Abstract
The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with man...Continue Reading
References
Oct 10, 1979·Nucleic Acids Research·M LheureuxM Steinert
Aug 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·S GelvinS H Howell
Jan 1, 1977·Nucleic Acids Research·G SchutzP Feigelson
Oct 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·Z PalfiA Bindereif
Mar 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·R A Young, R W Davis
Sep 1, 1980·Proceedings of the National Academy of Sciences of the United States of America·R T MacGillivrayE W Davie
Jun 1, 1980·Molecular and Biochemical Parasitology·J S Cordingley, M J Turner
Jan 4, 1994·Proceedings of the National Academy of Sciences of the United States of America·A HehlI Roditi
Aug 1, 1997·Nucleic Acids Research·H R HotzC Clayton
Aug 1, 1997·Molecular and Cellular Biology·A FurgerI Roditi
Dec 13, 2002·Proceedings of the National Academy of Sciences of the United States of America·Klaus HartmuthReinhard Lührmann
Mar 15, 2003·The Journal of Biological Chemistry·Javier G De GaudenziAlberto C C Frasch
Sep 18, 2007·Eukaryotic Cell·Claudia HartmannChristine E Clayton
Nov 10, 2007·The Journal of Biological Chemistry·Jurgen R HaanstraBarbara M Bakker
Jul 10, 2008·Nucleic Acids Research·Antonio M Estévez
Aug 21, 2008·Journal of Cell Science·Susanne KramerMark Carrington
Nov 5, 2008·Methods in Molecular Biology·Scott C WalkerDavid R Engelke
Feb 17, 2009·RNA·Michael Zeev SternShulamit Michaeli
Apr 8, 2010·Cell·Markus HafnerThomas Tuschl
Sep 30, 2010·RNA·Boris Slobodin, Jeffrey E Gerst
Oct 26, 2010·Biophysical Chemistry·Michael BusbyJason J Davis
Feb 3, 2012·Molecular Microbiology·Martin WurstChristine Clayton
Apr 18, 2013·PLoS Pathogens·Dorothea DrollChristine Clayton
Oct 26, 2013·Nucleic Acids Research·Kathrin Leppek, Georg Stoecklin
Jan 29, 2014·Nucleic Acids Research·Aditi SinghEsteban Erben
Jun 20, 2014·Molecular Microbiology·Ana C PenaLuisa M Figueiredo
Aug 26, 2014·Molecular Microbiology·Abeer FaddaChristine Clayton
Apr 7, 2015·Cell·Ci ChuHoward Y Chang
Apr 29, 2015·Nature·Colleen A McHughMitchell Guttman
Aug 20, 2015·PloS One·Cornelia KleinChristine Clayton