Protein expression from exogenous mRNA: uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway

RNA Biol. 2011 Jul-Aug;8(4):627-36. doi: 10.4161/rna.8.4.15394. Epub 2011 Jul 1.

Abstract

Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport
  • Carbocyanines
  • Caveolae / metabolism
  • Endocytosis*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Lysosomes / metabolism*
  • Membrane Microdomains / metabolism
  • Microscopy, Fluorescence
  • Protein Processing, Post-Translational
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Receptors, Scavenger / metabolism

Substances

  • Alexa Fluor 647
  • Carbocyanines
  • RNA, Messenger
  • Receptors, Scavenger