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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 -
Cold Spring Harbor Protocols Oct 2019This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and...
This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and heated briefly to 65°C-70°C, followed by immediate cooling on ice to disrupt secondary structures. The RNA is subsequently annealed to the oligo(dT)-magnetic beads at room temperature; the high-salt binding buffer stabilizes the poly(A)-oligo(dT) complexes. A high-salt washing buffer is then used to wash away unbound RNAs while retaining oligo(dT)-bound poly(A) mRNAs. To elute the poly(A) mRNAs from the beads, a low-salt buffer (or water) is used to destabilize the poly(A)-oligo(dT) complexes. Alternatively, poly(A) mRNAs can be retained on the beads for downstream applications (e.g., solid-phase cDNA synthesis).
Topics: Cellulose; Chromatography, Affinity; DNA, Complementary; Magnetic Phenomena; Magnetics; Microspheres; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Poly A; RNA; RNA, Messenger
PubMed: 31575797
DOI: 10.1101/pdb.prot101733 -
Analytical and Bioanalytical Chemistry Feb 2018The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were...
The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.
Topics: Animals; Cell Line; Chromatography, High Pressure Liquid; Mass Spectrometry; Mice; Plasmids; Poly A; Polyadenylation; RNA, Messenger; Transcription, Genetic
PubMed: 29313076
DOI: 10.1007/s00216-017-0840-6 -
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 Structural & Molecular Biology Jan 2022Analogous to alternative splicing, alternative polyadenylation (APA) has long been thought to occur independently at proximal and distal polyA sites. Using...
Analogous to alternative splicing, alternative polyadenylation (APA) has long been thought to occur independently at proximal and distal polyA sites. Using fractionation-seq, we unexpectedly identified several hundred APA genes in human cells whose distal polyA isoforms are retained in chromatin/nuclear matrix and whose proximal polyA isoforms are released into the cytoplasm. Global metabolic PAS-seq and Nanopore long-read RNA-sequencing provide further evidence that the strong distal polyA sites are processed first and the resulting transcripts are subsequently anchored in chromatin/nuclear matrix to serve as precursors for further processing at proximal polyA sites. Inserting an autocleavable ribozyme between the proximal and distal polyA sites, coupled with a Cleave-seq approach that we describe here, confirms that the distal polyA isoform is indeed the precursor to the proximal polyA isoform. Therefore, unlike alternative splicing, APA sites are recognized independently, and in many cases, in a sequential manner. This provides a versatile strategy to regulate gene expression in mammalian cells.
Topics: 3' Untranslated Regions; Alternative Splicing; HeLa Cells; Humans; Introns; Nuclear Matrix; Poly A; Polyadenylation; RNA; RNA Polymerase II
PubMed: 35013598
DOI: 10.1038/s41594-021-00709-z -
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