Cap-driven Internal Translation Initiation – MITIC

A combination of functional and structural methods is here used to understand the newly described translation initiation mechanism of histone H4. RNA probing, chemical and UV crosslinking experiments, and mutagenesis are used to decipher the functional aspects of the mechanism. CryoEM investigations are performed on the 80S particle assembled on H4 mRNA, as well as on the intermediate particles 43S and 48S stalled with specific blocking compounds. Whereas cryoEM is focused on the topology and low resolution of the initiating particles, the X-ray study is intended to solve the 3D structure of the mRNA at atomic resolution. Ribosome profiling on cell lines expressing variants of initiation factor eIF4E unable to initiate translation internally is performed in order to observe their translation landscape and determine the fraction of mRNAs translated as histone H4 mRNA.

Histone H4 mRNA exhibits a unique cap riboswitch that controls translation H4 translation.
Based on UV-cross linking results, we have established a preliminary model of the cap interaction into the riboswitch. In parallel, we started ribosome profiling experiments to quantify the relevance of initiation by tethering as observed with histone H4 mRNA. As tethering is based on the N-terminal domain of eIF4E, we established two stable cell lines expressing wild-type eIF4E or a truncated form of eIF4E deleted from the N-domain. Deep sequencing of mRNA fragments protected by ribosomes will highlight the differences in the cell translation landscape when initiation by tethering is prevented. The structure of the 80S ribosomal particle assembled on histone H4 mRNA has been solved by cryoEM investigation. A clear extra density is observed at the entrance of the mRNA path of the small ribosomal subunit. The actual 10 angstrom-resolution of the complex will be improved with the new Titan Krios cryoEM microscope recently installed in partner 2 lab. Investigations on the 48S complex assembled on H4 mRNA are also under investigation. 18 mRNA fragments have been purified, however crystallization attempts were unsuccessful. SAXS experiments have been performed on DESY synchrotron of Hambourg. Preliminary models of mRNA structure suggest that the molecule is not compact but elongated and branched.

The exhaustive listing of cellular mRNAs that bind the cap-binding factor eIF4E internally to secondary RNA structures may impact the research studies on cancer. Indeed, eIF4E is strongly up-regulated in most cancers and it has been shown that this deregulation induces over expression of a category of mRNAs involved in cell cycle and tumor progression. These mRNAs often contain highly structured sequences which suggests that they may bind eIF4E internally to stimulate translation more efficiently in a process of tethering similar to the histone H4 process. Identifying all these potential mRNAs targets would open the way of development of new inhibitors of these RNAs, with potential applications in cancer treatment.

Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA-DNA chimeras
Prongidi-Fix, L., Schaeffer, L., Simonetti, A., Barends, S., Ménétret, J.F., Klaholz, B., Eriani, G. and Martin, F. (2013)
BIOCHEM J. 449, 719-728.
The paper describes the new method we have developed to isolate 80S ribosomal particles assembled on histone H4 mRNA. The method can be adapted to isolate 80S and 48S particles assembled on any mRNA.

Cap analogs containing 6-thioguanosine–reagents for the synthesis of mRNAs selectively photo-crosslinkable with cap-binding biomolecules.
Nowakowska, M., Kowalska, J., Martin, F., d’Orchymont, A., Zuberek, J., Lukaszewicz, M., Darzynkiewicza, E. and Jemielity, J.
ORG. BIOMOL. CHEM. (2014) 12, 4841-4847.
This paper describes the crosslink capacity of new cap analogs synthesized in collaboration with the lab of Jacek Jemielity. With these results, the orientation of 5’ cap end of histone H4 mRNA could be oriented over the cap-binding pocket of the mRNA.

Hypermethylated-capped selenoprotein mRNAs in mammals
Wurth, L., Gribling-Burrer, A.S., Verheggen, C., Leichter, M., Takeuchi, A., Baudrey, S., Martin, F., Krol, A., Bertrand, E. and Allmang, C.
NUCL. ACIDS RES. (2014) 42, 8663-8677 first published online July 10, 2014 doi:10.1093/nar/gku580
In the broader framework of our project, we have explored translation initiation of selenocysteine-containing protein mRNAs. These mRNAs are hypermethylated in a way reminiscent of small nuclear RNAs. They do not bind the cap-binding initiation factor eIF4E but are found in polysomes. Further experiments will be performed to understand this unusual mode of translation initiation.