scholarly journals Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Matthew J Shurtleff ◽  
Morayma M Temoche-Diaz ◽  
Kate V Karfilis ◽  
Sayaka Ri ◽  
Randy Schekman
Keyword(s):  
Cell Migration ◽  
Membrane Proteins ◽  
Small Rnas ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Target Cells ◽  
Rna Packaging ◽  
Exosome Biogenesis ◽  
Purification Scheme ◽  
A Cell

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.

scholarly journals Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

2016 ◽  
Author(s):  
Matthew Shurtleff ◽  
Kate V. Karfilis ◽  
Morayma Temoche-Diaz ◽  
Sayaka Ri ◽  
Randy Schekman
Keyword(s):  
Cell Migration ◽  
Membrane Proteins ◽  
Small Rnas ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Target Cells ◽  
Rna Packaging ◽  
Exosome Biogenesis ◽  
Purification Scheme ◽  
A Cell

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of microRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.

scholarly journals CRISPR-mediated isogenic cell-SELEX approach for generating highly specific aptamers against native membrane proteins

2020 ◽  
Author(s):  
Jonah C. Rosch ◽  
Emma H. Neal ◽  
Daniel A. Balikov ◽  
Mohsin Rahim ◽  
Ethan S. Lippmann
Keyword(s):  
Membrane Proteins ◽  
Glucose Transporter ◽  
High Specificity ◽  
Epithelial Cell Line ◽  
Target Cells ◽  
Wild Type ◽  
Simultaneous Exposure ◽  
Affinity Reagents ◽  
A Cell

AbstractIntroductionThe generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques.MethodsUsing a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.Results10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.ConclusionsOur data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach should be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

scholarly journals The RNA binding protein Larp1 regulates cell division, apoptosis and cell migration

2010 ◽  
Vol 38 (16) ◽  
pp. 5542-5553 ◽  
Author(s):  
C. Burrows ◽  
N. Abd Latip ◽  
S.-J. Lam ◽  
L. Carpenter ◽  
K. Sawicka ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Division ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein

scholarly journals Feed-Forward Regulation of a Cell Fate Determinant by an RNA-Binding Protein Generates Asymmetry in Yeast

Genetics ◽  
2010 ◽  
Vol 185 (2) ◽  
pp. 513-522 ◽  
Author(s):  
Joshua J. Wolf ◽  
Robin D. Dowell ◽  
Shaun Mahony ◽  
Michal Rabani ◽  
David K. Gifford ◽  
...  
Keyword(s):  
Cell Fate ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Feed Forward ◽  
Cell Fate Determinant ◽  
A Cell

scholarly journals PSGL-1 Inhibits the Incorporation of SARS-CoV and SARS-CoV-2 Spike Glycoproteins into Pseudovirions and Impairs Pseudovirus Attachment and Infectivity

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 46
Author(s):  
Sijia He ◽  
Abdul A. Waheed ◽  
Brian Hetrick ◽  
Deemah Dabbagh ◽  
Ivan V. Akhrymuk ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Surface ◽  
Immune Cells ◽  
Interferon Γ ◽  
Surface Glycoprotein ◽  
Target Cells ◽  
Cell Surface Glycoprotein ◽  
A Cell ◽  
Antiviral Activities ◽  
Hiv 1

P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-γ (INF-γ)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks pseudovirus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1+ cells

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