Depletion of METTL3 alters cellular and extracellular levels of miRNAs containing m6A consensus sequences
Jessica J. Abner,
Jeffrey L. Franklin,
Margaret A. Clement,
Scott A. Hinger,
Ryan M. Allen,
Xiao Liu,
Stefanie Kellner,
Junzhou Wu,
John Karijolich,
Qi Liu,
Kasey C. Vickers,
Peter Dedon,
Alissa M. Weaver,
Robert J. Coffey,
James G. Patton
Affiliations
Jessica J. Abner
Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37235, USA
Jeffrey L. Franklin
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37235, USA; Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, 37235, USA
Margaret A. Clement
Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37235, USA
Scott A. Hinger
Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37235, USA
Ryan M. Allen
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37235, USA
Xiao Liu
Department of Biostatistics, Vanderbilt University Medical Center, Nashville, TN, 37235, USA
Stefanie Kellner
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
Junzhou Wu
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
John Karijolich
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, 37235, USA
Qi Liu
Department of Biostatistics, Vanderbilt University Medical Center, Nashville, TN, 37235, USA
Kasey C. Vickers
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37235, USA
Peter Dedon
Singapore-MIT Alliance for Research and Technology, Singapore
Alissa M. Weaver
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, 37235, USA
Robert J. Coffey
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37235, USA; Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, 37235, USA
James G. Patton
Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37235, USA; Corresponding author.
Extracellular vesicles (EVs) are capable of transferring cargo from donor to recipient cells, but precisely how cargo content is regulated for export is mostly unknown. For miRNA cargo, we previously showed that when compared to isogenic colorectal cancer (CRC) cells expressing wild-type KRAS, a distinct subset of miRNAs are differentially enriched in EVs from KRAS mutant active CRC cells, with miR-100 being one of the most enriched. The mechanisms that could explain how miR-100 and other miRNAs are differentially exported into EVs have not been fully elucidated. Here, we tested the effect of N6-methyladenosine (m6A) modification on miRNA export into EVs by depletion of METTL3 and ALKBH5, a writer and eraser of m6A modification, respectively. While the effects of ALKBH5 knockdown were quite modest, decreased levels of METTL3 led to reduced cellular and extracellular levels of a subset of miRNAs that contain consensus sequences for m6A modification. Functional testing of EVs prepared from cells expressing shRNAs against METTL3 showed that they were less capable of conferring colony growth in 3D to wild-type KRAS cells and were also largely incapable of conferring the spread of cetuximab resistance. Our data support a role for METTL3 modification on cellular miRNA levels and export of specific miRNAs.
