ABSTRACT
INTRODUCTION: Patients with familial early-onset dementia (EOD) pose a unique opportunity for gene identification studies. METHODS: We present the phenotype and whole-exome sequencing (WES) study of an autosomal dominant EOD family. Candidate genes were examined in a set of dementia cases and controls (n = 3712). Western blotting was conducted of the wild-type and mutant protein of the final candidate. RESULTS: Age at disease onset was 60 years (range 56 to 63). The phenotype comprised mixed amnestic and behavioral features, and parkinsonism. Cerebrospinal fluid and plasma biomarkers, and a positron emission tomography amyloid study suggested Alzheimer's disease. WES and the segregation pattern pointed to a nonsense mutation in the TRIM25 gene (p.C168*), coding for an E3 ubiquitin ligase, which was absent in the cohorts studied. Protein studies supported a loss-of-function mechanism. DISCUSSION: This study supports a new physiopathological mechanism for brain amyloidosis. Furthermore, it extends the role of E3 ubiquitin ligases dysfunction in the development of neurodegenerative diseases. HIGHLIGHTS: A TRIM25 nonsense mutation (p.C168*) is associated with autosomal dominant early-onset dementia and parkinsonism with biomarkers suggestive of Alzheimer's disease. TRIM25 protein studies support that the mutation exerts its effect through loss of function. TRIM25, an E3 ubiquitin ligase, is known for its role in the innate immune response but this is the first report of association with neurodegeneration. The role of TRIM25 dysfunction in development of amyloidosis and neurodegeneration merits a new line of research.
Subject(s)
Alzheimer Disease , Amyloidosis , Dementia , Parkinsonian Disorders , Humans , Alzheimer Disease/diagnostic imaging , Alzheimer Disease/genetics , Codon, Nonsense , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Parkinsonian Disorders/diagnostic imaging , Parkinsonian Disorders/genetics , Amyloidogenic Proteins , Biomarkers , Tripartite Motif Proteins/genetics , Transcription Factors/geneticsABSTRACT
Fragile X-associated premature ovarian insufficiency (FXPOI) is among a family of disorders caused by expansion of a CGG trinucleotide repeat sequence located in the 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene on the X chromosome. Women with FXPOI have a depleted ovarian follicle population, resulting in amenorrhea, hypoestrogenism, and loss of fertility before the age of 40. FXPOI is caused by expansions of the CGG sequence to lengths between 55 and 200 repeats, known as a FMRI premutation, however the mechanism by which the premutation drives disease pathogenesis remains unclear. Two main hypotheses exist, which describe an mRNA toxic gain-of-function mechanism or a protein-based mechanism, where repeat-associated non-AUG (RAN) translation results in the production of an abnormal protein, called FMRpolyG. Here, we have developed an in vitro granulosa cell model of the FMR1 premutation by ectopically expressing CGG-repeat RNA and FMRpolyG protein. We show that expanded CGG-repeat RNA accumulated in intranuclear RNA structures, and these aggregates were able to cause significant granulosa cell death independent of FMRpolyG expression. Using an innovative RNA pulldown, mass spectrometry-based approach we have identified proteins that are specifically sequestered by CGG RNA aggregates in granulosa cells in vitro, and thus may be deregulated as consequence of this interaction. Furthermore, we have demonstrated reduced expression of three proteins identified via our RNA pulldown (FUS, PA2G4 and TRA2ß) in ovarian follicles in a FMR1 premutation mouse model. Collectively, these data provide evidence for the contribution of an mRNA gain-of-function mechanism to FXPOI disease biology.
Subject(s)
Fragile X Mental Retardation Protein , Fragile X Syndrome , Menopause, Premature , Primary Ovarian Insufficiency , Animals , Female , Humans , Mice , 5' Untranslated Regions/genetics , Adaptor Proteins, Signal Transducing/metabolism , Fragile X Mental Retardation Protein/genetics , Fragile X Syndrome/complications , Fragile X Syndrome/genetics , Gain of Function Mutation , Menopause, Premature/genetics , Primary Ovarian Insufficiency/etiology , Primary Ovarian Insufficiency/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Trinucleotide Repeat Expansion/geneticsABSTRACT
The E3 ubiquitin ligase TRIM25 is a key factor in the innate immune response to RNA viruses. TRIM25 has been shown to play a role in the retinoic-acid-inducible gene-1 (RIG-I) pathway, which triggers expression of type 1 interferons upon viral infection. We and others have shown that TRIM25 is an RNA-binding protein; however, the role of TRIM25 RNA-binding in the innate immune response to RNA viruses is unclear. Here, we demonstrate that influenza A virus (IAV A/PR/8/34_NS1(R38A/K41A)) infection is inhibited by TRIM25. Surprisingly, previously identified RNA-binding deficient mutant TRIM25ΔRBD and E3 ubiquitin ligase mutant TRIM25ΔRING, which lack E3 ubiquitin ligase activity, still inhibited IAV replication. Furthermore, we show that in human-derived cultured cells, activation of the RIG-I/interferon type 1 pathway mediated by either an IAV-derived 5'-triphosphate RNA or by IAV itself does not require TRIM25 activity. Additionally, we present new evidence that instead of TRIM25 directly inhibiting IAV transcription it binds and destabilizes IAV mRNAs. Finally, we show that direct tethering of TRIM25 to RNA is sufficient to downregulate the targeted RNA. In summary, our results uncover a potential mechanism that TRIM25 uses to inhibit IAV infection and regulate RNA metabolism.
Subject(s)
Influenza A virus , Humans , RNA, Messenger/genetics , Influenza A virus/genetics , Ubiquitin-Protein Ligases/genetics , Tripartite Motif Proteins/genetics , Transcription FactorsABSTRACT
RNA-protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA-protein interactions have shown promising effects on some diseases that are previously regarded as 'incurable'. Here, we developed a fluorescent on-bead screening platform, RNA Pull-Down COnfocal NAnoscanning (RP-CONA), to identify RNA-protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7-1. Importantly, miR-7's primary target is an mRNA of α-synuclein, which contributes to the aetiology of Parkinson's disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-synuclein. This opens up new therapeutic avenues towards treatment of Parkinson's disease as well as provides a novel methodology to search for modulators of RNA-protein interaction.
Subject(s)
ELAV-Like Protein 1/antagonists & inhibitors , MicroRNAs/antagonists & inhibitors , Quercetin/pharmacology , alpha-Synuclein/metabolism , Drug Evaluation, Preclinical/methods , ELAV-Like Protein 1/metabolism , HEK293 Cells , HeLa Cells , Humans , MicroRNAs/metabolism , Microscopy, Confocal , RNA, Messenger/metabolism , alpha-Synuclein/geneticsABSTRACT
The innate immune system is the body's first line of defense against viruses, with pattern recognition receptors (PRRs) recognizing molecules unique to viruses and triggering the expression of interferons and other anti-viral cytokines, leading to the formation of an anti-viral state. The tripartite motif containing 25 (TRIM25) is an E3 ubiquitin ligase thought to be a key component in the activation of signaling by the PRR retinoic acid-inducible gene I protein (RIG-I). TRIM25 has recently been identified as an RNA-binding protein, raising the question of whether its RNA-binding activity is important for its role in innate immunity. Here, we review TRIM25's mechanisms and pathways in noninfected and infected cells. We also introduce models that explain how TRIM25 binding to RNA could modulate its functions and play part in the antiviral response. These findings have opened new lines of investigations into functional and molecular roles of TRIM25 and other E3 ubiquitin ligases in cell biology and control of pathogenic infections. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition.
Subject(s)
Antiviral Agents/metabolism , Immunity, Innate , RNA/immunology , RNA/metabolism , Transcription Factors/metabolism , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Viruses/immunology , Binding Sites/immunology , Humans , Immunity, Innate/immunologyABSTRACT
The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (m5C) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of m5C deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for m5C in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation.
Subject(s)
5-Methylcytosine/metabolism , DNA Methylation , Epidermal Cells/cytology , Methyltransferases/metabolism , RNA/metabolism , Vault Ribonucleoprotein Particles/genetics , Cell Differentiation , Cell Line , Cytosine/metabolism , Epidermal Cells/metabolism , HEK293 Cells , HeLa Cells , Human Embryonic Stem Cells/cytology , Humans , Methyltransferases/genetics , RNA/genetics , Vault Ribonucleoprotein Particles/metabolismABSTRACT
RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells.
Subject(s)
Isotope Labeling/methods , Mass Spectrometry/methods , MicroRNAs/biosynthesis , RNA-Binding Proteins/chemistry , Cell Culture Techniques , HeLa Cells , Humans , MicroRNAs/chemistry , MicroRNAs/metabolism , RNA-Binding Proteins/isolation & purificationABSTRACT
Post-transcriptional mechanisms play a predominant role in the control of microRNA (miRNA) production. Recognition of the terminal loop of precursor miRNAs by RNA-binding proteins (RBPs) influences their processing; however, the mechanistic basis for how levels of individual or subsets of miRNAs are regulated is mostly unexplored. We previously showed that hnRNP A1, an RBP implicated in many aspects of RNA processing, acts as an auxiliary factor that promotes the Microprocessor-mediated processing of pri-mir-18a. Here, by using an integrative structural biology approach, we show that hnRNP A1 forms a 1:1 complex with pri-mir-18a where both RNA recognition motifs (RRMs) bind to cognate RNA sequence motifs in the terminal loop of pri-mir-18a. Terminal loop binding induces an allosteric destabilization of base-pairing in the pri-mir-18a stem that promotes its downstream processing. Our results highlight terminal loop RNA recognition by RBPs as a potential general principle of miRNA biogenesis and regulation.
Subject(s)
Heterogeneous Nuclear Ribonucleoprotein A1/chemistry , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , MicroRNAs/chemistry , MicroRNAs/metabolism , Base Sequence , Binding Sites , Biophysical Phenomena , Crystallography, X-Ray , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1/genetics , Humans , MicroRNAs/genetics , Models, Molecular , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Protein Binding , Protein Domains , RNA Processing, Post-Transcriptional , RNA StabilityABSTRACT
BACKGROUND: TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. RESULTS: Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25's endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. CONCLUSIONS: Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.
Subject(s)
B30.2-SPRY Domain , RNA/metabolism , Transcription Factors/genetics , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transcription Factors/metabolism , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing.
Subject(s)
Exoribonucleases/genetics , MicroRNAs/genetics , Neurons/metabolism , RNA Precursors/genetics , RNA-Binding Proteins/genetics , Animals , Base Pairing , Base Sequence , Cell Differentiation/drug effects , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Exoribonucleases/metabolism , Gene Expression Regulation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Mice , MicroRNAs/metabolism , Neurons/cytology , Neurons/drug effects , Nucleic Acid Conformation , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/metabolism , Poly U/metabolism , Protein Binding , RNA Cleavage , RNA Nucleotidyltransferases/genetics , RNA Nucleotidyltransferases/metabolism , RNA Precursors/metabolism , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tretinoin/pharmacologyABSTRACT
Trim25 is a member of the tripartite motif family of E3 ubiquitin ligases. It plays major roles in innate immunity and defence against viral infection, control of cell proliferation and migration of cancer cells. Recent work identified Trim25 as being able to bind to RNA and to regulate Lin28a-mediated uridylation of pre-let-7. Here we review the current knowledge of the role of Trim25 in development, disease and RNA metabolism.
Subject(s)
Disease/genetics , RNA/genetics , Transcription Factors/genetics , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Uterus/metabolism , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Humans , Mice , Models, Genetic , RNA/metabolism , Transcription Factors/metabolism , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Uterus/growth & developmentABSTRACT
RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity.
Subject(s)
MicroRNAs/metabolism , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Uridine/metabolism , Animals , Base Sequence , HeLa Cells , Humans , Isotope Labeling , Mass Spectrometry , Mice , MicroRNAs/chemistry , MicroRNAs/genetics , Nucleic Acid Conformation , Nucleotide Motifs/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Binding , RNA Processing, Post-Transcriptional/genetics , Tripartite Motif Proteins , Ubiquitin-Activating Enzymes/metabolismABSTRACT
microRNAs shape the identity and function of cells by regulating gene expression. It is known that brain-specific miR-9 is controlled transcriptionally; however, it is unknown whether post-transcriptional processes contribute to establishing its levels. Here we show that miR-9 is regulated transcriptionally and post-transcriptionally during neuronal differentiation of the embryonic carcinoma cell line P19. We demonstrate that miR-9 is more efficiently processed in differentiated than in undifferentiated cells. We reveal that Lin28a affects miR-9 by inducing the degradation of its precursor through a uridylation-independent mechanism. Furthermore, we show that constitutively expressed untagged but not GFP-tagged Lin28a decreases differentiation capacity of P19 cells, which coincides with reduced miR-9 levels. Finally, using an inducible system we demonstrate that Lin28a can also reduce miR-9 levels in differentiated P19 cells. Together, our results shed light on the role of Lin28a in neuronal differentiation and increase our understanding of the mechanisms regulating the level of brain-specific microRNAs.
Subject(s)
Cell Differentiation/physiology , Gene Expression Regulation/physiology , MicroRNAs/metabolism , Neurogenesis/physiology , RNA-Binding Proteins/metabolism , Animals , Blotting, Northern , Blotting, Western , Electrophoretic Mobility Shift Assay , HeLa Cells , Humans , Mass Spectrometry , Mice , RNA Interference , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
MicroRNA (miRNA) biogenesis is a highly regulated process in eukaryotic cells. Several mature miRNAs exhibit a tissue-specific pattern of expression without an apparent tissue-specific pattern for their corresponding primary transcripts. This discrepancy is suggestive of post-transcriptional regulation of miRNA abundance. Here, we demonstrate that the brain-enriched expression of miR-7, which is processed from the ubiquitous hnRNP K pre-mRNA transcript, is achieved by inhibition of its biogenesis in nonbrain cells in both human and mouse systems. Using stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry combined with RNase-assisted RNA pull-down, we identified Musashi homolog 2 (MSI2) and Hu antigen R (HuR) proteins as inhibitors of miR-7 processing in nonneural cells. This is achieved through HuR-mediated binding of MSI2 to the conserved terminal loop of pri-miR-7. Footprinting and electrophoretic gel mobility shift analysis (EMSA) provide further evidence for a direct interaction between pri-miR-7-1 and the HuR/MSI2 complex, resulting in stabilization of the pri-miR-7-1 structure. We also confirmed the physiological relevance of this inhibitory mechanism in a neuronal differentiation system using human SH-SY5Y cells. Finally, we show elevated levels of miR-7 in selected tissues from MSI2 knockout (KO) mice without apparent changes in the abundance of the pri-miR-7 transcript. Altogether, our data provide the first insight into the regulation of brain-enriched miRNA processing by defined tissue-specific factors.
Subject(s)
Gene Expression Regulation , MicroRNAs/biosynthesis , MicroRNAs/genetics , Animals , Brain/metabolism , Cell Differentiation , Cell Line , Mice , Mice, Knockout , MicroRNAs/metabolism , Neurons/cytology , Neurons/metabolism , Protein Binding , Protein Stability , RNA-Binding Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Regulation of miRNA (microRNA) biogenesis shapes the profiles of miRNAs in the living cell, contributing to cell identity and function. Importantly, aberrant miRNA levels have been linked to a variety of human pathological states. In recent years, a number of proteins have been shown to regulate the miRNA biogenesis at the level of Drosha and Dicer cleavage. A large proportion of these factors regulate miRNA production through binding to the TL (terminal loop) regions of miRNA progenitors. In the present paper, we review the current knowledge about pri-miRNA (primary miRNA) and pre-miRNA (precursor miRNA) TL involvement in the regulation of miRNA biogenesis.
Subject(s)
MicroRNAs/biosynthesis , MicroRNAs/genetics , Animals , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Humans , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleases , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Although the functional parameters of microRNAs (miRNAs) have been explored in some depth, the roles of these molecules in viral infections remain elusive. Here we report a general method for global analysis of miRNA function that compares the significance of both overexpressing and inhibiting each mouse miRNA on the growth properties of different viruses. Our comparative analysis of representatives of all three herpesvirus subfamilies identified host miRNAs with broad anti- and proviral properties which extend to a single-stranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual host-encoded miRNA regulates multiple pathways required and/or activated by viruses, including PI3K/AKT and ERK/MAPK signaling, oxidative stress signaling, and prostaglandin synthesis. Global miRNA expression analysis further demonstrated that the miR-199a/miR-214 cluster is down-regulated in both murine and human cytomegalovirus infection and manifests similar antiviral properties in mouse and human cells. Overall, we report a general strategy for examining the contributions of individual host miRNAs in viral infection and provide evidence that these molecules confer broad inhibitory potential against multiple viruses.