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Biosensors & Bioelectronics Oct 2022MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs...
MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease.
Topics: Biosensing Techniques; CRISPR-Cas Systems; Humans; MicroRNAs; Nucleic Acid Amplification Techniques; Poly A
PubMed: 35797934
DOI: 10.1016/j.bios.2022.114497 -
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 -
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 -
Science (New York, N.Y.) Feb 2021Polyadenylate [poly(A)] tail addition to the 3' end of a wide range of RNAs is a highly conserved modification that plays a central role in cellular RNA function....
Polyadenylate [poly(A)] tail addition to the 3' end of a wide range of RNAs is a highly conserved modification that plays a central role in cellular RNA function. Elements for nuclear expression (ENEs) are cis-acting RNA elements that stabilize poly(A) tails by sequestering them in RNA triplex structures. A crystal structure of a double ENE from a rice hAT transposon messenger RNA complexed with poly(A) at a resolution of 2.89 angstroms reveals multiple modes of interaction with poly(A), including major-groove triple helices, extended minor-groove interactions with RNA double helices, a quintuple-base motif that transitions poly(A) from minor-groove associations to major-groove triple helices, and a poly(A) 3'-end binding pocket. Our findings both expand the repertoire of motifs involved in long-range RNA interactions and provide insights into how polyadenylation can protect an RNA's extreme 3' end.
Topics: Crystallization; Nucleic Acid Conformation; Oryza; Poly A; Polyadenylation; RNA Stability; RNA, Messenger
PubMed: 33414189
DOI: 10.1126/science.abe6523 -
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 -
Methods in Molecular Biology (Clifton,... 2024Poly(A) tails are added to most eukaryotic mRNA and have essential regulatory functions. However, due to its homopolymeric nature, the sequence information in poly(A)...
Poly(A) tails are added to most eukaryotic mRNA and have essential regulatory functions. However, due to its homopolymeric nature, the sequence information in poly(A) tails is challenging to obtain in transcriptome measurement studies. In this chapter, we describe the detailed procedures of poly(A) inclusive full-length RNA isoform-sequencing (PAIso-seq), a method that can measure transcriptome-wide poly(A) tails from as low as nanogram level of total RNA based on the PacBio HiFi sequencing platform. The accurate length and base composition of poly(A) tails can be obtained along with the full-length cDNA.
Topics: Transcriptome; Sequence Analysis, RNA; Polyadenylation; DNA, Complementary; RNA, Messenger; Poly A; High-Throughput Nucleotide Sequencing
PubMed: 37824073
DOI: 10.1007/978-1-0716-3481-3_13 -
Nature Structural & Molecular Biology Jun 2019The 3' poly(A) tail of messenger RNA is fundamental to regulating eukaryotic gene expression. Shortening of the poly(A) tail, termed deadenylation, reduces transcript...
The 3' poly(A) tail of messenger RNA is fundamental to regulating eukaryotic gene expression. Shortening of the poly(A) tail, termed deadenylation, reduces transcript stability and inhibits translation. Nonetheless, the mechanism for poly(A) recognition by the conserved deadenylase complexes Pan2-Pan3 and Ccr4-Not is poorly understood. Here we provide a model for poly(A) RNA recognition by two DEDD-family deadenylase enzymes, Pan2 and the Ccr4-Not nuclease Caf1. Crystal structures of Saccharomyces cerevisiae Pan2 in complex with RNA show that, surprisingly, Pan2 does not form canonical base-specific contacts. Instead, it recognizes the intrinsic stacked, helical conformation of poly(A) RNA. Using a fully reconstituted biochemical system, we show that disruption of this structure-for example, by incorporation of guanosine into poly(A)-inhibits deadenylation by both Pan2 and Caf1. Together, these data establish a paradigm for specific recognition of the conformation of poly(A) RNA by proteins that regulate gene expression.
Topics: Crystallography, X-Ray; Exoribonucleases; Models, Molecular; Multiprotein Complexes; Poly A; RNA, Messenger; Ribonucleases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31110294
DOI: 10.1038/s41594-019-0227-9 -
International Journal of Molecular... Oct 2021Mitochondria have their own double-stranded DNA genomes and systems to regulate transcription, mRNA processing, and translation. These systems differ from those... (Review)
Review
Mitochondria have their own double-stranded DNA genomes and systems to regulate transcription, mRNA processing, and translation. These systems differ from those operating in the host cell, and among eukaryotes. In recent decades, studies have revealed several plant-specific features of mitochondrial gene regulation. The polyadenylation status of mRNA is critical for its stability and translation in mitochondria. In this short review, I focus on recent advances in understanding the mechanisms regulating mRNA polyadenylation in plant mitochondria, including the role of poly(A)-specific ribonuclease-like proteins (PARNs). Accumulating evidence suggests that plant mitochondria have unique regulatory systems for mRNA poly(A) status and that PARNs play pivotal roles in these systems.
Topics: Embryophyta; Exoribonucleases; Gene Expression Regulation, Plant; Mitochondria; Poly A; Polyadenylation; RNA Stability; RNA, Messenger; RNA, Mitochondrial
PubMed: 34639116
DOI: 10.3390/ijms221910776 -
Journal of Inorganic Biochemistry Jul 2022To comprehend the binding properties of η-arene Ru(II) complexes with poly(U)*poly(A)•poly(U) triplex, two arene Ru(II) complexes with different fluorine substituent...
To comprehend the binding properties of η-arene Ru(II) complexes with poly(U)*poly(A)•poly(U) triplex, two arene Ru(II) complexes with different fluorine substituent positions, [(η-CH)Ru(o-fpip)Cl]PF (Ru1,η-CH = benzene ring, o-fpip = 2-(2'‑fluorine) imidazo [4,5-f] Biver et al. (2008), Gupta et al. (2012) [1, 10] phenanthroline) and [(η-CH)Ru(p-fpip)Cl]PF (Ru2,η-CH = benzene ring, o-fpip = 2-(4'‑fluorine) imidazo [4,5-f] Biver et al. (2008), Gupta et al. (2012) [1, 10] phenanthroline), have been synthesized and characterized in this study. The binding of Ru1 and Ru2 with poly(U)*poly(A)•poly(U) triplex has been investigated by viscosity measurement and spectroscopic methods. Analysis of UV-Vis absorption spectral titrations suggests that Ru1 and Ru2 bind to the triplex through an intercalative mode, but the binding affinity of Ru2 is slightly higher than that of Ru1, which is also verified by viscosity and EB (ethidium bromide) competition measurements. Furthermore, the thermal denaturation experiment shows that Ru1 and Ru2 increase the third-strand stabilization to a similar extent. Interestingly, the two complexes have essentially no effect on the stabilization of the template duplex. Considering the structure of Ru1 and Ru2, conceivably besides the intercalation of ligand, the force stabilizing the triplex should also involve covalent binding and electrostatic interaction. The obtained results will contribute to our understanding of the interaction of arene Ru(II) complexes with the poly(U)*poly(A)•poly(U) triplex.
Topics: Benzene; Fluorine; Nucleic Acid Conformation; Phenanthrolines; Poly A; Poly U; RNA; Ruthenium
PubMed: 35405487
DOI: 10.1016/j.jinorgbio.2022.111813