Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide

PloS One
Diana Inchaustegui Gil, Christine Clayton

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

Aug 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·S GelvinS H Howell
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
Dec 13, 2002·Proceedings of the National Academy of Sciences of the United States of America·Klaus HartmuthReinhard Lührmann
Nov 5, 2008·Methods in Molecular Biology·Scott C WalkerDavid R Engelke
Oct 26, 2010·Biophysical Chemistry·Michael BusbyJason J Davis
Apr 7, 2015·Cell·Ci ChuHoward Y Chang

❮ Previous
Next ❯

Citations


❮ Previous
Next ❯

Methods Mentioned

BETA
transgenic
affinity purification
pull-down
PCR

Software Mentioned

ImageJ

Related Concepts

Related Feeds

African Trypanosomiasis

African trypanosomiasis, also known as sleeping sickness, is an insect-borne parasitic disease of humans and other animals. It is caused by protozoa of the species Trypanosoma brucei and almost invariably progresses to death unless treated. Discover the latest research on African trypanosomiasis here.