Benchmarking sequencing methods and tools that facilitate the study of alternative polyadenylation

Background Alternative cleavage and polyadenylation (APA), an RNA processing event, occurs in over 70% of human protein-coding genes. APA results in mRNA transcripts with distinct 3′ ends. Most APA occurs within 3′ UTRs, which harbor regulatory elements that can impact mRNA stability, translation, and localization. Results APA can be profiled using a number of established computational tools that infer polyadenylation sites from standard, short-read RNA-seq datasets. Here, we benchmarked a number of such tools—TAPAS, QAPA, DaPars2, GETUTR, and APATrap— against 3′-Seq, a specialized RNA-seq protocol that enriches for reads at the 3′ ends of genes, and Iso-Seq, a Pacific Biosciences (PacBio) single-molecule full-length RNA-seq method in their ability to identify polyadenylation sites and quantify polyadenylation site usage. We demonstrate that 3′-Seq and Iso-Seq are able to identify and quantify the usage of polyadenylation sites more reliably than computational tools that take short-read RNA-seq as input. However, we find that running one such tool, QAPA, with a set of polyadenylation site annotations derived from small quantities of 3′-Seq or Iso-Seq can reliably quantify variation in APA across conditions, such asacross genotypes, as demonstrated by the successful mapping of alternative polyadenylation quantitative trait loci (apaQTL). Conclusions We envisage that our analyses will shed light on the advantages of studying APA with more specialized sequencing protocols, such as 3′-Seq or Iso-Seq, and the limitations of studying APA with short-read RNA-seq. We provide a computational pipeline to aid in the identification of polyadenylation sites and quantification of polyadenylation site usages using Iso-Seq data as input.

Androgen signaling connects short isoform production to breakpoint formation at Ewing sarcoma breakpoint region 1

Ewing sarcoma breakpoint region 1 (EWSR1) encodes a multifunctional protein that can cooperate with the transcription factor ERG to promote prostate cancer. The EWSR1 gene is also commonly involved in oncogenic gene rearrangements in Ewing sarcoma. Despite the cancer relevance of EWSR1, its regulation is poorly understood. Here we find that in prostate cancer, androgen signaling upregulates a 5′ EWSR1 isoform by promoting usage of an intronic polyadenylation site. This isoform encodes a cytoplasmic protein that can strongly promote cell migration and clonogenic growth. Deletion of an Androgen Receptor (AR) binding site near the 5′ EWSR1 polyadenylation site abolished androgen-dependent upregulation. This polyadenylation site is also near the Ewing sarcoma breakpoint hotspot, and androgen signaling promoted R-loop and breakpoint formation. RNase H overexpression reduced breakage and 5′ EWSR1 isoform expression suggesting an R-loop dependent mechanism. These data suggest that androgen signaling can promote R-loops internal to the EWSR1 gene leading to either early transcription termination, or breakpoint formation.

Streamlining differential exon and 3’ UTR usage with diffUTR

BackgroundDespite the importance of alternative poly-adenylation and 3’ UTR length for a variety of biological phenomena, there are limited means of detecting UTR changes from standard transcriptomic data.ResultsWe present the diffUTR Bioconductor package which streamlines and improves upon differential exon usage (DEU) analyses, and leverages existing DEU tools and alternative polyadenylation site databases to enable differential 3’ UTR usage analysis. We demonstrate the diffUTR features and show that it is more flexible and more accurate than state-of-the-art alternatives, both in simulations and in real data.ConclusionsdiffUTR enables differential 3’ UTR analysis and more generally facilitates DEU and the exploration of their results.

Enhancers regulate polyadenylation site cleavage and control 3’UTR isoform expression

SummaryEnhancers are DNA elements that increase gene expression. mRNA production is determined by transcript production and polyadenylation site (PAS) cleavage activity. We established an assay to measure enhancer-dependent PAS cleavage activity in human cells because PAS cleavage may control alternative 3’UTR isoform expression. We found that enhancers are widespread regulators of PAS cleavage and consistently increase cleavage of proximal and weak PAS. Half of tested transcription factors exclusively regulated PAS cleavage without affecting transcript production, whereas co-activators changed both parameters. Deletion of an endogenous enhancer of PTEN did not change gene-level mRNA or protein abundance but affected expression of alternative mRNA transcripts, thus preventing 3’UTR shortening. Our data reveal that in addition to controlling transcript production, enhancers also regulate PAS cleavage, thus changing 3’UTR isoform usage and protein activity, as PTEN proteins translated from the alternative 3’UTR isoforms differ in intrinsic lipid phosphatase activity despite having identical amino acid sequences.

Androgen signaling connects short isoform production to breakpoint formation at Ewing sarcoma breakpoint region 1 via an R-loop-dependent mechanism

SummaryEwing sarcoma breakpoint region 1 (EWSR1) encodes a multifunctional protein that can cooperate with the transcription factor ERG to promote prostate cancer. The EWSR1 gene is also commonly involved in oncogenic gene rearrangements in Ewing sarcoma. Despite the cancer relevance of EWSR1, its regulation is poorly understood. Here we find that in prostate cancer, androgen signaling upregulates a 5’ EWSR1 isoform by promoting usage of an intronic polyadenylation site. This isoform encodes a cytoplasmic protein that can strongly promote cell migration and clonogenic growth. Deletion of an Androgen Receptor (AR) binding site near the 5’ EWSR1 polyadenylation site abolished androgen-dependent upregulation. This polyadenylation site is also near the Ewing sarcoma breakpoint hotspot, and androgen signaling promoted R-loop and breakpoint formation. RNase H overexpression reduced breakage and 5’ EWSR1 isoform expression suggesting an R-loop dependent mechanism. These data suggest that androgen signaling can promote R-loops internal to the EWSR1 gene leading to early transcription termination and breakpoint formation.