title: An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins.
abstract: Determining the composition of messenger ribonucleoprotein (mRNP) particles is essential for a comprehensive understanding of the complex mechanisms underlying mRNA regulation, but is technically challenging. Here we present an RNA-based method to identify RNP components using a modified streptavidin (SA)-binding RNA aptamer termed S1m. By optimizing the RNA aptamer S1 in structure and repeat conformation, we improved its affinity for SA and found a 4-fold repeat of S1m (4×S1m) to be more efficient than the established MS2 and PP7 systems from bacteriophages. We then attached the AU-rich element (ARE) of tumor necrosis factor alpha (TNFα), a well-known RNA motif that induces mRNA degradation, via 4×S1m to a SA matrix, and used the resulting RNA affinity column to purify ARE-binding proteins (BPs) from cellular extracts. By quantitative mass spectrometry using differential dimethyl labeling, we identified the majority of established ARE-BPs and detected several RNA-BPs that had previously not been associated with AREs. For two of these proteins, Rbms1 and Roxan, we confirmed specific binding to the TNFα ARE. The optimized 4×S1m aptamer, therefore, provides a powerful tool for the discovery of mRNP components in a single affinity purification step.
authors.affiliation: Helmholtz Junior Research Group Posttranscriptional Control of Gene Expression, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany and DKFZ-ZMBH Alliance.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Leppek
authors.forename: Georg
authors.initials: G
authors.lastname: Stoecklin
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001710:Biotin; D019556:COS Cells; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D012260:Ribonucleases; D012261:Ribonucleoproteins / Q000032:analysis / Q000302:isolation & purification*; D019809:Streptavidin; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D052157:Aptamers, Nucleotide; D012261:Ribonucleoproteins; D014409:Tumor Necrosis Factor-alpha; D001710:Biotin; D019809:Streptavidin; D012260:Ribonucleases