Structural and biochemical characterization of the human mRNA polyadenylation machinery

Todesca, Sofia (2025): Structural and biochemical characterization of the human mRNA polyadenylation machinery. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

The maturation of precursor mRNAs (pre-mRNAs) involves multiple co-transcriptional processing steps, with nearly all 3′ ends generated through a two-step mechanism: cleavage and polyadenylation. This process forms the poly(A) tail, which influences various aspects of an mRNA transcript’s lifetime, including stability and translation. While the enzymes and protein factors involved—which assemble into a large multi-subunit machinery—have been extensively studied in yeast and higher eukaryotes, key mechanistic details remain unclear. Moreover, despite its evolutionary conservation, notable differences exist in compositional complexity, poly(A) polymerase association stability, and poly(A) tail length across species. The first study in this thesis investigates human poly(A) polymerase α (PAPOA) recruitment by the mammalian polyadenylation specificity factor (mPSF) using computational predictions, biochemical assays, and cryo-electron microscopy (cryo-EM). We show that PAPOA is anchored by the FIP1 subunit via an evolutionarily conserved interaction involving the structured core of the polymerase and a short FIP1 peptide. Notably, in higher eukaryotes an additional PAPOA C-terminal region competes for the same binding surface of FIP1 on the core, forming a potential autoregulatory conformation. Additionally, we identify a previously unknown interaction between this C-terminal region and CPSF160, another mPSF subunit. These findings support a dual recognition mechanism where FIP1 and CPSF160 mediate PAPOA recruitment through mutually exclusive interactions, potentially regulating polymerase stability on the complex. Beyond canonical polyadenylation, in the second study of this thesis, we explore links between mRNA 3′ end maturation and degradation. The nuclear exosome can degrade mRNA transcripts via the NEXT and PAXT pathways. We show that ZC3H18, a NEXT-associated protein, localizes at transcription termination sites and interacts with CPSF160. ZC3H18 also binds ZFC3H1, a key PAXT component that recruits auxiliary factors such as ZC3H3, which intriguingly shares a five-zinc-finger (ZF1-5) organization with CPSF30 of mPSF. We observed that while CPSF30 ZF4 and ZF5 interact with distinct FIP1 copies, a ZC3H3 mutation in ZF5 might restrict its binding to a single FIP1 unit. At last, our cryo-EM analysis further reveals that FIP1, in complex with CPSF30, forms a stabilizing interface with CPSF160, allowing visualization of previously unresolved CPSF30 ZF4-5 in the context of a full complex assembly. Overall, our findings suggest that mPSF not only drives mRNA maturation but also contributes to RNA degradation via PAXT interactions. We propose an alternative mPSF composition that may regulate transcription termination-coupled RNA decay, providing insights into mRNA processing and turnover.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: 3′ end processing, pre-mRNA, polyadenylation, cleavage and polyadenylation, poly(A) polymerase, mRNA maturation, RNA degradation, RNA decay, NEXT, PAXT
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 20. May 2025
• 1. Referee: Conti, Elena
• Signature of the printed copy: 0001/UMC 31992