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Advances in Pharmacology (San Diego,... 2023Canonical histone messenger RNAs (mRNAs) are transcribed during S phase and do not terminate with a poly(A) tail at the 3' end. Instead, the histone mRNAs display a...
Canonical histone messenger RNAs (mRNAs) are transcribed during S phase and do not terminate with a poly(A) tail at the 3' end. Instead, the histone mRNAs display a stem-loop structure at their 3-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. We previously demonstrated that exposure to arsenic, an environmental carcinogen, induces polyadenylation of canonical histone H3.1 mRNA, causing transformation of human cells in vitro. Arsenic decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Similarly, we also reported that nickel and arsenic have similar effects on canonical histone mRNA transcription and translation. Most recently, we further demonstrated that bisphenols' exposure increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. This facilitates the abnormal stability of at least one canonical histone isoform (H3.1), and also increases H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic, nickel and bisphenols exposure may contribute to metal and bisphenol-induced carcinogenesis.
Topics: Humans; Histones; Polyadenylation; Arsenic; Nickel; Carcinogenesis
PubMed: 36858776
DOI: 10.1016/bs.apha.2022.08.003 -
Trends in Genetics : TIG Aug 2019The concept of early termination as an important means of transcriptional control has long been established. Even so, its role in metazoan gene expression is... (Review)
Review
The concept of early termination as an important means of transcriptional control has long been established. Even so, its role in metazoan gene expression is underappreciated. Recent technological advances provide novel insights into premature transcription termination (PTT). This process is frequent, widespread, and can occur close to the transcription start site (TSS), or within the gene body. Stable prematurely terminated transcripts contribute to the transcriptome as instances of alternative polyadenylation (APA). Independently of transcript stability and function, premature termination opposes the formation of full-length transcripts, thereby negatively regulating gene expression, especially of transcriptional regulators. Premature termination can be beneficial or harmful, depending on its context. As a result, multiple factors have evolved to control this process.
Topics: Animals; Bacteria; Codon, Nonsense; Exons; Gene Expression Regulation; Introns; Plants; Polyadenylation; RNA, Messenger; RNA, Untranslated; Transcription Initiation Site; Transcription Termination, Genetic; Transcription, Genetic; Transcriptome; Yeasts
PubMed: 31213387
DOI: 10.1016/j.tig.2019.05.005 -
Scientific Reports Oct 2023The ATP-binding cassette transporter (ABCC1) is associated with poor survival and chemotherapy drug resistance in high grade serous ovarian cancer (HGSOC). The...
The role and impact of alternative polyadenylation and miRNA regulation on the expression of the multidrug resistance-associated protein 1 (MRP-1/ABCC1) in epithelial ovarian cancer.
The ATP-binding cassette transporter (ABCC1) is associated with poor survival and chemotherapy drug resistance in high grade serous ovarian cancer (HGSOC). The mechanisms driving ABCC1 expression are poorly understood. Alternative polyadenylation (APA) can give rise to ABCC1 mRNAs which differ only in the length of their 3'untranslated regions (3'UTRs) in a process known as 3'UTR-APA. Like other ABC transporters, shortening of the 3'UTR of ABCC1 through 3'UTR-APA would eliminate microRNA binding sites found within the longer 3'UTRs, hence eliminating miRNA regulation and altering gene expression. We found that the HGSOC cell lines Caov-3 and Ovcar-3 express higher levels of ABCC1 protein than normal cells. APA of ABCC1 occurs in all three cell lines resulting in mRNAs with both short and long 3'UTRs. In Ovcar-3, mRNAs with shorter 3'UTRs dominate resulting in a six-fold increase in protein expression. We were able to show that miR-185-5p and miR-326 both target the ABCC1 3'UTR. Hence, 3'UTR-APA should be considered as an important regulator of ABCC1 expression in HGSOC. Both HGSOC cell lines are cisplatin resistant, and we used erastin to induce ferroptosis, an alternative form of cell death. We showed that we could induce ferroptosis and sensitize the cisplatin resistant cells to cisplatin by using erastin. Knocking down ABCC1 resulted in decreased cell viability, but did not contribute to erastin induced ferroptosis.
Topics: Humans; Female; MicroRNAs; Polyadenylation; 3' Untranslated Regions; Cisplatin; Carcinoma, Ovarian Epithelial; Apoptosis; Cell Line, Tumor; Ovarian Neoplasms
PubMed: 37838788
DOI: 10.1038/s41598-023-44548-y -
Nature Communications Mar 2021RNA-mediated chromatin silencing is central to genome regulation in many organisms. However, how nascent non-coding transcripts regulate chromatin is poorly understood....
RNA-mediated chromatin silencing is central to genome regulation in many organisms. However, how nascent non-coding transcripts regulate chromatin is poorly understood. Here, through analysis of Arabidopsis FLC, we show that resolution of a nascent-transcript-induced R-loop promotes chromatin silencing. Stabilization of an antisense-induced R-loop at the 3' end of FLC enables an RNA binding protein FCA, with its direct partner FY/WDR33 and other 3'-end processing factors, to polyadenylate the nascent antisense transcript. This clears the R-loop and recruits the chromatin modifiers demethylating H3K4me1. FCA immunoprecipitates with components of the mA writer complex, and mA modification affects dynamics of FCA nuclear condensates, and promotes FLC chromatin silencing. This mechanism also targets other loci in the Arabidopsis genome, and consistent with this fca and fy are hypersensitive to a DNA damage-inducing drug. These results show how modulation of R-loop stability by co-transcriptional RNA processing can trigger chromatin silencing.
Topics: Arabidopsis; Arabidopsis Proteins; Chromatin; Flowers; Gene Expression Regulation, Plant; Gene Silencing; MADS Domain Proteins; Polyadenylation; Protein Binding; R-Loop Structures; RNA Stability; RNA, Plant; RNA-Binding Proteins; Transcription, Genetic; mRNA Cleavage and Polyadenylation Factors
PubMed: 33741984
DOI: 10.1038/s41467-021-22083-6 -
Genome Biology Oct 2021Alternative cleavage and polyadenylation (APA), an RNA processing event, occurs in over 70% of human protein-coding genes. APA results in mRNA transcripts with distinct...
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.
Topics: Benchmarking; Cell Line; Genome, Human; Humans; Polyadenylation; RNA-Seq; Software
PubMed: 34649612
DOI: 10.1186/s13059-021-02502-z -
Methods in Enzymology 2021Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is...
Transcription of mRNAs culminates in RNA cleavage and a coordinated polyadenylation event at the 3' end. In its journey to be translated, the resulting transcript is under constant regulation by cap-binding proteins, miRNAs, and RNA binding proteins, including poly(A) binding proteins (PABPs). The interplay between all these factors determines whether nuclear or cytoplasmic exoribonucleases will gain access to and remove the poly(A) tail, which is so critical to the stability and translation capacity of the mRNA. In this chapter, we present an overview of two of the key features of the mRNA life-cycle: cleavage/polyadenylation and deadenylation, and describe biochemical assays that have been generated to study the activity of each of these enzymatic reactions. Finally, we also provide protocols to investigate mRNA's poly(A) length. The importance of these assays is highlighted by the dynamic and essential role the poly(A) tail length plays in controlling gene expression.
Topics: Exoribonucleases; Poly A; Polyadenylation; RNA, Messenger; RNA-Binding Proteins
PubMed: 34183126
DOI: 10.1016/bs.mie.2021.04.005 -
Genome Biology Nov 20223'-end processing by cleavage and polyadenylation is an important and finely tuned regulatory process during mRNA maturation. Numerous genetic variants are known to...
BACKGROUND
3'-end processing by cleavage and polyadenylation is an important and finely tuned regulatory process during mRNA maturation. Numerous genetic variants are known to cause or contribute to human disorders by disrupting the cis-regulatory code of polyadenylation signals. Yet, due to the complexity of this code, variant interpretation remains challenging.
RESULTS
We introduce a residual neural network model, APARENT2, that can infer 3'-cleavage and polyadenylation from DNA sequence more accurately than any previous model. This model generalizes to the case of alternative polyadenylation (APA) for a variable number of polyadenylation signals. We demonstrate APARENT2's performance on several variant datasets, including functional reporter data and human 3' aQTLs from GTEx. We apply neural network interpretation methods to gain insights into disrupted or protective higher-order features of polyadenylation. We fine-tune APARENT2 on human tissue-resolved transcriptomic data to elucidate tissue-specific variant effects. By combining APARENT2 with models of mRNA stability, we extend aQTL effect size predictions to the entire 3' untranslated region. Finally, we perform in silico saturation mutagenesis of all human polyadenylation signals and compare the predicted effects of [Formula: see text] million variants against gnomAD. While loss-of-function variants were generally selected against, we also find specific clinical conditions linked to gain-of-function mutations. For example, we detect an association between gain-of-function mutations in the 3'-end and autism spectrum disorder. To experimentally validate APARENT2's predictions, we assayed clinically relevant variants in multiple cell lines, including microglia-derived cells.
CONCLUSIONS
A sequence-to-function model based on deep residual learning enables accurate functional interpretation of genetic variants in polyadenylation signals and, when coupled with large human variation databases, elucidates the link between functional 3'-end mutations and human health.
Topics: Humans; Polyadenylation; Autism Spectrum Disorder; RNA Stability; Transcriptome; Genetic Variation; 3' Untranslated Regions
PubMed: 36335397
DOI: 10.1186/s13059-022-02799-4 -
Scientific Reports Apr 2022Colorectal cancer (CRC) is among the most widely spread cancers globally. Aberrant alternative polyadenylation (APA) plays a role in cancer onset and its progression....
Colorectal cancer (CRC) is among the most widely spread cancers globally. Aberrant alternative polyadenylation (APA) plays a role in cancer onset and its progression. Consequently, this study focused on highlighting the role of APA events and signals in the prognosis of patients with CRC. The APA events, RNA sequencing (RNA-seq), somatic mutations, copy number variants (CNVs), and clinical information of the CRC cohort were obtained from The Cancer Genome Atlas (TCGA) database and UCSC (University of California-Santa Cruz) Xena database. The whole set was sorted into two sets: a training set and a test set in a ratio of 7:3. 197 prognosis-related APA events were collected by performing univariate Cox regression signature in patients with CRC. Subsequently, a signature for APA events was established by least absolute shrinkage and selection operator (LASSO) and multivariate Cox analysis. The risk scores were measured for individual patients on the basis of the signature and patients were sorted into two groups; the high-risk group and the low-risk group as per their median risk scores. Kaplan-Meier curves, principal component analysis (PCA), and time-dependent receiver operator characteristic (ROC) curves revealed that the signature was able to predict patient prognosis effectively and further validation was provided in the test set and the whole set. The high-risk and low-risk groups displayed various distributions of mutations and CNVs. Tumor mutation burden (TMB) alone and in combination with the signature predicted the prognosis of CRC patients, but the gene frequencies of TMBs and CNVs did not change in the low- and high-risk groups. Moreover, immunotherapy and chemotherapy treatments showed different responses to PD-1 inhibitors and multiple chemotherapeutic agents in the low and high-risk groups based on the tumor immune dysfunction and exclusion (TIDE) and genomics of drugs sensitivity in cancer (GDSC) databases. This study may help in understanding the potential roles of APA in CRC, and the signature for prognosis-related APA events can work as a potential predictor for survival and treatment in patients with CRC.
Topics: Colorectal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Polyadenylation; Prognosis
PubMed: 35487956
DOI: 10.1038/s41598-022-11089-9 -
BMC Genomics Nov 2019Alternative polyadenylation is commonly examined using cDNA sequencing, which is known to be affected by template-switching artifacts. However, the effects of such...
BACKGROUND
Alternative polyadenylation is commonly examined using cDNA sequencing, which is known to be affected by template-switching artifacts. However, the effects of such template-switching artifacts on alternative polyadenylation are generally disregarded, while alternative polyadenylation artifacts are attributed to internal priming.
RESULTS
Here, we analyzed both long-read cDNA sequencing and direct RNA sequencing data of two organisms, generated by different sequencing platforms. We developed a filtering algorithm which takes into consideration that template-switching can be a source of artifactual polyadenylation when filtering out spurious polyadenylation sites. The algorithm outperformed the conventional internal priming filters based on comparison to direct RNA sequencing data. We also showed that the polyadenylation artifacts arise in cDNA sequencing at consecutive stretches of as few as three adenines. There was no substantial difference between the lengths of poly(A) tails at the artifactual and the true transcriptional end sites even though it is expected that internal priming artifacts have shorter poly(A) tails than genuine polyadenylated reads.
CONCLUSIONS
Our findings suggest that template switching plays an important role in the generation of spurious polyadenylation and support the need for more rigorous filtering of artifactual polyadenylation sites in cDNA data, or that alternative polyadenylation should be annotated using native RNA sequencing.
Topics: Artifacts; DNA, Complementary; Polyadenylation; Sequence Analysis, DNA; Transcription, Genetic
PubMed: 31703623
DOI: 10.1186/s12864-019-6199-7 -
RNA Biology Jan 2024Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well...
Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3' end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3'UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3' untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3'UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.
Topics: Humans; Polyadenylation; Gene Expression Regulation; Epithelial-Mesenchymal Transition; Base Sequence; RNA-Binding Proteins; 3' Untranslated Regions
PubMed: 38112323
DOI: 10.1080/15476286.2023.2294222