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The Journal of Physical Chemistry. B Jun 2024Microscopic understanding of protein-RNA interactions is important for different biological activities, such as RNA transport, translation, splicing, silencing, etc....
Microscopic understanding of protein-RNA interactions is important for different biological activities, such as RNA transport, translation, splicing, silencing, etc. Polyadenine (Poly(A)) binding proteins (PABPs) make up a class of regulatory proteins that play critical roles in protecting the poly(A) tails of cellular mRNAs from nuclease degradation. In this work, we performed molecular dynamics simulations to investigate the conformational modifications of human PABP protein and poly(A) RNA that occur during complexation. It is demonstrated that the intermediate linker domain of the protein transforms from a disordered coil-like structure to a helical form during the recognition process, leading to the formation of the complex. On the other hand, disordered collapsed coil-like RNA on complexation has been found to transform into a rigid extended conformation. Importantly, the binding free energy calculation showed that the thermodynamic stability of the complex is primarily guided by favorable hydrophobic interactions between the protein and the RNA.
PubMed: 38941243
DOI: 10.1021/acs.jpcb.4c00704 -
Frontiers in Genetics 2024Breast cancer (BC), as a highly prevalent malignant tumor worldwide, is still unclear in its pathogenesis and has poor therapeutic outcomes. Alternative polyadenylation... (Review)
Review
Breast cancer (BC), as a highly prevalent malignant tumor worldwide, is still unclear in its pathogenesis and has poor therapeutic outcomes. Alternative polyadenylation (APA) is a post-transcriptional regulatory mechanism widely found in eukaryotes. Precursor mRNA (pre-mRNA) undergoes the APA process to generate multiple mRNA isoforms with different coding regions or 3'UTRs, thereby greatly increasing the diversity and complexity of the eukaryotic transcriptome and proteome. Studies have shown that APA is involved in the progression of various diseases, including cancer, and plays a crucial role. Therefore, clarifying the biological mechanisms of APA and its regulators in breast cancer will help to comprehensively understand the pathogenesis of breast cancer and provide new ideas for its prevention and treatment.
PubMed: 38939531
DOI: 10.3389/fgene.2024.1377275 -
Vaccines Jun 2024Current mRNA vaccines are mainly administered via intramuscular injection, which induces good systemic immunity but limited mucosal immunity. Achieving mucosal immunity... (Review)
Review
Current mRNA vaccines are mainly administered via intramuscular injection, which induces good systemic immunity but limited mucosal immunity. Achieving mucosal immunity through mRNA vaccination could diminish pathogen replication at the entry site and reduce interhuman transmission. However, delivering mRNA vaccines to mucosae faces challenges like mRNA degradation, poor entry into cells, and reactogenicity. Encapsulating mRNA in extracellular vesicles may protect the mRNA and reduce reactogenicity, making mucosal mRNA vaccines possible. Plant-derived extracellular vesicles from edible fruits have been investigated as mRNA carriers. Studies in animals show that mRNA vehiculated in orange-derived extracellular vesicles can elicit both systemic and mucosal immune responses when administered by the oral, nasal, or intramuscular routes. Once lyophilized, these products show remarkable stability. The optimization of mRNA to improve translation efficiency, immunogenicity, reactogenicity, and stability can be obtained through adjustments of the 5'cap region, poly-A tail, codons selection, and the use of nucleoside analogues. Recent studies have also proposed self-amplifying RNA vaccines containing an RNA polymerase as well as circular mRNA constructs. Data from parenterally primed animals demonstrate the efficacy of nasal immunization with non-adjuvanted protein, and studies in humans indicate that the combination of a parenteral vaccine with the natural exposure of mucosae to the same antigen provides protection and reduces transmission. Hence, mucosal mRNA vaccination would be beneficial at least in organisms pre-treated with parenteral vaccines. This practice could have wide applications for the treatment of infectious diseases.
PubMed: 38932399
DOI: 10.3390/vaccines12060670 -
Viruses May 2024Hepatitis E virus (HEV) can cause self-limiting acute and chronic hepatitis infections, particularly in immunocompromised individuals. In developing countries, HEV is...
The Full-Genome Analysis and Generation of an Infectious cDNA Clone of a Genotype 6 Hepatitis E Virus Variant Obtained from a Japanese Wild Boar: In Vitro Cultivation in Human Cell Lines.
Hepatitis E virus (HEV) can cause self-limiting acute and chronic hepatitis infections, particularly in immunocompromised individuals. In developing countries, HEV is mainly transmitted via drinking contaminated water, whereas zoonotic transmission dominates the route of infection in developed countries, including Japan. Pigs are an important reservoir for HEV infection. Wild boars, which share the same genus and species as domestic pigs, are also an HEV reservoir. During our nationwide study of HEV infection in wild boar populations in Japan, a genotype 6 (HEV-6) strain, wbJHG_23, was isolated in Hyogo Prefecture in 2023. The genomic length was 7244 nucleotides, excluding the poly(A) tract. The wbJHG_23 strain exhibited the highest nucleotide identity throughout its genome with two previously reported HEV-6 strains (80.3-80.9%). Conversely, it displayed lower similarity (73.3-78.1%) with the HEV-1-5, HEV-7, and HEV-8 strains, indicating that, although closely related, the wbJHG_23 strain differs significantly from the reported HEV-6 strains and might represent a novel subtype. The wbJHG_23 strain successfully infected the human-derived cancer cell lines, PLC/PRF/5 and A549 1-1H8 cells, suggesting that HEV-6 has the potential for zoonotic infection. An infectious cDNA clone was constructed using a reverse genetics system, and a cell culture system supporting the efficient propagation of the HEV-6 strain was established, providing important tools for further studies on this genotype. Using this cell culture system, we evaluated the sensitivity of the wbJHG_23 strain to ribavirin treatment. Its good response to this treatment suggested that it could be used to treat human infections caused by HEV-6.
Topics: Animals; Cell Line; DNA, Complementary; Genome, Viral; Genotype; Hepatitis E; Hepatitis E virus; Japan; Phylogeny; RNA, Viral; Sus scrofa; Swine; Swine Diseases
PubMed: 38932135
DOI: 10.3390/v16060842 -
Viruses May 2024Hepatitis delta virus (HDV), an RNA virus with two forms of the delta antigen (HDAg), relies on hepatitis B virus (HBV) for envelope proteins essential for hepatocyte...
Hepatitis delta virus (HDV), an RNA virus with two forms of the delta antigen (HDAg), relies on hepatitis B virus (HBV) for envelope proteins essential for hepatocyte entry. Hepatocellular carcinoma (HCC) ranks third in global cancer deaths, yet HDV's involvement remains uncertain. Among 300 HBV-associated HCC serum samples from Taiwan's National Health Research Institutes, 2.7% (8/300) tested anti-HDV positive, with 62.7% (5/8) of these also HDV RNA positive. Genotyping revealed HDV-2 in one sample, HDV-4 in two, and two samples showed mixed HDV-2/HDV-4 infection with RNA recombination. A mixed-genotype infection revealed novel mutations at the polyadenylation signal, coinciding with the ochre termination codon for the L-HDAg. To delve deeper into the possible oncogenic properties of HDV-2, the predominant genotype in Taiwan, which was previously thought to be less associated with severe disease outcomes, an HDV-2 cDNA clone was isolated from HCC for study. It demonstrated a replication level reaching up to 74% of that observed for a widely used HDV-1 strain in transfected cultured cells. Surprisingly, both forms of HDV-2 HDAg promoted cell migration and invasion, affecting the rearrangement of actin cytoskeleton and the expression of epithelial-mesenchymal transition markers. In summary, this study underscores the prevalence of HDV-2, HDV-4, and their mixed infections in HCC, highlighting the genetic diversity in HCC as well as the potential role of both forms of the HDAg in HCC oncogenesis.
Topics: Carcinoma, Hepatocellular; Hepatitis Delta Virus; Humans; Liver Neoplasms; Genetic Variation; Genotype; Male; Middle Aged; Carcinogenesis; Female; Taiwan; Evolution, Molecular; Virus Replication; Phylogeny; RNA, Viral; Hepatitis D; Aged; Hepatitis B virus
PubMed: 38932110
DOI: 10.3390/v16060817 -
Cell Jun 2024Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery....
Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.
PubMed: 38925112
DOI: 10.1016/j.cell.2024.06.005 -
Experimental Gerontology Jun 2024This study aimed to conduct an umbrella review of meta-analyses to synthesize the existing evidence regarding the relationship between vitamin D receptor (VDR)... (Review)
Review
This study aimed to conduct an umbrella review of meta-analyses to synthesize the existing evidence regarding the relationship between vitamin D receptor (VDR) polymorphism and breast cancer (BC) risk. A comprehensive search was performed across multiple databases, including Embase, PubMed, Scopus, the Cochrane Database of Systematic Reviews, and the Web of Science. The investigation included 17 meta-analyses for the BsmI polymorphism, 6 for the Cdx2 polymorphism, and 6 for the Poly (A) polymorphism. Among the 119 datasets analyzed, only 6 (5 %) reported statistically significant outcomes (p < 0.05), comprising 2 comparisons for VDR BsmI polymorphism (3 %), 1 for VDR Cdx-2 polymorphism (4 %), and 3 for VDR Poly (A) polymorphism (14 %), across various genetic models. Notably, significant heterogeneity was observed in 82 comparisons, and publication bias was detected in 16 comparisons. Furthermore, a substantial proportion (86 %) of the included studies exhibited critically low methodological quality. In conclusion, our findings suggest that VDR polymorphism (BsmI, Cdx-2, and Poly (A)) is not strongly associated with BC risk in the general population.
PubMed: 38917941
DOI: 10.1016/j.exger.2024.112502 -
Proceedings of the National Academy of... Jul 2024The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO)...
The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO) specifically targeting U4 snRNA to achieve functional knockdown of U4 snRNP in HeLa cells. Our results showed that this knockdown resulted in global intronic premature cleavage and polyadenylation (PCPA) events, comparable to the effects observed with U1 AMO treatment, as demonstrated by mRNA 3'-seq analysis. Furthermore, our study suggested that this may be a common phenomenon in both human and mouse cell lines. Additionally, we showed that U4 AMO treatment disrupted transcription elongation, as evidenced by chromatin immunoprecipitation sequencing (ChIP-seq) analysis for RNAPII. Collectively, our results identified a unique role for U4 snRNP in the inhibition of PCPA and indicated a model wherein splicing intrinsically inhibits intronic cleavage and polyadenylation in the context of cotranscriptional mRNA processing.
Topics: Humans; Polyadenylation; RNA Precursors; HeLa Cells; Mice; Animals; RNA Splicing; Ribonucleoprotein, U4-U6 Small Nuclear; RNA, Messenger; Introns
PubMed: 38917004
DOI: 10.1073/pnas.2406710121 -
BioRxiv : the Preprint Server For... Jun 2024Zinc knuckle (ZCCHC) motif-containing proteins are present in unicellular and multicellular eukaryotes and most ZCCHC proteins with known functions participate in the...
Zinc knuckle (ZCCHC) motif-containing proteins are present in unicellular and multicellular eukaryotes and most ZCCHC proteins with known functions participate in the metabolism of various classes of RNA, such as mRNAs, ribosomal RNAs, and microRNAs. The Arabidopsis () genome encodes 69 ZCCHC-containing proteins, but the functions of most remain unclear. One of these proteins is CAX-INTERACTING PROTEIN 4 (CXIP4), which has been classified as a PTHR31437 family member, along with human SREK1-interacting protein 1 (SREK1IP1), which is thought to function in pre-mRNA splicing and RNA methylation. Metazoan SREK1IP1-like and plant CXIP4-like proteins only share a ZCCHC motif, and their functions remain almost entirely unknown. We studied two loss-of-function alleles of Arabidopsis , the first mutations in PTHR31437 family genes described to date: is likely null and shows early lethality, and is hypomorphic and viable, with pleiotropic morphological defects. The mutant exhibited deregulation of defense genes and upregulation of transcription factor encoding genes, some of which might explain its developmental defects. This mutant also exhibited increased intron retention events, and the specific functions of misspliced genes, such as those involved in "gene silencing by DNA methylation" and "mRNA polyadenylation factor" suggest that CXIP4 has additional functions. The CXIP4 protein localizes to the nucleus in a pattern resembling nuclear speckles, which are rich in splicing factors. Therefore, is required for plant survival and proper development, and mRNA maturation.
PubMed: 38915646
DOI: 10.1101/2024.06.06.597795 -
BioRxiv : the Preprint Server For... Jun 2024Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The...
Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIB's role in termination, we performed GRO-seq analyses in mutant (TFIIB ) and the isogenic wild type (TFIIB ) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIB mutant compared to the TFIIB cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIB and TFIIB cells. TFIIB purified from the chromatin fraction of TFIIB cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIB mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.
PubMed: 38915573
DOI: 10.1101/2024.02.22.581640