-
Nature Communications May 2024The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization...
The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.
Topics: Animals; Transcription Factors; DNA Helicases; Nuclear Proteins; Mice; Nucleosomes; Indoleacetic Acids; RNA Polymerase II; Fibroblasts; Gene Knock-In Techniques; Hepatocytes; E1A-Associated p300 Protein; Transcriptional Activation; Transcription, Genetic; Histones; Deoxyribonuclease I; Chromatin; Humans
PubMed: 38811575
DOI: 10.1038/s41467-024-48911-z -
Signal Transduction and Targeted Therapy May 2024Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2'-O-MTase activity is essential for efficient viral replication and immune...
Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2'-O-MTase activity is essential for efficient viral replication and immune evasion. However, the K-D-K-E catalytic motif of 2'-O-MTase is highly conserved across numerous viruses, including flaviviruses, vaccinia viruses, coronaviruses, and extends even to mammals. Here, we observed a stronger 2'-O-MTase activity in SARS-CoV-2 compared to SARS-CoV, despite the presence of a consistently active catalytic center. We further identified critical residues (Leu-36, Asn-138 and Ile-153) which served as determinants of discrepancy in 2'-O-MTase activity between SARS-CoV-2 and SARS-CoV. These residues significantly enhanced the RNA binding affinity of 2'-O-MTase and boosted its versatility toward RNA substrates. Of interest, a triple substitution (Leu → Ile, Asn → His, Ile → Leu, from SARS-CoV-2 to SARS-CoV) within nsp16 resulted in a proportional reduction in viral 2'-O-methylation and impaired viral replication. Furthermore, it led to a significant upregulation of type I interferon (IFN-I) and proinflammatory cytokines both in vitro and vivo, relying on the cooperative sensing of melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). In conclusion, our findings demonstrated that alterations in residues other than K-D-K-E of 2'-O-MTase may affect viral replication and subsequently influence pathogenesis. Monitoring changes in nsp16 residues is crucial as it may aid in identifying and assessing future alteration in viral pathogenicity resulting from natural mutations occurring in nsp16.
Topics: Humans; SARS-CoV-2; COVID-19; Methyltransferases; Virus Replication; RNA, Viral; Viral Nonstructural Proteins; Severe acute respiratory syndrome-related coronavirus; Animals; Interferon-Induced Helicase, IFIH1
PubMed: 38811528
DOI: 10.1038/s41392-024-01860-x -
The Journal of Biological Chemistry May 2024Ataxin-2 (Atx2) is a polyglutamine (polyQ) tract-containing RNA-binding protein, while its polyQ expansion may cause protein aggregation that is implicated in the...
Ataxin-2 (Atx2) is a polyglutamine (polyQ) tract-containing RNA-binding protein, while its polyQ expansion may cause protein aggregation that is implicated in the pathogenesis of neurodegenerative diseases such as spinocerebellar ataxia type 2 (SCA2). However, the molecular mechanism underlying how Atx2 aggregation contributes to the proteinopathies remains elusive. Here, we investigated the influence of Atx2 aggregation on the assembly and functionality of cellular processing bodies (P-bodies) by using biochemical and fluorescence imaging approaches. We have revealed that polyQ-expanded (PQE) Atx2 sequesters the DEAD-box RNA helicase (DDX6), an essential component of P-bodies, into aggregates or puncta via some RNA sequences. The N-terminal like-Sm (LSm) domain of Atx2 (residues 82-184) and the C-terminal helicase domain of DDX6 are responsible for the interaction and specific sequestration. Moreover, sequestration of DDX6 may aggravate pre-mRNA mis-splicing, and interfere with the assembly of cellular P-bodies, releasing the endoribonuclease MARF1 that promotes mRNA decay and translational repression. Rescuing the DDX6 protein level can recover the assembly and functionality of P-bodies preventing targeted mRNA from degradation. This study provides a line of evidence for sequestration of the P-body components and impairment of the P-body homeostasis in dysregulating RNA metabolism, which is implicated in the disease pathologies and a potential therapeutic target.
PubMed: 38810698
DOI: 10.1016/j.jbc.2024.107413 -
Cell Reports May 2024Protein aggregation, which can sometimes spread in a prion-like manner, is a hallmark of neurodegenerative diseases. However, whether prion-like aggregates form during...
Protein aggregation, which can sometimes spread in a prion-like manner, is a hallmark of neurodegenerative diseases. However, whether prion-like aggregates form during normal brain aging remains unknown. Here, we use quantitative proteomics in the African turquoise killifish to identify protein aggregates that accumulate in old vertebrate brains. These aggregates are enriched for prion-like RNA-binding proteins, notably the ATP-dependent RNA helicase DDX5. We validate that DDX5 forms aggregate-like puncta in the brains of old killifish and mice. Interestingly, DDX5's prion-like domain allows these aggregates to propagate across many generations in yeast. In vitro, DDX5 phase separates into condensates. Mutations that abolish DDX5 prion propagation also impair the protein's ability to phase separate. DDX5 condensates exhibit enhanced enzymatic activity, but they can mature into inactive, solid aggregates. Our findings suggest that protein aggregates with prion-like properties form during normal brain aging, which could have implications for the age-dependency of cognitive decline.
PubMed: 38810650
DOI: 10.1016/j.celrep.2023.112787 -
BioMed Research International 2024Epigenetic alterations have been observed in many hematological malignancies, including acute myeloid leukemia (AML). Many of these alterations result from mutations in...
Epigenetic alterations have been observed in many hematological malignancies, including acute myeloid leukemia (AML). Many of these alterations result from mutations in DNA methyl transferase (DNMT) enzymes, disabling them to methylate target genes in a proper way. In this case-control study, we investigated the association between R882H mutation in gene and gene methylation in patients with AML. 47 AML patients and 6 controls were included in this study. After DNA extraction, amplification refractory mutation system (ARMS)-PCR was used to evaluate R882H mutations in gene. The high-resolution melting (HRM) method was used to determine the methylation changes of the gene promoter. R882H mutation was only found in 10.6% (5 out of 47) of AML patients. The frequency of gene methylation was significantly higher in patients without R882H mutations compared to patients with R882H mutations ( < 0.05). The DNMT3A R882H mutation is typically present in a minority of AML patients. Nevertheless, this mutation is associated with a reduced frequency of methylation in the DDX43 promoter region.
Topics: Humans; Leukemia, Myeloid, Acute; DNA Methyltransferase 3A; Promoter Regions, Genetic; DEAD-box RNA Helicases; DNA Methylation; Male; DNA (Cytosine-5-)-Methyltransferases; Female; Middle Aged; Adult; Mutation; Aged; Case-Control Studies; Neoplasm Proteins
PubMed: 38807916
DOI: 10.1155/2024/9625043 -
Life Science Alliance Aug 2024IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in...
IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing , we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.
Topics: Humans; Protein Biosynthesis; Transcription Factors; Stress, Physiological; DNA-Binding Proteins; K562 Cells; Activating Transcription Factor 4; Gene Deletion; Gene Expression Regulation; RNA, Transfer
PubMed: 38803225
DOI: 10.26508/lsa.202302554 -
Molecular Genetics and Genomics : MGG May 2024RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and...
RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast Saccharomyces cerevisiae, which does not have a RECQL5 ortholog. We found that RECQL5 expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in S. cerevisiae.
Topics: Saccharomyces cerevisiae; Genomic Instability; RecQ Helicases; Humans; Transcription, Genetic; RNA Polymerase II
PubMed: 38796829
DOI: 10.1007/s00438-024-02152-3 -
Molecules (Basel, Switzerland) May 2024Nsp13, a non-structural protein belonging to the coronavirus family 1B (SF1B) helicase, exhibits 5'-3' polarity-dependent DNA or RNA unwinding using NTPs. Crucially, it...
Nsp13, a non-structural protein belonging to the coronavirus family 1B (SF1B) helicase, exhibits 5'-3' polarity-dependent DNA or RNA unwinding using NTPs. Crucially, it serves as a key component of the viral replication-transcription complex (RTC), playing an indispensable role in the coronavirus life cycle and thereby making it a promising target for broad-spectrum antiviral therapies. The imidazole scaffold, known for its antiviral potential, has been proposed as a potential scaffold. In this study, a fluorescence-based assay was designed by labeling dsDNA substrates with a commercial fluorophore and monitoring signal changes upon Nsp13 helicase activity. Optimization and high-throughput screening validated the feasibility of this approach. In accordance with the structural characteristics of ADP, we employed a structural-based design strategy to synthesize three classes of imidazole-based compounds through substitution reaction. Through in vitro activity research, pharmacokinetic parameter analysis, and molecular docking simulation, we identified compounds A16 (IC = 1.25 μM) and B3 (IC = 0.98 μM) as potential lead antiviral compounds for further targeted drug research.
Topics: Imidazoles; SARS-CoV-2; Antiviral Agents; Molecular Docking Simulation; Viral Nonstructural Proteins; Humans; COVID-19 Drug Treatment; RNA Helicases; Fluorescent Dyes; Methyltransferases
PubMed: 38792162
DOI: 10.3390/molecules29102301 -
International Journal of Molecular... May 2024Mitochondrial genomes of land plants are large and exhibit a complex mode of gene organization and expression, particularly at the post-transcriptional level. The... (Review)
Review
Mitochondrial genomes of land plants are large and exhibit a complex mode of gene organization and expression, particularly at the post-transcriptional level. The primary organellar transcripts in plants undergo extensive maturation steps, including - and/or -nucleolytic cleavage, RNA-base modifications (mostly C-to-U deaminations) and both ''- and ''-splicing events. These essential processing steps rely on the activities of a large set of nuclear-encoded factors. RNA helicases serve as key players in RNA metabolism, participating in the regulation of transcription, mRNA processing and translation. They unwind RNA secondary structures and facilitate the formation of ribonucleoprotein complexes crucial for various stages of gene expression. Furthermore, RNA helicases are involved in RNA metabolism by modulating pre-mRNA maturation, transport and degradation processes. These enzymes are, therefore, pivotal in RNA quality-control mechanisms, ensuring the fidelity and efficiency of RNA processing and turnover in plant mitochondria. This review summarizes the significant roles played by helicases in regulating the highly dynamic processes of mitochondrial transcription, RNA processing and translation in plants. We further discuss recent advancements in understanding how dysregulation of mitochondrial RNA helicases affects the splicing of organellar genes, leading to respiratory dysfunctions, and consequently, altered growth, development and physiology of land plants.
Topics: RNA Splicing; Gene Expression Regulation, Plant; RNA Helicases; Mitochondria; RNA, Plant; Plants; Plant Proteins
PubMed: 38791540
DOI: 10.3390/ijms25105502 -
Journal of Translational Medicine May 2024N6-methyladenosine (m6A) stands as the most prevalent modified form of RNA in eukaryotes, pivotal in various biological processes such as regulating RNA stability,... (Review)
Review
N6-methyladenosine (m6A) stands as the most prevalent modified form of RNA in eukaryotes, pivotal in various biological processes such as regulating RNA stability, translation, and transcription. All members within the YT521-B homology (YTH) gene family are categorized as m6A reading proteins, capable of identifying and binding m6A modifications on RNA, thereby regulating RNA metabolism and functioning across diverse physiological processes. YTH domain-containing 2 (YTHDC2), identified as the latest member of the YTH family, has only recently started to emerge for its biological function. Numerous studies have underscored the significance of YTHDC2 in human physiology, highlighting its involvement in both tumor progression and non-tumor diseases. Consequently, this review aims to further elucidate the pathological mechanisms of YTHDC2 by summarizing its functions and roles in tumors and other diseases, with a particular focus on its downstream molecular targets and signaling pathways.
Topics: Humans; Adenosine; Neoplasms; RNA-Binding Proteins; Animals; Disease; Signal Transduction; RNA Helicases
PubMed: 38790013
DOI: 10.1186/s12967-024-05293-6