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

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.

Primary human myoblasts display only minor alternative polyadenylation compared to the transformed C2C12model of muscle differentiation

“…Previous studies have linked the differential expression of CPA factors to proliferative potential (Gruber et al 2014). Highly proliferative cells, such as cancer cells, tend toward shorter 3’ UTRs (Sandberg et al 2008) such that the 3’ UTR length is inversely proportional to the level of available CPA in most cell types where it has been measured (Sommerkamp et al 2021; Turner et al 2021). To determine whether the observed difference between APA in the two myotube models could be linked to the available CPA machinery, the expression of 33 previously identified CPA genes (Gruber et al 2014) was analysed.…”

Seeking a Role for Translational Control by Alternative Polyadenylation in Saccharomyces cerevisiae

“…S. cerevisiae represents a key model organism for basic biological research and has aided in a better understanding of human biology and disease. Though the majority of proteins that make up the yeast core cleavage and polyadenylation machinery have been identified, and much has been learnt about how alternative cleavage sites are selected [ 2 ], the role of this APA remains a relative mystery. The discovery of whether changes to the length of the 3′ UTR alter the translational efficiency of an mRNA transcript has been compromised by the techniques used to study the translatome.…”

“…This suggests a possibility that APA may exercise only a limited impact on translation in budding yeast. However, nutritional and pharmacological changes to cellular metabolism and transcription cause abundant changes to 3′ UTR lengths [ 2 , 3 , 4 ]. An option is that that this APA functions primarily as a transcription termination safety mechanism.…”

“…In such cases, the longer isoform tends to have a higher potential for regulation, due to additional regulatory elements present within the extended 3′ UTR. It is now clear from multiple gene-by-gene and transcriptome-wide studies that the relative concentration of the cleavage and polyadenylation factors, the rate of transcription, and chromatin architecture can influence poly(A)-choice [ 2 , 3 , 4 , 5 , 6 ]. However, whilst progress has been made towards understanding the effects of such alternative polyadenylation (APA) in mammalian systems [ 7 ], surprisingly little is known about its consequences in yeast.…”

Yeast cleavage factor Hrp1 is a novel guard protein that surveils pre-mRNA 3’ processing

“…Malfunctioning cleavage factors usually lead to production of 3'-extended mRNAs in budding yeast (Torchet et al, 2002;Singh et al, 2021;Mapendano et al, 2010;Turner et al, 2021;Al-Husini et al, 2017). Brodsky and others have shown that mRNAs in the CPF-CF mutants are retained in the nucleus, indicating that the readthrough mRNAs are surveilled in the nucleus (Brodsky and Silver, 2000;Carneiro et al, 2008;Hammell et al, 2002).…”