scholarly journals An Xist-dependent protein assembly mediates Xist localization and gene silencing

2020 ◽  
Author(s):  
Amy Pandya-Jones ◽  
Yolanda Markaki ◽  
Jacques Serizay ◽  
Tsotne Chitiashvilli ◽  
Walter Mancia ◽  
...  
Keyword(s):  
Gene Silencing ◽  
X Chromosome ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Repeat Sequence ◽  
Inactive X Chromosome ◽  
Dependent Protein ◽  
Self Association ◽  
Molecular Forces

SummaryNuclear compartments play diverse roles in regulating gene expression, yet the molecular forces and components driving compartment formation are not well understood. Studying how the lncRNA Xist establishes the inactive-X-chromosome (Xi)-compartment, we found that the Xist RNA-binding-proteins PTBP1, MATR3, TDP43, and CELF1 form a condensate to create an Xi-domain that can be sustained in the absence of Xist. The E-repeat-sequence of Xist serves a multivalent binding-platform for these proteins. Without the E-repeat, Xist initially coats the X-chromosome during XCI onset but subsequently disperses across the nucleus with loss of gene silencing. Recruitment of PTBP1, MATR3, TDP-43 or CELF1 to ΔE-Xist rescues these phenotypes, and requires both self-association of MATR3 and TDP-43 and a heterotypic PTBP1-MATR3-interaction. Together, our data reveal that Xist sequesters itself within the Xi-territory and perpetuates gene silencing by seeding a protein-condensate. Our findings uncover an unanticipated mechanism for epigenetic memory and elucidate the interplay between RNA and RNA-binding-proteins in creating compartments for gene regulation.

Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains

2013 ◽  
Vol 394 (8) ◽  
pp. 1077-1090 ◽  
Author(s):  
Kristin Wächter ◽  
Marcel Köhn ◽  
Nadine Stöhr ◽  
Stefan Hüttelmaier
Keyword(s):  
Subcellular Localization ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Accumulation ◽  
Structural Constraints ◽  
Cellular Functions ◽  
Dependent Protein ◽  
Mrna Binding

Abstract The IGF2 mRNA-binding protein family (IGF2BPs) directs the cytoplasmic fate of various target mRNAs and controls essential cellular functions. The three IGF2BP paralogues expressed in mammals comprise two RNA-recognition motifs (RRM) as well as four KH domains. How these domains direct IGF2BP paralogue-dependent protein function remains largely elusive. In this study, we analyze the role of KH domains in IGF2BPs by the mutational GXXG-GEEG conversion of single KH domain loops in the context of full-length polypeptides. These analyses reveal that all four KH domains of IGF2BP1 and IGF2BP2 are essentially involved in RNA-binding in vitro and the cellular association with RNA-binding proteins (RBPs). Moreover the KH domains prevent the nuclear accumulation of these two paralogues and facilitate their recruitment to stress granules. The role of KH domains appears less pronounced in IGF2BP3, because GxxG-GEEG conversion in all four KH domains only modestly affects RNA-binding, subcellular localization and RNA-dependent protein association of this paralogue. These findings indicate paralogue-dependent RNA-binding properties of IGF2BPs which likely direct distinct cellular functions. Our findings suggest that IGF2BPs contact target RNAs via all four KH domains. This implies significant structural constraints, which presumably allow the formation of exceedingly stable protein-RNA complexes.

scholarly journals Polycomb complexes in X chromosome inactivation

2017 ◽  
Vol 372 (1733) ◽  
pp. 20170021 ◽  
Author(s):  
Neil Brockdorff
Keyword(s):  
X Chromosome ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Histone H2a ◽  
Post Translational Modifications ◽  
Link Type ◽  
Polycomb Proteins ◽  
Inactive X Chromosome

Identifying the critical RNA binding proteins (RBPs) that elicit Xist mediated silencing has been a key goal in X inactivation research. Early studies implicated the Polycomb proteins, a family of factors linked to one of two major multiprotein complexes, PRC1 and PRC2 (Wang 2001 Nat. Genet. 28 , 371–375 ( doi:10.1038/ng574 ); Silva 2003 Dev. Cell 4 , 481–495 ( doi:10.1016/S1534-5807(03)00068-6 ); de Napoles 2004 Dev. Cell 7 , 663–676 ( doi:10.1016/j.devcel.2004.10.005 ); Plath 2003 Science 300 , 131–135 ( doi:10.1126/science.1084274 )). PRC1 and PRC2 complexes catalyse specific histone post-translational modifications (PTMs), ubiquitylation of histone H2A at position lysine 119 (H2AK119u1) and methylation of histone H3 at position lysine 27 (H3K27me3), respectively, and accordingly, these modifications are highly enriched over the length of the inactive X chromosome (Xi). A key study proposed that PRC2 subunits bind directly to Xist RNA A-repeat element, a region located at the 5′ end of the transcript known to be required for Xist mediated silencing (Zhao 2008 Science 322 , 750–756 ( doi:10.1126/science.1163045 )). Subsequent recruitment of PRC1 was assumed to occur via recognition of PRC2 mediated H3K27me3 by the CBX subunit of PRC1, as has been shown to be the case at other Polycomb target loci (Cao 2002 Science 298 , 1039–1043 ( doi:10.1126/science.1076997 )). More recently, several reports have questioned aspects of the prevailing view, both in relation to the mechanism for Polycomb recruitment by Xist RNA and the contribution of the Polycomb pathway to Xist mediated silencing. In this article I provide an overview of our recent progress towards resolving these discrepancies. This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’.

scholarly journals Periphilin self-association underpins epigenetic silencing by the HUSH complex

2019 ◽  
Author(s):  
Daniil M. Prigozhin ◽  
Anna Albecka ◽  
Christopher H. Douse ◽  
Iva A. Tchasovnikarova ◽  
Richard T. Timms ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Low Complexity ◽  
Core Complex ◽  
Terminal Domain ◽  
Genome Wide ◽  
Repressive Mark ◽  
Self Association ◽  
Nascent Transcripts

AbstractTranscription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin forms α-helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts.

scholarly journals The B-side of Xist

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 55 ◽  
Author(s):  
Asun Monfort ◽  
Anton Wutz
Keyword(s):  
X Chromosome ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Functional Characterization ◽  
Repeated Sequence ◽  
Effector Proteins ◽  
Barr Body ◽  
Inactive X Chromosome ◽  
Chromatin Configuration

Female mammals express the long noncoding X inactivation-specific transcript (Xist) RNA to initiate X chromosome inactivation (XCI) that eventually results in the formation of the Barr body. Xist encompasses half a dozen repeated sequence stretches containing motifs for RNA-binding proteins that recruit effector complexes with functions for silencing genes and establishing a repressive chromatin configuration. Functional characterization of these effector proteins unveils the cooperation of a number of pathways to repress genes on the inactive X chromosome. Mechanistic insights can be extended to other noncoding RNAs with similar structure and open avenues for the design of new therapies to switch off gene expression. Here we review recent advances in the understanding of Xist and on this basis try to synthesize a model for the initiation of XCI.

scholarly journals Periphilin self-association underpins epigenetic silencing by the HUSH complex

2020 ◽  
Vol 48 (18) ◽  
pp. 10313-10328
Author(s):  
Daniil M Prigozhin ◽  
Christopher H Douse ◽  
Laura E Farleigh ◽  
Anna Albecka ◽  
Iva A Tchasovnikarova ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Low Complexity ◽  
Epigenetic Landscape ◽  
Core Complex ◽  
Terminal Domain ◽  
Genome Wide ◽  
Repressive Mark ◽  
Self Association

Abstract Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.

scholarly journals Musashi-1: An Example of How Polyalanine Tracts Contribute to Self-Association in the Intrinsically Disordered Regions of RNA-Binding Proteins

2020 ◽  
Vol 21 (7) ◽  
pp. 2289 ◽  
Author(s):  
Tsai-Chen Chen ◽  
Jie-rong Huang
Keyword(s):  
Nmr Spectroscopy ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Heterogeneity ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Self Association ◽  
As Stress ◽  
Disordered Regions

RNA-binding proteins (RBPs) have intrinsically disordered regions (IDRs) whose biophysical properties have yet to be explored to the same extent as those of the folded RNA interacting domains. These IDRs are essential to the formation of biomolecular condensates, such as stress and RNA granules, but dysregulated assembly can be pathological. Because of their structural heterogeneity, IDRs are best studied by NMR spectroscopy. In this study, we used NMR spectroscopy to investigate the structural propensity and self-association of the IDR of the RBP Musashi-1. We identified two transient α-helical regions (residues ~208–218 and ~270–284 in the IDR, the latter with a polyalanine tract). Strong NMR line broadening in these regions and circular dichroism and micrography data suggest that the two α-helical elements and the hydrophobic residues in between may contribute to the formation of oligomers found in stress granules and implicated in Alzheimer’s disease. Bioinformatics analysis suggests that polyalanine stretches in the IDRs of RBPs may have evolved to promote RBP assembly.

scholarly journals A post-transcriptional respiratome regulon in trypanosomes

2019 ◽  
Vol 47 (13) ◽  
pp. 7063-7077 ◽  
Author(s):  
Anna Trenaman ◽  
Lucy Glover ◽  
Sebastian Hutchinson ◽  
David Horn
Keyword(s):  
Gene Silencing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Positive Control ◽  
Surface Glycoprotein ◽  
Chain Complexes ◽  
Transport Chain ◽  
The Impact ◽  
Fof1 Atp Synthase

Abstract Post-transcriptional regulons coordinate the expression of groups of genes in eukaryotic cells, yet relatively few have been characterized. Parasitic trypanosomatids are particularly good models for studies on such mechanisms because they exhibit almost exclusive polycistronic, and unregulated, transcription. Here, we identify the Trypanosoma brucei ZC3H39/40 RNA-binding proteins as regulators of the respiratome; the mitochondrial electron transport chain (complexes I–IV) and the FoF1-ATP synthase (complex V). A high-throughput RNAi screen initially implicated both ZC3H proteins in variant surface glycoprotein (VSG) gene silencing. This link was confirmed and both proteins were shown to form a cytoplasmic ZC3H39/40 complex. Transcriptome and mRNA-interactome analyses indicated that the impact on VSG silencing was indirect, while the ZC3H39/40 complex specifically bound and stabilized transcripts encoding respiratome-complexes. Quantitative proteomic analyses revealed specific positive control of >20 components from complexes I, II and V. Our findings establish a link between the mitochondrial respiratome and VSG gene silencing in bloodstream form T. brucei. They also reveal a major respiratome regulon controlled by the conserved trypanosomatid ZC3H39/40 RNA-binding proteins.

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