An in vitro reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands

Abstract U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon–intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.

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Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

Nature Communications ◽

10.1038/s41467-021-21035-4 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 3

Author(s):

Vikas A. Tillu ◽

James Rae ◽

Ya Gao ◽

Nicholas Ariotti ◽

Matthias Floetenmeyer ◽

...

Keyword(s):

Electrostatic Interactions ◽

Membrane Lipid ◽

Membrane Curvature ◽

Caveolin 1 ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Solution Generation ◽

Endosomal System ◽

Disordered Regions

AbstractCaveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

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Bioinformatics-based study on prokaryotic, archaeal and eukaryotic nucleic acid-binding proteins for identification of low-complexity and intrinsically disordered regions

Frontiers in Life Science ◽

10.1080/21553769.2015.1075433 ◽

2015 ◽

Vol 9 (1) ◽

pp. 2-16

Author(s):

Birendra Singh Yadav ◽

Swati Singh ◽

Prashant Kumar ◽

Devika Mathur ◽

Raj Kumar Meena ◽

...

Keyword(s):

Nucleic Acid ◽

Binding Proteins ◽

Low Complexity ◽

Nucleic Acid Binding ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Nucleic Acid Binding Proteins ◽

Disordered Regions

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Ebola Virus VP30 Is an RNA Binding Protein

Journal of Virology ◽

10.1128/jvi.02523-06 ◽

2007 ◽

Vol 81 (17) ◽

pp. 8967-8976 ◽

Cited By ~ 44

Author(s):

Sinu P. John ◽

Tan Wang ◽

Scott Steffen ◽

Sonia Longhi ◽

Connie S. Schmaljohn ◽

...

Keyword(s):

Binding Affinity ◽

Ebola Virus ◽

Rna Binding ◽

Transcriptional Start Site ◽

Direct Interaction ◽

Binding Activity ◽

Stem Loop ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions

ABSTRACT The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21072289 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2289 ◽

Cited By ~ 2

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.

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Intrinsically disordered regions of Tristetraprolin and DCP2 directly interact to mediate decay of ARE-mRNA

10.1101/2021.11.07.467627 ◽

2021 ◽

Author(s):

Vincent D. Maciej ◽

Nevena Mateva ◽

Theresa Dittmers ◽

Sutapa Chakrabarti

Keyword(s):

Mrna Decay ◽

Rna Binding ◽

Weak Interactions ◽

Direct Interaction ◽

Interaction Network ◽

Cellular Interaction ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

The RNA binding protein Tristetraprolin (TTP) is a potent activator of mRNA decay, specifically for transcripts bearing AU-rich elements (AREs) in their 3'-untranslated regions. TTP functions as a mediator for mRNA decay by interacting with the decay machinery and recruiting it to the target ARE-mRNA. In this study, we report a weak, but direct interaction between TTP and the human decapping enzyme DCP2, which impacts the stability of ARE-transcripts. The TTP-DCP2 interaction is unusual as it involves intrinsically disordered regions (IDRs) of both binding partners. We show that the IDR of DCP2 has a propensity for oligomerization and liquid-liquid phase separation (LLPS) in vitro. Binding of TTP to DCP2 leads to its partitioning into phase-separated droplets formed by DCP2, suggesting that molecular crowding might facilitate the weak interaction between the two proteins and enable assembly of a decapping-competent mRNA-protein complex on TTP-bound transcripts in cells. Our studies underline the role of weak interactions in the cellular interaction network and their contribution towards cellular functionality.

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Promiscuity as a functional trait: intrinsically disordered regions as central players of interactomes

Biochemical Journal ◽

10.1042/bj20130545 ◽

2013 ◽

Vol 454 (3) ◽

pp. 361-369 ◽

Cited By ~ 108

Author(s):

Alexander Cumberworth ◽

Guillaume Lamour ◽

M. Madan Babu ◽

Jörg Gsponer

Keyword(s):

Low Complexity ◽

Functional Trait ◽

Protein Interaction Networks ◽

Altered Expression ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Linear Motifs ◽

Eukaryotic Genomes ◽

Molecular Recognition Features ◽

Disordered Regions

Because of their pervasiveness in eukaryotic genomes and their unique properties, understanding the role that ID (intrinsically disordered) regions in proteins play in the interactome is essential for gaining a better understanding of the network. Especially critical in determining this role is their ability to bind more than one partner using the same region. Studies have revealed that proteins containing ID regions tend to take a central role in protein interaction networks; specifically, they act as hubs, interacting with multiple different partners across time and space, allowing for the co-ordination of many cellular activities. There appear to be three different modules within ID regions responsible for their functionally promiscuous behaviour: MoRFs (molecular recognition features), SLiMs (small linear motifs) and LCRs (low complexity regions). These regions allow for functionality such as engaging in the formation of dynamic heteromeric structures which can serve to increase local activity of an enzyme or store a collection of functionally related molecules for later use. However, the use of promiscuity does not come without a cost: a number of diseases that have been associated with ID-containing proteins seem to be caused by undesirable interactions occurring upon altered expression of the ID-containing protein.

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