Exosomes are predominantly loaded with mRNA transcript encoding cytoplasmic proteins and exclude mRNA transcript encoding nuclear proteins

Abstract

Exosomes are nanovesicles ([~]30-150 nm diameters) released via an endocytic pathway in almost all mammalian cell types. Exosomes are composed of a lipid bilayer membrane that encloses RNA, miRNA, proteins and DNA. This manuscript unravels how exosome cargo is collected by a highly precise process delineating two separate mRNA transcript entities encoding cytoplasmic and nuclear proteins separately. Ultracentrifuge isolated exosomes were directly converted into cDNA (Exo-cDNA), by a method developed in our laboratory. Cellular RNA was extracted from each cell line and cDNA was prepared (Cell-cDNA). We amplified mRNA transcripts translating cytoplasmic proteins CD10 and CXCR4 and mRNA transcripts translating nuclear proteins such as proliferating cell nuclear antigen (PCNA), CREB-BP, activation induced cytidine deaminase (AID), and terminal deoxynucleotidyl transferase (TdT). We amplified all four different mRNA transcripts (PCNA, CREB-BP, AID, and TdT) from cellular cDNA but none from exosomal cDNA (Exo-cDNA). These findings suggest that exosomes carry mRNA transcripts encoding cytoplasmic proteins only but mRNA transcripts encoding nuclear proteins could not be detected. This important observation could prove to be crucial for the exosome research community since it sheds light on one of the limitations relating to the use of exosomes as biomarkers in cancer biology and other diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/227223v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@102c790org.highwire.dtl.DTLVardef@17bcabdorg.highwire.dtl.DTLVardef@3b5ac0org.highwire.dtl.DTLVardef@c303c8_HPS_FORMAT_FIGEXP M_FIG C_FIG