The role of 4E-T in translational regulation and mRNA decay

Abstract

The eukaryotic initiation factor 4E (eIF4E) binds the cap structure of an mRNA as the first step of cap dependent translation. Together with the RNA helicase eIF4A and the scaffolding protein eIF4G, eIF4E forms the eIF4F complex, which recruits the 43S pre- initiation complex to initiate translation. The eIF4E-transporter (4E-T) is an eIF4E-binding protein (4E-BP) responsible for the nucleocytoplasmic shuttling of eIF4E. Additionally, 4E-T prevents translation initiation by competing with eIF4G for binding to eIF4E. 4E-T is also a P- body protein that contributes to mRNA decay triggered by AU-rich elements (ARE) and microRNAs (miRNAs).

The work described in this thesis focuses on the molecular mechanisms and protein interaction partners used by 4E-T in the regulation of mRNA decay. To study the effects of 4E- T on mRNA stability and translation, tethering assays with reporter transcripts coupled to northern blotting were performed in human cells. Binding of 4E-T to a reporter promoted CCR4-NOT dependent mRNA deadenylation. Unlike typical 5′-3′ mRNA decay, the 4E-T bound mRNA was not decapped and degraded but remained stable as a deadenylated decay intermediate. Binding assays indicated that recruitment of the CCR4-NOT complex to an mRNA by 4E-T is mediated by the so far uncharacterized middle region of 4E-T and independent of the interaction partners described to date.

The studies reported here also indicate that the interaction of 4E-T with the cap binding proteins eIF4E or 4EHP prevented decapping of the deadenylated mRNA. Furthermore, we found that inhibition of decapping by 4E-T participates in the regulation of gene expression by the microRNA effector protein TNRC6B, the CCR4-NOT scaffolding protein NOT1 and in the turnover of ARE containing reporter mRNAs. These results show that 4E-T can potentially stabilize mRNAs targeted for decay in a wide range of biological processes, including microRNA-mediated gene silencing.

This work highlights that inhibition of decapping by 4E-T allows the storage of deadenylated and silenced mRNAs. To date, this is the first insight into the molecular mechanism of mRNA storage in human cells, which plays important yet poorly understood functions in oocyte and neuronal development.