| First Author | Leppek K | Year | 2014 |
| Journal | Nucleic Acids Res | Volume | 42 |
| Issue | 2 | Pages | e13 |
| PubMed ID | 24157833 | Mgi Jnum | J:211904 |
| Mgi Id | MGI:5576861 | Doi | 10.1093/nar/gkt956 |
| Citation | Leppek K, et al. (2014) An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins. Nucleic Acids Res 42(2):e13 |
| abstractText | 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 (4xS1m) 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 (TNFalpha), a well-known RNA motif that induces mRNA degradation, via 4xS1m 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 TNFalpha ARE. The optimized 4xS1m aptamer, therefore, provides a powerful tool for the discovery of mRNP components in a single affinity purification step. |
