Complexity and function of messenger ribonucleoprotein particles
Final Report Abstract
The major achievement of the trypanosome part of the project was to develop a method to purify reporter mRNPs from polysomes. The purification factor was 1000-fold at the protein level, and 10-fold at the RNA level, with a yield of about 8%. The method should be sufficient to detect proteins that are bound with high specificity to the reporter mRNA, if very few other mRNAs bind the same protein; chances of detection by mass spectrometry will be enhanced if several copies of the protein are bound to the reporter. The yield and purity are better than any previously reported. However, the reporter mRNP is still only 10% of the final preparation. The method cannot be used to determine the overall protein composition of a specific mRNP because the background from other mRNPs is too high. The mammalian part of the project focused on optimization of a streptavidin-binding aptamer. When introduced into reporter genes and expressed in mammalian cell cultures, the optimized aptamer strategy was able to recover 4.5% of the mRNA. While the recovery was too low for purifying sufficient amounts of specific mRNPs from cells for mass spectrometry, the optimized aptamer could be used very efficiently for affinity purification of RNP complexes using in vitro transcribed RNA. The in vitro purification strategy was used very successfully for the identification of proteins bound to AU-rich RNA motifs, which was published together with a detailed description of the method. Moreover, the aptamer-based in vitro approach allowed us to identify Roquin as the major trans-acting factor that recognizes a novel class of stem-loop RNA degradation motifs.
Publications
- (2013) Roquin promotes constitutive mRNA decay via a conserved class of stem-loop recognition motifs. Cell 153:869
Leppek K, Schott J, Reitter S, Poetz F, Hammond MC, Stoecklin G
(See online at https://doi.org/10.1016/j.cell.2013.04.016) - (2014) An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins. Nucleic Acids Res 42:e13
Leppek K, Stoecklin G
(See online at https://doi.org/10.1093/nar/gkt956) - (2016) Purification of messenger ribonucleoprotein particles via a tagged nascent polypeptide. PLoS ONE 11: e0148131
Inchaustegui Gil D, Clayton C
(See online at https://doi.org/10.1371/journal.pone.0148131)
