BME PhD Dissertation Defense - Yingjie Sun
- 2:00 pm on Wednesday, July 31, 2013
- 44 Cummington Mall, Room 203
Prof. Amit Meller (Academic Advisor, BU, BME& Physics)
Prof. Sandor Vajda ( Chair, BU, BME)
Prof. Mark Grinstaff (BU, BME)
Prof. Kenneth Rothschild (BU, Physics& Physiology)
Prof. Adam Cohen (Harvard, Chemistry and Chemical Biology and of Physics)
The PI3K/Akt/mTOR pathway regulates several cellular functions, including cellular proliferation, growth, and survival. Components of this pathway are frequently abnormal in a variety of cancer cells. The PI3K/Akt/mTOR pathway converges on the eukaryotic translation initiation factor 4F (eIF4F), making it an attractive molecular target for anti-cancer therapies. As a subunit of eIF4F, the translation initiation factor 4A (eIF4A) is known to facilitate binding and scanning of the ribosome by unwinding secondary structures in the 5' untranslated region (UTR) of mRNAs during translation initiation. However, the molecular mechanisms of eIF4A activity have remained elusive.
Single-molecule Fluorescence Resonance Energy Transfer (sm-FRET) can probe structural changes and interactions of biological systems in real time, which cannot be observed using bulk techniques. When complemented with bulk assays, such as gel-shift and fluorescence spectroscopy, a powerful approach for the investigation of translation initiation can be realized. First we directly observe and quantify the helicase activity of eIF4A in the presence of the ancillary RNA-binding factor eIF4H, using sm-FRET. We show that eIF4H can significantly enhance the helicase activity of eIF4A by strongly binding both to loop structures within the RNA substrate as well as to eIF4A. Electrophoretic mobility shift assay (EMSA) shows that eIF4H binds to the amino-terminal domain (NTD) but not to the carboxy-terminal domain (CTD) of eIF4A. In the presence of ATP, the eIF4A/eIF4H complex exhibits rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. Second, we directly probe the conformational dynamics of double-labeled eIF4A, in real time, during RNA unwinding using sm-FRET. We demonstrate that the eIF4A in the presence of eIF4H can repetitively unwind the RNA hairpin substrate by transitioning between an “open” and a “closed” conformation using the energy from ATP hydrolysis. Upon binding of an RNA hairpin and ATP, which is mediated by eIF4H, eIF4A adopts a closed conformation; after ATP hydrolysis, eIF4A returns to the open conformation, the RNA duplex is completely unwound, and the RNA is released, quickly reannealing to reform the hairpin. Third, we find that both an RNA aptamer and the small molecule hippuristanol can inhibit the helicase activity of eIF4A/eIF4H with different mechanisms: the RNA aptamer can directly compete with an RNA hairpin for binding to both eIF4A and eIF4H, while hippuristanol does not interfere with the binding of eIF4A/eIF4H to the loop structure of RNA. Instead, hippuristanol inhibits helicase activity by blocking the conformational change of eIF4A.
To explain our observations, we propose that the eIF4A/eIF4H complex binds directly to mRNA loop structures and then destabilizes nearby duplexes. Duplex unwinding is mediated by the dynamic conformational transitions of the eIF4A, while maintaining contact with the eIF4H at its amino-terminal domain. The proposed molecular mechanism of eIF4A’s activity may shed new light on cancer therapies that use small-molecules or RNA aptamers to regulate translation at the initiation step.