Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast
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Evaluation Summary:
This study aims to provide a comprehensive analysis of factors governing polyadenylation site selection in yeast. Overall, the authors reveal that multiple but distinct inputs including polyadenylation machinery integrity, transcription elongation rate, nucleotide availability and chromatin landscape all contribute to controlling cleavage and polyadenylation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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.
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Author Response:
Reviewer #2:
The authors investigated how alternative polyadenylation (APA) is modulated in yeast using appropriate transcriptomic methodologies.
The authors found that mutants for mRNA 3' end formation factors and cordycepin treatment alter alternative polyadenylation in the same manner, generating transcripts with longer 3'UTRs, due to a switch to distal polyadenylation sites (PAS). Most mutants analyzed cause a PAS switch, in particular mutants for RNA14, PCF11, YSH1, FIP1, NAB4 and PAP1. They also found that MPA and a rpb1 mutant, with a slower transcription elongation rate, reverts the cordycepin effect of distal PAS selection. This implies that in yeast, as in higher organisms, APA is modulated by RNAPII elongation. There is nucleosome depletion in the 3' end of convergent genes that undergo cordycepin-driven …
Author Response:
Reviewer #2:
The authors investigated how alternative polyadenylation (APA) is modulated in yeast using appropriate transcriptomic methodologies.
The authors found that mutants for mRNA 3' end formation factors and cordycepin treatment alter alternative polyadenylation in the same manner, generating transcripts with longer 3'UTRs, due to a switch to distal polyadenylation sites (PAS). Most mutants analyzed cause a PAS switch, in particular mutants for RNA14, PCF11, YSH1, FIP1, NAB4 and PAP1. They also found that MPA and a rpb1 mutant, with a slower transcription elongation rate, reverts the cordycepin effect of distal PAS selection. This implies that in yeast, as in higher organisms, APA is modulated by RNAPII elongation. There is nucleosome depletion in the 3' end of convergent genes that undergo cordycepin-driven APA alterations, which is a new finding.
On the basis of their data, the authors propose a kinetic model for APA in yeast that is regulated by the concentration of core mRNA 3' end factors and nucleotide levels, which in turn modulates RNAPII elongation. This integrative model has been already described in higher organisms, but not in yeast, and overall this study covers an impressive body of work that makes an important contribution to the field.
- The authors show that cordycepin have the same effect in APA as most of the 3' end factors mutants used, but there is a lack of integration between the two sets of PAS-seq data. The cordycepin APA effect may be due to decreased expression of mRNA 3' end factors but this hypothesis was not fully explored. Treating those mRNA 3' end mutants with cordycepin could shed some light on this.
The effect of cordycepin on cleavage factor expression is explored in supplemental Figure 2A. If anything, the increased expression of CFIA and CFIB encoding genes PCF11, RNA14 and HRP1/NAB4 support our model. I.e., that due to an increased transcriptional rate in cordycepin treated cells, the level of the cleavage and polyadenylation machinery fails to meet transcriptional demand. Their expression therefore increased to re-balance the connection between transcription and 3’ end formation.
- A new role for SEN1 in APA for a subset of protein coding was observed. The SEN1 mechanism could be clarified if the authors show that SEN1 is within the subset of convergent genes analyzed, and also if SEN1 expression changes upon cordycepin treatment.
SEN1 expression changes upon cordycepin treatment were shown in supplemental figure 2. There was an increase in SEN1 expression. As mentioned in the text “this might reflect a need to process the elevated level of cryptic unstable transcripts (CUTs) seen following cordycepin treatment (Holbein et al., 2009), or suggest a compensatory mechanism whereby cells under 3’ end stress co- opt an alternative pathway to appropriately cleave mRNA transcripts (Rondon et al., 2009).” Sen1 is also included within Supplemental file 5. as a convergent gene that does not undergo significant APA following cordycepin treatment.
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Reviewer #3 (Public Review):
The manuscript by Turner et al. employs a transcriptome-wide approach to study the effects of mutants of the 3'-end processing machinery and the anti-cancer drug cordycepin (3' deoxyadenosine) on alternative poly(A) site selection in budding yeast to better understand alternative polyadenylation (APA) mechanism(s). In particular, poly(A) test sequencing (PAT-seq), a 3'-end focused deep sequencing technique, is employed to determine cleavage/poly(A) site choice in seven mutants of the core 3'-end processing machinery – three cleavage factor IA (CFIA) mutants (rna14-1, pcf11-2, clp1-pm), one cleavage factor IB (CFIB) mutant (nab4-1), and three cleavage and polyadenylation factor (CPF) mutants (ysh1-13, fip1-1, pap1-1). Six of the 3'-end processing factor mutants exhibit increased distal poly(A) site usage and …
Reviewer #3 (Public Review):
The manuscript by Turner et al. employs a transcriptome-wide approach to study the effects of mutants of the 3'-end processing machinery and the anti-cancer drug cordycepin (3' deoxyadenosine) on alternative poly(A) site selection in budding yeast to better understand alternative polyadenylation (APA) mechanism(s). In particular, poly(A) test sequencing (PAT-seq), a 3'-end focused deep sequencing technique, is employed to determine cleavage/poly(A) site choice in seven mutants of the core 3'-end processing machinery – three cleavage factor IA (CFIA) mutants (rna14-1, pcf11-2, clp1-pm), one cleavage factor IB (CFIB) mutant (nab4-1), and three cleavage and polyadenylation factor (CPF) mutants (ysh1-13, fip1-1, pap1-1). Six of the 3'-end processing factor mutants exhibit increased distal poly(A) site usage and lengthening of 3'-UTRs, with rna14-1 and pcf11-2 showing the greatest effect, but clp1-pm exhibiting little effect. Notably, 3511/7091 genomic annotations (49.5%) have two or more poly(A) sites and 422 genes have significantly changed poly(A) sites in all the 3'-end processing factors mutants except clp1-pm. APA is also examined in 41 genes in a full spectrum of 3'-end processing mutants (22) using a multiplexed poly(A) test (mPAT) method and most of the mutants alter poly(A) site choice, with a predominant shift to distal site usage. In addition, APA analysis of cells treated with cordycepin using PAT-seq indicates that cordycepin alters poly(A) site choice in 1959 genes, with predominant distal cleavage site usage and lengthening of 3'-UTRs. Cordycepin is also shown to increase nucleotide abundance. Interestingly, impairment of transcription elongation, using mycophenolic acid (MPA), which reduces GTP levels, or an RNA polymerase II mutant, rpb1-H1085Y, in cells treated with cordycepin promotes proximal poly(A) site usage and shorter 3'-UTRs, reversing the effects of cordycepin. Finally, comparison of genes altered in APA by cordycepin to a dataset of yeast nucleosome occupancy suggests that 3'-end nucleosome positioning and length of intergenic regions in convergent genes correlates with cordycepin responsiveness. The data presented in the paper suggest a kinetic model for cleavage/poly(A) site selection in yeast that involves a balance between the concentration/availability of the cleavage and polyadenylation machinery and transcription elongation rate.
The strengths of the study include the generation of transcriptome-wide datasets for poly(A) site usage in numerous mutants of evolutionarily conserved, essential cleavage and polyadenylation factors using the PAT-seq method. In addition, the study indicates that almost 50% of the annotated genes in budding yeast exhibit alternative polyadenylation. The study also indicates that impairment of numerous 3'-end processing factors, irrespective of subcomplex, predominantly causes an increase in distal poly(A) site usage and lengthening of 3'-UTRs. Interestingly, the study also suggests that the choice of poly(A) site is regulated by the availability of cleavage and polyadenylation factors and transcription elongation. Finally, the study shows that anticancer drug cordycepin causes transcriptome-wide changes in alternative polyadenylation, predominantly elevating distal poly(A) site usage.
The weaknesses of the study revolve around basing some conclusions solely on the transcriptome-wide data without additional small-scale experiments. In addition, the effects of 3'-end processing mutants and cordycepin on alternative polyadenylation have been examined in two different strain backgrounds, which could impact direct comparisons of the data. The proposed kinetic model for cleavage site choice in yeast seems only to be tested in cells treated with cordycepin.
Overall, the authors achieved their aims of providing greater insight into the mechanism of alternative polyadenylation and its links to transcription and more understanding of the biological effects of cordycepin in cells. At present, most of the conclusions are supported by the results, but some conclusions require additional experiments.
This study will be of enormous interest to the RNA processing field and to the wider community, especially given that alternative polyadenylation regulates so many aspects of mRNA function, the 3'-end processing factors studied are evolutionary conserved, and cordycepin is an anti-cancer agent.
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Reviewer #2 (Public Review):
The authors investigated how alternative polyadenylation (APA) is modulated in yeast using appropriate transcriptomic methodologies.
The authors found that mutants for mRNA 3' end formation factors and cordycepin treatment alter alternative polyadenylation in the same manner, generating transcripts with longer 3'UTRs, due to a switch to distal polyadenylation sites (PAS). Most mutants analyzed cause a PAS switch, in particular mutants for RNA14, PCF11, YSH1, FIP1, NAB4 and PAP1. They also found that MPA and a rpb1 mutant, with a slower transcription elongation rate, reverts the cordycepin effect of distal PAS selection. This implies that in yeast, as in higher organisms, APA is modulated by RNAPII elongation. There is nucleosome depletion in the 3' end of convergent genes that undergo cordycepin-driven APA …
Reviewer #2 (Public Review):
The authors investigated how alternative polyadenylation (APA) is modulated in yeast using appropriate transcriptomic methodologies.
The authors found that mutants for mRNA 3' end formation factors and cordycepin treatment alter alternative polyadenylation in the same manner, generating transcripts with longer 3'UTRs, due to a switch to distal polyadenylation sites (PAS). Most mutants analyzed cause a PAS switch, in particular mutants for RNA14, PCF11, YSH1, FIP1, NAB4 and PAP1. They also found that MPA and a rpb1 mutant, with a slower transcription elongation rate, reverts the cordycepin effect of distal PAS selection. This implies that in yeast, as in higher organisms, APA is modulated by RNAPII elongation. There is nucleosome depletion in the 3' end of convergent genes that undergo cordycepin-driven APA alterations, which is a new finding.
On the basis of their data, the authors propose a kinetic model for APA in yeast that is regulated by the concentration of core mRNA 3' end factors and nucleotide levels, which in turn modulates RNAPII elongation. This integrative model has been already described in higher organisms, but not in yeast, and overall this study covers an impressive body of work that makes an important contribution to the field.
The authors show that cordycepin have the same effect in APA as most of the 3' end factors mutants used, but there is a lack of integration between the two sets of PAS-seq data. The cordycepin APA effect may be due to decreased expression of mRNA 3' end factors but this hypothesis was not fully explored. Treating those mRNA 3' end mutants with cordycepin could shed some light on this.
A new role for SEN1 in APA for a subset of protein coding was observed. The SEN1 mechanism could be clarified if the authors show that SEN1 is within the subset of convergent genes analyzed, and also if SEN1 expression changes upon cordycepin treatment.
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Reviewer #1 (Public Review):
The authors set out to test a variety of factors that could impact poladenylation site (PAS) selection in yeast. To that end, they rigorously tested a collection of temperature-sensitive mutations in polyadenylation machinery components and utilized a custom 3'-end sequencing method to assess PAS selection genome-wide. The most common result associated with polyadenylation machinery dysfunction was global switching to a more distal PAS. Further, the authors test an interesting phenomenon of cordecypin-induced switching to the distal PAS and reveal through metabolomics that enhanced nucleotide biosynthesis may be the root cause. The enhanced nucleotide pools was found to alter elongation rate leading to alterations in PAS choice. Finally, the authors find that convergent genes are influenced by the nucleosome …
Reviewer #1 (Public Review):
The authors set out to test a variety of factors that could impact poladenylation site (PAS) selection in yeast. To that end, they rigorously tested a collection of temperature-sensitive mutations in polyadenylation machinery components and utilized a custom 3'-end sequencing method to assess PAS selection genome-wide. The most common result associated with polyadenylation machinery dysfunction was global switching to a more distal PAS. Further, the authors test an interesting phenomenon of cordecypin-induced switching to the distal PAS and reveal through metabolomics that enhanced nucleotide biosynthesis may be the root cause. The enhanced nucleotide pools was found to alter elongation rate leading to alterations in PAS choice. Finally, the authors find that convergent genes are influenced by the nucleosome landscape to impact APA events.
Overall, this is a rigorous and thorough study that brings together multiple regulatory components that impact PAS selection. The model presented by the authors is supported by their work and provides the field with a clear picture of the complex nature of cleavage and polyadenylation in yeast.
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Evaluation Summary:
This study aims to provide a comprehensive analysis of factors governing polyadenylation site selection in yeast. Overall, the authors reveal that multiple but distinct inputs including polyadenylation machinery integrity, transcription elongation rate, nucleotide availability and chromatin landscape all contribute to controlling cleavage and polyadenylation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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