RNA-binding Proteins Required to Sort Small RNAs for Secretion in Exosomes from Human Cultured Cells
Abstract
Highly purified extracellular vesicles (EVs) isolated from human cell lines display a small number of substantially (~ 1000 fold) enriched miRNAs that differ from one cell line to another. In spite of the small number of such species, no single RNA sorting sequence is evident. In order to explore the mechanism of RNA sorting, with the establishment of a cell-free reaction that reproduces the selective incorporation of synthetic, mature miRNAs (miR223 and miR122) into vesicles formed in a reaction containing membranes and cytosol from mechanically disrupted HEK293 cells. The sorting reaction requires both membrane and cytosol and is stimulated by hydrolysable ATP and incubation at a physiologic temperature. Using biotinylated derivatives of two different miRNAs, different sets of RNA binding proteins incorporated along with each species were found, among which the proteins Ybx1 and Lupus La are required to sort mir223 and miR122, respectively. EVs also contain more abundant major species of small RNA including full-length tRNA, Y-RNA and vault RNA, and each requires the Ybx1 protein for selective sorting into exosomes secreted by cells and into vesicles in the cell-free reaction. The La antigen binds to both 5’ and 3’ signals on miR122 to promote packaging of the RNA into vesicles in the cell-free vesicle biogenesis reaction.
Note: This lecture is a part of IAS Symposium on Biogenesis and Trafficking of Intracellular and Extracellular Vesicles.
About the speaker
Prof. Randy Schekman received his BA in Molecular Biology from the University of California, Los Angeles in 1970 and his PhD in Biochemistry from Stanford University in 1975. He spent two years as a Postdoctoral Fellow at the University of California, San Diego before joining the College of Letters & Science of the University of California, Berkeley in 1976. In 1990-1994, he was appointed the Head of the Division of Biochemistry and Molecular Biology and currently, he is a Professor of Cell and Developmental Biology. He has also been an Investigator at Howard Hughes Medical Institute (HHMI) since 1991.
Prof. Schekman's research is focused on the process of membrane assembly, vesicular transport, and membrane fusion among organelles of the secretory pathway. At UC Berkeley, he developed a genetic and biochemical approach to the study of eukaryotic membrane traffic. Basic principles that emerged from his past studies in yeast are now being applied to studies of genetic diseases of protein transport, as well to the study of extracellular vesicles. He was appointed the Editor-in-Chief of the Annual Review of Cell and Developmental Biology in 2002 and the Proceedings of the National Academy of Sciences in 2006. In 2012-2019, he served as the Editor-in-Chief in eLife, a peer-reviewed open access scientific journal established in 2012 with sponsorships from HHMI, Wellcome Trust and the Max Planck Society.
Prof. Schekman is a Member of the US National Academy of Sciences, the Institute of Medicine, the American Academy of Arts and Sciences, the American Philosophical Society, a Foreign Associate of the Accademia Nazionale dei Lincei, a Foreign Associate of the Royal Society of London and an Honorary Academician of the Academia Sinica. In 1999, he was elected President of the American Society for Cell Biology. Since 2017, he has been chairing the Advisory Council of the Aligning Science Across Parkinson's Initiative. Among his awards are the Eli Lilly and Company-Elanco Research Award (1987), the Lewis S. Rosenstiel Award for Distinguished Work in Basic Medical Research (1993), the Canada Gairdner International Award (1996), the Amgen Award of the Protein Society (1999), the Albert Lasker Basic Medical Research Award (2002), the Massry Prize (2010) and the E.B. Wilson Medal (2010). He is one of the laureates of the 2013 Nobel Prize in Physiology or Medicine for the "discoveries of machinery regulating vesicle traffic, a major transport system in our cells".
