The FXR2P low complexity domain drives assembly of multiple fibril types with differing ribosome association in neurons

RNA binding proteins (RBPs) typically function in higher order assemblages to regulate RNA localization and translation. The Fragile X homolog FXR2P is an RBP essential for formation of Fragile X granules, which associate with axonal mRNA and ribosomes in the intact brain. Here we performed an unbiased EGFP insertional mutagenesis screen to probe for FXR2P domains important for assembly into higher order structural states in neurons. Fifteen of the 18 unique in-frame FXR2PEGFP fusions tested formed cytosolic granules. However, EGFP insertion within a 23 amino acid region of the low complexity (LC) domain induced formation of distinct FXR2PEGFP fibrils (A and B) that were found in isolation or assembled into highly ordered bundles. Type A and B complexes exhibited different developmental timelines, ultrastructure and ribosome association with ribosomes absent from bundled Type B fibrils. The formation of both fibril types was dependent on an intact RNA binding domain. We conclude that formation of these higher order FXR2P assemblages with alternative structural and compositional states in neurons requires collaboration between the LC and RNA binding domains.

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Integrated multi-omics reveals common properties underlying stress granule and P-body formation

10.1101/2020.05.18.102517 ◽

2020 ◽

Cited By ~ 2

Author(s):

Christopher J. Kershaw ◽

Michael G. Nelson ◽

Jennifer Lui ◽

Christian P. Bates ◽

Martin D. Jennings ◽

...

Keyword(s):

Phase Separation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulation Of Gene Expression ◽

Low Complexity ◽

P Bodies ◽

Binding Domains ◽

Glucose Depletion ◽

Before And After ◽

Mrna Content

ABSTRACTNon-membrane-bound compartments such as P-bodies (PBs) and stress granules (SGs) play important roles in the regulation of gene expression following environmental stresses. We have systematically determined the protein and mRNA composition of PBs and SGs formed in response to a common stress condition imposed by glucose depletion. We find that high molecular weight (HMW) complexes exist prior to glucose depletion that may act as seeds for the further condensation of proteins forming mature PBs and SGs. Both before and after glucose depletion, these HMW complexes are enriched for proteins containing low complexity and RNA binding domains. The mRNA content of these HMW complexes is enriched for long, structured mRNAs that become more poorly translated following glucose depletion. Many proteins and mRNAs are shared between PBs and SGs including several multivalent RNA binding proteins that may promote condensate interactions during liquid-liquid phase separation. Even where the precise identity of mRNAs and proteins localizing to PBs and SGs is distinct, the mRNAs and proteins share common biophysical and chemical features that likely trigger their phase separation.

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RNA-Binding Proteins and Translation in Neurodegenerative Disease

The Oxford Handbook of Neuronal Protein Synthesis ◽

10.1093/oxfordhb/9780190686307.013.25 ◽

2020 ◽

Author(s):

Kent E. Duncan

Keyword(s):

Neurodegenerative Diseases ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fragile X ◽

Potential Contribution ◽

Fused In Sarcoma ◽

Specific Mrnas ◽

Ataxia Syndrome ◽

Research Frontiers

Both RNA-binding proteins (RBPs) and translation are increasingly implicated in several neurodegenerative diseases, but their specific roles in promoting disease are not yet fully defined. This chapter critically evaluates the evidence that altered translation of specific mRNAs mediated by RNA-binding proteins plays an important role in driving specific neurodegenerative diseases. First, diseases are discussed where a causal role for RNA-binding proteins in disease appears solid, but whether this involves altered translation is less clear. The main foci here are TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Subsequently, diseases are presented where altered translation is believed to contribute, but involvement of RNA-binding proteins is less clear. These include Huntington’s and other repeat expansion disorders such as fragile X tremor/ataxia syndrome (FXTAS), where repeat-induced non-AUG-initiated (RAN) translation is a focus. The potential contribution of both canonical and non-canonical RBPs to altered translation in Parkinson’s disease is discussed. The chapter closes by proposing key research frontiers for the field to explore and outlining methodological advances that could help to address them.

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Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

Scientific Reports ◽

10.1038/s41598-021-87097-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yogeeshwar Ajjugal ◽

Narendar Kolimi ◽

Thenmalarchelvi Rathinavelan

Keyword(s):

Rna Binding ◽

Trinucleotide Repeat ◽

Rna Binding Proteins ◽

Fragile X ◽

Repeat Expansion ◽

Mobility Shift ◽

Cgg Repeat ◽

Dna And Rna ◽

Structural Choice ◽

Sense Strand

AbstractCGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

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RNA Binding Proteins and the Pathogenesis of Frontotemporal Lobar Degeneration

Annual Review of Pathology Mechanisms of Disease ◽

10.1146/annurev-pathmechdis-012418-012955 ◽

2019 ◽

Vol 14 (1) ◽

pp. 469-495 ◽

Cited By ~ 9

Author(s):

Jeffrey W. Hofmann ◽

William W. Seeley ◽

Eric J. Huang

Keyword(s):

Frontotemporal Lobar Degeneration ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Low Complexity ◽

Compelling Evidence ◽

Abnormal Protein ◽

Early Onset Dementia ◽

Cellular Pathways ◽

Liquid Liquid Phase Separation

Frontotemporal dementia is a group of early onset dementia syndromes linked to underlying frontotemporal lobar degeneration (FTLD) pathology that can be classified based on the formation of abnormal protein aggregates involving tau and two RNA binding proteins, TDP-43 and FUS. Although elucidation of the mechanisms leading to FTLD pathology is in progress, recent advances in genetics and neuropathology indicate that a majority of FTLD cases with proteinopathy involving RNA binding proteins show highly congruent genotype–phenotype correlations. Specifically, recent studies have uncovered the unique properties of the low-complexity domains in RNA binding proteins that can facilitate liquid–liquid phase separation in the formation of membraneless organelles. Furthermore, there is compelling evidence that mutations in FTLD genes lead to dysfunction in diverse cellular pathways that converge on the endolysosomal pathway, autophagy, and neuroinflammation. Together, these results provide key mechanistic insights into the pathogenesis and potential therapeutic targets of FTLD.

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The Role of RNA-Binding Proteins in Vertebrate Neural Crest and Craniofacial Development

Journal of Developmental Biology ◽

10.3390/jdb9030034 ◽

2021 ◽

Vol 9 (3) ◽

pp. 34

Author(s):

Thomas E. Forman ◽

Brenna J. C. Dennison ◽

Katherine A. Fantauzzo

Keyword(s):

Gene Expression ◽

Neural Crest ◽

Binding Proteins ◽

Rna Binding ◽

Gene Expression Regulation ◽

Rna Binding Proteins ◽

Craniofacial Development ◽

Specific Sequence ◽

Altered Expression ◽

Binding Domains

Cranial neural crest (NC) cells delaminate from the neural folds in the forebrain to the hindbrain during mammalian embryogenesis and migrate into the frontonasal prominence and pharyngeal arches. These cells generate the bone and cartilage of the frontonasal skeleton, among other diverse derivatives. RNA-binding proteins (RBPs) have emerged as critical regulators of NC and craniofacial development in mammals. Conventional RBPs bind to specific sequence and/or structural motifs in a target RNA via one or more RNA-binding domains to regulate multiple aspects of RNA metabolism and ultimately affect gene expression. In this review, we discuss the roles of RBPs other than core spliceosome components during human and mouse NC and craniofacial development. Where applicable, we review data on these same RBPs from additional vertebrate species, including chicken, Xenopus and zebrafish models. Knockdown or ablation of several RBPs discussed here results in altered expression of transcripts encoding components of developmental signaling pathways, as well as reduced cell proliferation and/or increased cell death, indicating that these are common mechanisms contributing to the observed phenotypes. The study of these proteins offers a relatively untapped opportunity to provide significant insight into the mechanisms underlying gene expression regulation during craniofacial morphogenesis.

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PUB1: a major yeast poly(A)+ RNA-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6114-6123.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6114-6123

Author(s):

M J Matunis ◽

E L Matunis ◽

G Dreyfuss

Keyword(s):

Rna Polymerase Ii ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Uv Light ◽

Binding Protein ◽

Gene Replacement ◽

Yeast Saccharomyces Cerevisiae ◽

Binding Domains ◽

Development And Differentiation

The expression of RNA polymerase II transcripts can be regulated at the posttranscriptional level by RNA-binding proteins. Although extensively characterized in metazoans, relatively few RNA-binding proteins have been characterized in the yeast Saccharomyces cerevisiae. Three major proteins are cross-linked by UV light to poly(A)+ RNA in living S. cerevisiae cells. These are the 72-kDa poly(A)-binding protein and proteins of 60 and 50 kDa (S.A. Adam, T.Y. Nakagawa, M.S. Swanson, T. Woodruff, and G. Dreyfuss, Mol. Cell. Biol. 6:2932-2943, 1986). Here, we describe the 60-kDa protein, one of the major poly(A)+ RNA-binding proteins in S. cerevisiae. This protein, PUB1 [for poly(U)-binding protein 1], was purified by affinity chromatography on immobilized poly(rU), and specific monoclonal antibodies to it were produced. UV cross-linking demonstrated that PUB1 is bound to poly(A)+ RNA (mRNA or pre-mRNA) in living cells, and it was detected primarily in the cytoplasm by indirect immunofluorescence. The gene for PUB1 was cloned and sequenced, and the sequence was found to predict a 51-kDa protein with three ribonucleoprotein consensus RNA-binding domains and three glutamine- and asparagine-rich auxiliary domains. This overall structure is remarkably similar to the structures of the Drosophila melanogaster elav gene product, the human neuronal antigen HuD, and the cytolytic lymphocyte protein TIA-1. Each of these proteins has an important role in development and differentiation, potentially by affecting RNA processing. PUB1 was found to be nonessential in S. cerevisiae by gene replacement; however, further genetic analysis should reveal important features of this class of RNA-binding proteins.

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The structural basis for RNA selectivity by the IMP family of RNA-binding proteins

Nature Communications ◽

10.1038/s41467-019-12193-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Jeetayu Biswas ◽

Vivek L. Patel ◽

Varun Bhaskar ◽

Jeffrey A. Chao ◽

Robert H. Singer ◽

...

Keyword(s):

Neural Development ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Stability ◽

Underlying Mechanism ◽

Structural Basis ◽

General Mechanism ◽

Binding Domains ◽

Variable Loops

Abstract The IGF2 mRNA-binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine systematic evolution of ligands by exponential enrichment (SELEX) and NMR spectroscopy to demonstrate that the major RNA-binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.

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RNA-binding proteins with prion-like domains in health and disease

Biochemical Journal ◽

10.1042/bcj20160499 ◽

2017 ◽

Vol 474 (8) ◽

pp. 1417-1438 ◽

Cited By ~ 152

Author(s):

Alice Ford Harrison ◽

James Shorter

Keyword(s):

Phase Transitions ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Low Complexity ◽

Fused In Sarcoma ◽

Heterogeneous Nuclear Ribonucleoproteins ◽

Health And Disease ◽

Lateral Sclerosis

Approximately 70 human RNA-binding proteins (RBPs) contain a prion-like domain (PrLD). PrLDs are low-complexity domains that possess a similar amino acid composition to prion domains in yeast, which enable several proteins, including Sup35 and Rnq1, to form infectious conformers, termed prions. In humans, PrLDs contribute to RBP function and enable RBPs to undergo liquid–liquid phase transitions that underlie the biogenesis of various membraneless organelles. However, this activity appears to render RBPs prone to misfolding and aggregation connected to neurodegenerative disease. Indeed, numerous RBPs with PrLDs, including TDP-43 (transactivation response element DNA-binding protein 43), FUS (fused in sarcoma), TAF15 (TATA-binding protein-associated factor 15), EWSR1 (Ewing sarcoma breakpoint region 1), and heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNPA1 and hnRNPA2), have now been connected via pathology and genetics to the etiology of several neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Here, we review the physiological and pathological roles of the most prominent RBPs with PrLDs. We also highlight the potential of protein disaggregases, including Hsp104, as a therapeutic strategy to combat the aberrant phase transitions of RBPs with PrLDs that likely underpin neurodegeneration.

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Lack of a Clear Behavioral Phenotype in an Inducible FXTAS Mouse Model Despite the Presence of Neuronal FMRpolyG-Positive Aggregates

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.599101 ◽

2020 ◽

Vol 7 ◽

Author(s):

Saif N. Haify ◽

Ruchira S. D. Mankoe ◽

Valerie Boumeester ◽

Esmay C. van der Toorn ◽

Rob F. M. Verhagen ◽

...

Keyword(s):

Mouse Model ◽

Rna Binding ◽

Neurodegenerative Disorder ◽

Rna Binding Proteins ◽

Fragile X ◽

Brain Regions ◽

Intranuclear Inclusions ◽

Behavioral Deficits ◽

Cgg Repeat ◽

Progressive Cerebellar Ataxia

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a rare neurodegenerative disorder caused by a 55–200 CGG repeat expansion in the 5′ untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. FXTAS is characterized by progressive cerebellar ataxia, Parkinsonism, intention tremors and cognitive decline. The main neuropathological hallmark of FXTAS is the presence of ubiquitin-positive intranuclear inclusions in neurons and astrocytes throughout the brain. The molecular pathology of FXTAS involves the presence of 2 to 8-fold elevated levels of FMR1 mRNA, and of a repeat-associated non-AUG (RAN) translated polyglycine peptide (FMRpolyG). Increased levels of FMR1 mRNA containing an expanded CGG repeat can result in cellular toxicity by an RNA gain-of-function mechanism. The increased levels of CGG repeat-expanded FMR1 transcripts may create RNA foci that sequester important cellular proteins, including RNA-binding proteins and FMRpolyG, in intranuclear inclusions. To date, it is unclear whether the FMRpolyG-positive intranuclear inclusions are a cause or a consequence of FXTAS disease pathology. In this report we studied the relation between the presence of neuronal intranuclear inclusions and behavioral deficits using an inducible mouse model for FXTAS. Neuronal intranuclear inclusions were observed 4 weeks after dox-induction. After 12 weeks, high numbers of FMRpolyG-positive intranuclear inclusions could be detected in the hippocampus and striatum, but no clear signs of behavioral deficits related to these specific brain regions were found. In conclusion, the observations in our inducible mouse model for FXTAS suggest a lack of correlation between the presence of intranuclear FMRpolyG-positive aggregates in brain regions and specific behavioral phenotypes.

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