Physiological and pathological mechanisms of wobble adenosine to inosine modification in tRNAs – InotRNA

In the last decades, our understanding of the RNA world and its complexity has drastically enlarged with the characterization of numerous non-coding RNA species that participate together with mRNAs and tRNAs to the regulated development of cells and organisms. Notably, many RNA posttranscriptional modifications (PTM), which exceed by far the number of protein PTM, expand the functions of RNAs. Accordingly, dysregulations of these RNA PTM are linked with the etiology of a wide range of human diseases.

In tRNAs, the most modified RNA macromolecules, at least 50% of the PTM are linked to human diseases, strongly reviving the interest for tRNAs as effector molecules. Strikingly, an astounding 86% of the diseases linked to mutations in genes coding for tRNA-modifying enzymes are neurological disorders, of which 90% are neurodevelopmental disorders (NDDs), highlighting the importance of tRNAs modification for proper brain development. Yet, the high sensitivity of the human brain to perturbations in tRNAs PTM is not understood and it is therefore crucial to understand how these modifications are essential for brain development, and how defects on tRNA editing provoke neurological disorders.

Among RNA modifications, adenosine to inosine (A-to-I) is of major interest as inosine is found in most RNA species where it plays many essential roles, including in decoding at the ribosomes (wobble hypothesis). In eukaryotes, tRNAs wobble A-to-I is catalysed by the essential ADAT complex that, when mutated, causes wobble inosine (I34) decrease and leads to human NDDs, including microcephaly, intellectual disability and epilepsy. The biological importance of the ADAT complex and of I34 remains however elusive, calling for the precise characterization in molecular and functional terms of the wobble inosine modification pathways.

Building on its strong preliminary data (e.g. ADAT 3D structure, mouse models) that ensure its feasibility, the InotRNA project will tackle this challenge by combining, in a unique integrated manner, in vitro biochemical, biophysical and structural analyzes with in vivo manipulation of gene expression in mouse brain and in cellulo genome-wide omics approaches.

InotRNA will address the following questions:

1. How does ADAT recognize tRNAs?
InotRNA will characterize by tRNA binding, footprinting and structural analyses the specific recognition by ADAT of its cognate tRNAs, notably how ADAT modifies tRNAs having different sequences and tertiary structures.

2. How do ADAT and I34 regulate cortical development?
InotRNA will (i) investigate ADAT dysregulation in defined cell types and developmental stages in mouse models to pinpoint the developmental processes perturbed by I34 imbalance, and (ii) quantitate mRNA-specific translation rates by ribosome profiling in mouse cortices to define how I34 regulates translational programs.

3. How does I34 loss affect tRNAs stability and processing?
InotRNA will determine by omics analyses to which extent I34 loss affects the stability, abundance, modification and aminoacylation of ADAT cognate tRNAs in mouse cortices.

4. How do ADAT disease variants affect ADAT structure, mechanism and developmental function?
InotRNA will look at the effects of novel NDD-causing mutations on ADAT structure, tRNA binding and activity, and will test in vivo for pathological mechanisms of action of these ADAT variants.

The InotRNA project will therefore unravel the catalytic mechanism of ADAT, the effect of the wobble inosine on tRNA stability and function, and the consequences of the wobble inosine decrease on brain development. The unique and complementary expertise of the InotRNA partners, including in vivo analyses in mouse cortices to keep with the most relevant biological environment, will provide this consortium an exclusive and crucial competitive edge to tackle this challenging project in epitranscriptomics.