scholarly journals Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in Drosophila

Science ◽  
2020 ◽  
Vol 367 (6483) ◽  
pp. 1230-1234 ◽  
Author(s):  
Ruben Hervas ◽  
Michael J. Rau ◽  
Younshim Park ◽  
Wenjuan Zhang ◽  
Alexey G. Murzin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Amino Acid ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Fundamental Question ◽  
Hydrophobic Core ◽  
Filament Formation ◽  
Cytoplasmic Polyadenylation ◽  
Cryo Electron Microscopy ◽  
Memory Persistence

How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element–binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo–electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and “seed” for further translationally active aggregation. The 31–amino acid protofilament core adopted a cross-β unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory.

scholarly journals Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Joseph Atherton ◽  
Irene Farabella ◽  
I-Mei Yu ◽  
Steven S Rosenfeld ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Catalytic Site ◽  
Fundamental Question ◽  
Force Generation ◽  
Atp Binding ◽  
Cellular Functions ◽  
Microtubule Binding ◽  
Cryo Electron Microscopy ◽  
Adp Release

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.

scholarly journals A continuous model of physiological prion aggregation suggests a role for Orb2 in gating long-term synaptic information

2018 ◽  
Vol 5 (12) ◽  
pp. 180336
Author(s):  
Michele Sanguanini ◽  
Antonino Cattaneo
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Mrna Translation ◽  
Continuous Model ◽  
Cellular Level ◽  
Long Term Memory ◽  
Cytoplasmic Polyadenylation ◽  
Differential Model

The regulation of mRNA translation at the level of the synapse is believed to be fundamental in memory and learning at the cellular level. The family of cytoplasmic polyadenylation element binding (CPEB) proteins emerged as an important RNA-binding protein family during development and in adult neurons. Drosophila Orb2 (homologue of mouse CPEB3 protein and of the neural isoform of Aplysia CPEB) has been found to be involved in the translation of plasticity-dependent mRNAs and has been associated with long-term memory. Orb2 protein presents two main isoforms, Orb2A and Orb2B, which form an activity-induced amyloid-like functional aggregate, thought to be the translation-inducing state of the RNA-binding protein. Here we present a first two-states continuous differential model for Orb2A–Orb2B aggregation. This model provides new working hypotheses for studying the role of prion-like CPEB proteins in long-term synaptic plasticity. Moreover, this model can be used as a first step to integrate translation- and protein aggregation-dependent phenomena in synaptic facilitation rules.

scholarly journals Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

2015 ◽  
Vol 112 (43) ◽  
pp. 13237-13242 ◽  
Author(s):  
Lorenzo Sborgi ◽  
Francesco Ravotti ◽  
Venkata P. Dandey ◽  
Mathias S. Dick ◽  
Adam Mazur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Specific Binding ◽  
3D Structure ◽  
High Mobility ◽  
Atomic Resolution ◽  
Receptor Protein ◽  
Filament Formation ◽  
Cryo Electron Microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

scholarly journals Ataxin-2, Twenty-four and Dicer-2 are components of a non-canonical cytoplasmic polyadenylation complex

2021 ◽  
Author(s):  
Hima Priyanka Nadimpalli ◽  
Tanit Guitart ◽  
Olga Coll ◽  
Fátima Gebauer
Keyword(s):  
Large Scale ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Mrna Translation ◽  
Early Embryogenesis ◽  
High Confidence ◽  
Interference Factor ◽  
Cytoplasmic Polyadenylation ◽  
Mrna Targets ◽  
Ataxin 2

Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts. A canonical complex centered around the conserved RNA-binding protein family CPEB has been shown to be responsible for this process. We have previously reported evidence for an alternative non-canonical, CPEB-independent complex in Drosophila, of which the RNA-interference factor Dicer-2 is a component. Here, we investigate Dicer-2 mRNA targets and protein co-factors in cytoplasmic polyadenylation. Using RIP-Seq analysis we identify hundreds of novel Dicer-2 target transcripts, ~50% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Functional analysis indicate that both factors form an RNA-independent complex with Dicer-2, and are required for cytoplasmic polyadenylation of Dicer-2 targets. Our results reveal the composition of a novel cytoplasmic polyadenylation complex that operates during Drosophila early embryogenesis.

scholarly journals Elongational stalling activates mitoribosome-associated quality control

Science ◽  
2020 ◽  
Vol 370 (6520) ◽  
pp. 1105-1110
Author(s):  
Nirupa Desai ◽  
Hanting Yang ◽  
Viswanathan Chandrasekaran ◽  
Razina Kazi ◽  
Michal Minczuk ◽  
...  
Keyword(s):  
Quality Control ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Release Factor ◽  
Peptidyl Transfer ◽  
Cryo Electron Microscopy ◽  
Mitochondrial Ribosome ◽  
Nascent Chain ◽  
Associated Proteins ◽  
Guanosine Triphosphatase

The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo–electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.

scholarly journals A 3.8 Å Resolution Cryo-EM Structure of a Small Protein Bound to a Modular Imaging Scaffold

2018 ◽  
Author(s):  
Yuxi Liu ◽  
Duc Huynh ◽  
Todd O Yeates
Keyword(s):  
Electron Microscopy ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Structural Features ◽  
Cryo Electron Microscopy ◽  
Cargo Protein ◽  
Size Limitation ◽  
Protein Molecules ◽  
Lower Size ◽  
General Method

Proteins smaller than about 50 kDa are currently too small to be imaged by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein. We show that the bound cargo protein is held rigidly enough to visualize it at a resolution of 3.8 Å by cryo-EM, where basic structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM.

scholarly journals ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Charlotte Lässig ◽  
Sarah Matheisl ◽  
Konstantin MJ Sparrer ◽  
Carina C de Oliveira Mann ◽  
Manuela Moldt ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Innate Immune ◽  
Viral Rna ◽  
Wild Type ◽  
Cryo Electron Microscopy ◽  
Biochemical Studies ◽  
Infected Cells ◽  
System Disorder

The cytosolic antiviral innate immune sensor RIG-I distinguishes 5′ tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding.

scholarly journals The RNA-Binding Protein YBX3 Controls Amino Acid Levels by Regulating SLC mRNA Abundance

Cell Reports ◽  
2019 ◽  
Vol 27 (11) ◽  
pp. 3097-3106.e5 ◽  
Author(s):  
Amy Cooke ◽  
Thomas Schwarzl ◽  
Ina Huppertz ◽  
Gertjan Kramer ◽  
Panagiotis Mantas ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Mrna Abundance ◽  
Amino Acid Levels

scholarly journals Cryo-EM structure of the tetracycline resistance protein TetM in complex with a translating ribosome at 3.9-Å resolution

2015 ◽  
Vol 112 (17) ◽  
pp. 5401-5406 ◽  
Author(s):  
Stefan Arenz ◽  
Fabian Nguyen ◽  
Roland Beckmann ◽  
Daniel N. Wilson
Keyword(s):  
Electron Microscopy ◽  
Amino Acid ◽  
16S Rrna ◽  
Binding Site ◽  
Tetracycline Resistance ◽  
Small Subunit ◽  
Amino Acid Residues ◽  
Tyrosine Residues ◽  
Cryo Electron Microscopy ◽  
Resistance Protein

Ribosome protection proteins (RPPs) confer resistance to tetracycline by binding to the ribosome and chasing the drug from its binding site. Current models for RPP action are derived from 7.2- to 16-Å resolution structures of RPPs bound to vacant or nontranslating ribosomes. Here we present a cryo-electron microscopy reconstruction of the RPP TetM in complex with a translating ribosome at 3.9-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM with a unique splayed conformation of nucleotides A1492 and A1493 at the decoding center of the small subunit. The resolution enables us to unambiguously model the side chains of the amino acid residues comprising loop III in domain IV of TetM, revealing that the tyrosine residues Y506 and Y507 are not responsible for drug-release as suggested previously but rather for intrafactor contacts that appear to stabilize the conformation of loop III. Instead, Pro509 at the tip of loop III is located directly within the tetracycline binding site where it interacts with nucleotide C1054 of the 16S rRNA, such that RPP action uses Pro509, rather than Y506/Y507, to directly dislodge and release tetracycline from the ribosome.

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