Nucleotide-level linkage of transcriptional elongation and polyadenylation
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Giesberg and colleagues provide evidence both in yeast and human cells that fast elongation speeds of RNA polymerases result in a "downstream-shifted" poly(A) profile while the opposite is true for slower speeds of elongating polymerases. GC content of sequences downstream of poly(A) clusters influences the cluster profiles by affecting elongation and thus allowing more time for the 3'-cleavage complex to find the poly(A) site and form the transcript terminus. Although the findings presented in this manuscript are not surprising, they are new and contribute a missing piece to our knowledge of how the transcription machinery determines which poly(A) site to utilize at the end of genes.
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Abstract
Alternative polyadenylation yields many mRNA isoforms whose 3’ termini occur disproportionately in clusters within 3’ untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters – from the last isoform of one cluster to the first isoform of the next – is much less pronounced, even over large distances. GC content in a region 13–30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.
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eLife assessment
Giesberg and colleagues provide evidence both in yeast and human cells that fast elongation speeds of RNA polymerases result in a "downstream-shifted" poly(A) profile while the opposite is true for slower speeds of elongating polymerases. GC content of sequences downstream of poly(A) clusters influences the cluster profiles by affecting elongation and thus allowing more time for the 3'-cleavage complex to find the poly(A) site and form the transcript terminus. Although the findings presented in this manuscript are not surprising, they are new and contribute a missing piece to our knowledge of how the transcription machinery determines which poly(A) site to utilize at the end of genes.
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Reviewer #1 (Public Review):
In this paper from Geisberg et al., the authors examined cleavage site usage in yeast and human cells that express Pol II mutants with faster or slower elongation rates. The authors focused on two types of alternative cleavage sites, one being multiple sites clustered within a short range (called within cluster sites) and the other being multiple sites that are distant from one another (called between cluster sites). The authors identified polarity site usage of within a cluster in cells expressing mutant Pol II. Slower Pol II leads to more proximal site usage whereas faster Pol II mutant to distal site usage. In contrast, these trends were not observed with sites between clusters. The authors made four conclusions based on these observations. Overall this is a very well-written paper revealing some …
Reviewer #1 (Public Review):
In this paper from Geisberg et al., the authors examined cleavage site usage in yeast and human cells that express Pol II mutants with faster or slower elongation rates. The authors focused on two types of alternative cleavage sites, one being multiple sites clustered within a short range (called within cluster sites) and the other being multiple sites that are distant from one another (called between cluster sites). The authors identified polarity site usage of within a cluster in cells expressing mutant Pol II. Slower Pol II leads to more proximal site usage whereas faster Pol II mutant to distal site usage. In contrast, these trends were not observed with sites between clusters. The authors made four conclusions based on these observations. Overall this is a very well-written paper revealing some fundamental features associated with cleavage site choice. Most conclusions are supported by their data. I do, however, have some concerns about their between-cluster analysis.
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Reviewer #2 (Public Review):
In this manuscript, Geisberg et al. present profiles of poly(A) site usage in cells with RNA Polymerase II variants transcribing at different elongation rates. It was known that transcript termination sites in cell populations occur as clusters at the 3'UTR of genes but how the choice of poly(A) site may be influenced by transcription elongation speed was not known.
The strength of their study involves using 3' READ technologies and data analyses that they have previously developed. A weakness of the study is that since the speed of elongation of Pol II is central for the data obtained and conclusions drawn, it would be important to actually measure the speed of elongation by the slow, fast, and wt Pol II used in these studies within the genes analyzed. Although the findings presented in this manuscript are …
Reviewer #2 (Public Review):
In this manuscript, Geisberg et al. present profiles of poly(A) site usage in cells with RNA Polymerase II variants transcribing at different elongation rates. It was known that transcript termination sites in cell populations occur as clusters at the 3'UTR of genes but how the choice of poly(A) site may be influenced by transcription elongation speed was not known.
The strength of their study involves using 3' READ technologies and data analyses that they have previously developed. A weakness of the study is that since the speed of elongation of Pol II is central for the data obtained and conclusions drawn, it would be important to actually measure the speed of elongation by the slow, fast, and wt Pol II used in these studies within the genes analyzed. Although the findings presented in this manuscript are not surprising, they are novel and contribute a missing piece of how the transcription machinery determines which poly(A) site to utilize at the end of genes.
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Reviewer #3 (Public Review):
In this study, the authors explore an under-studied but widely observed phenomenon that polyA site selection often occurs in clusters leading to the excepted interpretation that cleavage and polyadenylation are imprecise. Here, the authors use 3READS to map polyA sites in yeast and human cells to define trends in intra-cluster polyA site usage as it relates to RNAPII speed. They observe clear trends in cleavage events that correlate with either increased or decreased RNAPII elongation rate and make a further identification that downstream GC content also correlates with these trends. The potential impact of this work is to explain the imprecise behavior of cleavage and Polyadenylation as a component of local elongation rates that are influenced by nucleotide content.
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