Repertoire of mRNAs with hypermodified caps and study of their translation – ReCAPtrans

Epigenetic RNA modifications play important roles in the regulation of gene expression. Canonical cap-dependent translation relies on the presence of a 7-methylguanosine (m7G cap) at the 5’ end of mRNAs to recruit the initiation factor eIF4E. The strict dependence on the eIF4E/m7G interaction is called into question by multiple transcriptomic analyses, challenging the classical cap-dependent translation model. We reported the first example of epigenetic modification of the cap of certain mRNAs in mammals and showed that several stress-related selenoprotein mRNAs have a hypermodified m32,2,7G cap (or TMG cap). These mRNAs are not recognized by eIF4E but are nevertheless translated by a mechanism that needs to be elucidated. We recently identified the entire repertoire of TMG-capped mRNAs at the transcriptome level by TMG immunoprecipitation coupled to RNA-seq (TMG RIP-seq). TMG-capped mRNA genes encode proteins involved in translation, stress response, antioxidant protection, and cancer prevention.
Our hypothesis is that the TMG cap may allow mRNAs to be directed to specific translation pathways. The aim of the ReCAPtrans project is to determine the role of the TMG cap in mRNA translation and localization, identify associated translation factors, and characterize the repertoire of TMG-capped mRNAs under stress conditions to understand the role of this modification in the regulation of translation.

The role of the TMG cap in endogenous TMG-capped mRNA translation will be studied by the combination of TMG IP and polysome fractionation in canonical conditions and torin-1 inhibition that inactivates eIF4E-dependent translation. Translation of selected synthetic TMG- or m7G-capped mRNA transcripts will be used to decipher translation mechanisms in vitro using cell-free translation assays, or in vivo. TMG caps identified in noncoding RNAs play a role in their functional localization. The impact of the TMG cap on mRNA localization will be analyzed by subcellular fractionation and immunolocalization. The potential role of TMG-cap binding proteins, known for their role in nucleocytoplasmic trafficking, will be evaluated.
To capture native initiation factors involved in TMG-capped mRNA translation, we will use a ribosome/mRNA particle purification method dedicated to in-depth proteomic analysis. This will provide access to the composition of the translation complexes used by these different mRNAs, cap-binding factors and auxiliary proteins. Validation of trans-acting factors will be performed by classical mRNA-protein or protein-protein interaction assays. Their impact on the translation of TMG-capped mRNAs will be tested by siRNA. Particular attention will be paid to RNA binding proteins previously identified to support alternative cap-dependent translation such as initiation factors eIF4G2, eIF3 and cap binding proteins.
TMG-RIP seq experiments will be performed in different cell types (HEK293FT, HeLa or HEPG2) and under various conditions of stress (oxidative stress, hypoxic conditions, amino acid starvation) to determine whether the repertoire of TMG-capped mRNAs is regulated and could thus modulate translation under certain conditions.

This will indicate if a common model for their translation emerges or if specific mRNA pathways are engaged. Epigenetic modifications of the mRNA cap have never been studied. ReCAPtrans should elucidate a new mechanism of translation regulation in eukaryotes. Translational responses triggered by cellular stress will be particularly relevant for diseases such as cancer.

Wether translational responses are triggered by cellular stress will be particularly relevant for diseases such as cancer. The ReCAPtrans proposal should represent a step forward in the characterization of new post-transcriptional control mechanisms in higher eukaryotes. Failure to ensure translation fidelity is often associated with stress related diseases and cancer.