RNA–protein interactions: disorder, moonlighting and junk contribute to eukaryotic complexity

RNA–protein interactions are crucial for most biological processes in all organisms. However, it appears that the complexity of RNA-based regulation increases with the complexity of the organism, creating additional regulatory circuits, the scope of which is only now being revealed. It is becoming apparent that previously unappreciated features, such as disordered structural regions in proteins or non-coding regions in DNA leading to higher plasticity and pliability in RNA–protein complexes, are in fact essential for complex, precise and fine-tuned regulation. This review addresses the issue of the role of RNA–protein interactions in generating eukaryotic complexity, focusing on the newly characterized disordered RNA-binding motifs, moonlighting of metabolic enzymes, RNA-binding proteins interactions with different RNA species and their participation in regulatory networks of higher order.

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RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

Non-Coding RNA ◽

10.3390/ncrna7010011 ◽

2021 ◽

Vol 7 (1) ◽

pp. 11 ◽

Cited By ~ 1

Author(s):

André P. Gerber

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Regulatory Networks ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Cell Protein ◽

Transcriptional Regulatory Networks ◽

Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

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An exon DNA element modulates heterochromatin spreading in the master regulator for sexual commitment in malaria parasites

10.1101/2020.06.26.163105 ◽

2020 ◽

Author(s):

Carlos Cordon-Obras ◽

Anna Barcons-Simon ◽

Christine Scheidig-Benatar ◽

Aurelie Claës ◽

Valentin Sabatet ◽

...

Keyword(s):

Molecular Mechanisms ◽

Rna Binding ◽

Antigenic Variation ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Heterochromatin Protein 1 ◽

Master Regulator ◽

Heterochromatin Protein

ABSTRACTHeterochromatin is essential in all eukaryotes to maintain genome integrity, long-term gene repression and to help chromosome segregation during mitosis. However, heterochromatin regions must be restricted by boundary elements to avoid its spreading over actively transcribed loci. In Plasmodium falciparum, facultative heterochromatin is important to regulate parasite virulence, antigenic variation and transmission. However, the underlying molecular mechanisms regulating repressive regions remain unknown. To investigate this topic, we chose the ap2-g gene, which forms a strictly delimited and independent heterochromatin island. Using electrophoretic motility shift assay (EMSA) we identified an ap2-g exon element at the 3’ end binding nuclear protein complexes. Upon replacement of this region by a gfp gene, we observed a shift in the heterochromatin boundary resulting in HP1 (Heterochromatin Protein 1) spreading over ∼2 additional kb downstream. We used this DNA element to purify candidate proteins followed by proteomic analysis. The identified complexes were found to be enriched in RNA-binding proteins, pointing to a potential role of RNA in the regulation of the ap2-g 3’ heterochromatin boundary. Our results provide insight into the unexplored topic of heterochromatin biology in P. falciparum and identify a DNA element within the master regulator of sexual commitment modulating heterochromatin spreading.

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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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CLIPreg: Constructing translational regulatory networks from CLIP-, Ribo- and RNA-seq

10.1101/2021.12.06.470871 ◽

2021 ◽

Author(s):

Baptiste Kerouanton ◽

Sebastian Schafer ◽

Lena Ho ◽

Sonia Chothani ◽

Owen JL Rackham

Keyword(s):

Protein Interactions ◽

Translational Regulation ◽

Regulatory Networks ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Role ◽

Sequencing Data ◽

Data Types ◽

Network Resources ◽

Gene Regulatory

Motivation: The creation and analysis of gene regulatory networks have been the focus of bioinformatic research and underpins much of what is known about gene regulation. However, as a result of a bias in the availability of data-types that are collected, the vast majority of gene regulatory network resources and tools have focused on either transcriptional regulation or protein-protein interactions. This has left other areas of regulation, for instance translational regulation, vastly underrepresented despite them having been shown to play a critical role in both health and disease. Results: In order to address this we have developed CLIPreg, a package that integrates RNA, Ribo and CLIP- sequencing data in order to construct translational regulatory networks coordinated by RNA-binding proteins. This is the first tool of its type to be created, allowing for detailed investigation into a previously unseen layer of regulation.

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The role of disorder in RNA binding affinity and specificity

Open Biology ◽

10.1098/rsob.200328 ◽

2020 ◽

Vol 10 (12) ◽

pp. 200328

Author(s):

Diana S. M. Ottoz ◽

Luke E. Berchowitz

Keyword(s):

Protein Interactions ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Multiple Partners ◽

Cellular Environment ◽

Rna Targets

Most RNA-binding modules are small and bind few nucleotides. RNA-binding proteins typically attain the physiological specificity and affinity for their RNA targets by combining several RNA-binding modules. Here, we review how disordered linkers connecting RNA-binding modules govern the specificity and affinity of RNA–protein interactions by regulating the effective concentration of these modules and their relative orientation. RNA-binding proteins also often contain extended intrinsically disordered regions that mediate protein–protein and RNA–protein interactions with multiple partners. We discuss how these regions can connect proteins and RNA resulting in heterogeneous higher-order assemblies such as membrane-less compartments and amyloid-like structures that have the characteristics of multi-modular entities. The assembled state generates additional RNA-binding specificity and affinity properties that contribute to further the function of RNA-binding proteins within the cellular environment.

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System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

10.1101/748194 ◽

2019 ◽

Cited By ~ 1

Author(s):

Cornelia Kilchert ◽

Tea Kecman ◽

Emily Priest ◽

Svenja Hester ◽

Krzysztof Kus ◽

...

Keyword(s):

Fission Yeast ◽

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Rna Degradation ◽

Binding Activity ◽

Rna Exosome ◽

And Function ◽

Rna Protein Complexes

AbstractProduction, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNA-binding activity and provide key information about the RNA-binding properties of large multi-protein complexes, such as the mRNA 3’ end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.

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RNA Proximity Labeling: A New Detection Tool for RNA–Protein Interactions

Molecules ◽

10.3390/molecules26082270 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2270

Author(s):

Ronja Weissinger ◽

Lisa Heinold ◽

Saira Akram ◽

Ralf-Peter Jansen ◽

Orit Hermesh

Keyword(s):

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Affinity Purification ◽

Cellular Functions ◽

Cellular Compartments ◽

Rna Protein Complexes

Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners. To this end, multiple techniques have been developed to characterize the interaction between RNAs and proteins. In this review, we briefly summarize the common methods to study RNA–protein interaction including crosslinking and immunoprecipitation (CLIP), and aptamer- or antisense oligonucleotide-based RNA affinity purification. Following this, we focus on in vivo proximity labeling to study RNA–protein interactions. In proximity labeling, a labeling enzyme like ascorbate peroxidase or biotin ligase is targeted to specific RNAs, RNA-binding proteins, or even cellular compartments and uses biotin to label the proteins and RNAs in its vicinity. The tagged molecules are then enriched and analyzed by mass spectrometry or RNA-Seq. We highlight the latest studies that exemplify the strength of this approach for the characterization of RNA protein complexes and distribution of RNAs in vivo.

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Translation repression via modulation of the cytoplasmic poly(A)-binding protein in the inflammatory response

eLife ◽

10.7554/elife.27786 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 13

Author(s):

Xu Zhang ◽

Xiaoli Chen ◽

Qiuying Liu ◽

Shaojie Zhang ◽

Wenqian Hu

Keyword(s):

Gene Expression ◽

Inflammatory Response ◽

Regulatory Networks ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Mrna Translation ◽

Translational Repression ◽

Binding Motifs ◽

Biologically Relevant

Gene expression is precisely regulated during the inflammatory response to control infection and limit the detrimental effects of inflammation. Here, we profiled global mRNA translation dynamics in the mouse primary macrophage-mediated inflammatory response and identified hundreds of differentially translated mRNAs. These mRNAs’ 3’UTRs have enriched binding motifs for several RNA-binding proteins, which implies extensive translational regulatory networks. We characterized one such protein, Zfp36, as a translation repressor. Using primary macrophages from a Zfp36-V5 epitope tagged knock-in mouse generated by CRISPR/Cas9-mediated genome editing, we found that the endogenous Zfp36 directly interacts with the cytoplasmic poly(A)-binding protein. Importantly, this interaction is required for the translational repression of Zfp36’s target mRNAs in resolving inflammation. Altogether, these results uncovered critical roles of translational regulations in controlling appropriate gene expression during the inflammatory response and revealed a new biologically relevant molecular mechanism of translational repression via modulating the cytoplasmic poly(A)-binding protein.

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Recent advances in proximity-based labeling methods for interactome mapping

F1000Research ◽

10.12688/f1000research.16903.1 ◽

2019 ◽

Vol 8 ◽

pp. 135 ◽

Cited By ~ 29

Author(s):

Laura Trinkle-Mulcahy

Keyword(s):

Protein Interactions ◽

Large Scale ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Affinity Purification ◽

Small Scale ◽

Protein Protein Interactions ◽

Large Scale Network ◽

Complementary Approach

Proximity-based labeling has emerged as a powerful complementary approach to classic affinity purification of multiprotein complexes in the mapping of protein–protein interactions. Ongoing optimization of enzyme tags and delivery methods has improved both temporal and spatial resolution, and the technique has been successfully employed in numerous small-scale (single complex mapping) and large-scale (network mapping) initiatives. When paired with quantitative proteomic approaches, the ability of these assays to provide snapshots of stable and transient interactions over time greatly facilitates the mapping of dynamic interactomes. Furthermore, recent innovations have extended biotin-based proximity labeling techniques such as BioID and APEX beyond classic protein-centric assays (tag a protein to label neighboring proteins) to include RNA-centric (tag an RNA species to label RNA-binding proteins) and DNA-centric (tag a gene locus to label associated protein complexes) assays.

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RNA-Binding Proteins and the Complex Pathophysiology of ALS

International Journal of Molecular Sciences ◽

10.3390/ijms22052598 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2598

Author(s):

Wanil Kim ◽

Do-Yeon Kim ◽

Kyung-Ha Lee

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Current Knowledge ◽

Protein Complexes ◽

Dynamic Function ◽

Disease Related Genes ◽

Cellular Dysfunction ◽

Lateral Sclerosis

Genetic analyses of patients with amyotrophic lateral sclerosis (ALS) have identified disease-causing mutations and accelerated the unveiling of complex molecular pathogenic mechanisms, which may be important for understanding the disease and developing therapeutic strategies. Many disease-related genes encode RNA-binding proteins, and most of the disease-causing RNA or proteins encoded by these genes form aggregates and disrupt cellular function related to RNA metabolism. Disease-related RNA or proteins interact or sequester other RNA-binding proteins. Eventually, many disease-causing mutations lead to the dysregulation of nucleocytoplasmic shuttling, the dysfunction of stress granules, and the altered dynamic function of the nucleolus as well as other membrane-less organelles. As RNA-binding proteins are usually components of several RNA-binding protein complexes that have other roles, the dysregulation of RNA-binding proteins tends to cause diverse forms of cellular dysfunction. Therefore, understanding the role of RNA-binding proteins will help elucidate the complex pathophysiology of ALS. Here, we summarize the current knowledge regarding the function of disease-associated RNA-binding proteins and their role in the dysfunction of membrane-less organelles.

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