Mechanistic insights into endoribonuclease activation of the mRNA 3' end processing machinery by Cleavage Factor IA

Pre-mRNAs must undergo a series of cotranscriptional processing steps for efficient export from the nucleus, translation and mRNA stability. Near the 3' end of the nascent transcript, the pre-mRNA is cleaved at specific RNA sequences, releasing the pre- mRNA from the transcribing RNA polymerase II. The new 3' end is subsequently polyadenylated with a tail of ~60 adenosines in Saccharomyces cerevisiae. In yeast, 3' end processing is conducted by a large 14-protein complex of ~850 kDa called Cleavage and Polyadenylation Factor (CPF). CPF contains three enzymatic activities that each form the core of one of three interconnected modules: the cleavage module, the polymerase module and the phosphatase module. The endoribonuclease within CPF is held in an inactive form and must be activated to maintain fidelity. The specific mechanism of activation remains unknown, but it is thought that the endoribonuclease requires additional protein factors within CPF and an auxiliary complex called Cleavage Factor IA (CFIA). CFIA is an RNA binding complex of ~350 kDa consisting of 4 different proteins: Rna14, Rna15, Pcf11 and Clp1. Within this thesis, I use a biochemical and structural approach with an in vitro reconstituted system to understand the activation mechanism of the endoribonuclease, with a particular focus on CFIA. This provides new insights into the interactions both within CPF, CFIA and between the two complexes that are required for efficient cleavage. I show that the interactions between the complexes are multifaceted and that Rna14 forms a hub for multiple similar motifs within CPF to bind. Mutagenesis of these motifs indicate a cumulative reduction in cleavage. I have further demonstrated a role for Rna15 of CFIA in RNA binding and the requirement of a conserved zinc finger within Pcf11. Structural analysis of the endoribonuclease reveals a conserved pseudodimer conformation reminiscent of other active RNA processing complexes. Finally, I utilise the reconstituted 3' end processing machinery to establish single molecule assays using novel RNA based substrates. Together these results define the multiple roles of CFIA as an essential complex in 3' end processing.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Fagarasan, Holly

Advisor dc:contributor.advisor

• Passmore, Lori

• mRNA processing
• Protein biochemistry
• Cleavage and Polyadenylation
• RNA Biology

Licence

• https://www.repository.cam.ac.uk/bitstreams/50cc6995-f9fe-4675-93ac-f38ddb268134/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.124262

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/394211