Abstract
Protein–RNA interactions are implicated in a number of physiological roles as well as diseases, with molecular mechanisms ranging from defects in RNA splicing, localization and translation ...to the formation of aggregates. Currently, ∼1400 human proteins have experimental evidence of RNA-binding activity. However, only ∼250 of these proteins currently have experimental data on their target RNAs from various sequencing-based methods such as eCLIP. To bridge this gap, we used an established, computationally expensive protein–RNA interaction prediction method, catRAPID, to populate a large database, RNAct. RNAct allows easy lookup of known and predicted interactions and enables global views of the human, mouse and yeast protein–RNA interactomes, expanding them in a genome-wide manner far beyond experimental data (http://rnact.crg.eu).
Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) ...domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular β-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.
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•Intermolecular β sheet hydrogen bonding and cation-π interactions drive FUS demixing•Cation-π interactions form between C-terminal arginines and N-terminal tyrosines•Cation-π strength is regulated by arginine methylation and interacting proteins•FUS hypomethylation in FTLD induces FUS gelation and impairs RNP granule function
Phase transition of the RNA-binding protein FUS is mediated by cation-π interactions between C-terminal arginines and N-terminal tyrosines and is modulated by arginine methlylation.
Advances in the characterization of RNA-binding proteins Marchese, Domenica; de Groot, Natalia Sanchez; Lorenzo Gotor, Nieves ...
Wiley interdisciplinary reviews. RNA,
November/December 2016, Letnik:
7, Številka:
6
Journal Article
Recenzirano
Odprti dostop
From transcription, to transport, storage, and translation, RNA depends on association with different RNA‐binding proteins (RBPs). Methods based on next‐generation sequencing and protein ...mass‐spectrometry have started to unveil genome‐wide interactions of RBPs but many aspects still remain out of sight. How many of the binding sites identified in high‐throughput screenings are functional? A number of computational methods have been developed to analyze experimental data and to obtain insights into the specificity of protein–RNA interactions. How can theoretical models be exploited to identify RBPs? In addition to oligomeric complexes, protein and RNA molecules can associate into granular assemblies whose physical properties are still poorly understood. What protein features promote granule formation and what effects do these assemblies have on cell function? Here, we describe the newest in silico, in vitro, and in vivo advances in the field of protein–RNA interactions. We also present the challenges that experimental and computational approaches will have to face in future studies. WIREs RNA 2016, 7:793–810. doi: 10.1002/wrna.1378
This article is categorized under:
RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA in Disease and Development > RNA in Disease
Protein and RNA molecules interact in vivo to accomplish a number of essential functions (e.g., transcription, translation, etc.). Deciphering these interactions is crucial to understand the mechanisms related to cell function and dysfunction. All these fundamental questions have started to be addressed thanks to the recent development of new experimental and computational techniques.
Breast cancer is a heterogeneous disease classified into four main subtypes with different clinical outcomes, such as patient survival, prognosis, and relapse. Current genetic tests for the ...differential diagnosis of BC subtypes showed a poor reproducibility. Therefore, an early and correct diagnosis of molecular subtypes is one of the challenges in the clinic. In the present study, we identified differentially expressed genes, long non-coding RNAs and RNA binding proteins for each BC subtype from a public dataset applying bioinformatics algorithms. In addition, we investigated their interactions and we proposed interacting biomarkers as potential signature specific for each BC subtype. We found a network of only 2 RBPs (RBM20 and PCDH20) and 2 genes (HOXB3 and RASSF7) for luminal A, a network of 21 RBPs and 53 genes for luminal B, a HER2-specific network of 14 RBPs and 30 genes, and a network of 54 RBPs and 302 genes for basal BC. We validated the signature considering their expression levels on an independent dataset evaluating their ability to classify the different molecular subtypes with a machine learning approach. Overall, we achieved good performances of classification with an accuracy >0.80. In addition, we found some interesting novel prognostic biomarkers such as RASSF7 for luminal A, DCTPP1 for luminal B, DHRS11, KLC3, NAGS, and TMEM98 for HER2, and ABHD14A and ADSSL1 for basal. The findings could provide preliminary evidence to identify putative new prognostic biomarkers and therapeutic targets for individual breast cancer subtypes.
Protein aggregation is linked with neurodegeneration and numerous other diseases by mechanisms that are not well understood. Here, we have analyzed the gain-of-function toxicity of artificial β sheet ...proteins that were designed to form amyloid-like fibrils. Using quantitative proteomics, we found that the toxicity of these proteins in human cells correlates with the capacity of their aggregates to promote aberrant protein interactions and to deregulate the cytosolic stress response. The endogenous proteins that are sequestered by the aggregates share distinct physicochemical properties: They are relatively large in size and significantly enriched in predicted unstructured regions, features that are strongly linked with multifunctionality. Many of the interacting proteins occupy essential hub positions in cellular protein networks, with key roles in chromatin organization, transcription, translation, maintenance of cell architecture and protein quality control. We suggest that amyloidogenic aggregation targets a metastable subproteome, thereby causing multifactorial toxicity and, eventually, the collapse of essential cellular functions.
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► Designed β sheet proteins form amyloid-like aggregates and are toxic in human cells ► The aggregates sequester numerous preexistent and newly synthesized proteins ► The affected proteins are enriched in disordered regions and are multifunctional ► Protein coaggregation may contribute to toxicity in neurodegenerative diseases.
Abstract
Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine ...hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5′untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1–UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.
Abstract
Post-transcriptional methylation of N6-adenine and N1-adenine can affect transcriptome turnover and translation. Furthermore, the regulatory function of N6-methyladenine (m6A) during heat ...shock has been uncovered, including the enhancement of the phase separation potential of RNAs. In response to acute stress, e.g. heat shock, the orderly sequestration of mRNAs in stress granules (SGs) is considered important to protect transcripts from the irreversible aggregation. Until recently, the role of N1-methyladenine (m1A) on mRNAs during acute stress response remains largely unknown. Here we show that the methyltransferase complex TRMT6/61A, which generates the m1A tag, is involved in transcriptome protection during heat shock. Our bioinformatics analysis indicates that occurrence of the m1A motif is increased in mRNAs known to be enriched in SGs. Accordingly, the m1A-generating methyltransferase TRMT6/61A accumulated in SGs and mass spectrometry confirmed enrichment of m1A in the SG RNAs. The insertion of a single methylation motif in the untranslated region of a reporter RNA leads to more efficient recovery of protein synthesis from that transcript after the return to normal temperature. Our results demonstrate far-reaching functional consequences of a minimal RNA modification on N1-adenine during acute proteostasis stress.
Abstract
Syndesmos (SDOS) is a functionally poorly characterized protein that directly interacts with p53 binding protein 1 (53BP1) and regulates its recruitment to chromatin. We show here that SDOS ...interacts with another important cancer-linked protein, the chaperone TRAP1, associates with actively translating polyribosomes and represses translation. Moreover, we demonstrate that SDOS directly binds RNA in living cells. Combining individual gene expression profiling, nucleotide crosslinking and immunoprecipitation (iCLIP), and ribosome profiling, we discover several crucial pathways regulated post-transcriptionally by SDOS. Among them, we identify a small subset of mRNAs responsible for the biogenesis of primary cilium that have been linked to developmental and degenerative diseases, known as ciliopathies, and cancer. We discover that SDOS binds and regulates the translation of several of these mRNAs, controlling cilia development.
Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease affecting motor neurons and characterized by microglia-mediated neurotoxic inflammation whose underlying ...mechanisms remain incompletely understood. In this work, we reveal that MAPK/MAK/MRK overlapping kinase (MOK), with an unknown physiological substrate, displays an immune function by controlling inflammatory and type-I interferon (IFN) responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as an effector protein regulated by MOK, by promoting Ser
-phospho-Brd4 levels. We further demonstrate that MOK regulates Brd4 functions by supporting its binding to cytokine gene promoters, therefore enabling innate immune responses. Remarkably, we show that MOK levels are increased in the ALS spinal cord, particularly in microglial cells, and that administration of a chemical MOK inhibitor to ALS model mice can modulate Ser
-phospho-Brd4 levels, suppress microglial activation, and modify the disease course, indicating a pathophysiological role of MOK kinase in ALS and neuroinflammation.
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