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Nucleic Acids Research Jul 2023RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However,...
RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However, transcriptome-wide methods to profile RNA-protein interactions in vivo remain technically challenging and require large amounts of starting material. Herein, we present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that is based on tailing and ligation of cDNA molecules (TLC). TLC involves the generation of solid-phase cDNA, followed by ribotailing to significantly enhance the efficiency of subsequent adapter ligation. These modifications result in a streamlined, fully bead-based library preparation strategy, which eliminates time-consuming purification procedures and drastically reduces sample loss. As a result, TLC-CLIP displays unparalleled sensitivity, enabling the profiling of RNA-protein interactions from as few as 1000 cells. To demonstrate the effectiveness of TLC-CLIP, we profiled four endogenous RNA-binding proteins, showcasing its reproducibility and improved precision resulting from a higher occurrence of crosslinking-induced deletions. These deletions serve as an intrinsic quality metric and increase both specificity and nucleotide-resolution.
Topics: RNA; DNA, Complementary; Reproducibility of Results; RNA-Binding Proteins; Immunoprecipitation; High-Throughput Nucleotide Sequencing; Binding Sites
PubMed: 37283087
DOI: 10.1093/nar/gkad466 -
Bioengineering (Basel, Switzerland) Sep 2023Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses...
Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.
PubMed: 37892846
DOI: 10.3390/bioengineering10101116 -
Frontiers in Cell and Developmental... 2024Mammalian genomic DNA is packed in a small nucleus, and its folding and organization in the nucleus are critical for gene regulation and cell fate determination. In... (Review)
Review
Mammalian genomic DNA is packed in a small nucleus, and its folding and organization in the nucleus are critical for gene regulation and cell fate determination. In interphase, chromosomes are compartmentalized into certain nuclear spaces and territories that are considered incompatible with each other. The regulation of gene expression is influenced by the epigenetic characteristics of topologically associated domains and A/B compartments within chromosomes (intrachromosomal). Previously, interactions among chromosomes detected via chromosome conformation capture-based methods were considered noise or artificial errors. However, recent studies based on newly developed ligation-independent methods have shown that inter-chromosomal interactions play important roles in gene regulation. This review summarizes the recent understanding of spatial genomic organization in mammalian interphase nuclei and discusses the potential mechanisms that determine cell identity. In addition, this review highlights the potential role of inter-chromosomal interactions in early mouse development.
PubMed: 38774644
DOI: 10.3389/fcell.2024.1397807 -
International Journal of Molecular... Sep 2023Neurofibromatosis type 1 (NF1) is a clinically heterogeneous neurocutaneous disorder inherited in autosomal dominant manner. Approximately 5-10% of the cases are caused...
Neurofibromatosis type 1 (NF1) is a clinically heterogeneous neurocutaneous disorder inherited in autosomal dominant manner. Approximately 5-10% of the cases are caused by microdeletions involving the gene and its flanking regions. Microdeletions, which lead to more severe clinical manifestations, can be subclassified into four different types (type 1, 2, 3 and atypical) according to their size, the genomic location of the breakpoints and the number of genes included within the deletion. Besides the prominent hallmarks of NF1, patients with microdeletions frequently exhibit specific additional clinical manifestations like dysmorphic facial features, macrocephaly, overgrowth, global developmental delay, cognitive disability and an increased risk of malignancies. It is important to identify the genes co-deleted with , because they are likely to have an effect on the clinical manifestation. Multiplex ligation-dependent probe amplification (MLPA) and microarray analysis are the primary techniques for the investigation of microdeletions. However, based on previous research, optical genome mapping (OGM) could also serve as an alternative method to identify copy number variations (CNVs). Here, we present a case with microdeletion identified by means of OGM and demonstrate that this novel technology is a suitable tool for the identification and classification of the microdeletions.
Topics: Humans; Neurofibromatosis 1; DNA Copy Number Variations; Genes, Neurofibromatosis 1; Megalencephaly; Chromosome Mapping
PubMed: 37686382
DOI: 10.3390/ijms241713580 -
Nature Communications Nov 2023Bdelloid rotifers are part of the restricted circle of multicellular animals that can withstand a wide range of genotoxic stresses at any stage of their life cycle. In...
Bdelloid rotifers are part of the restricted circle of multicellular animals that can withstand a wide range of genotoxic stresses at any stage of their life cycle. In this study, bdelloid rotifer Adineta vaga is used as a model to decipher the molecular basis of their extreme tolerance. Proteomic analysis shows that a specific DNA ligase, different from those usually involved in DNA repair in eukaryotes, is strongly over-represented upon ionizing radiation. A phylogenetic analysis reveals its orthology to prokaryotic DNA ligase E, and its horizontal acquisition by bdelloid rotifers and plausibly other eukaryotes. The fungus Mortierella verticillata, having a single copy of this DNA Ligase E homolog, also exhibits an increased radiation tolerance with an over-expression of this DNA ligase E following X-ray exposure. We also provide evidence that A. vaga ligase E is a major contributor of DNA breaks ligation activity, which is a common step of all important DNA repair pathways. Consistently, its heterologous expression in human cell lines significantly improves their radio-tolerance. Overall, this study highlights the potential of horizontal gene transfers in eukaryotes, and their contribution to the adaptation to extreme conditions.
Topics: Animals; Humans; Eukaryota; Phylogeny; DNA Ligases; Ligases; Proteomics; Rotifera; DNA Damage; DNA Ligase ATP
PubMed: 37993452
DOI: 10.1038/s41467-023-43075-8 -
Burns & Trauma 2023Immunosuppression is an important characteristic of sepsis and is closely related to poor outcomes. Regulatory T cells (Tregs) contribute to immune suppression by...
BACKGROUND
Immunosuppression is an important characteristic of sepsis and is closely related to poor outcomes. Regulatory T cells (Tregs) contribute to immune suppression by inhibiting effector T cell (Teff) proliferation and differentiation. We aimed to investigate the role of p53 in Treg expansion after sepsis.
METHODS
We constructed a sepsis model in wild-type (WT) and p53/CD4-Cre mice by cecal ligation and puncture (CLP) and evaluated the proportions of CD4CD25 Foxp3 Tregs by flow cytometry. The expression levels of forkhead/winged helix transcription factor p3 (Foxp3), DNA methyltransferase enzyme (DMNT)3a and ten-eleven translocation (TET)2 were examined using quantitative real-time PCR and Western blot analysis. Treg-specific demethylation region (TSDR) methylation sites in cells were analyzed by bisulfite-sequencing PCR. Furthermore, the direct binding of p53 to the Dnmt3a and TET2 promoters was illustrated using a luciferase assay. The suppressive ability of Tregs was indicated by enzyme-linked immunosorbent assay analysis of cytokine levels and the proliferation of cocultured Teffs. Finally, mortality rates after CLP were compared among WT and p53/CD4-Cre mice.
RESULTS
The proportion of CD4CD25 Foxp3 Tregs was significantly reduced in p53/CD4-Cre mice compared to WT mice after CLP. The enhanced expression of Foxp3 in WT mice was downregulated in the p53/CD4-Cre group. We found decreased DMNT3a and increased TET2 levels after CLP. However, the dysregulation of DNMT3a and TET2 was significantly reversed in p53/CD4-Cre mice. TSDR underwent increased demethylation in p53/CD4-Cre mice. Luciferase activity indicated direct binding of p53 to the promoter regions of DNMT3a and TET2 to regulate their transcription. Consequently, Tregs from p53/CD4-Cre CLP mice exhibited limited suppressive ability, as indicated by the reduced production of transforming growth factor-β and interleukin 10 (IL-10). In the coculture system, Teffs showed preserved production of IL-2, differentiation into Th1 cells and proliferation in the presence of Tregs isolated from p53/CD4-Cre CLP mice. Finally, the mortality rate of the p53/CD4-Cre group after CLP was significantly reduced in comparison to that of the WT group.
CONCLUSION
p53 appears to be critical for Foxp3 expression and consequent Treg expansion by regulating the induction of DNMT3a and TET2, thereby resulting in Foxp3-TSDR demethylation in the context of sepsis.
PubMed: 37564681
DOI: 10.1093/burnst/tkad021 -
International Journal of Analytical... 2023A novel ligation-based loop-mediated isothermal amplification has been developed for miRNA detection. Two stem-loop structure DNA linker A/B probes which hybridized with...
A novel ligation-based loop-mediated isothermal amplification has been developed for miRNA detection. Two stem-loop structure DNA linker A/B probes which hybridized with miRNA were designed to establish a rapid and ultrasensitive miRNA-LAMP system for miRNA detection. Target miR-200a was used to template the ligation of Linker A/B probes with SplintR Ligase and used as a dumbbell-shaped amplicon. By adding BIP/FIP and Bst 2.0 DNA polymerase, the LAMP reaction was carried out, which brought greatly improved amplification efficiency. The double-stranded DNA fluorescent dye EvaGreen was added for the detection of amplification product to achieve the quantification of the target miRNA. This method can detect miRNA in a linear range of seven orders of magnitude, with a detection limit of 100 fM. Therefore, this ultrasensitive miRNA-LAMP assay provides a new path for the highly sensitive quantitative analysis of miRNA, thereby bringing convenience to clinical diagnosis and prognostic research.
PubMed: 37732283
DOI: 10.1155/2023/6624884 -
STAR Protocols Sep 2023Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g.,...
Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria). Here, we present an in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments. We describe steps for crRNA design and preparation, genomic DNA isolation, and CRISPR-Cas12a cleavage and capture plasmid construction and linearization. We then detail target BGC and plasmid DNA ligation and transformation and screening for positive clones. For complete details on the use and execution of this protocol, please refer to Liang et al..
Topics: CRISPR-Cas Systems; DNA; Cloning, Molecular; Genomics
PubMed: 37432853
DOI: 10.1016/j.xpro.2023.102435 -
Scientific Reports Sep 2023Bone marrow-derived human mesenchymal stem cells (hMSCs) can differentiate into various lineages, such as chondrocytes, adipocytes, osteoblasts, and neuronal lineages....
Bone marrow-derived human mesenchymal stem cells (hMSCs) can differentiate into various lineages, such as chondrocytes, adipocytes, osteoblasts, and neuronal lineages. It has been shown that the high-efficiency DNA-repair capacity of hMSCs is decreased during their differentiation. However, the underlying its mechanism during adipogenesis and osteogenesis is unknown. Herein, we investigated how alkyl-damage repair is modulated during adipogenic and osteogenic differentiation, especially focusing on the base excision repair (BER) pathway. Response to an alkylation agent was assessed via quantification of the double-strand break (DSB) foci and activities of BER-related enzymes during differentiation in hMSCs. Adipocytes showed high resistance against methyl methanesulfonate (MMS)-induced alkyl damage, whereas osteoblasts were more sensitive than hMSCs. During the differentiation, activities, and protein levels of uracil-DNA glycosylase were found to be regulated. In addition, ligation-related proteins, such as X-ray repair cross-complementing protein 1 (XRCC1) and DNA polymerase β, were upregulated in adipocytes, whereas their levels and recruitment declined during osteogenesis. These modulations of BER enzyme activity during differentiation influenced DNA repair efficiency and the accumulation of DSBs as repair intermediates in the nucleus. Taken together, we suggest that BER enzymatic activity is regulated in adipogenic and osteogenic differentiation and these alterations in the BER pathway led to different responses to alkyl damage from those in hMSCs.
Topics: Humans; Adipogenesis; Osteogenesis; Bone Marrow; Cells, Cultured; Cell Differentiation; DNA Repair; Mesenchymal Stem Cells; X-ray Repair Cross Complementing Protein 1
PubMed: 37773206
DOI: 10.1038/s41598-023-43737-z -
Molecular Brain Jun 2024Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double-strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that the TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complementary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 h treatment of 10 μM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS-affected neurons.
Topics: Humans; DNA-Binding Proteins; MutL Protein Homolog 1; DNA Damage; Protein Binding; Cell Line, Tumor; Amyotrophic Lateral Sclerosis; Neurons; Middle Aged; Male
PubMed: 38840222
DOI: 10.1186/s13041-024-01108-3