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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 2023The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific...
The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.
Topics: Humans; Alternative Splicing; Polyadenylation; Promoter Regions, Genetic; RNA Isoforms; RNA, Messenger; Transcription Initiation Site
PubMed: 37178687
DOI: 10.1016/j.cell.2023.04.012 -
Nature Reviews. Molecular Cell Biology Dec 2022Alternative cleavage and polyadenylation (APA) is a widespread mechanism to generate mRNA isoforms with alternative 3' untranslated regions (UTRs). The expression of... (Review)
Review
Alternative cleavage and polyadenylation (APA) is a widespread mechanism to generate mRNA isoforms with alternative 3' untranslated regions (UTRs). The expression of alternative 3' UTR isoforms is highly cell type specific and is further controlled in a gene-specific manner by environmental cues. In this Review, we discuss how the dynamic, fine-grained regulation of APA is accomplished by several mechanisms, including cis-regulatory elements in RNA and DNA and factors that control transcription, pre-mRNA cleavage and post-transcriptional processes. Furthermore, signalling pathways modulate the activity of these factors and integrate APA into gene regulatory programmes. Dysregulation of APA can reprogramme the outcome of signalling pathways and thus can control cellular responses to environmental changes. In addition to the regulation of protein abundance, APA has emerged as a major regulator of mRNA localization and the spatial organization of protein synthesis. This role enables the regulation of protein function through the addition of post-translational modifications or the formation of protein-protein interactions. We further discuss recent transformative advances in single-cell RNA sequencing and CRISPR-Cas technologies, which enable the mapping and functional characterization of alternative 3' UTRs in any biological context. Finally, we discuss new APA-based RNA therapeutics, including compounds that target APA in cancer and therapeutic genome editing of degenerative diseases.
Topics: Polyadenylation; 3' Untranslated Regions; RNA, Messenger; Gene Expression Regulation; Protein Biosynthesis
PubMed: 35798852
DOI: 10.1038/s41580-022-00507-5 -
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 -
Cell Reports Oct 2023Cancer cells usually exhibit shortened 3' untranslated regions (UTRs) due to alternative polyadenylation (APA) to promote cell proliferation and migration. Upregulated...
Cancer cells usually exhibit shortened 3' untranslated regions (UTRs) due to alternative polyadenylation (APA) to promote cell proliferation and migration. Upregulated CPSF6 leads to a systematic prolongation of 3' UTRs, but CPSF6 expression in tumors is typically higher than that in healthy tissues. This contradictory observation suggests that it is necessary to investigate the underlying mechanism by which CPSF6 regulates APA switching in cancer. Here, we find that CPSF6 can undergo liquid-liquid phase separation (LLPS), and elevated LLPS is associated with the preferential usage of the distal poly(A) sites. CLK2, a kinase upregulated in cancer cells, destructs CPSF6 LLPS by phosphorylating its arginine/serine-like domain. The reduction of CPSF6 LLPS can lead to a shortened 3' UTR of cell-cycle-related genes and accelerate cell proliferation. These results suggest that CPSF6 LLPS, rather than its expression level, may be responsible for APA regulation in cancer cells.
Topics: 3' Untranslated Regions; Cell Proliferation; Gene Expression Regulation; mRNA Cleavage and Polyadenylation Factors; Neoplasms; Polyadenylation; Humans; Cell Line, Tumor
PubMed: 37777964
DOI: 10.1016/j.celrep.2023.113197 -
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 -
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 Methods Aug 2023Capture array-based spatial transcriptomics methods have been widely used to resolve gene expression in tissues; however, their spatial resolution is limited by the...
Capture array-based spatial transcriptomics methods have been widely used to resolve gene expression in tissues; however, their spatial resolution is limited by the density of the array. Here we present expansion spatial transcriptomics to overcome this limitation by clearing and expanding tissue prior to capturing the entire polyadenylated transcriptome with an enhanced protocol. This approach enables us to achieve higher spatial resolution while retaining high library quality, which we demonstrate using mouse brain samples.
Topics: Animals; Mice; Gene Expression Profiling; Gene Library; Poly A; Transcriptome
PubMed: 37349575
DOI: 10.1038/s41592-023-01911-1 -
Science Advances Oct 2022Growing oocytes store a large amount of maternal mRNA to support the subsequent "maternal-zygotic transition" process. At present, it is not clear how the growing...
Growing oocytes store a large amount of maternal mRNA to support the subsequent "maternal-zygotic transition" process. At present, it is not clear how the growing oocytes store and process the newly transcribed mRNA under physiological conditions. In this study, we report non-membrane-bound compartments, nuclear poly(A) domains (NPADs), as the hub for newly transcribed mRNA, in developing mouse oocytes. The RNA binding protein PABPN1 promotes the formation of NPAD through its N-terminal disordered domain and RNA-recognized motif by means of liquid phase separation. -null growing oocytes cannot form NPAD normally in vivo and have defects in stability of oocyte growing-related transcripts and formation of long 3' untranslated region isoform transcripts. Ultimately, mice are completely sterile with primary ovarian insufficiency. These results demonstrate that NPAD formed by the phase separation properties of PABPN1-mRNA are the hub of the newly transcribed mRNA and essential for the development of oocytes and female reproduction.
Topics: Animals; Female; Mice; Cell Nucleus; Oocytes; Poly A; RNA, Messenger; RNA-Binding Proteins
PubMed: 36306357
DOI: 10.1126/sciadv.abn9016 -
Current Opinion in Structural Biology Dec 2019The polyadenosine (poly(A)) tail found on the 3'-end of almost all eukaryotic mRNAs is important for mRNA stability and regulation of translation. mRNA 3'-end processing... (Review)
Review
The polyadenosine (poly(A)) tail found on the 3'-end of almost all eukaryotic mRNAs is important for mRNA stability and regulation of translation. mRNA 3'-end processing occurs co-transcriptionally and involves more than 20 proteins to specifically recognize the polyadenylation site, cleave the pre-mRNA, add a poly(A) tail, and trigger transcription termination. The polyadenylation site (PAS) defines the end of the 3'-untranslated region (3'-UTR) and, therefore, selection of the cleavage site is a critical event in regulating gene expression. Integrated structural biology approaches including biochemical reconstitution of multi-subunit complexes, cross-linking mass spectrometry, and structural analyses by X- ray crystallography and single-particle electron cryo-microscopy (cryoEM) have enabled recent progress in understanding the molecular mechanisms of the mRNA 3'-end processing machinery. Here, we describe new molecular insights into pre-mRNA recognition, cleavage and polyadenylation.
Topics: 3' Untranslated Regions; Binding Sites; Eukaryota; Models, Molecular; Polyadenylation; Protein Binding; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA-Binding Proteins; Structure-Activity Relationship
PubMed: 31499460
DOI: 10.1016/j.sbi.2019.08.001