating myoblasts or differentiated myotubes to regulate distinct aspects of RNA metabolism in a stage-dependent manner ( Hiller et al, 2020 ). As will be discussed below, this is often correlated with their dynamic expression and subcellular localization during myogenesis.…”

Section: An Overview Of Post-transcriptional Regulation In Muscle Developmentmentioning

confidence: 99%

“…Transcriptome and proteome studies have not only identified hundreds of muscle-specific RBPs but also revealed changes in the interactome during muscle differentiation ( Hiller et al, 2020 ). With so many RBPs dynamically implicated in different steps of myogenesis and in multiple aspects of post-transcriptional regulation of gene expression, it is not surprising that they functionally interact in a tight network to coordinate muscle development and maintain muscle homeostasis.…”

Section: Functional Interactions Between Rna-binding Proteins Coordinate Gene Expression During Myogenesismentioning

confidence: 99%

RNA-Binding Proteins in the Post-transcriptional Control of Skeletal Muscle Development, Regeneration and Disease

Shi

Grifone

2021

Front. Cell Dev. Biol.

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Embryonic myogenesis is a temporally and spatially regulated process that generates skeletal muscle of the trunk and limbs. During this process, mononucleated myoblasts derived from myogenic progenitor cells within the somites undergo proliferation, migration and differentiation to elongate and fuse into multinucleated functional myofibers. Skeletal muscle is the most abundant tissue of the body and has the remarkable ability to self-repair by re-activating the myogenic program in muscle stem cells, known as satellite cells. Post-transcriptional regulation of gene expression mediated by RNA-binding proteins is critically required for muscle development during embryogenesis and for muscle homeostasis in the adult. Differential subcellular localization and activity of RNA-binding proteins orchestrates target gene expression at multiple levels to regulate different steps of myogenesis. Dysfunctions of these post-transcriptional regulators impair muscle development and homeostasis, but also cause defects in motor neurons or the neuromuscular junction, resulting in muscle degeneration and neuromuscular disease. Many RNA-binding proteins, such as members of the muscle blind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) families, display both overlapping and distinct targets in muscle cells. Thus they function either cooperatively or antagonistically to coordinate myoblast proliferation and differentiation. Evidence is accumulating that the dynamic interplay of their regulatory activity may control the progression of myogenic program as well as stem cell quiescence and activation. Moreover, the role of RNA-binding proteins that regulate post-transcriptional modification in the myogenic program is far less understood as compared with transcription factors involved in myogenic specification and differentiation. Here we review past achievements and recent advances in understanding the functions of RNA-binding proteins during skeletal muscle development, regeneration and disease, with the aim to identify the fundamental questions that are still open for further investigations.

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“…CLIP and RIC have provided us with useful catalogs of RBPs and their RNA-binding sites (Gerstberger, Hafner and Tuschl, 2014; Maatz et al ., 2014; Hentze et al ., 2018; Zhu et al ., 2019; Van Nostrand et al ., 2020). Also, comparative RIC studies provided information about how the interaction of RBPs with RNA changes upon perturbation (Milek et al ., 2017; Perez-Perri et al ., 2018; Garcia-Moreno et al ., 2019; Hiller et al ., 2020). However, the impact of PTMs in RBPs on their interaction with RNAs has not yet been investigated systematically.…”

Section: Introductionmentioning

confidence: 99%

Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20

Vieira-Vieira

Dauksaite

Gotthardt

et al. 2021

Preprint

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SummaryRNA-binding proteins (RBPs) are major regulators of gene expression at the post-transcriptional level. While many posttranslational modification sites in RBPs have been identified, little is known about how these modifications regulate RBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of cellular RBPs pulled down with polyadenylated mRNAs. Applying qRIC to HEK293T cells quantified pull-down efficiencies of over 300 RBPs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of RBPs. Over hundred phosphorylation events increased or decreased pull-down efficiency compared to the unmodified RBPs and thus have regulatory potential. Our data captures known regulatory phosphorylation sites in ELAVL1, SF3B1 and UPF1 and identifies new potentially regulatory sites. Follow-up experiments on the cardiac splicing regulator RBM20 revealed that multiple phosphorylation sites in the C-terminal disordered region affect nucleo-cytoplasmic localization, association with cytosolic RNA granules and alternative splicing. Together, we show that qRIC is a scalable method to identify functional posttranslational modification sites in RBPs.HighlightsqRIC globally quantifies the fraction of RNA-binding proteins pulled down with mRNACombining qRIC with phosphoproteomics identifies sites that affect RNA bindingPhosphorylation sites in RBM20 regulate its function in splicing

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The mRNA Binding Proteome of Proliferating and Differentiated Muscle Cells

Cited by 5 publications

References 38 publications

Opportunities and Challenges in Global Quantification of RNA-Protein Interaction via UV Cross-Linking

Opportunities and Challenges in Global Quantification of RNA-Protein Interaction via UV Cross-Linking

RNA-Binding Proteins in the Post-transcriptional Control of Skeletal Muscle Development, Regeneration and Disease

Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20

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