ARCHITECTURE AND RNA RECOGNITION BY THE EUKARYOTIC M6A METHYLTRANSFERASE COMPLEX

RNA modifications are critical post-transcriptional regulators of gene expression, influencing diverse biological functions. Among them, N6-methyladenosine (m6A) is one of the most abundant mRNA modifications and contributes to key cellular processes, including mRNA processing, translation, and decay. In eukaryotes, m6A is installed by the m6A Methyltransferase Complex (MTC), a multi-protein assembly composed of seven subunits in humans and six in Saccharomyces cerevisiae. However, the complete composition and architecture of the active MTC remain incompletely defined. This dissertation presents the in vitro reconstitution of an active core m6A Methyltransferase Complex from budding yeast (ycMTC), composed of five subunits conserved across eukaryotes: Ime4, Kar4, Mum2, Vir1, and Slz1 (homologous to human METTL3, METTL14, WTAP, VIRMA, and ZC3H13). Using an integrative structural biology centred on cryo-electron microscopy, the organisation and stoichiometry of the ycMTC were elucidated. The 2.6 Å cryo-EM structure reveals a compact assembly with an unexpected stoichiometry, featuring four copies of Mum2 and two copies of Vir1. Beyond its catalytic role, Ime4 also acts as a central scaffold, and its interactions with Mum2 are essential for m6A deposition in vivo. Additionally, this work clarified how the ycMTC recognises RNA. Using a natural REC8 mRNA substrate, a 2.7 Å cryo-EM structure, supported by RNA-binding assays, defines the molecular basis of substrate engagement and DRACH motif selectivity, including the coordination of the target adenosine. Together, these findings provide mechanistic insights into m6A installation by the MTC and establish a structural framework for understanding the evolution and functional regulation of this key epitranscriptomic machinery.

Grantor dc:publisher

Università degli Studi di Milano

Year dc:date

2025

Author dc:creator

• LASSANDRO, MICHELANGELO

Contributors dc:contributor

• tutor: A. Casañal ; co-tutor: S. Polo ; internal advisor: A. Vannini ; external advisor: M. Sattler ; coordinator: D. Pasini
• M. Lassandro
• POLO, SIMONA LAURA ANNA
• PASINI, DIEGO

• Settore BIOS-07/A - Biochimica

Statement dc:rights

• info:eu-repo/semantics/embargoedAccess
• license:Creative commons
• license uri:http://creativecommons.org/licenses/by-sa/4.0/

Language dc:language

eng

Handle dc:identifier

https://hdl.handle.net/2434/1198323

OAI identifier oai:identifier

oai:air.unimi.it:2434/1198323