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Briefings in Bioinformatics Sep 2023Dynamic tuning of the poly(A) tail is a crucial mechanism for controlling translation and stability of eukaryotic mRNA. Achieving a comprehensive understanding of how...
Dynamic tuning of the poly(A) tail is a crucial mechanism for controlling translation and stability of eukaryotic mRNA. Achieving a comprehensive understanding of how this regulation occurs requires unbiased abundance quantification of poly(A)-tail transcripts and simple poly(A)-length measurement using high-throughput sequencing platforms. Current methods have limitations due to complicated setups and elaborate library preparation plans. To address this, we introduce central limit theorem (CLT)-managed RNA-seq (CLT-seq), a simple and straightforward homopolymer-sequencing method. In CLT-seq, an anchor-free oligo(dT) primer rapidly binds to and unbinds from anywhere along the poly(A) tail string, leading to position-directed reverse transcription with equal probability. The CLT mechanism enables the synthesized poly(T) lengths, which correspond to the templated segment of the poly(A) tail, to distribute normally. Based on a well-fitted pseudogaussian-derived poly(A)-poly(T) conversion model, the actual poly(A)-tail profile is reconstructed from the acquired poly(T)-length profile through matrix operations. CLT-seq follows a simple procedure without requiring RNA-related pre-treatment, enrichment or selection, and the CLT-shortened poly(T) stretches are more compatible with existing sequencing platforms. This proof-of-concept approach facilitates direct homopolymer base-calling and features unbiased RNA-seq. Therefore, CLT-seq provides unbiased, robust and cost-efficient transcriptome-wide poly(A)-tail profiling. We demonstrate that CLT-seq on the most common Illumina platform delivers reliable poly(A)-tail profiling at a transcriptome-wide scale in human cellular contexts. We find that the poly(A)-tail-tuned ncRNA regulation undergoes a dynamic, complex process similar to mRNA regulation. Overall, CLT-seq offers a simplified, effective and economical approach to investigate poly(A)-tail regulation, with potential implications for understanding gene expression and identifying therapeutic targets.
Topics: Humans; Sequence Analysis, RNA; Polyadenylation; RNA, Messenger; Gene Expression Profiling; Transcriptome; High-Throughput Nucleotide Sequencing; RNA, Untranslated
PubMed: 37874949
DOI: 10.1093/bib/bbad374 -
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 -
Biomedicine & Pharmacotherapy =... Feb 2023As an emerging tumor therapy, ideal oncolytic viruses preferentially replicate in malignant cells, reverse the immunosuppressive tumor microenvironment, and eventually...
As an emerging tumor therapy, ideal oncolytic viruses preferentially replicate in malignant cells, reverse the immunosuppressive tumor microenvironment, and eventually can be eliminated by the patient. It is of great significance for cancer treatment to discover new excellent oncolytic viruses. Here, we found that WNV live attenuated vaccine WNV-poly(A) could be developed as a novel ideal oncolytic agent against several types of cancers. Mechanistically, due to its high sensitivity to type Ι interferon (IFN-Ι), WNV-poly(A) could specifically kill tumor cells rather than normal cells. At the same time, WNV-poly(A) could activate Dendritic cells (DCs) and trigger tumor antigen specific response mediated by CD8 + T cell, which contributed to inhibit the propagation of original and distal tumor cells. Like intratumoral injection, intravenous injection with WNV-poly(A) also markedly delays Huh7 hepatic carcinoma (HCC) transplanted tumor progression. Most importantly, in addition to an array of mouse xenograft tumor models, WNV-poly(A) also has a significant inhibitory effect on many different types of patient-derived tumor tissues and HCC patient-derived xenograft (PDX) tumor models. Our studies reveal that WNV-poly(A) is a potent and excellent oncolytic agent against many types of tumors and may have a role in metastatic and recurrent tumors.
Topics: Animals; Mice; Carcinoma, Hepatocellular; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Immunity; Liver Neoplasms; Neoplasm Recurrence, Local; Oncolytic Viruses; Tumor Microenvironment; Virus Replication
PubMed: 36502755
DOI: 10.1016/j.biopha.2022.114094 -
Cell Systems Jun 2024Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small...
Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small library of identical mRNAs that differ only in their poly(A)-tail length to examine their behavior in human embryonic kidney cells. We find that tail length strongly correlates with mRNA degradation rates but is decoupled from translation. Interestingly, an optimal tail length of ∼100 nt displays the highest translation rate, which is identical to the average endogenous tail length measured by nanopore sequencing. Furthermore, poly(A)-tail length variability-a feature of endogenous mRNAs-impacts translation efficiency but not mRNA degradation rates. Stochastic modeling combined with single-cell tracking reveals that poly(A) tails provide cells with an independent handle to tune gene expression fluctuations by decoupling mRNA degradation and translation. Together, this work contributes to the basic understanding of gene expression regulation and has potential applications in nucleic acid therapeutics.
Topics: Humans; RNA, Messenger; Poly A; Protein Biosynthesis; RNA Stability; HEK293 Cells; Gene Expression Regulation
PubMed: 38901403
DOI: 10.1016/j.cels.2024.05.004 -
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 -
Nucleic Acids Research Sep 2022La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem...
La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts. Here, we characterize the La-module of LARP1, which plays an important role in regulating synthesis of ribosomal proteins in response to mTOR signaling and mRNA stabilization. LARP1 has been well characterized functionally but no structural information exists for its La-module. We show that unlike other LARPs, the La-module in LARP1 does not contain an RRM domain. The LaM alone is sufficient for binding poly(A) RNA with submicromolar affinity and specificity. Multiple high-resolution crystal structures of the LARP1 LaM domain in complex with poly(A) show that it is highly specific for the RNA 3'-end, and identify LaM residues Q333, Y336 and F348 as the most critical for binding. Use of a quantitative mRNA stabilization assay and poly(A) tail-sequencing demonstrate functional relevance of LARP1 RNA binding in cells and provide novel insight into its poly(A) 3' protection activity.
Topics: Ribonucleoproteins; Autoantigens; RNA, Messenger; RNA-Binding Proteins; Poly A; RNA; Protein Binding
PubMed: 35979957
DOI: 10.1093/nar/gkac696 -
International Journal of Molecular... Aug 2023This study evaluated the wound-healing activity of a polymer, Poly(3,4-ethylenedioxythiophene):poly-(styrene sulfonate) (PEDOT: PSS), and determined its mechanism based...
This study evaluated the wound-healing activity of a polymer, Poly(3,4-ethylenedioxythiophene):poly-(styrene sulfonate) (PEDOT: PSS), and determined its mechanism based on angiogenic activity in a full-thickness excision wound model in Spraque Dawley (SD) rats. Administering PEDOT: PSS (1.6) 1.5 ppm at a dose of 50 mg/kg/day significantly improved wound healing in the SD rats on the eleventh day after the incision was created. PEDOT: PSS-treated animals presented no anti-inflammatory skin effects; however, there was an increase in angiogenic behavior. VEGF was found to be significantly elevated in the PEDOT: PSS-treated groups seven days post-incision. However, only a higher concentration of PEDOT: PSS increased TGF-β1 expression within the same time frame. Our results showed that PEDOT: PSS enhances wound healing activity, mainly in terms of its angiogenic effects. In this paper, we describe the highly conductive macromolecular material PEDOT: PSS, which demonstrated accelerated wound-healing activity in the animal incision model. The results will further provide information regarding the application of PEDOT: PSS as a dressing for medical use.
Topics: Animals; Rats; Polystyrenes; Wound Healing; Thiophenes; Skin; Poly A; Surgical Wound
PubMed: 37628719
DOI: 10.3390/ijms241612539 -
Nucleic Acids Research Apr 2023During oocyte development in mice, transcripts accumulate in the growth phase and are subsequently degraded during maturation. At the transition point between growth and...
During oocyte development in mice, transcripts accumulate in the growth phase and are subsequently degraded during maturation. At the transition point between growth and maturation, oocytes have an intact nucleus or germinal vesicle (GV), and terminal uridylation labels RNA for degradation in meiosis I. By profiling the transcriptome using single-oocyte long-read PacBio RNA sequencing, we document that a small cohort of mRNAs are polyadenylated after terminal uridylation in GV oocytes [designated uridylated-poly(A) RNA]. Because DIS3L2 ribonuclease is known to degrade uridylated transcripts, we established oocyte-specific Dis3l2 knockout mice (Dis3l2cKO). Upon DIS3L2 depletion, uridylated-poly(A) RNAs remain intact which increases their abundance, and they predominate in the transcriptome of Dis3l2cKO oocytes. The abundance of uridylated-poly(A) RNA in Dis3l2cKO oocytes arises not only from insufficient degradation, but also from the stabilizing effect of subsequent polyadenylation. Uridylated-poly(A) RNAs have shorter poly(A) tails and their translation activity decreases in Dis3l2cKO oocytes. Almost all Dis3l2cKO oocytes arrest at the GV stage, and female mice are infertile. Our study demonstrates multiple fates for RNA after terminal uridylation and highlights the role of DIS3L2 ribonuclease in safeguarding the transcriptome and ensuring female fertility.
Topics: Animals; Female; Mice; Fertility; Oocytes; RNA; RNA, Messenger; Exoribonucleases
PubMed: 36727488
DOI: 10.1093/nar/gkad061 -
Genes Jun 2020The intensity of the merle pattern is determined by the length of the poly(A) tail of a repeat element which has been inserted into the boundary of intron 10 and exon 11... (Review)
Review
The intensity of the merle pattern is determined by the length of the poly(A) tail of a repeat element which has been inserted into the boundary of intron 10 and exon 11 of the locus in reverse orientation. This poly(A) tail behaves as a microsatellite, and due to replication slippage, longer and shorter alleles of it might be generated during cell divisions. The length of the poly(A) tail regulates the splicing mechanism. In the case of shorter tails, the removal of intron 10 takes place at the original splicing, resulting in a normal premelanosome protein (PMEL). Longer tails generate larger insertions, forcing splicing to a cryptic splice site, thereby coding for an abnormal PMEL protein, which is unable to form the normal fibrillar matrix of the eumelanosomes. Thus, eumelanin deposition ensuring the dark color formation is reduced. In summary, the longer the poly(A) tail, the lighter the coat color intensity of the melanocytes. These mutations can occur in the somatic cells and the resulting cell clones will shape the merle pattern of the coat. When they take place in the germ line, they occasionally produce offspring with unexpected color variations which are different from those of their parents.
Topics: Animals; Dogs; Genetic Background; Hair Color; Melanins; Melanocytes; Mutation; RNA Splice Sites; RNA Splicing; gp100 Melanoma Antigen
PubMed: 32560567
DOI: 10.3390/genes11060660 -
ACS Applied Materials & Interfaces Apr 2020The near-field effects in the vicinity of metallic nanoparticle surfaces, as induced by electromagnetic radiation with specific wavelength, give rise to a variety of... (Review)
Review
The near-field effects in the vicinity of metallic nanoparticle surfaces, as induced by electromagnetic radiation with specific wavelength, give rise to a variety of novel optical properties and attractive applications because of surface plasmons, which are the coherent oscillations of conduction electrons on a metal surface. The interdisciplinary field of plasmonics has witnessed vigorous growth, promoting research on the modulation of plasmon resonance by constructing advanced plasmonic nanoarchitectures with controllable size, morphology, or interparticle coupling. Among diversified tools, deoxyribonucleic nucleic acid (DNA) possesses prominent superiority as a result of its designability, programmability, addressability, and ease of nanomaterial modification. In this review, we focus on the methods and optical applications of plasmon resonance modulation accomplished by DNA nanotechnology. Recent developments in the construction of DNA-mediated plasmonic nanoarchitecture and key ongoing research directions utilizing unique optical features are highlighted. Obstacles and challenges in this field are pointed out, followed by preliminary suggestions on some areas of opportunity that deserve attention.
Topics: Biosensing Techniques; DNA; Gold; Metal Nanoparticles; Nanotubes; Optical Imaging; Poly A; Quantum Theory; Surface Plasmon Resonance
PubMed: 32154704
DOI: 10.1021/acsami.9b23608