Purification of messenger ribonucleoprotein particles via a tagged nascent polypeptide

• Verfasst von: Inchaustegui Gil, Diana Patricia [VerfasserIn]
• Verfasst von: Clayton, Christine [VerfasserIn]
• Titel: Purification of messenger ribonucleoprotein particles via a tagged nascent polypeptide
• Verf.angabe: Diana P. Inchaustegui Gil, Christine Clayton
• E-Jahr: 2016
• Jahr: 2016 Jan 25
• Umfang: 13 S.
• Fussnoten: Gesehen am 10.06.2020
• Titel Quelle: Enthalten in: PLOS ONE
• Ort Quelle: San Francisco, California, US : PLOS, 2006
• Jahr Quelle: 2016
• Band/Heft Quelle: 11(2016,1) Artikel-Nummer e0148131, 13 Seiten
• ISSN Quelle: 1932-6203
• DOI: doi:10.1371/journal.pone.0148131
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1700315307

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 many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA.