Building Complexity In Vitro: Single Molecule Reconstitution of ASH1 mRNA Transport by a Class V Myosin from Budding Yeast

Abstract Eukaryotic mRNAs are predominantly translated via the cap-dependent pathway. Initiation is a rate-limiting step in cap-dependent translation and is the main target of translational control mechanisms. There is a lack of high-resolution techniques for characterizing the cap-dependent initiation kinetics. Here, we report an in vitro single-molecule assay that allows characterization of both initiation and peptide chain elongation kinetics for cap-dependent translation. Surprisingly, the histogram of the first-round initiation time is highly asymmetrical and spans a large time range that is several-fold greater than the average peptide synthesis time in translation reactions with a firefly luciferase-encoding mRNA. Both the histogram and single-molecule trajectories reveal an unexpected high-degree of asynchrony in translation activity between mRNA molecules. Furthermore, by inserting a small stem-loop (ΔG = −4.8 kcal/mol) in the middle of the mRNA 5′ untranslated region (UTR), our assay robustly detects small changes in budding yeast initiation kinetics, which could not be resolved by bulk luminescence kinetics. Lastly, we demonstrate the general applicability of this assay to distinct cell-free translation systems by using extracts prepared from budding yeast, wheat germ, and rabbit reticulocyte lysates. This assay should facilitate mechanistic studies of eukaryotic cap-dependent translation initiation and translational control.

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Single-molecule reconstitution of mRNA transport by a class V myosin

Nature Structural & Molecular Biology ◽

10.1038/nsmb.2614 ◽

2013 ◽

Vol 20 (8) ◽

pp. 952-957 ◽

Cited By ~ 30

Author(s):

Thomas E Sladewski ◽

Carol S Bookwalter ◽

Myoung-Soon Hong ◽

Kathleen M Trybus

Keyword(s):

Single Molecule ◽

Mrna Transport ◽

Class V

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Multiple Myo4 motors enhance ASH1 mRNA transport in Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.200912011 ◽

2010 ◽

Vol 189 (4) ◽

pp. 755-767 ◽

Cited By ~ 36

Author(s):

Sunglan Chung ◽

Peter A. Takizawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Binding ◽

Purification Method ◽

Class V ◽

Transport Unit ◽

Multiple Copies ◽

Ash1 Mrna ◽

Localization Element

In Saccharomyces cerevisiae, ASH1 mRNA is transported to the bud tip by the class V myosin Myo4. In vivo, Myo4 moves RNA in a rapid and continuous fashion, but in vitro Myo4 is a nonprocessive, monomeric motor that forms a complex with She3. To understand how nonprocessive motors generate continuous transport, we used a novel purification method to show that Myo4, She3, and the RNA-binding protein She2 are the sole major components of an active ribonucleoprotein transport unit. We demonstrate that a single localization element contains multiple copies of Myo4 and a tetramer of She2, which suggests that She2 may recruit multiple motors to an RNA. Furthermore, we show that increasing the number of Myo4–She3 molecules bound to ASH1 RNA in the absence of She2 increases the efficiency of RNA transport to the bud. Our data suggest that multiple, nonprocessive Myo4 motors can generate continuous transport of mRNA to the bud tip.

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Essential Features of the Class V Myosin from Budding Yeast for ASH1 mRNA Transport

Molecular Biology of the Cell ◽

10.1091/mbc.e08-08-0801 ◽

2009 ◽

Vol 20 (14) ◽

pp. 3414-3421 ◽

Cited By ~ 16

Author(s):

Carol S. Bookwalter ◽

Matthew Lord ◽

Kathleen M. Trybus

Keyword(s):

Daughter Cell ◽

Leucine Zipper ◽

Budding Yeast ◽

Binding Protein ◽

Adapter Protein ◽

Class V ◽

Mrna Binding Protein ◽

Ash1 Mrna ◽

Mrna Binding ◽

Zip Code

Myo4p, a single-headed and nonprocessive class V myosin in budding yeast, transports >20 different mRNAs asymmetrically to the bud. Here, we determine the features of the Myo4p motor that are necessary for correct localization of ASH1 mRNA to the daughter cell, a process that also requires the adapter protein She3p and the dimeric mRNA-binding protein She2p. The rod region of Myo4p, but not the globular tail, is essential for correct localization of ASH1 mRNA, confirming that the rod contains the primary binding site for She3p. The requirement for both the rod region and She3p can be bypassed by directly coupling the mRNA-binding protein She2p to Myo4p. ASH1 mRNA was also correctly localized when one motor was bound per dimeric She2p, or when two motors were joined together by a leucine zipper. Because multiple mRNAs are cotransported to the bud, it is likely that this process involves multiple motor transport regardless of the number of motors per zip code. Our results show that the most important feature for correct localization is the retention of coupling between all the members of the complex (Myo4p–She3p–She2p–ASH1 mRNA), which is aided by She3p being a tightly bound subunit of Myo4p.

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Stu2p binds tubulin and undergoes an open-to-closed conformational change

The Journal of Cell Biology ◽

10.1083/jcb.200511010 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1009-1022 ◽

Cited By ~ 87

Author(s):

Jawdat Al-Bassam ◽

Mark van Breugel ◽

Stephen C. Harrison ◽

Anthony Hyman

Keyword(s):

Conformational Change ◽

Conformational Transition ◽

Budding Yeast ◽

Microtubule Dynamics ◽

Open Structure ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

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The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Biomolecules ◽

10.3390/biom11030360 ◽

2021 ◽

Vol 11 (3) ◽

pp. 360

Author(s):

Pieterjan Debie ◽

Noemi B. Declerck ◽

Danny van Willigen ◽

Celine M. Huygen ◽

Bieke De Sloovere ◽

...

Keyword(s):

Single Molecule ◽

Gamma Ray ◽

Nuclear Imaging ◽

Primary Amines ◽

Resection Margins ◽

Fluorescent Light ◽

Single Structure ◽

Fluorescence Images

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

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DNA replication machinery: Insights from in vitro single-molecule approaches

Computational and Structural Biotechnology Journal ◽

10.1016/j.csbj.2021.04.013 ◽

2021 ◽

Vol 19 ◽

pp. 2057-2069

Author(s):

Rebeca Bocanegra ◽

G.A. Ismael Plaza ◽

Carlos R. Pulido ◽

Borja Ibarra

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Replication Machinery

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Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single Aβ42 oligomers

Nature Communications ◽

10.1038/s41467-020-20782-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Francesco Simone Ruggeri ◽

Johnny Habchi ◽

Sean Chia ◽

Robert I. Horne ◽

Michele Vendruscolo ◽

...

Keyword(s):

Small Molecules ◽

Single Molecule ◽

Protein Misfolding ◽

Molecular Mechanisms ◽

Chemical Sensitivity ◽

Incomplete Knowledge ◽

Parkinson’S Diseases ◽

Aggregation Inhibitor ◽

Misfolding Diseases

AbstractSignificant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer’s and Parkinson’s diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single Aβ42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aβ42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer’s disease. Our results demonstrate that the carboxyl group of this compound interacts with Aβ42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.

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