BPS2026 – Tracking polyadenylation of single pre-mRNA molecules using fluorescent probes

Cleavage and polyadenylation are essential reactions that occur during pre-mRNA 3′ end maturation, which is crucial for gene expression in eukaryotes. In budding yeast, cleavage and polyadenylation are catalyzed by the cleavage and polyadenylation factor (CPF) complex, cleavage factors (CF) CF IA, and CF IB. Pre-mRNA cleavage separates the messenger RNA from the chromatin-bound transcription machinery and allows polyadenylation to occur on the cleaved RNA fragment upstream of the cleavage site. Using a home-built total internal reflection fluorescence (TIRF) microscope along with fully purified and reconstituted CPF and cleavage factors, we have observed cleavage and polyadenylation of single RNA molecules in real-time. Using fluorescently labeled locked nucleic acid (LNA) probes with repeating thymine nucleotides that bind to polyadenylate (poly(A)) tails, we are gaining new insights into the coupling of cleavage and polyadenylation reactions. Using this system, we show that individual RNAs are heterogenous regarding their onset of polyadenylation after cleavage, suggesting the potential of additional regulatory steps between these reactions. We have also used in vitro transcription to create RNA standards with poly(A) tails of known lengths. With these, we have been able to investigate LNA probe lifetime and the relationship between probe binding and tail length. There are many outstanding questions regarding cleavage and polyadenylation that can be studied using single-molecule fluorescence experiments. Using these methods pre-mRNA 3′ end mutations linked to cancers, neurological diseases, and developmental disorders can be studied at a fundamental level to better understand their molecular underpinnings. This work enhances our ability to study poly(A) tail formation and allows us to directly visualize the activities of this complex macromolecular machine in real-time.