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Methods in Molecular Biology (Clifton,... 2014mRNA polyadenylation functions in nuclear export, translation, and stability. We describe an efficient protocol designed to assess poly(A) tail length that is based on...
mRNA polyadenylation functions in nuclear export, translation, and stability. We describe an efficient protocol designed to assess poly(A) tail length that is based on 3' tailing by yeast poly(A) polymerase and product analysis to single-nucleotide resolution by capillary electrophoresis.
Topics: Electrophoresis, Capillary; Genetic Techniques; Poly A; Polynucleotide Adenylyltransferase; RNA, Messenger
PubMed: 24590776
DOI: 10.1007/978-1-62703-971-0_2 -
RNA (New York, N.Y.) Oct 2019Polyadenylation at the 3'-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only...
Polyadenylation at the 3'-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3'-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present , an R package to estimate poly(A) tail length on ONT long-read sequencing data. operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess 's performance across different poly(A) lengths, demonstrating that is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.
Topics: Nanopores; Poly A; Poly T; Polyadenylation; Sequence Analysis, DNA; Sequence Analysis, RNA
PubMed: 31266821
DOI: 10.1261/rna.071332.119 -
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 -
Journal of Inorganic Biochemistry Jul 2022Two Ru(II) complexes, [Ru(phen)(11-F-dppz)] (Ru1, phen = 1,10-phenanthroline, 11-F-dppz = 11-fluorodipyrido[3,2-a:2',3'-c]phenazine) and [Ru(phen)(11-CN-dppz)] (Ru2,...
Two Ru(II) complexes, [Ru(phen)(11-F-dppz)] (Ru1, phen = 1,10-phenanthroline, 11-F-dppz = 11-fluorodipyrido[3,2-a:2',3'-c]phenazine) and [Ru(phen)(11-CN-dppz)] (Ru2, 11-CN-dppz = 11-cyanodipyrido[3,2-a:2',3'-c]phenazine), have been synthesized and characterized in this work. The binding properties of Ru1 and Ru2 with poly(A)•poly(U) RNA duplex have been investigated by spectroscopic methods and viscosity measurements. UV-vis absorption spectra and viscosity experiments demonstrate that the binding modes of Ru1 and Ru2 with poly(A)•poly(U) RNA duplex are intercalation, while the binding affinity for Ru2 is greater than that for Ru1. In addition, thermal denaturation studies reveal that both complexes significantly improve the stability of poly(A)•poly(U) duplex RNA. However, fluorescence titrations indicate that Ru1, unlike Ru2, can act as a molecular "light switch" for the poly(A)•poly(U) duplex RNA. The obtained results of this work indicate that the electron-withdrawing effect of substituents on the main ligands can significantly affect the binding of Ru(II) polypyridyl complexes with poly(A)•poly(U).
Topics: Coordination Complexes; Phenazines; Poly A; RNA; Ruthenium
PubMed: 35462128
DOI: 10.1016/j.jinorgbio.2022.111833 -
RNA (New York, N.Y.) Jul 2022The poly(A) tail enhances translation and transcript stability, and tail length is under dynamic control during cell state transitions. Tail regulation plays essential...
The poly(A) tail enhances translation and transcript stability, and tail length is under dynamic control during cell state transitions. Tail regulation plays essential roles in translational timing and fertilization in early development, but poly(A) tail dynamics have not been fully explored in post-embryonic systems. Here, we examined the landscape and impact of tail length control during macrophage activation. Upon activation, more than 1500 mRNAs, including proinflammatory genes, underwent distinctive changes in tail lengths. Increases in tail length correlated with mRNA levels regardless of transcriptional activity, and many mRNAs that underwent tail extension encode proteins necessary for immune function and post-transcriptional regulation. Strikingly, we found that , whose protein product destabilizes target transcripts, undergoes tail extension. Our analyses indicate that many mRNAs undergoing tail lengthening are, in turn, degraded by elevated levels of ZFP36, constituting a post-transcriptional feedback loop that ensures transient regulation of transcripts integral to macrophage activation. Taken together, this study establishes the complexity, relevance, and widespread nature of poly(A) tail dynamics, and the resulting post-transcriptional regulation during macrophage activation.
Topics: Gene Expression Regulation; Macrophage Activation; Poly A; Polyadenylation; RNA, Messenger
PubMed: 35512831
DOI: 10.1261/rna.078918.121 -
IUBMB Life Dec 1999Arguments are presented in favor of capability of poly(A)-tracts of cellular RNA to form double helices in vivo. It is suggested that formation of the double helix in... (Review)
Review
Arguments are presented in favor of capability of poly(A)-tracts of cellular RNA to form double helices in vivo. It is suggested that formation of the double helix in the mRNA poly(A) tall provides the basis for such processes as polyadenylation termination, PAB I synthesis autoregulation, and stabilization of ARE-containing mRNA by ELAV-like proteins.
Topics: Nucleic Acid Conformation; Poly A; RNA, Double-Stranded; RNA, Messenger
PubMed: 10683761
DOI: 10.1080/713803577 -
RNA (New York, N.Y.) Jun 2022Neurons provide a rich setting for studying post-transcriptional control. Here, we investigate the landscape of translational control in neurons and search for mRNA...
Neurons provide a rich setting for studying post-transcriptional control. Here, we investigate the landscape of translational control in neurons and search for mRNA features that explain differences in translational efficiency (TE), considering the interplay between TE, mRNA poly(A)-tail lengths, microRNAs, and neuronal activation. In neurons and brain tissues, TE correlates with tail length, and a few dozen mRNAs appear to undergo cytoplasmic polyadenylation upon light or chemical stimulation. However, the correlation between TE and tail length is modest, explaining <5% of TE variance, and even this modest relationship diminishes when accounting for other mRNA features. Thus, tail length appears to affect TE only minimally. Accordingly, miRNAs, which accelerate deadenylation of their mRNA targets, primarily influence target mRNA levels, with no detectable effect on either steady-state tail lengths or TE. Larger correlates with TE include codon composition and predicted mRNA folding energy. When combined in a model, the identified correlates explain 38%-45% of TE variance. These results provide a framework for considering the relative impact of factors that contribute to translational control in neurons. They indicate that when examined in bulk, translational control in neurons largely resembles that of other types of post-embryonic cells. Thus, detection of more specialized control might require analyses that can distinguish translation occurring in neuronal processes from that occurring in cell bodies.
Topics: Gene Expression Regulation; MicroRNAs; Neurons; Poly A; Polyadenylation; Protein Biosynthesis; RNA, Messenger
PubMed: 35273099
DOI: 10.1261/rna.079046.121 -
Nature Structural & Molecular Biology Dec 2017
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Gene Expression Regulation; Poly A; Poly(A)-Binding Proteins; RNA, Messenger; Transcription, Genetic
PubMed: 29215636
DOI: 10.1038/nsmb.3509 -
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 -
Journal of Inorganic Biochemistry Dec 2022To further determine the factors that affect the binding properties of ruthenium(II) polypyridine complexes with RNA duplex and to find excellent RNA-binding agents, the...
To further determine the factors that affect the binding properties of ruthenium(II) polypyridine complexes with RNA duplex and to find excellent RNA-binding agents, the binding properties of ruthenium(II) complexes [Ru(phen)(7-OCH-dppz)] (Ru1, phen = 1,10-phenan- throline, 7-OCH-dppz = 7-methoxy-dipyrido-[3,2-a,2',3'-c]-phenazine) and [Ru(phen)(7-NO- dppz)] (Ru2, 7-NO-dppz = 7-nitro-dipyrido-[3,2-a,2',3'-c]-phenazine) with RNA poly(A)•poly(U) duplex have been investigated by spectroscopic methods and viscosity measurements in this work. The results show that complexes Ru1 and Ru2 bind to poly(A)•poly(U) through intercalation and the binding affinity between Ru2 and poly(A)•poly(U) is greater than that of Ru1. Thermal denaturation experiments suggest that both ruthenium(II) complexes exhibit a significant stabilizing effect on poly(A)•poly(U) duplex. Moreover, fluorescence emission spectra exhibit that, deviating from Ru2, Ru1 exhibits a "light switch" effect for poly(A)•poly(U). This effect can be observed by the naked eye under UV light and adjusted by pH, meaning that Ru1 may act as a reversible pH controlled molecular "light switch". The results obtained in this work will contribute to our understanding of the significant influence of the intercalative ligand substituent effect in the binding process of ruthenium(II) complexes with RNA duplex.
Topics: Poly A; Ruthenium; Nitrogen Dioxide; RNA; Phenazines
PubMed: 36115329
DOI: 10.1016/j.jinorgbio.2022.111991