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RNA Biology Nov 2017Asymmetric localization of mRNAs is a widespread gene regulatory mechanism that is crucial for many cellular processes. The localization of a transcript involves... (Review)
Review
Asymmetric localization of mRNAs is a widespread gene regulatory mechanism that is crucial for many cellular processes. The localization of a transcript involves multiple steps and requires several protein factors to mediate transport, anchoring and translational repression of the mRNA. Specific recognition of the localizing transcript is a key step that depends on linear or structured localization signals, which are bound by RNA-binding proteins. Genetic studies have identified many components involved in mRNA localization. However, mechanistic aspects of the pathway are still poorly understood. Here we provide an overview of structural studies that contributed to our understanding of the mechanisms underlying mRNA localization, highlighting open questions and future challenges.
Topics: Animals; Cell Cycle Proteins; Drosophila Proteins; Drosophila melanogaster; Eukaryotic Initiation Factor-4E; Gene Expression Regulation; Models, Molecular; Poly(A)-Binding Proteins; Protein Binding; Protein Biosynthesis; Protein Structure, Secondary; RNA Stability; RNA, Messenger
PubMed: 28640665
DOI: 10.1080/15476286.2017.1338231 -
Nucleic Acids Research Jan 2022Poly(A)-binding protein (PABP) is a translation initiation factor that interacts with the poly(A) tail of mRNAs. PABP bound to poly(A) stimulates translation by...
Poly(A)-binding protein (PABP) is a translation initiation factor that interacts with the poly(A) tail of mRNAs. PABP bound to poly(A) stimulates translation by interacting with the eukaryotic initiation factor 4G (eIF4G), which brings the 3' end of an mRNA close to its 5' m7G cap structure through consecutive interactions of the 3'-poly(A)-PABP-eIF4G-eIF4E-5' m7G cap. PABP is a highly abundant translation factor present in considerably larger quantities than mRNA and eIF4G in cells. However, it has not been elucidated how eIF4G, present in limited cellular concentrations, is not sequestered by mRNA-free PABP, present at high cellular concentrations, but associates with PABP complexed with the poly(A) tail of an mRNA. Here, we report that RNA-free PABPs dimerize with a head-to-head type configuration of PABP, which interferes in the interaction between PABP and eIF4G. We identified the domains of PABP responsible for PABP-PABP interaction. Poly(A) RNA was shown to convert the PABP-PABP complex into a poly(A)-PABP complex, with a head-to-tail-type configuration of PABP that facilitates the interaction between PABP and eIF4G. Lastly, we showed that the transition from the PABP dimer to the poly(A)-PABP complex is necessary for the translational activation function.
Topics: Cell Line, Tumor; Eukaryotic Initiation Factor-4G; Humans; Poly(A)-Binding Proteins; Protein Binding; Protein Multimerization; RNA, Messenger
PubMed: 34904669
DOI: 10.1093/nar/gkab1205 -
ELife Jul 2021Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic...
Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.
Topics: 3' Untranslated Regions; DNA Helicases; Kinetics; Poly A; Polyadenylation; RNA Helicases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34232857
DOI: 10.7554/eLife.65331 -
RNA (New York, N.Y.) Sep 2020Polyriboadenylic [poly(rA)] strands of sufficient length form parallel double helices in acidic and/or ammonium-containing conditions. Poly(rA) duplexes in acidic...
Polyriboadenylic [poly(rA)] strands of sufficient length form parallel double helices in acidic and/or ammonium-containing conditions. Poly(rA) duplexes in acidic conditions are held together by A-A base-pairing also involving base interactions with the phosphate backbone. Traditional UV-melting studies of parallel poly(A) duplexes have typically examined homo-duplex formation of a single nucleic acid species in solution. We have adapted a technique utilizing a DNA nanoswitch that detects interaction of two different strands either with similar or differing lengths or modifications. Our method detected parallel duplex formation as a function of length, chemical modifications, and pH, and at a sensitivity that required over 100-fold less concentration of sample than prior UV-melting methods. While parallel polyriboadenylic acid and poly-2'-O-methyl-adenylic acid homo-duplexes formed, we did not detect homo-duplexes of polydeoxyriboadenylic acid strands or poly-locked nucleic acid (LNA)-adenylic strands. Importantly however, a poly-locked nucleic acid (LNA)-adenylic strand, as well as a poly-2'-O-methyl-adenylic strand, formed a hetero-duplex with a polyriboadenylic strand. Overall, our work validates a new tool for studying parallel duplexes and reveals fundamental properties of poly(A) parallel duplex formation. Parallel duplexes may find use in DNA nanotechnology and in molecular biology applications such as a potential poly(rA) tail capture tool as an alternative to traditional oligo(dT) based purification.
Topics: Base Pairing; DNA; Nucleic Acid Conformation; Oligonucleotides; Poly A
PubMed: 32414856
DOI: 10.1261/rna.075408.120 -
Nature Communications Jan 2017Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate...
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.
Topics: Genetic Techniques; Mutation; Poly A; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; RNA Stability
PubMed: 28106166
DOI: 10.1038/ncomms14112 -
Nature Structural & Molecular Biology May 2024Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of...
Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or 'mixed tailing', has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4-NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4-NOT stalls at the 0, -1 and -2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability.
Topics: Humans; Kinetics; RNA Stability; Poly A; RNA, Messenger; Adenosine; Receptors, CCR4; Exoribonucleases; RNA Nucleotidyltransferases
PubMed: 38374449
DOI: 10.1038/s41594-023-01187-1 -
ELife Jul 2021In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail...
In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, strong coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.
Topics: Animals; Gene Expression Regulation; Poly A; Poly(A)-Binding Proteins; RNA, Messenger; Xenopus Proteins; Xenopus laevis
PubMed: 34213414
DOI: 10.7554/eLife.66493 -
Nature Structural & Molecular Biology Dec 2019Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein...
Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome's decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.
Topics: HEK293 Cells; Humans; Models, Molecular; Nucleic Acid Conformation; Peptides; Poly A; Polyadenylation; Polylysine; Protein Biosynthesis; RNA Stability; RNA, Messenger; RNA, Transfer; RNA, Transfer, Amino Acyl; Ribosomes
PubMed: 31768042
DOI: 10.1038/s41594-019-0331-x -
Analytical Chemistry Sep 2023Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this...
Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.
Topics: RNA, Messenger; Chromatography, Liquid; Chromatography, Reverse-Phase; Chromatography, Gel; Quality Control
PubMed: 37696042
DOI: 10.1021/acs.analchem.3c02552 -
Nature Methods Jan 2023RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the...
RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
Topics: Animals; Mice; DNA, Complementary; Transcriptome; Nanopores; Zebrafish; Poly A; Gene Expression Profiling; RNA; RNA, Messenger; Sequence Analysis, RNA
PubMed: 36536091
DOI: 10.1038/s41592-022-01714-w