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Next generation sequencing to determine alternative cleavage and polyadenylation events among prostate and prostate cancer cell lines

ABSTRACT: Alternative cleavage and polyadenylation (APA) is a form of transcriptional regulation via shifts in how frequently multiple polyadenylation (polyA) sites within genes are used across biological conditions. APA can result in transcripts with varying length and information content, and has been linked to gene regulation in both cancer and development. To provide a reference for investigating APA in the context of prostate cancer, we performed a genome-wide quantification of polyA site usage using a next generation sequencing technique. By priming the polyA-tails of an RNA library, we mapped cleavage sites and abundances with base-pair resolution across the genomes of one normal prostate and two prostate cancer cell lines. This data can be used to detect APA events among these cell line models, and integration of these data with APA events from clinical prostate cancer samples can lead to candidate genes for future mechanistic studies of the regulation and function of APA in cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE86180 | GEO | 2022/12/29

REPOSITORIES: GEO

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Next generation sequencing to determine polyadenylation events in DICER hypomorph cells

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DNA Methylation Regulates Alternative Polyadenylation via CTCF and the Cohesin Complex

Project description:Alternative cleavage and polyadenylation (APA) is a form of transcriptional regulation via shifts in how frequently multiple polyadenylation (polyA) sites within genes are used across biological conditions. APA can result in transcripts with varying length and information content, and has been linked to gene regulation in both cancer and development. While trans-acting regulatory factors can cause changes in APA, the process of cleavage and polyadenylation is often co-transcriptional. We hypothesized that epigenetic changes, such as DNA methylation, may regulate APA co-transcriptionally. To identify genes where DNA methylation may regulate APA, we performed a genome-wide quantification of polyA site usage using a next generation sequencing technique. By priming the polyA-tails of an RNA library, we mapped cleavage sites and abundances with base-pair resolution across the genomes of two cell lines. The HCT116 cell line is derived from colon cancer, and contains cancer-associated DNA hypermethylation. The DKO cell line is derived from the parental HCT116 line, but has a near-complete depletion of DNA methylation due to mutations in DNA methyltransferase enzymes. Previous studies have used this sytem to detect genes where promoter DNA methylation regulates the gene expression of tumor suppressors. Analogously, detecting APA between HCT116 and DKO reveals candidate genes where DNA methylation may regulate the process of APA. Mechanistic experiments can further interrogate cross-talk between DNA methylation and APA at these loci, and contribute to the understanding of the function of non-promoter differentially methylated regions.

2020-03-18 | GSE86178 | GEO

The transcriptional terminator XRN2 and the RNA binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Project description:Alternative cleavage and polyadenylation (APA) of pre-mRNAs yields multiple transcripts differing in their 3' end and its regulation is often aberrant in human cancers, including prostate cancer (PC). In this study, we have uncovered a novel mechanism of APA regulation which impinges on the functional interaction between the 5'-3' exonuclease XRN2 and the A/U-rich motif RNA binding protein Sam68 in PC cells. XRN2 promotes recruitment of Sam68 to its target transcripts, where it competes with the cleavage and polyadenylation specificity factor (CPSF) for the recognition of the AAUAAA core polyadenylation signal (PAS) motif. In particular, the Sam68/XRN2 complex suppresses the usage of strong canonical PASs at distal ends of genes and, consequently, promotes the usage of suboptimal proximal PASs.

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Regulation of alternative cleavage and polyadenylation by 3’ end processing and splicing factors

Project description:Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage and polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1, and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly influencing C/P events in 5’ introns and U2 impacting those in efficiently spliced introns. Furthermore, PABPN1 regulates expression of transcripts with pAs near the transcription start site, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results indicate that the abundance of different C/P factors and splicing factors plays diverse roles in APA, and is relevant to APA regulation in biological conditions. knockdown experiments of 23 C/P factors, 3 splicing factors and U1D in mouse C2C12 myoblast cells

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Project description:Prostate cancer (PC) onset and progression relies on the androgen receptor (AR) signaling. Anti-androgenic hormonal therapy (HT) is initially efficacious, but most patients evolve to a castration resistant stage (CRPC) for which no cure is currently available. Most proposed mechanisms of acquired resistance to HT focus on the transcriptional activity in CRPC cells. In this study, however, we have uncovered a new role of AR in alternative cleavage and polyadenylation (APA) regulation.

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A compendium of conserved cleavage and polyadenylation events in mammalian genes

Project description:Cleavage and polyadenylation is essential for 3’ end processing of almost all eukaryotic mRNAs. Recent studies have shown widespread alternative cleavage and polyadenylation (APA) events leading to mRNA isoforms with different 3’UTRs and/or coding sequences. Here we present a compendium of conserved cleavage and polyadenylation sites (PASs) in mammalian genes, based on ~1.2 billion 3’ end sequencing reads from over 360 human, mouse and rat samples. We show that ~80% of mammalian mRNA genes contain at least one conserved PAS, and ~50% have conserved APA events. PAS conservation generally reduces promiscuous 3’ end processing, stabling gene expression levels across species. Conservation of APA correlates with gene age, gene expression features, and gene functions. Genes with certain functions, such as cell morphology, cell proliferation, and mRNA metabolism, are particularly enriched with APA events. While tissue-specific genes typically have a low APA rate, brain-specific genes tend to evolve APA. We show enrichment of mRNA destabilizing motifs in alternative 3’UTR sequences, leading to substantial differences in mRNA stability between 3’UTR APA isoforms. Using conserved PASs, we reveal sequence motifs surrounding APA sites and a preference of adenosine at the cleavage site. Mutations of the U-rich motif around the PAS often accompany APA profile changes between species. Analysis of lncRNA PASs indicates a mechanism of PAS fixation involving evolution of A-rich motifs. Taken together, our results present a comprehensive view of PAS evolution in mammals, and a phylogenic understanding of APA functions.

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The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking [PAT-seq]

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2023-12-21 | GSE228929 | GEO

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