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Nature Communications Aug 2023Cleavage and polyadenylation (CPA) is responsible for 3' end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets...
Cleavage and polyadenylation (CPA) is responsible for 3' end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets CPSF-73, is the first known CPA inhibitor (CPAi) in mammalian cells. Here we show that JTE-607 perturbs gene expression through both transcriptional readthrough and alternative polyadenylation (APA). Sensitive genes are associated with features similar to those previously identified for PCF11 knockdown, underscoring a unified transcriptomic signature of CPAi. The degree of inhibition of an APA site by JTE-607 correlates with its usage level and, consistently, cells with elevated CPA activities, such as those with induced overexpression of FIP1, display greater transcriptomic disturbances when treated with JTE-607. Moreover, JTE-607 causes S phase crisis and is hence synergistic with inhibitors of DNA damage repair pathways. Together, our data reveal CPA activity and proliferation rate as determinants of CPAi-mediated cell death, raising the possibility of using CPAi as an adjunct therapy to suppress certain cancers.
Topics: Animals; Polyadenylation; RNA Precursors; mRNA Cleavage and Polyadenylation Factors; RNA, Messenger; Mammals; Neoplasms
PubMed: 37528120
DOI: 10.1038/s41467-023-39793-8 -
International Journal of Molecular... Aug 2023Potential oncogene cleavage and polyadenylation specific factor 4 (CPSF4) has been linked to several cancer types. However, little research has been conducted on its...
Potential oncogene cleavage and polyadenylation specific factor 4 (CPSF4) has been linked to several cancer types. However, little research has been conducted on its function in prostate cancer (PCa). In benign, incidental, advanced, and castrate resistant PCa (CRPCa) patient samples, protein expression of CPSF4 was examined on tissue microarray (TMAs) of 353 PCa patients using immunohistochemistry. Using the 'The Cancer Genome Atlas' Prostate Adenocarcinoma (TCGA PRAD) database, significant correlations were found between high CPSF4 expression and high-risk genomic abnormalities such as ERG-fusion, ETV1-fusion, and SPOP mutations. Gene Set Enrichment Analysis (GSEA) of CPSF4 revealed evidence for the increase in biological processes such as cellular proliferation and metastasis. We further examined the function of CPSF4 in vitro and confirmed CPSF4 clinical outcomes and its underlying mechanism. Our findings showed a substantial correlation between Gleason groups and CPSF4 protein expression. In vitro, CPSF4 knockdown reduced cell invasion and migration while also causing G1 and G2 arrest in PC3 cell lines. Our findings demonstrate that CPSF4 may be used as a possible biomarker in PCa and support its oncogenic function in cellular proliferation and metastasis.
Topics: Humans; Male; Cell Cycle; Cell Division; Cell Movement; Hyperplasia; Nuclear Proteins; Polyadenylation; Prostatic Neoplasms; Repressor Proteins
PubMed: 37629142
DOI: 10.3390/ijms241612961 -
Genome Research Oct 2019The cleavage and polyadenylation reaction is a crucial step in transcription termination and pre-mRNA maturation in human cells. Despite extensive research, the encoding...
The cleavage and polyadenylation reaction is a crucial step in transcription termination and pre-mRNA maturation in human cells. Despite extensive research, the encoding of polyadenylation-mediated regulation of gene expression within the DNA sequence is not well understood. Here, we utilized a massively parallel reporter assay to inspect the effect of over 12,000 rationally designed polyadenylation sequences (PASs) on reporter gene expression and cleavage efficiency. We find that the PAS sequence can modulate gene expression by over five orders of magnitude. By using a uniquely designed scanning mutagenesis data set, we gain mechanistic insight into various modes of action by which the cleavage efficiency affects the sensitivity or robustness of the PAS to mutation. Furthermore, we employ motif discovery to identify both known and novel sequence motifs associated with PAS-mediated regulation. By leveraging the large scale of our data, we train a deep learning model for the highly accurate prediction of RNA levels from DNA sequence alone ( = 0.83). Moreover, we devise unique approaches for predicting exact cleavage sites for our reporter constructs and for endogenous transcripts. Taken together, our results expand our understanding of PAS-mediated regulation, and provide an unprecedented resource for analyzing and predicting PAS for regulatory genomics applications.
Topics: 3' Untranslated Regions; Deep Learning; Gene Expression Regulation; Genes, Reporter; Genomics; High-Throughput Nucleotide Sequencing; Humans; Mutagenesis; Polyadenylation; RNA Precursors; RNA, Messenger; Transcription Termination, Genetic
PubMed: 31530582
DOI: 10.1101/gr.247312.118 -
Clinical and Translational Medicine Jul 2022Cell-free messenger RNA (cf-mRNA) and long non-coding RNA (cf-lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease...
BACKGROUND
Cell-free messenger RNA (cf-mRNA) and long non-coding RNA (cf-lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease prediction, diagnosis and prognosis, but the simultaneous characterization of coding and non-coding RNAs in human biofluids remains challenging.
METHODS
Here, we developed polyadenylation ligation-mediated sequencing (PALM-Seq), an RNA sequencing strategy employing treatment of RNA with T4 polynucleotide kinase to generate cell-free RNA (cfRNA) fragments with 5' phosphate and 3' hydroxyl and RNase H to deplete abundant RNAs, achieving simultaneous quantification and characterization of cfRNAs.
RESULTS
Using PALM-Seq, we successfully identified well-known differentially abundant mRNA, lncRNA and microRNA in the blood plasma of pregnant women. We further characterized cfRNAs in blood plasma, saliva, urine, seminal plasma and amniotic fluid and found that the detected numbers of different RNA biotypes varied with body fluids. The profiles of cf-mRNA reflected the function of originated tissues, and immune cells significantly contributed RNA to blood plasma and saliva. Short fragments (<50 nt) of mRNA and lncRNA were major in biofluids, whereas seminal plasma and amniotic fluid tended to retain long RNA. Body fluids showed distinct preferences of pyrimidine at the 3' end and adenine at the 5' end of cf-mRNA and cf-lncRNA, which were correlated with the proportions of short fragments.
CONCLUSION
Together, PALM-Seq enables a simultaneous characterization of cf-mRNA and cf-lncRNA, contributing to elucidating the biology and promoting the application of cfRNAs.
Topics: Cell-Free Nucleic Acids; Female; Humans; MicroRNAs; Polyadenylation; Pregnancy; RNA, Long Noncoding; RNA, Messenger; Sequence Analysis, RNA
PubMed: 35858042
DOI: 10.1002/ctm2.987 -
International Journal of Molecular... Sep 2020Mature mRNA is generated by the 3' end cleavage and polyadenylation of its precursor pre-mRNA. Eukaryotic genes frequently have multiple polyadenylation sites, resulting... (Review)
Review
Mature mRNA is generated by the 3' end cleavage and polyadenylation of its precursor pre-mRNA. Eukaryotic genes frequently have multiple polyadenylation sites, resulting in mRNA isoforms with different 3'-UTR lengths that often encode different C-terminal amino acid sequences. It is well-known that this form of post-transcriptional modification, termed alternative polyadenylation, can affect mRNA stability, localization, translation, and nuclear export. We focus on the alternative polyadenylation of pre-mRNA for vascular endothelial growth factor receptor-1 (VEGFR-1), the receptor for VEGF. VEGFR-1 is a transmembrane protein with a tyrosine kinase in the intracellular region. Secreted forms of VEGFR-1 (sVEGFR-1) are also produced from the same gene by alternative polyadenylation, and sVEGFR-1 has a function opposite to that of VEGFR-1 because it acts as a decoy receptor for VEGF. However, the mechanism that regulates the production of sVEGFR-1 by alternative polyadenylation remains poorly understood. In this review, we introduce and discuss the mechanism of alternative polyadenylation of VEGFR-1 mediated by protein arginine methylation.
Topics: Arginine; Methylation; Polyadenylation; RNA Precursors; RNA Stability; RNA, Messenger; Vascular Endothelial Growth Factor Receptor-1
PubMed: 32899690
DOI: 10.3390/ijms21186460 -
Molecules and Cells Jan 2023In eukaryotic cells, a key RNA processing step to generate mature mRNA is the coupled reaction for cleavage and polyadenylation (CPA) at the 3' end of individual... (Review)
Review
In eukaryotic cells, a key RNA processing step to generate mature mRNA is the coupled reaction for cleavage and polyadenylation (CPA) at the 3' end of individual transcripts. Many transcripts are alternatively polyadenylated (APA) to produce mRNAs with different 3' ends that may either alter protein coding sequence (CDS-APA) or create different lengths of 3'UTR (tandem-APA). As the CPA reaction is intimately associated with transcriptional termination, it has been widely assumed that APA is regulated cotranscriptionally. Isoforms terminated at different regions may have distinct RNA stability under different conditions, thus altering the ratio of APA isoforms. Such differential impacts on different isoforms have been considered as post-transcriptional APA, but strictly speaking, this can only be considered "apparent" APA, as the choice is not made during the CPA reaction. Interestingly, a recent study reveals sequential APA as a new mechanism for post-transcriptional APA. This minireview will focus on this new mechanism to provide insights into various documented regulatory paradigms.
Topics: Polyadenylation; Transcription, Genetic; Protein Isoforms; RNA, Messenger; 3' Untranslated Regions
PubMed: 36697238
DOI: 10.14348/molcells.2023.2176 -
Cellular and Molecular Life Sciences :... Aug 2015MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression by altering the translation efficiency and/or stability of targeted... (Review)
Review
MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression by altering the translation efficiency and/or stability of targeted mRNAs. In vertebrates, more than 50% of all protein-coding RNAs are assumed to be subject to miRNA-mediated control, but current high-throughput methods that reliably measure miRNA-mRNA interactions either require prior knowledge of target mRNAs or elaborate preparation procedures. Consequently, experimentally validated interactions are relatively rare. Furthermore, in silico prediction based on sequence complementarity of miRNAs and their corresponding target sites suffers from extremely high false positive rates. Apparently, sequence complementarity alone is often insufficient to reflect the complex post-transcriptional regulation of mRNAs by miRNAs, which is especially true for animals. Therefore, combined analysis of small non-coding and protein-coding RNAs is indispensable to better understand and predict the complex dynamics of miRNA-regulated gene expression. Single-nucleotide polymorphisms (SNPs) and alternative polyadenylation (APA) can affect miRNA binding of a given transcript from different individuals and tissues, and especially APA is currently emerging as a major factor that contributes to variations in miRNA-mRNA interplay in animals. In this review, we focus on the influence of APA and SNPs on miRNA-mediated gene regulation and discuss the computational approaches that take these mechanisms into account.
Topics: Humans; MicroRNAs; Models, Biological; Polyadenylation; Polymorphism, Single Nucleotide; RNA Processing, Post-Transcriptional; RNA, Messenger
PubMed: 26037721
DOI: 10.1007/s00018-015-1922-2 -
Biomolecules Jun 2015Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3' end is a key step in the... (Review)
Review
Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3' end is a key step in the expression of genetic information. Alternative polyadenylation (APA) serves as an important check point for the regulation of gene expression. Recent studies have shown widespread prevalence of APA in diverse systems. A considerable amount of research has been done in characterizing different subunits of so-called Cleavage and Polyadenylation Specificity Factor (CPSF). In plants, CPSF30, an ortholog of the 30 kD subunit of mammalian CPSF is a key polyadenylation factor. CPSF30 in the model plant Arabidopsis thaliana was reported to possess unique biochemical properties. It was also demonstrated that poly(A) site choice in a vast majority of genes in Arabidopsis are CPSF30 dependent, suggesting a pivotal role of this gene in APA and subsequent regulation of gene expression. There are also indications of this gene being involved in oxidative stress and defense responses and in cellular signaling, suggesting a role of CPSF30 in connecting physiological processes and APA. This review will summarize the biochemical features of CPSF30, its role in regulating APA, and possible links with cellular signaling and stress response modules.
Topics: Arabidopsis Proteins; Cleavage And Polyadenylation Specificity Factor; Gene Expression Regulation, Plant; Plants; Polyadenylation; Signal Transduction; Stress, Physiological
PubMed: 26061761
DOI: 10.3390/biom5021151 -
The New Phytologist Jul 2022Alternative splicing (AS) and alternative polyadenylation (APA) contribute significantly to the regulation of gene expression in higher eukaryotes. Their biological...
Alternative splicing (AS) and alternative polyadenylation (APA) contribute significantly to the regulation of gene expression in higher eukaryotes. Their biological impact in filamentous fungi, however, is largely unknown. Here we combine PacBio Isoform-Sequencing and strand-specific RNA-sequencing of multiple tissues and mutant characterization to reveal the landscape and regulation of AS and APA in Fusarium graminearum. We generated a transcript annotation comprising 51 617 isoforms from 17 189 genes. In total, 4997 and 11 133 genes are alternatively spliced and polyadenylated, respectively. Majority of the AS events alter coding sequences. Unexpectedly, the AS transcripts containing premature-termination codons are not sensitive to nonsense-mediated messenger RNA decay. Unlike in yeasts and animals, distal APA sites have strong signals, but proximal APA isoforms are highly expressed in F. graminearum. The 3'-end processing factors FgRNA15, FgHRP1, and FgFIP1 play roles in promoting proximal APA site usage and intron splicing. A genome-wide increase in intron inclusion and distal APA site usage and downregulation of the spliceosomal and 3'-end processing factors were observed in older and quiescent tissues, indicating intron inclusion and 3'-untranslated region lengthening as novel mechanisms in regulating aging and dormancy in fungi. This study provides new insights into the complexity and regulation of AS and APA in filamentous fungi.
Topics: 3' Untranslated Regions; Alternative Splicing; Animals; Fungi; Polyadenylation; Protein Isoforms
PubMed: 35451076
DOI: 10.1111/nph.18164 -
Molecules and Cells Jan 2023Genomic information stored in the DNA is transcribed to the mRNA and translated to proteins. The 3' untranslated regions (3'UTRs) of the mRNA serve pivotal roles in... (Review)
Review
Genomic information stored in the DNA is transcribed to the mRNA and translated to proteins. The 3' untranslated regions (3'UTRs) of the mRNA serve pivotal roles in posttranscriptional gene expression, regulating mRNA stability, translation, and localization. Similar to DNA mutations producing aberrant proteins, RNA alterations expand the transcriptome landscape and change the cellular proteome. Recent global analyses reveal that many genes express various forms of altered RNAs, including 3'UTR length variants. Alternative polyadenylation and alternative splicing are involved in diversifying 3'UTRs, which could act as a hidden layer of eukaryotic gene expression control. In this review, we summarize the functions and regulations of 3'UTRs and elaborate on the generation and functional consequences of 3'UTR diversity. Given that dynamic 3'UTR length control contributes to phenotypic complexity, dysregulated 3'UTR diversity might be relevant to disease development, including cancers. Thus, 3'UTR diversity in cancer could open exciting new research areas and provide avenues for novel cancer theragnostics.
Topics: Humans; 3' Untranslated Regions; RNA, Messenger; Polyadenylation; Transcriptome; Alternative Splicing
PubMed: 36697237
DOI: 10.14348/molcells.2023.0003