RNA metabolism, RNA processing: This proposal aims to define cellular RNA targets of the essential, highly conserved DEAD-box helicase Ded1p.
Final Report Abstract
In summary, I developed a systematic approach to define RNA substrates of RNA helicases in yeast which revealed the first glimpse of the cellular RNA targets of Ded1p. We obtained a target RNA profile for Ded1p in which is consistent with a role as a general translation initiation factor but which is different from the profile of the very similar DEAD-box protein Dbp2p. Ded1p binds to multiple sites on mRNAs preferably in the vicinity hairpin secondary structures. Consistently, Ded1p exhibits a higher RNA binding affinity for those structures in vitro. Ded1p binds in a position-dependent manner to mRNAs and those pronounced binding sites are at or downstream of the AUG. The results of this study give unprecedented insights into the universal capacity of DEAD box proteins to regulate a wide range of RNA targets. Moreover, this data marks the first step to link RNA binding to Ded1p’s function in translation initiation and provides the base for setting up experimental systems that can illuminate the functional connection between translation, Ded1p and the binding site on the mRNA in more detail. In order to publish these results, we are predominantly focusing on the reproduction of Ded1p binding sites. As mentioned above, our experiments depend on a very inefficient crosslinking step which we are currently trying to improve. However, we are also in the process of determining of the functional significance of Ded1p binding sites on mRNAs. Initial experiments to manipulate the binding sites through mutations gave mixed results thus not leading to a testable hypothesis. We further aim to test whether Ded1p binds to the position close to the AUG only during active translation initiation in the presence of the ribosomal subunits. To that, we are purifying crosslinked Ded1p complexes from polysome gradients from the polysome vs the free mRNP fractions and determining Ded1p binding to the AUG by primer extension assays.
