Middle-down proteomics reveals dense sites of methylation and phosphorylation in arginine-rich RNA-binding proteins

AbstractArginine (Arg)-rich RNA-binding proteins play an integral role in RNA metabolism. Post-translational modifications (PTMs) within Arg-rich domains, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these proteins. Here, we report a middle-down proteomic approach coupled with electron transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the Arg-rich domains of U1-70K and structurally similar RNA-binding proteins from nuclear extracts of HEK293 cells. Remarkably, the Arg-rich domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with di-methylation and phosphorylation favoring RSRS motifs. Although they favor a common motif, analysis of combinatorial PTMs within RSRS motifs indicate that phosphorylation and methylation do not often co-occur, suggesting they may functionally oppose one another. Collectively, these findings suggest that the level of PTMs within Arg-rich domains may be among the highest in the proteome, and a possible unexplored regulator of RNA metabolism. These data also serve as a resource to facilitate future mechanistic studies of the role of PTMs in RNA-binding protein structure and function.BriefsMiddle-down proteomics reveals arginine-rich RNA-binding proteins contain many sites of methylation and phosphorylation.

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RNA Is a Double-Edged Sword in ALS Pathogenesis

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.708181 ◽

2021 ◽

Vol 15 ◽

Author(s):

Benjamin L. Zaepfel ◽

Jeffrey D. Rothstein

Keyword(s):

Motor Neurons ◽

Cortical Neurons ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Metabolism ◽

Small Subset ◽

Toxic Rna ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that affects upper and lower motor neurons. Familial ALS accounts for a small subset of cases (<10–15%) and is caused by dominant mutations in one of more than 10 known genes. Multiple genes have been causally or pathologically linked to both ALS and frontotemporal dementia (FTD). Many of these genes encode RNA-binding proteins, so the role of dysregulated RNA metabolism in neurodegeneration is being actively investigated. In addition to defects in RNA metabolism, recent studies provide emerging evidence into how RNA itself can contribute to the degeneration of both motor and cortical neurons. In this review, we discuss the roles of altered RNA metabolism and RNA-mediated toxicity in the context of TARDBP, FUS, and C9ORF72 mutations. Specifically, we focus on recent studies that describe toxic RNA as the potential initiator of disease, disease-associated defects in specific RNA metabolism pathways, as well as how RNA-based approaches can be used as potential therapies. Altogether, we highlight the importance of RNA-based investigations into the molecular progression of ALS, as well as the need for RNA-dependent structural studies of disease-linked RNA-binding proteins to identify clear therapeutic targets.

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The expanding world of metabolic enzymes moonlighting as RNA binding proteins

Biochemical Society Transactions ◽

10.1042/bst20200664 ◽

2021 ◽

Author(s):

Nicole J. Curtis ◽

Constance J. Jeffery

Keyword(s):

Gene Expression ◽

Regulatory Networks ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Molecular Structures ◽

Intermediary Metabolism ◽

The Many ◽

And Function

RNA binding proteins play key roles in many aspects of RNA metabolism and function, including splicing, transport, translation, localization, stability and degradation. Within the past few years, proteomics studies have identified dozens of enzymes in intermediary metabolism that bind to RNA. The wide occurrence and conservation of RNA binding ability across distant branches of the evolutionary tree suggest that these moonlighting enzymes are involved in connections between intermediary metabolism and gene expression that comprise far more extensive regulatory networks than previously thought. There are many outstanding questions about the molecular structures and mechanisms involved, the effects of these interactions on enzyme and RNA functions, and the factors that regulate the interactions. The effects on RNA function are likely to be wider than regulation of translation, and some enzyme–RNA interactions have been found to regulate the enzyme's catalytic activity. Several enzyme–RNA interactions have been shown to be affected by cellular factors that change under different intracellular and environmental conditions, including concentrations of substrates and cofactors. Understanding the molecular mechanisms involved in the interactions between the enzymes and RNA, the factors involved in regulation, and the effects of the enzyme–RNA interactions on both the enzyme and RNA functions will lead to a better understanding of the role of the many newly identified enzyme–RNA interactions in connecting intermediary metabolism and gene expression.

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Roles of Organellar RNA-Binding Proteins in Plant Growth, Development, and Abiotic Stress Responses

International Journal of Molecular Sciences ◽

10.3390/ijms21124548 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4548 ◽

Cited By ~ 1

Author(s):

Kwanuk Lee ◽

Hunseung Kang

Keyword(s):

Abiotic Stress ◽

Plant Growth ◽

Stress Responses ◽

Translational Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Metabolism ◽

Functional Roles ◽

Growth Development

Organellar gene expression (OGE) in chloroplasts and mitochondria is primarily modulated at post-transcriptional levels, including RNA processing, intron splicing, RNA stability, editing, and translational control. Nucleus-encoded Chloroplast or Mitochondrial RNA-Binding Proteins (nCMRBPs) are key regulatory factors that are crucial for the fine-tuned regulation of post-transcriptional RNA metabolism in organelles. Although the functional roles of nCMRBPs have been studied in plants, their cellular and physiological functions remain largely unknown. Nevertheless, existing studies that have characterized the functions of nCMRBP families, such as chloroplast ribosome maturation and splicing domain (CRM) proteins, pentatricopeptide repeat (PPR) proteins, DEAD-Box RNA helicase (DBRH) proteins, and S1-domain containing proteins (SDPs), have begun to shed light on the role of nCMRBPs in plant growth, development, and stress responses. Here, we review the latest research developments regarding the functional roles of organellar RBPs in RNA metabolism during growth, development, and abiotic stress responses in plants.

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Identification of hemicatenane-specific binding proteins by fractionation of HeLa nuclei extracts

Biochemical Journal ◽

10.1042/bcj20190855 ◽

2020 ◽

Vol 477 (2) ◽

pp. 509-524

Author(s):

Oumayma Rouis ◽

Cédric Broussard ◽

François Guillonneau ◽

Jean-Baptiste Boulé ◽

Emmanuelle Delagoutte

Keyword(s):

Spatial Organization ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Binding ◽

Regulation Of Gene Expression ◽

Double Stranded Dna ◽

Nuclear Extracts ◽

Interesting Possibility

DNA hemicatenanes (HCs) are four-way junctions in which one strand of a double-stranded helix is catenated with one strand of another double-stranded DNA. Frequently mentioned as DNA replication, recombination and repair intermediates, they have been proposed to participate in the spatial organization of chromosomes and in the regulation of gene expression. To explore potential roles of HCs in genome metabolism, we sought to purify proteins capable of binding specifically HCs by fractionating nuclear extracts from HeLa cells. This approach identified three RNA-binding proteins: the Tudor-staphylococcal nuclease domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing family, the paraspeckle protein component 1 and the splicing factor proline- and glutamine-rich protein. Since these proteins were partially pure after fractionation, truncated forms of these proteins were expressed in Escherichia coli and purified to near homogeneity. The specificity of their interaction with HCs was re-examined in vitro. The two truncated purified SND1 proteins exhibited specificity for HCs, opening the interesting possibility of a link between the basic transcription machinery and HC structures via SND1.

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The Fine Art of Writing a Message: RNA Metabolism in the Shaping and Remodeling of the Nervous System

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.755686 ◽

2021 ◽

Vol 14 ◽

Author(s):

María Landínez-Macías ◽

Olivier Urwyler

Keyword(s):

Nervous System ◽

Binding Proteins ◽

Neuronal Development ◽

Rna Binding ◽

Rna Binding Proteins ◽

Axon Growth ◽

Rna Metabolism ◽

Nervous System Development ◽

Protein Isoforms ◽

Growth Guidance

Neuronal morphogenesis, integration into circuits, and remodeling of synaptic connections occur in temporally and spatially defined steps. Accordingly, the expression of proteins and specific protein isoforms that contribute to these processes must be controlled quantitatively in time and space. A wide variety of post-transcriptional regulatory mechanisms, which act on pre-mRNA and mRNA molecules contribute to this control. They are thereby critically involved in physiological and pathophysiological nervous system development, function, and maintenance. Here, we review recent findings on how mRNA metabolism contributes to neuronal development, from neural stem cell maintenance to synapse specification, with a particular focus on axon growth, guidance, branching, and synapse formation. We emphasize the role of RNA-binding proteins, and highlight their emerging roles in the poorly understood molecular processes of RNA editing, alternative polyadenylation, and temporal control of splicing, while also discussing alternative splicing, RNA localization, and local translation. We illustrate with the example of the evolutionary conserved Musashi protein family how individual RNA-binding proteins are, on the one hand, acting in different processes of RNA metabolism, and, on the other hand, impacting multiple steps in neuronal development and circuit formation. Finally, we provide links to diseases that have been associated with the malfunction of RNA-binding proteins and disrupted post-transcriptional regulation.

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Circular RNAs: Biogenesis, Mechanism, and Function in Human Cancers

International Journal of Molecular Sciences ◽

10.3390/ijms20163926 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3926 ◽

Cited By ~ 25

Author(s):

Xing Zhao ◽

Yujie Cai ◽

Jianzhen Xu

Keyword(s):

Noncoding Rna ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cancer Development ◽

Circular Rnas ◽

Open Questions ◽

Circular Configuration ◽

And Function

CircRNAs are a class of noncoding RNA species with a circular configuration that is formed by either typical spliceosome-mediated or lariat-type splicing. The expression of circRNAs is usually abnormal in many cancers. Several circRNAs have been demonstrated to play important roles in carcinogenesis. In this review, we will first provide an introduction of circRNAs biogenesis, especially the regulation of circRNA by RNA-binding proteins, then we will focus on the recent findings of circRNA molecular mechanisms and functions in cancer development. Finally, some open questions are also discussed.

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Plasticity of nuclear and cytoplasmic stress responses of RNA-binding proteins

Nucleic Acids Research ◽

10.1093/nar/gkaa256 ◽

2020 ◽

Vol 48 (9) ◽

pp. 4725-4740 ◽

Cited By ~ 3

Author(s):

Michael Backlund ◽

Frank Stein ◽

Mandy Rettel ◽

Thomas Schwarzl ◽

Joel I Perez-Perri ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Stress Responses ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Environmental Cues ◽

Adaptive Responses ◽

Stressed Cells ◽

And Function

Abstract Cellular stress causes multifaceted reactions to trigger adaptive responses to environmental cues at all levels of the gene expression pathway. RNA-binding proteins (RBP) are key contributors to stress-induced regulation of RNA fate and function. Here, we uncover the plasticity of the RNA interactome in stressed cells, differentiating between responses in the nucleus and in the cytoplasm. We applied enhanced RNA interactome capture (eRIC) analysis preceded by nucleo-cytoplasmic fractionation following arsenite-induced oxidative stress. The data reveal unexpectedly compartmentalized RNA interactomes and their responses to stress, including differential responses of RBPs in the nucleus versus the cytoplasm, which would have been missed by whole cell analyses.

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