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Nature Reviews. Molecular Cell Biology Feb 2022In eukaryotes, poly(A) tails are present on almost every mRNA. Early experiments led to the hypothesis that poly(A) tails and the cytoplasmic polyadenylate-binding... (Review)
Review
In eukaryotes, poly(A) tails are present on almost every mRNA. Early experiments led to the hypothesis that poly(A) tails and the cytoplasmic polyadenylate-binding protein (PABPC) promote translation and prevent mRNA degradation, but the details remained unclear. More recent data suggest that the role of poly(A) tails is much more complex: poly(A)-binding protein can stimulate poly(A) tail removal (deadenylation) and the poly(A) tails of stable, highly translated mRNAs at steady state are much shorter than expected. Furthermore, the rate of translation elongation affects deadenylation. Consequently, the interplay between poly(A) tails, PABPC, translation and mRNA decay has a major role in gene regulation. In this Review, we discuss recent work that is revolutionizing our understanding of the roles of poly(A) tails in the cytoplasm. Specifically, we discuss the roles of poly(A) tails in translation and control of mRNA stability and how poly(A) tails are removed by exonucleases (deadenylases), including CCR4-NOT and PAN2-PAN3. We also discuss how deadenylation rate is determined, the integration of deadenylation with other cellular processes and the function of PABPC. We conclude with an outlook for the future of research in this field.
Topics: Animals; Eukaryota; Gene Expression Regulation; Humans; Poly A; Protein Biosynthesis; RNA Stability; RNA, Messenger
PubMed: 34594027
DOI: 10.1038/s41580-021-00417-y -
Cell May 2020SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown....
SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3' tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.
Topics: Animals; Betacoronavirus; Chlorocebus aethiops; Epigenesis, Genetic; RNA Processing, Post-Transcriptional; RNA, Viral; SARS-CoV-2; Sequence Analysis, RNA; Transcriptome; Vero Cells
PubMed: 32330414
DOI: 10.1016/j.cell.2020.04.011 -
Nature Methods Dec 2019High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose...
High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.
Topics: Cells, Cultured; Humans; Nanopore Sequencing; Poly A; Sequence Analysis, RNA; Transcriptome
PubMed: 31740818
DOI: 10.1038/s41592-019-0617-2 -
Nature Chemical Biology Jun 2021Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development...
Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.
Topics: Animals; Cyclin-Dependent Kinases; DNA Damage; Drug Design; Drug Discovery; Drug Resistance; Humans; Poly A; Poly Adenosine Diphosphate Ribose; Protein Kinase Inhibitors; Proteomics
PubMed: 33753926
DOI: 10.1038/s41589-021-00765-y -
Molecular Cell Sep 2022Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with...
Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.
Topics: 3' Untranslated Regions; Humans; Poly A; Polyadenylation; RNA, Messenger; Sp1 Transcription Factor; Zinc
PubMed: 35914531
DOI: 10.1016/j.molcel.2022.06.031 -
Annual Review of Biochemistry Jun 2023Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave... (Review)
Review
Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3'-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.
Topics: Humans; RNA, Messenger; RNA Precursors; mRNA Cleavage and Polyadenylation Factors; Saccharomyces cerevisiae; Gene Expression
PubMed: 37001138
DOI: 10.1146/annurev-biochem-052521-012445 -
Cell Aug 2022Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit...
Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome.
Topics: Animals; Eukaryotic Initiation Factor-4G; Eukaryotic Initiation Factors; Poly(A)-Binding Proteins; Protein Biosynthesis; Purines; RNA, Messenger
PubMed: 35907403
DOI: 10.1016/j.cell.2022.06.037 -
Cell Sep 2021Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in...
Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.
Topics: 3' Untranslated Regions; Base Sequence; Cell Nucleus; DNA-Binding Proteins; HEK293 Cells; HeLa Cells; Homeostasis; Humans; Mutation; Nucleotide Motifs; Phase Transition; Point Mutation; Poly A; Protein Binding; Protein Multimerization; RNA; RNA, Messenger; RNA-Binding Proteins; Sequence Deletion
PubMed: 34380047
DOI: 10.1016/j.cell.2021.07.018 -
Cancer Letters Feb 2023Bladder cancer (BCa), characterized by high invasion, metastasis, recurrence, and chemoresistance, is one of the most prevalent urologic malignant tumors. Recent studies...
Bladder cancer (BCa), characterized by high invasion, metastasis, recurrence, and chemoresistance, is one of the most prevalent urologic malignant tumors. Recent studies have highlighted the potential impact of the circRNAs-protein complex in tumorigenesis. However, the mechanisms by which the circRNAs-protein complex regulates BCa metastasis and chemoresistance remain elusive. Herein, we identified an upregulated circRNA, circPTK2, which could regulate SETDB1 expression by analyzing the transcriptome by RNA-sequencing. Importantly, using circRNA pulldown assay and RNA-binding protein immunoprecipitation, we identified PABPC1 as a robust novel interacting protein of circPTK2. Mechanistically, circPTK2 could bind to PABPC1 and enhance its ability to stabilize SETDB1 mRNA, thereby specifically promoting SETDB1 expression and facilitating SETDB1-mediated epithelial-mesenchymal transition (EMT). Functionally, overexpression of the circPTK2-SETDB1 axis markedly promoted migration, invasion, and gemcitabine resistance in vitro and enhanced lymph node metastasis in vivo. Collectively, our findings clarified a hitherto unexplored mechanism of the circPTK2/PABPC1/SETDB1 axis in EMT-mediated tumor metastasis and gemcitabine resistance in BCa.
Topics: Humans; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Gemcitabine; Gene Expression Regulation, Neoplastic; Histone-Lysine N-Methyltransferase; MicroRNAs; RNA, Circular; Urinary Bladder Neoplasms; Poly(A)-Binding Protein I
PubMed: 36436682
DOI: 10.1016/j.canlet.2022.216023 -
ELife Jul 2021Longer poly(A) tails improve translation in early development, but not in mature cells that have higher levels of the protein PABPC.
Longer poly(A) tails improve translation in early development, but not in mature cells that have higher levels of the protein PABPC.
Topics: Oocytes; RNA, Messenger
PubMed: 34213415
DOI: 10.7554/eLife.70757