Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

3’ end processing is a fundamental step of gene expression that precedes RNA polyadenylation and that is intimately associated with transcription termination. 3’ end processing is a co-transcriptional process that involves the recruitment of RNA maturation factors by the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Despite a somewhat fair assessment of the players implicated in RNA processing, much is still unknown about the regulatory mechanisms underlying their integration with the transcription cycle. This is particularly true for 3’ end processing and transcription termination. Indeed, recent transcriptome-wide studies indicate that multiple polyadenylation sites (PAS) are used at most eukaryotic genes, as demonstrated in humans, mouse, plants, flies, and yeast. This process, known as alternative polyadenylation (APA), is emerging as a major layer of gene regulation, allowing the inclusion or exclusion of sequences that control the localization, stability, and translation of mRNAs. However, the mechanism of PAS recognition and how PAS selection is coordinated with transcription termination remains unclear. The long-term goal of our research program is therefore to elucidate the molecular basis by which co-transcriptional 3’ end processing and transcription termination determine the fate of cellular RNAs.

Our studies using the yeast Schizosaccharomyces pombe have recently contributed to this field by showing that the conserved RNA-binding protein Seb1 promotes poly(A) site selection and termination of RNAPII transcription at protein-coding and noncoding genes. Accordingly, the GENERAL OBJECTIVE of this application is to determine the mechanism by which Seb1 controls 3’ end processing and transcription termination. Our central HYPOTHESIS , which is based on strong preliminary findings, is that binding of Seb1 to the CTD of RNAPII influences transcription elongation kinetics, thereby controlling the position of poly(A) site selection. We will test our hypothesis and accomplish the objective of this proposal by pursuing the following SPECIFIC AIMS :

1. Determine the mechanism by which Seb1 controls polyadenylation site selection;
2. Establish the link between RNAPII phosphorylation and co-transcriptional 3’ end processing;
3. Define the mechanism of snoRNA 3’ end processing and transcription termination in S. pombe.

The ORIGINALITY of this proposal relies on studying a protein that binds to both the CTD of RNAPII and to nascent transcripts to elucidate how transcription coordinates 3’ end processing and termination. Given the similarities between S. pombe and human polyadenylation signals, including use of the canonical AAUAAA hexamer, we predict that the links between transcription and polyadenylation site selection described by our studies in fission yeast will apply to all eukaryotes.