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RNA (New York, N.Y.) Dec 2020Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of...
Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5',3'-phosphodiester bonds in the mRNA body and poly(A) tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogs modified at the α-phosphate (containing either O-to-S or O-to-BH substitutions) and three different RNA polymerases-SP6, T7, and poly(A) polymerase. To verify the efficiency of incorporation of ATP analogs in the presence of ATP, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5'-mononucleotides. The method also estimated the average poly(A) tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the poly(A) tail were substantially less susceptible to degradation by 3'-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.
Topics: Adenosine Triphosphate; Animals; DNA-Directed RNA Polymerases; Dendritic Cells; HeLa Cells; Humans; Mice; Phosphorothioate Oligonucleotides; Poly A; Protein Biosynthesis; Protein Processing, Post-Translational; RNA, Messenger; Transcription, Genetic
PubMed: 32820035
DOI: 10.1261/rna.077099.120 -
Methods in Molecular Biology (Clifton,... 1995
Topics: Gene Expression; Plants; Poly A; RNA, Messenger; RNA, Plant
PubMed: 8563805
DOI: 10.1385/0-89603-321-X:203 -
Bioorganic Chemistry Jun 2023To further explore the binding properties of Ru(Ⅱ) polypyridine complexes with RNA, three Ru(Ⅱ) complexes [Ru(phen)(PIP)] (Ru1), [Ru(phen)(p-HPIP)] (Ru2), and...
Interaction of ruthenium(Ⅱ) polypyridyl complexes [Ru(phen)(L)] (L = PIP, p-HPIP and m-HPIP) with RNA poly(A)•poly(U): Each complex unexpectedly exhibiting a destabilizing effect on RNA.
To further explore the binding properties of Ru(Ⅱ) polypyridine complexes with RNA, three Ru(Ⅱ) complexes [Ru(phen)(PIP)] (Ru1), [Ru(phen)(p-HPIP)] (Ru2), and [Ru(phen)(m- HPIP)] (Ru3) have been synthesized and characterized in this work. The binding properties of three Ru(Ⅱ) complexes with RNA duplex poly(A)•poly(U) have been investigated by spectral and viscosity experiments. These studies all support that these three Ru(Ⅱ) complexes bind to poly RNA duplex poly(A)•poly(U) by intercalation, and Ru1 without substituents has a stronger binding affinity for poly(A)•poly(U). Interestingly, the thermal melting experiments show that these three Ru(Ⅱ) complexes all destabilize RNA duplex poly(A)•poly(U), and the destabilizing effect can be explained by the conformational changes of duplex structure induced by intercalating agents. To the best of our knowledge, this work report for the first time a small molecule capable of destabilizing an RNA duplex, which reflects that the substitution effect of intercalated ligands has an important influence on the affinity of Ru(Ⅱ) complexes to RNA duplex, and that not all Ru(Ⅱ) complexes show thermal stability effects on an RNA duplex.
Topics: Poly A; Ruthenium; RNA
PubMed: 37027949
DOI: 10.1016/j.bioorg.2023.106523 -
Journal of Biological Inorganic... Aug 2023Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)(dppx)] (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)(dppx)]...
Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)(dppx)] (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)(dppx)] (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. Spectrophotometric titrations and viscosity measurements suggested that enantiomers Λ-1 and Δ-1 bind with the triplex through intercalation, while the binding strengths of the two enantiomers toward the triplex differed only slightly from each other. Fluorescence titrations showed that although enantiomers Λ-1 and Δ-1 exhibited molecular "light switch" effects toward the triplex, the effect of Δ-1 was more marked. Furthermore, Furthermore, thermal denaturation showed that the two enantiomers have significantly different stabilizing effects on the triplex. The obtained results indicate that the racemic complex [Ru(bpy)(dppx)] is similar to a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex. Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)(dppx)] (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)(dppx)] (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. The obtained results indicate that the racemic complex [Ru(bpy)(dppx)] is similar as a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex.
Topics: Poly A; Ruthenium; Poly U; 2,2'-Dipyridyl; RNA
PubMed: 37452869
DOI: 10.1007/s00775-023-02004-2 -
RNA (New York, N.Y.) May 2022During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The...
During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding cleavage sites for over 2600 genes. We find that uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in .
Topics: 3' Untranslated Regions; Animals; Giardia lamblia; Mammals; Poly A; Polyadenylation; RNA, Messenger
PubMed: 35110372
DOI: 10.1261/rna.078793.121 -
Methods in Molecular Biology (Clifton,... 2024Poly(A) tail metabolism is critical for various biological processes, including early embryogenesis and cell differentiation. While traditional biochemical methods to...
Poly(A) tail metabolism is critical for various biological processes, including early embryogenesis and cell differentiation. While traditional biochemical methods to measure poly(A) tail length allow for the study of selected transcripts, the advent of long-read sequencing technologies enabled the development of simple and robust protocols to measure poly(A) tail length at the transcriptome level. Here, we describe a direct RNA sequencing protocol to capture poly(A) tail terminal additions based on the splint ligation of barcoded oligos compatible with terminal guanylation and uridylation. We cover how to prepare the libraries and perform the bioinformatics analysis to simultaneously determine the length of the transcripts' poly(A) tails and detect the presence of terminal guanylation and uridylation.
Topics: RNA; RNA, Messenger; Base Sequence; Sequence Analysis, RNA; Poly A
PubMed: 37824075
DOI: 10.1007/978-1-0716-3481-3_15 -
Methods in Molecular Biology (Clifton,... 2024The polyadenylation of the 3' ends of messenger RNAs is an important regulator of stability and translation. We developed the single-molecule poly(A) tail sequencing...
The polyadenylation of the 3' ends of messenger RNAs is an important regulator of stability and translation. We developed the single-molecule poly(A) tail sequencing method, SM-PATseq, to assay tail lengths of the whole transcriptome at nucleotide resolution using long-read sequencing. This method generates cDNA using an oligo-dT 3' splint adaptor ligation to prime first-strand cDNA synthesis, followed by random hexamer priming for second-strand synthesis. By directly sequencing the cDNA on long-read platforms, we can resolve tail lengths at nucleotide resolution, identify non-A bases within the tail, and quantify transcript abundance analogous to traditional RNAseq methods. Here, we discuss the method for generating, sequencing, and primary analysis of poly(A) tail data from total RNA using the Pacific Biosciences Sequel platform.
Topics: DNA, Complementary; RNA, Messenger; Transcriptome; Sequence Analysis, RNA; High-Throughput Nucleotide Sequencing; Nucleotides; Polyadenylation; Poly A
PubMed: 37824077
DOI: 10.1007/978-1-0716-3481-3_17 -
Nature Plants Sep 2022Poly(A) tail is a hallmark of eukaryotic messenger RNA and its length plays an essential role in regulating mRNA metabolism. However, a comprehensive resource for plant...
Poly(A) tail is a hallmark of eukaryotic messenger RNA and its length plays an essential role in regulating mRNA metabolism. However, a comprehensive resource for plant poly(A) tail length has yet to be established. Here, we applied a poly(A)-enrichment-free, nanopore-based method to profile full-length RNA with poly(A) tail information in plants. Our atlas contains over 120 million polyadenylated mRNA molecules from seven different tissues of Arabidopsis, as well as the shoot tissue of maize, soybean and rice. In most tissues, the size of plant poly(A) tails shows peaks at approximately 20 and 45 nucleotides, while the poly(A) tails in pollen exhibit a distinct pattern with strong peaks centred at 55 and 80 nucleotides. Moreover, poly(A) tail length is regulated in a gene-specific manner-mRNAs with short half-lives in general have long poly(A) tails, while mRNAs with long half-lives are featured with relatively short poly(A) tails that peak at ~45 nucleotides. Across species, poly(A) tails in the nucleus are almost twice as long as in the cytoplasm. Our comprehensive dataset lays the groundwork for future functional and evolutionary studies on poly(A) tail length regulation in plants.
Topics: Arabidopsis; Cytoplasm; Poly A; RNA, Messenger; RNA, Plant
PubMed: 35982302
DOI: 10.1038/s41477-022-01224-9 -
Molecular BioSystems Jan 2010The use of small molecules to specifically control important cellular functions through binding to nucleic acids is an area of major current interest at the interface of... (Review)
Review
The use of small molecules to specifically control important cellular functions through binding to nucleic acids is an area of major current interest at the interface of chemical biology and medicinal chemistry. The polyadenylic acid [poly(A)] tail of mRNA has been recently established as a potential drug target due to its significant role in the initiation of translation, maturation and stability of mRNA as well as in the production of alternate proteins in eukaryotic cells. Very recently some small molecule alkaloids of the isoquinoline group have been found to bind poly(A) with remarkably high affinity leading to self-structure formation. Plant alkaloids are small molecules known to have important traditional roles in medicinal chemistry due to their extensive biological activity. Especially, noteworthy are the protoberberine alkaloids that are widely distributed in several botanical families exhibiting myriad therapeutic applications. This review focuses on the structural and biological significance of poly(A) and interaction of protoberberine alkaloids with this RNA structure for the development of new small molecule alkaloids targeted to poly(A) structures as futuristic therapeutic agents.
Topics: Berberine Alkaloids; Models, Biological; Molecular Structure; Poly A; RNA
PubMed: 20024069
DOI: 10.1039/b910706a -
Methods in Molecular Biology (Clifton,... 2024Deadenylation is a major process that regulates gene expression by shaping the length of mRNA poly(A) tails. Deadenylation is controlled by factors in trans that recruit...
Deadenylation is a major process that regulates gene expression by shaping the length of mRNA poly(A) tails. Deadenylation is controlled by factors in trans that recruit or impede deadenylases, by the incorporation of non-adenosines during poly(A) tail synthesis, and by the posttranscriptional addition of 3' nucleotides to poly(A) tails. Deciphering the regulation of poly(A) tail shortening requires both transcriptome-wide approaches and more targeted methodologies, allowing deep analyses of specific mRNAs. In this chapter, we present Nano3'RACE, a nanopore-based cDNA sequencing method that allows in-depth analysis to precisely measure poly(A) tail length and detect 3' terminal nucleotide addition, such as uridylation, for mRNAs of interest.
Topics: Nucleotides; RNA, Messenger; Nanopore Sequencing; Poly A
PubMed: 37824074
DOI: 10.1007/978-1-0716-3481-3_14