scholarly journals RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs

2018 ◽  
Author(s):  
Mark A. McClintock ◽  
Carly I. Dix ◽  
Christopher M. Johnson ◽  
Stephen H. McLaughlin ◽  
Rory J. Maizels ◽  
...  
Keyword(s):  
Binding Sites ◽  
Intracellular Transport ◽  
Rna Binding ◽  
Adaptor Protein ◽  
Cytoplasmic Dynein ◽  
Protein Assemblies ◽  
Mrna Transport ◽  
Long Distance ◽  
Spatial Control ◽  
Dynein Motor

Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis-acting RNA localisation signal stabilises the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport.

scholarly journals RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Mark A McClintock ◽  
Carly I Dix ◽  
Christopher M Johnson ◽  
Stephen H McLaughlin ◽  
Rory J Maizels ◽  
...  
Keyword(s):  
Binding Sites ◽  
Intracellular Transport ◽  
Rna Binding ◽  
Adaptor Protein ◽  
Cytoplasmic Dynein ◽  
Protein Assemblies ◽  
Mrna Transport ◽  
Long Distance ◽  
Cis Acting ◽  
Dynein Motor

Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis-acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport.

scholarly journals The Gene for the Intermediate Chain Subunit of Cytoplasmic Dynein Is Essential in Drosophila

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1211-1220 ◽  
Author(s):  
Kristin L M Boylan ◽  
Thomas S Hays
Keyword(s):  
Intracellular Transport ◽  
De Novo ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Wing Development ◽  
Dynein Motor ◽  
Dynein Intermediate Chain ◽  
Microtubule Motor ◽  
Intermediate Chain

Abstract The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, we have identified mutations in the gene for the cytoplasmic dynein intermediate chain, Dic19C. We used a functional Dic transgene in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued1, a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, we used complementation analysis to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene and revealing a novel role for dynein function during wing development.

scholarly journals DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

2021 ◽  
Vol 22 (8) ◽  
pp. 3985
Author(s):  
Manami Deshimaru ◽  
Mariko Kinoshita-Kawada ◽  
Kaori Kubota ◽  
Takuya Watanabe ◽  
Yasuyoshi Tanaka ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Rna Binding ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Terminal Region ◽  
Missense Mutations ◽  
Long Distance ◽  
Causative Gene ◽  
Dynactin Complex ◽  
Lateral Sclerosis

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.

scholarly journals Targeting Allostery in the Dynein Motor Domain with Small Molecule Inhibitors

2020 ◽  
Author(s):  
Cristina C. Santarossa ◽  
Keith J. Mickolajczyk ◽  
Jonathan B. Steinman ◽  
Linas Urnavicius ◽  
Nan Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Intracellular Transport ◽  
Aaa Atpase ◽  
Nucleotide Binding Sites ◽  
Allosteric Communication ◽  
Large Size ◽  
Dynein Motor ◽  
Chemical Inhibitors ◽  
Regulatory Sites

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein’s unusually large size, four distinct nucleotide-binding sites, and the existence of closely-related isoforms with different functions, pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with single molecule experiments and mutagenesis studies, indicate that the compounds likely inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein’s main catalytic site in the AAA1 domain. A double Walker B mutant in AAA3 and AAA4 is an inactive enzyme, suggesting that inhibiting these regulatory sites can have a similar effect to inhibiting AAA1. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein’s AAA domains.

scholarly journals Nanometer-Resolution Long-term Tracking of Single Cargos Reveals Dynein Motor Mechanisms

2022 ◽  
Author(s):  
Chunte Sam Peng ◽  
Yunxiang Zhang ◽  
Qian Liu ◽  
G. Edward Marti ◽  
Yu-Wen Alvin Huang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intracellular Transport ◽  
Molecular Model ◽  
Cytoplasmic Dynein ◽  
Single Particle Tracking ◽  
Thermally Activated ◽  
Dynein Motor ◽  
Biochemical Measurements ◽  
Hydrolysis Of

Cytoplasmic dynein is essential for intracellular transport, but because of its complexity, we still do not fully understand how this 1.5 megadalton protein works. Here, we used novel optical probes that enable single-particle tracking (SPT) of individual cargos transported by dynein motors in live neurons over 900 μm. Analyses using the Fluctuation Theorem (FT) showed that the number of dynein molecules switches between 1-5 motors during the transport. Clearly resolved single-molecular steps revealed that the dwell times between individual steps were accurately described by an enzymatic cycle dominated by two equal and thermally-activated rate constants. Based on these data, we propose a new molecular model whereby each step requires the hydrolysis of 2 ATPs. The model is consistent with extensive structural, single-molecule and biochemical measurements.

scholarly journals Microtubule-Dependent mRNA Transport in Fungi

2010 ◽  
Vol 9 (7) ◽  
pp. 982-990 ◽  
Author(s):  
Kathi Zarnack ◽  
Michael Feldbrügge
Keyword(s):  
Molecular Motors ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Hyphal Growth ◽  
Subcellular Targeting ◽  
Mrna Transport ◽  
Long Distance ◽  
Content Type ◽  
Long Distance Transport

ABSTRACT The localization and local translation of mRNAs constitute an important mechanism to promote the correct subcellular targeting of proteins. mRNA localization is mediated by the active transport of mRNPs, large assemblies consisting of mRNAs and associated factors such as RNA-binding proteins. Molecular motors move mRNPs along the actin or microtubule cytoskeleton for short-distance or long-distance trafficking, respectively. In filamentous fungi, microtubule-based long-distance transport of vesicles, which are involved in membrane and cell wall expansion, supports efficient hyphal growth. Recently, we discovered that the microtubule-mediated transport of mRNAs is essential for the fast polar growth of infectious filaments in the corn pathogen Ustilago maydis. Combining in vivo UV cross-linking and RNA live imaging revealed that the RNA-binding protein Rrm4, which constitutes an integral part of the mRNP transport machinery, mediates the transport of distinct mRNAs encoding polarity factors, protein synthesis factors, and mitochondrial proteins. Moreover, our results indicate that microtubule-dependent mRNA transport is evolutionarily conserved from fungi to higher eukaryotes. This raises the exciting possibility of U. maydis as a model system to uncover basic concepts of long-distance mRNA transport.

scholarly journals Egalitarian feeds forward to Staufen to inhibit Dynein during mRNP transport

2021 ◽  
Author(s):  
Imre Gáspár ◽  
Ly Jane Phea ◽  
Anne Ephrussi
Keyword(s):  
Intracellular Transport ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Localization ◽  
Posterior Pole ◽  
Protein Accumulation ◽  
Double Stranded Rna ◽  
Ribonucleoprotein Complexes ◽  
Dynein Motor

AbstractThe regulated recruitment and activity of motor proteins is crucial for intracellular transport of cargoes, including ribonucleoprotein complexes (RNP). Here we show that regulation of mRNP transport by the minus end-directed dynein motor complex in the Drosophila germline relies on the interplay of two double-stranded RNA binding proteins, Staufen and the dynein adaptor Egalitarian. Our quantitative in situ analysis shows that, in the nurse cells, Egalitarian associates with bicoid, oskar and staufen mRNAs, which consequently enrich in the oocyte. This results in ooplasmic accumulation of Staufen and its recruitment to the transcripts. We demonstrate that Staufen levels scale proportionately with RNA content in oskar mRNPs, and to a lesser extent in bicoid mRNPs. We show that enhanced recruitment of Staufen to oskar mRNPs results in dissociation of Egalitarian and a reduction in minus end-directed transport, although dynein remains associated with the RNPs. The downregulation of dynein activity prevents anterior accumulation of oskar mRNA, whose transport to the posterior is essential for localized production of Oskar protein. Our observations identify a feed-forward loop, whereby staufen mRNA localization and protein accumulation in the oocyte enable motor switching, promoting oskar mRNA localization to the posterior pole.

CSBPI_Site：Multi-Information Sources of Features to RNA Binding Sites Prediction

2021 ◽  
Vol 15 ◽  
Author(s):  
Lichao Zhang ◽  
Zihong Huang ◽  
Liang Kong
Keyword(s):  
Computational Model ◽  
Binding Sites ◽  
Noncoding Rna ◽  
Computational Models ◽  
Information Sources ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Gradient Boosting ◽  
Position Information ◽  
Rna Binding Sites

Background: RNA-binding proteins establish posttranscriptional gene regulation by coordinating the maturation, editing, transport, stability, and translation of cellular RNAs. The immunoprecipitation experiments could identify interaction between RNA and proteins, but they are limited due to the experimental environment and material. Therefore, it is essential to construct computational models to identify the function sites. Objective: Although some computational methods have been proposed to predict RNA binding sites, the accuracy could be further improved. Moreover, it is necessary to construct a dataset with more samples to design a reliable model. Here we present a computational model based on multi-information sources to identify RNA binding sites. Method: We construct an accurate computational model named CSBPI_Site, based on xtreme gradient boosting. The specifically designed 15-dimensional feature vector captures four types of information (chemical shift, chemical bond, chemical properties and position information). Results: The satisfied accuracy of 0.86 and AUC of 0.89 were obtained by leave-one-out cross validation. Meanwhile, the accuracies were slightly different (range from 0.83 to 0.85) among three classifiers algorithm, which showed the novel features are stable and fit to multiple classifiers. These results showed that the proposed method is effective and robust for noncoding RNA binding sites identification. Conclusion: Our method based on multi-information sources is effective to represent the binding sites information among ncRNAs. The satisfied prediction results of Diels-Alder riboz-yme based on CSBPI_Site indicates that our model is valuable to identify the function site.

scholarly journals Sam68 promotes hepatic gluconeogenesis via CRTC2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aijun Qiao ◽  
Junlan Zhou ◽  
Shiyue Xu ◽  
Wenxia Ma ◽  
Chan Boriboun ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Therapeutic Target ◽  
Rna Binding ◽  
Adaptor Protein ◽  
Diabetic Mouse ◽  
Mrna Levels ◽  
Hepatic Gluconeogenesis ◽  
Kinase Substrate ◽  
Glucose Levels ◽  
Patients With Diabetes

AbstractHepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes.

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