Understanding the role of RNA modifications University of Birmingham in United Kingdom

All classes of RNA are chemically modified in vivo. ~160 distinct chemical moieties can be appended to nucleotides’ functional groups in all four bases as well as the ribose to modulate RNA structure, dynamics and interactome (1). However, besides the very well-studied m6A modification, the mechanisms by which most chemical modifications impact RNA function are largely unknown. At the level of site-specific modifications, our knowledge is even more limited, as in vivo functional studies struggle to identify a phenotype for the depletion of modification at individual sites. Despite these challenges, the increasing demand of new drug targets, justifies the effort to establish the functional mechanisms of individual RNA modifications in health and disease contexts, to be able to establish the modified/unmodified RNAs as a novel and specific drug targets. Recently, the RNA modification N4-acetylcytidine (ac4C) has gained attention, with more than 6,000 acetylation sites having been identified in human messenger RNA (mRNA). It has been demonstrated that ac4C is induced upon several different stress stimuli and regulates translation efficiency and mRNA localization (2–4). Besides mRNA and ribosomal RNA, also long non-coding RNAs (lnc RNA) are subject to acetylation, but the function of this modification is unknown. In this project we will study one extensively acetylated lnc RNA transcript and dissect the role of acetylation of this RNA on the regulation of cellular metabolism.

This process is mediated by an acetylation dependent RNA–protein interaction, which we will illuminate using biochemistry, structural and cellular biology. First, we will identify which region of the lnc RNA binds to the target protein and which acetylation is necessary to sustain the interaction. Second, we will solve the three-dimensional structure of the RNA in its modified and unmodified forms to understand the impact of the modification on RNA structure and thus function, Third, we will solve the three-dimensional structure of the protein¬–RNA complex, to illuminate how acetylation-dependent RNA binding modulates protein activity. Third, we will verify the effect of selectively depleting this interaction in vivo (in collaboration with our partner laboratory in Denmark) on the cell metabolism.

To generate site-selective ac4C labelled RNAs we will use two approaches. First, we will develop a synthetic method to implement acetylation on the building blocks used for RNA solid-phase synthesis. Second, we will try to implement the modification enzymatically, using the only known ac4C writer, the nucleolar protein NAT10. Both approaches present challenges: in the chemical approach, the modification needs to be stable during the RNA solid-state synthesis reaction; in the enzymatic approach, NAT10 needs an adaptor to achieve RNA recognition and acetylation selectivity. This project will offer opportunities for training in molecular engineering (chemistry, chemical biology) and structural mechanistic studies (structural and molecular biology).

The overarching goals are:

1. Provide tools to introduce site-selective ac4C modifications in large RNAs.

2. Understand how ac4C modulates RNA structure.

3. Understand how ac4C modulates protein–RNA interactions.

4. Illuminate the mechanism through which the lnc RNA regulates cell metabolism through its acetylation-dependent interactome.

The techniques that the PGR will learn and apply are:

1. Chemical synthesis of oligonucleotides.

2. RNA engineering via enzymatic synthesis.

3. Molecular biology of both proteins and RNA.

4. Structural biology by NMR spectroscopy and cryo-EM.