scholarly journals Arginine-enriched mixed-charge domains provide cohesion for nuclear speckle condensation

2019 ◽  
Author(s):  
Jamie A. Greig ◽  
Tu Anh Nguyen ◽  
Michelle Lee ◽  
Alex S. Holehouse ◽  
Ammon E. Posey ◽  
...  
Keyword(s):  
Low Complexity ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Nuclear Speckle ◽  
Net Charge ◽  
Intrinsically Disordered ◽  
Recognition Motif ◽  
Condensate Formation ◽  
Dynamic Material Properties

AbstractLow-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine/aspartic-acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine’s guanidinium ion. MCDs can synergise with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behaviour is tuneable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. By contrast, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation, and link the speckle’s dynamic material properties with function in mRNA processing.

scholarly journals An autoinhibitory intramolecular interaction proof-reads RNA recognition by the essential splicing factor U2AF2

2020 ◽  
Vol 117 (13) ◽  
pp. 7140-7149 ◽  
Author(s):  
Hyun-Seo Kang ◽  
Carolina Sánchez-Rico ◽  
Stefanie Ebersberger ◽  
F. X. Reymond Sutandy ◽  
Anke Busch ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Intramolecular Interaction ◽  
Intramolecular Interactions ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Rna Motifs ◽  
Intrinsically Disordered ◽  
Molecular Features

The recognition of cis-regulatory RNA motifs in human transcripts by RNA binding proteins (RBPs) is essential for gene regulation. The molecular features that determine RBP specificity are often poorly understood. Here, we combined NMR structural biology with high-throughput iCLIP approaches to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker region connecting the two RNA recognition motif (RRM) domains of U2AF2 mediates autoinhibitory intramolecular interactions to reduce nonproductive binding to weak Py-tract RNAs. This proofreading favors binding of U2AF2 at stronger Py-tracts, as required to define 3′ splice sites at early stages of spliceosome assembly. Mutations that impair the linker autoinhibition enhance the affinity for weak Py-tracts result in promiscuous binding of U2AF2 along mRNAs and impact on splicing fidelity. Our findings highlight an important role of intrinsically disordered linkers to modulate RNA interactions of multidomain RBPs.

scholarly journals Tethering-induced destabilization and ATP-binding for tandem RRM domains of ALS-causing TDP-43 and hnRNPA1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mei Dang ◽  
Yifan Li ◽  
Jianxing Song
Keyword(s):  
Conformational Dynamics ◽  
Md Simulations ◽  
Atp Binding ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Nucleic Acid Binding ◽  
General Role ◽  
Nmr Characterization ◽  
Lateral Sclerosis

AbstractTDP-43 and hnRNPA1 contain tandemly-tethered RNA-recognition-motif (RRM) domains, which not only functionally bind an array of nucleic acids, but also participate in aggregation/fibrillation, a pathological hallmark of various human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), alzheimer's disease (AD) and Multisystem proteinopathy (MSP). Here, by DSF, NMR and MD simulations we systematically characterized stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RRM domains in both tethered and isolated forms. The results reveal three key findings: (1) upon tethering TDP-43 RRM domains become dramatically coupled and destabilized with Tm reduced to only 49 °C. (2) ATP specifically binds TDP-43 and hnRNPA1 RRM domains, in which ATP occupies the similar pockets within the conserved nucleic-acid-binding surfaces, with the affinity slightly higher to the tethered than isolated forms. (3) MD simulations indicate that the tethered RRM domains of TDP-43 and hnRNPA1 have higher conformational dynamics than the isolated forms. Two RRM domains become coupled as shown by NMR characterization and analysis of inter-domain correlation motions. The study explains the long-standing puzzle that the tethered TDP-43 RRM1–RRM2 is particularly prone to aggregation/fibrillation, and underscores the general role of ATP in inhibiting aggregation/fibrillation of RRM-containing proteins. The results also rationalize the observation that the risk of aggregation-causing diseases increases with aging.

scholarly journals Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

Crystals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 273
Author(s):  
Yoshita Srivastava ◽  
Rachel Bonn-Breach ◽  
Sai Shashank Chavali ◽  
Geoffrey M. Lippa ◽  
Jermaine L. Jenkins ◽  
...  
Keyword(s):  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Molecular Replacement ◽  
Hydrophobic Core ◽  
Anomalous Diffraction ◽  
Recognition Motif ◽  
Determination Process ◽  
Experimental Phasing ◽  
Crystal Contacts ◽  
Apical Loop

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

Identification and expression analysis of Dazl homologue in Cynops cyanurus

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Yinjiao Zhao ◽  
Ya Du ◽  
Qinglan Ge ◽  
Fang Yan ◽  
Shu Wei
Keyword(s):  
Phylogenetic Analysis ◽  
Comparative Studies ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Specific Expression ◽  
Recognition Motif ◽  
Deleted In Azoospermia ◽  
Specific Expression Pattern

Summary The Dazl (deleted in azoospermia-like) gene encodes an RNA-binding protein containing an RNA recognition motif (RRM) and a DAZ motif. Dazl is essential for gametogenesis in vertebrates. In this study, we report the cloning of Dazl cDNA from Cynops cyanurus. Ccdazl mRNA showed a germline-specific expression pattern as expected. Ccdazl expression gradually decreased during oogenesis, suggesting that it may be involved in oocyte development. Phylogenetic analysis revealed that the Ccdazl protein shares conserved motifs/domains with Dazl proteins from other species. Cloning of Ccdazl provides a new tool to carry out comparative studies of germ cell development in amphibians.

scholarly journals A Small Cyclic β‐Hairpin Peptide Mimics the Rbfox2 RNA Recognition Motif and Binds to the Precursor miRNA 20b

ChemBioChem ◽  
2019 ◽  
Vol 20 (7) ◽  
pp. 931-939 ◽  
Author(s):  
Yi‐Ting Sun ◽  
Matthew D. Shortridge ◽  
Gabriele Varani
Keyword(s):  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Peptide Mimics ◽  
Recognition Motif

scholarly journals The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Kathleen L McCann ◽  
Takamasa Teramoto ◽  
Jun Zhang ◽  
Traci M Tanaka Hall ◽  
Susan J Baserga
Keyword(s):  
Protein Interactions ◽  
Molecular Basis ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Rrna Processing ◽  
Protein Protein Interactions ◽  
Recognition Motif ◽  
Processing Defects

ANE syndrome is a ribosomopathy caused by a mutation in an RNA recognition motif of RBM28, a nucleolar protein conserved to yeast (Nop4). While patients with ANE syndrome have fewer mature ribosomes, it is unclear how this mutation disrupts ribosome assembly. Here we use yeast as a model system and show that the mutation confers growth and pre-rRNA processing defects. Recently, we found that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome. Here we demonstrate that the ANE syndrome mutation disrupts Nop4’s hub function by abrogating several of Nop4’s protein-protein interactions. Circular dichroism and NMR demonstrate that the ANE syndrome mutation in RRM3 of human RBM28 disrupts domain folding. We conclude that the ANE syndrome mutation generates defective protein folding which abrogates protein-protein interactions and causes faulty pre-LSU rRNA processing, thus revealing one aspect of the molecular basis of this human disease.

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