Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast.

Rachael Emily Turner,Calvin A Kraupner-Taylor,Ralf B Schittenhelm,Michael See,Angavai Swaminathan,Darren J Creek,Paul F Harrison,Amanda L Peterson,Belinda J Goldie,David R Powell,Traude H Beilharz,Bernhard Dichtl

doi:10.7554/elife.65331

Rachael Emily Turner, Calvin A Kraupner-Taylor + Show 10 more

Open Access

https://doi.org/10.7554/elife.65331

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Journal: eLife	Publication Date: Jul 7, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: Monash University, Deakin University

Abstract

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.

Highlights

We demonstrate that cordycepin treatment caused a global shift to distal poly (A) sites, an effect that we found to be accompanied by an accumulation of nucleotides and their biosynthetic intermediates
In agreement with the idea that changes in cellular nucleotide concentrations impacted the usage of alternative poly(A) sites following cordycepin treatment, we show that mycophenolic acid, which is known to reduce GTP levels, caused a shift to more proximal sites and combined treatment reversed the shifts observed with cordycepin alone
Since transcription elongation is sensitive to nucleotide levels, we propose that nucleotide metabolism plays a key role in transmitting kinetic coordination of RNA polymerase II (RNAP II) elongation and coupled pre-messenger RNA (mRNA) 3’ end processing in the regulation of alternative polyadenylation

Summary

Introduction

3’ end processing is a critical step in eukaryotic messenger RNA (mRNA) maturation. This two-step process involves co-transcriptional endonucleolytic cleavage of the pre-mRNA transcript and the subsequent addition of a non-templated polyadenosine (poly(A)) tail by poly(A) polymerase (Colgan and Manley, 1997). There is an A-rich positioning element (PE) located 10–30 nucleotides upstream of the cleavage site (Tian and Graber, 2012) This is the equivalent of the mammalian polyadenylation signal (PAS) and has the same optimal sequence of AAUAAA though this motif is less conserved in yeast (Zhao et al, 1999). This occurrence, termed alternative polyadenylation (APA), is pervasive in all studied eukaryotes and appears in up to 70% of human and yeast genes (Derti et al, 2012; Ozsolak et al, 2010) Use of such alternate cleavage sites has the potential to impact the stability, localisation and translational efficiency of an mRNA transcript. We propose that APA in yeast employs a balance between cleavage site strength, 3’ end processing factor availability, and transcriptional rate to establish a kinetic control of poly(A) site choice

Results

Discussion

Materials and methods

Funding Funder Australian Research Council