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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 -
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 -
Nature Mar 2017Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage...
Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (mA) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A) transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of mA in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA mA modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which mA RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.
Topics: Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Animals; Biocatalysis; Cell Line; Cell Survival; DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Methylation; Methyltransferases; Mice; Poly A; RNA; S Phase; Ultraviolet Rays
PubMed: 28297716
DOI: 10.1038/nature21671 -
Cell Mar 2017In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form...
In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.
Topics: Amino Acid Sequence; Cytoplasmic Granules; Hot Temperature; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Intrinsically Disordered Proteins; Mutagenesis; Poly(A)-Binding Proteins; Proline; Protein Domains; Ribonucleases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Stress, Physiological
PubMed: 28283059
DOI: 10.1016/j.cell.2017.02.027 -
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 -
RNA (New York, N.Y.) Apr 2019Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular...
Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in , resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in without any negative effect on mRNA half-life and protein expression. This effect was independent of the coding sequence. A segmented poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40-60 adenosines, separated by a spacer element of different length. Furthermore, reducing the spacer length between the poly(A) segments resulted in higher translation efficiencies compared to homogeneous poly(A) tail and reduced recombination (depending upon the choice of spacer nucleotide). Our results demonstrate the superior potential of segmented poly(A) tails compared to the conventionally used homogeneous poly(A) tails with respect to recombination of the plasmids and the resulting mRNA performance (half-life and translational efficiency).
Topics: A549 Cells; Animals; Base Sequence; Escherichia coli; Genetic Engineering; HEK293 Cells; Half-Life; Humans; Plasmids; Poly A; Protein Biosynthesis; RNA, Messenger; Recombination, Genetic; Transfection
PubMed: 30647100
DOI: 10.1261/rna.069286.118 -
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 -
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