-
Frontiers in Cellular and Infection... 2023The polysaccharide capsule of plays a major role in virulence, adherence to epithelial cells, and overall survival of the bacterium in the human host. Galactose,...
BACKGROUND
The polysaccharide capsule of plays a major role in virulence, adherence to epithelial cells, and overall survival of the bacterium in the human host. Galactose, mannose, and N-acetylglucosamine (GlcNAc) are likely to be relevant for metabolization in the nasopharynx, while glucose is the primary carbon source in the blood. In this study, we aim to further the understanding of the influence of carbon sources on pneumococcal growth, capsule biosynthesis, and subsequent adherence potential.
METHODS
We tested the growth behavior of clinical wild-type and capsule knockout strains, using galactose, GlcNAc, mannose, and glucose as carbon source for growth. We measured capsule thickness and quantified capsule precursors by fluorescein isothiocyanate (FITC)-dextran exclusion assays and P-nuclear magnetic resonance measurements, respectively. We also performed epithelial adherence assays using Detroit 562 cells and performed a transcriptome analysis (RNA sequencing).
RESULTS
We observed a reduced growth in galactose, mannose, and GlcNAc compared to growth in glucose and found capsular size reductions in mannose and GlcNAc compared to galactose and glucose. Additionally, capsular precursor measurements of uridine diphosphate-(UDP)-glucose and UDP-galactose showed less accumulation of precursors in GlcNAc or mannose than in glucose and galactose, indicating a possible link with the received capsular thickness measurements. Epithelial adherence assays showed an increase in adherence potential for a pneumococcal strain, when grown in mannose compared to glucose. Finally, transcriptome analysis of four clinical isolates revealed not only strain specific but also common carbon source-specific gene expression.
CONCLUSION
Our findings may indicate a careful adaption of the lifestyle of according to the monosaccharides encountered in the respective human niche.
Topics: Humans; Streptococcus pneumoniae; Galactose; Carbon; Mannose; Glucose; Uridine Diphosphate; Bacterial Capsules
PubMed: 38094742
DOI: 10.3389/fcimb.2023.1279119 -
Nucleic Acids Research Nov 2023Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational...
Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that 7-deazaguanine-derivatives such as queuosine are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.
Topics: Anticodon; Escherichia coli; Eukaryota; Nucleoside Q; RNA; RNA, Transfer; Schizosaccharomyces; Sequence Analysis, RNA
PubMed: 37811872
DOI: 10.1093/nar/gkad826 -
Nature Communications Jul 2023Regional bias of N-methyladenosine (mA) mRNA modification avoiding splice site region, calls for an open hypothesis whether exon-intron boundary could affect mA...
Regional bias of N-methyladenosine (mA) mRNA modification avoiding splice site region, calls for an open hypothesis whether exon-intron boundary could affect mA deposition. By deep learning modeling, we find that exon-intron boundary represses a proportion (12% to 34%) of mA deposition at adjacent exons (~100 nt to splice site). Experiments validate that mA signal increases once the host gene does not undergo pre-mRNA splicing to produce the same mRNA. Inhibited mA sites have higher mA enhancers and lower mA silencers locally and show high heterogeneity at different exons genome-widely, with only a small proportion (12% to 15%) of exons showing strong inhibition, enabling more stable mRNAs and flexible protein coding. mA is majorly responsible for why mRNAs with more exons be more stable. Exon junction complex (EJC) only partially contributes to this exon-intron boundary mA inhibition in some short internal exons, highlighting additional factors yet to be identified.
Topics: Introns; RNA, Messenger; Half-Life; Exons; RNA Splicing; RNA Precursors
PubMed: 37443320
DOI: 10.1038/s41467-023-39897-1 -
Nature Dec 2023The group II intron ribonucleoprotein is an archetypal splicing system with numerous mechanistic parallels to the spliceosome, including excision of lariat introns....
The group II intron ribonucleoprotein is an archetypal splicing system with numerous mechanistic parallels to the spliceosome, including excision of lariat introns. Despite the importance of branching in RNA metabolism, structural understanding of this process has remained elusive. Here we present a comprehensive analysis of three single-particle cryogenic electron microscopy structures captured along the splicing pathway. They reveal the network of molecular interactions that specifies the branchpoint adenosine and positions key functional groups to catalyse lariat formation and coordinate exon ligation. The structures also reveal conformational rearrangements of the branch helix and the mechanism of splice site exchange that facilitate the transition from branching to ligation. These findings shed light on the evolution of splicing and highlight the conservation of structural components, catalytic mechanism and dynamical strategies retained through time in premessenger RNA splicing machines.
Topics: Adenosine; Biocatalysis; Cryoelectron Microscopy; Exons; Introns; Nucleic Acid Conformation; RNA Precursors; RNA Splice Sites; RNA Splicing
PubMed: 37993708
DOI: 10.1038/s41586-023-06746-6 -
Proceedings of the National Academy of... Jul 2023The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core...
The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.
Topics: Male; Humans; Heterogeneous-Nuclear Ribonucleoprotein Group F-H; RNA Precursors; RNA Splicing; RNA-Binding Proteins; Exons; Alternative Splicing; Prostatic Neoplasms; Proto-Oncogene Proteins p21(ras)
PubMed: 37399401
DOI: 10.1073/pnas.2220190120 -
BioRxiv : the Preprint Server For... Nov 2023Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are...
Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation. In , these inherited small RNAs have poly (UG) tails, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop novel assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and microRNAs and their precursors. We also detect substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in pollen, and piwi-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are localized to sperm cells, and we found that (), the plant homolog of Exportin-t, interacts genetically with Ψ and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that Exportin-t is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, Ψ has a conserved role in marking inherited small RNAs in the germline.
PubMed: 37398006
DOI: 10.1101/2023.05.27.542553 -
Nucleic Acids Research Nov 2023Alternative Polyadenylation (APA) is an emerging mechanism for dynamic changes in gene expression. Previously, we described widespread APA occurrence in introns during...
Alternative Polyadenylation (APA) is an emerging mechanism for dynamic changes in gene expression. Previously, we described widespread APA occurrence in introns during the DNA damage response (DDR). Here, we show that a DDR-activated APA event occurs in the first intron of CDKN1A, inducing an alternate last exon-containing lncRNA. We named this lncRNA SPUD (Selective Polyadenylation Upon DNA Damage). SPUD localizes to polysomes in the cytoplasm and is detectable as multiple isoforms in available high-throughput studies. SPUD has low abundance compared to the CDKN1A full-length isoform under non-stress conditions, and SPUD is induced in cancer and normal cells under a variety of DNA damaging conditions in part through p53. The RNA binding protein HuR binds to and promotes the stability of SPUD precursor RNA. SPUD induction increases p21 protein, but not mRNA levels, affecting p21 functions in cell-cycle, CDK2 expression and cell growth. Like CDKN1A full-length isoform, SPUD can bind two competitive p21 translational regulators, the inhibitor calreticulin and the activator CUGBP1; SPUD alters their association with CDKN1A full-length in a DDR-dependent manner, promoting CDKN1A translation. Together, these results show a new regulatory mechanism by which a lncRNA controls p21 expression post-transcriptionally, highlighting lncRNA relevance in DDR progression and cell-cycle.
Topics: Cell Cycle; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Polyadenylation; Protein Isoforms; RNA, Long Noncoding; Tumor Suppressor Protein p53; Humans; Cell Line, Tumor
PubMed: 37870464
DOI: 10.1093/nar/gkad899 -
Nature Cancer Dec 2023Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared...
Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared expression across malignant and normal cells. Here, high-density immunophenotyping of AML coupled with proteogenomics identified unique expression of a variety of antigens, including the RNA helicase U5 snRNP200, on the surface of AML cells but not on normal hematopoietic precursors and skewed Fc receptor distribution in the AML immune microenvironment. Cell membrane localization of U5 snRNP200 was linked to surface expression of the Fcγ receptor IIIA (FcγIIIA, also known as CD32A) and correlated with expression of interferon-regulated immune response genes. Anti-U5 snRNP200 antibodies engaging activating Fcγ receptors were efficacious across immunocompetent AML models and were augmented by combination with azacitidine. These data provide a roadmap of AML-associated antigens with Fc receptor distribution in AML and highlight the potential for targeting the AML cell surface using Fc-optimized therapeutics.
Topics: Humans; Antibodies, Monoclonal; Antigens, Surface; Leukemia, Myeloid, Acute; Receptors, Fc; Receptors, IgG; Ribonucleoproteins, Small Nuclear; Tumor Microenvironment
PubMed: 37872381
DOI: 10.1038/s43018-023-00656-2 -
Neurobiology of Disease Oct 2023G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to...
G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to achieve fully-myelinating cells. However, whether and how these two systems are related to each other is still unknown. Of interest, both factors are dysregulated in developing and adult brain diseases, including white matter injury and cancer, making the understanding of their reciprocal interactions of potential importance for identifying new targets and strategies for myelin repair. Here, by a combined pharmacological and biotechnological approach, we examined regulatory mechanisms linking WNT signaling to GPR17 expression in OLs. We first analyzed the relative expression of mRNAs encoding for GPR17 and the T cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF) transcription factors of the canonical WNT/β-CATENIN pathway, in PDGFRα and O4 OLs during mouse post-natal development. In O4 cells, Gpr17 mRNA level peaked at post-natal day 14 and then decreased concomitantly to the physiological uprise of WNT tone, as shown by increased Lef1 mRNA level. The link between WNT signaling and GPR17 expression was further reinforced in vitro in primary PDGFRα cells and in Oli-neu cells. High WNT tone impaired OL differentiation and drastically reduced GPR17 mRNA and protein levels. In Oli-neu cells, WNT/β-CATENIN activation repressed Gpr17 promoter activity through both putative WNT response elements (WRE) and upregulation of the inhibitor of DNA-binding protein 2 (Id2). We conclude that the WNT pathway influences OL maturation by repressing GPR17, which could have implications in pathologies characterized by dysregulations of the OL lineage including multiple sclerosis and oligodendroglioma.
Topics: Mice; Animals; Wnt Signaling Pathway; beta Catenin; Oligodendrocyte Precursor Cells; Receptor, Platelet-Derived Growth Factor alpha; Nerve Tissue Proteins; Receptors, G-Protein-Coupled; Cell Differentiation; Oligodendroglia; RNA, Messenger
PubMed: 37783234
DOI: 10.1016/j.nbd.2023.106315 -
Cell Reports Oct 2023Pre-mRNA splicing is surveilled at different stages by quality control (QC) mechanisms. The leukemia-associated DExH-box family helicase hDHX15/scPrp43 is known to...
Pre-mRNA splicing is surveilled at different stages by quality control (QC) mechanisms. The leukemia-associated DExH-box family helicase hDHX15/scPrp43 is known to disassemble spliceosomes after splicing. Here, using rapid protein depletion and analysis of nascent and mature RNA to enrich for direct effects, we identify a widespread splicing QC function for DHX15 in human cells, consistent with recent in vitro studies. We find that suboptimal introns with weak splice sites, multiple branch points, and cryptic introns are repressed by DHX15, suggesting a general role in promoting splicing fidelity. We identify SUGP1 as a G-patch factor that activates DHX15's splicing QC function. This interaction is dependent on both DHX15's ATPase activity and on SUGP1's U2AF ligand motif (ULM) domain. Together, our results support a model in which DHX15 plays a major role in splicing QC when recruited and activated by SUGP1.
Topics: Humans; RNA; RNA Helicases; RNA Precursors; RNA Splicing; RNA Splicing Factors; Spliceosomes; Splicing Factor U2AF
PubMed: 37805921
DOI: 10.1016/j.celrep.2023.113223