Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction
- University of California, United States
- University of Oregon, United States
- Howard Hughes Medical Institute, University of California, United States
Figures
Figure 1 with 1 supplement
Purified CD63-positive exosomes contain RNA.
(a) Exosome purification schematic. (b–e) Representative electron micrographs of negative stained samples from the 100,000 ×g pellet fraction (b,d) and post-flotation fractions (c,e) at either 9300X …
Figure 1—figure supplement 1
Sub-cellular localization of C-terminal CD63-luciferase-FLAG fusion.
CD63-luciferase-FLAG cells induced for 48 hr were fixed with 4% paraformaldehyde blocked with 5% BSA in PBS and stained with M2-Flag antibody (1:500) and then Alexa-488 conjugated anti-mouse …
Figure 2 with 1 supplement
Enrichment of select miRNAs in exosomes.
(a) Venn diagram showing the number of total (above diagram), unique (inside red or green circles) and shared miRNAs (inside yellow) from each library. (b) Pie charts showing the relative proportion …
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Figure 2—source data 1
Mapping statistics for small RNA-seq libraries to the human genome (hg19).
Reads were processed (see Materials and methods) and mapped to the human genome (hg19) using Bowtie 2. Total counts for reads mapped to the genome, to rRNA and to miRNA (using miRdeep2 - see Materials and methods) are shown. Percent of total reads are shown in parenthesis.
- https://doi.org/10.7554/eLife.19276.006
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Figure 2—source data 2
miRNA counts from cell and exosomes libraries using miRdeep2.
Number of reads mapped to each miRNA annotated in miRBase version 21 using the quantifier program of the miRdeep2 package. Reads per million miRNA mapped reads (RPM) were calculated and the quotient was taken to determine enrichment in exosomes.
- https://doi.org/10.7554/eLife.19276.007
Figure 2—figure supplement 1
Read frequency distribution along miR-223 and miR-144 precursors.
All reads detected for miR-223 (a) and miR-144 (b) in the exosome small RNA-seq libraries were aligned to the hairpin precursor sequences and the read frequency distribution across the precursor was …
Figure 3
Cell-free exosome biogenesis reaction.
(a) Schematic illustrating the in vitro biogenesis reaction. (b) Exosome biogenesis measured by relative protected CD63-luciferase. Reactions with or without cytosol, 1%Tx-100 and incubation …
Figure 4
Cell-free selective sorting of miRNA into exosomes.
(a) Schematic illustrating the in vitro packaging reaction. (b) Cell-free packaging of miR-223 measured as percent protected by qRT-PCR. Reactions with or without membranes (15,000 ×g pellet), …
Figure 5
Identification of YBX1 as a candidate exosomal miRNA sorting protein.
(a) Scheme to identify candidate miRNA sorting proteins (b) Proteins identified by tandem mass spectroscopy from the experiment illustrated in (a). (c) Schematic of YBX1 protein. The cold-shock …
Figure 6
Lack of evidence for a specific role for Ago2 in sorting miR-223 into exosomes.
(a) Immunoblots for Ago2 and exosome markers TSG101 and CD9 in 100,000 ×g (100K) pellet and the 20/40% sucrose interface fractions. (b) Schematics showing 3' and internally biotinylated miR-223 …
Figure 7 with 2 supplements
YBX1 is necessary for exosomal miRNA packaging and secretion.
(a) Analysis of wild-type and CRISPR/Cas9 genome edited HEK293T clones by PCR flanking the genomic target site (top) and immunoblot for YBX1 (middle) and GAPDH (bottom). (b) In vitro miR-223 …
Figure 7—figure supplement 1
Partial redundancy for YBX2 for the secretion of miR-223 in cells.
Relative quantity of miR-223 secreted into the medium by WT and ΔYBX1 cells after 24 hr with or without transfection with control or YBX2 siRNA.
Figure 7—figure supplement 2
miR-223 association with Ago2 in WT and ΔYBX1 cells.
RNA immunoprecipitation was performed using mouse anti-Ago2 antibody or a mouse IgG isotype control antibody in lysates of WT or ΔYBX1 cells. Ago2-associated miR-223 was detected by qPCR. …
