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ACS Central Science Feb 2023The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic...
The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic acids fail to efficiently cross cell or endosomal membranes. The impact of biologic-based therapeutics would expand exponentially if proteins and nucleic acids could reliably evade endosomal degradation, escape endosomal vesicles, and remain functional. Using the cell-permeant mini-protein ZF5.3, here we report the efficient nuclear delivery of functional Methyl-CpG-binding-protein 2 (MeCP2), a transcriptional regulator whose mutation causes Rett syndrome (RTT). We report that ZF-MeCP2, a conjugate of ZF5.3 and MeCP2(Δaa13-71, 313-484), binds DNA in a methylation-dependent manner , and reaches the nucleus of model cell lines intact to achieve an average concentration of 700 nM. When delivered to live cells, ZF-MeCP2 engages the NCoR/SMRT corepressor complex, selectively represses transcription from methylated promoters, and colocalizes with heterochromatin in mouse primary cortical neurons. We also report that efficient nuclear delivery of ZF-MeCP2 relies on an endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-MeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of biologic-based therapeutics.
PubMed: 36844491
DOI: 10.1021/acscentsci.2c01226 -
Frontiers in Endocrinology 2023Extensive morphological, biochemical, and cellular changes occur during anuran metamorphosis, which is triggered by a single hormone, thyroid hormone (TH). The function...
INTRODUCTION
Extensive morphological, biochemical, and cellular changes occur during anuran metamorphosis, which is triggered by a single hormone, thyroid hormone (TH). The function of TH is mainly mediated through thyroid receptor (TR) by binding to the specific thyroid response elements (TREs) of direct response genes, in turn regulating the downstream genes in the cascade. The remodeling of dorsal skeletal muscle during anuran metamorphosis provides the perfect model to identify the immediate early and direct response genes that are important during apoptosis, proliferation, and differentiation of the muscle.
METHODS
In our current study, we performed Illumina sequencing combined with single-molecule real-time (SMRT) sequencing in the dorsal muscle of after TH, cycloheximide (CHX), and TH_CHX treatment.
RESULTS AND DISCUSSION
We first identified 1,245 differentially expressed transcripts (DETs) after TH exposure, many of which were involved in DNA replication, protein processing in the endoplasmic reticulum, cell cycle, apoptosis, p53 signaling pathway, and protein digestion and absorption. In the comparison of the TH group vs. control group and TH_CHX group vs. CHX group overlapping gene, 39 upregulated and 6 downregulated genes were identified as the TH directly induced genes. Further analysis indicated that AGGTCAnnTnAGGTCA is the optimal target sequence of target genes for TR/RXR heterodimers in . Future investigations on the function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian dorsal muscle remodeling. These full-length and high-quality transcriptomes in this study also provide an important foundation for future studies in M. fissipes metamorphosis.
Topics: Animals; Receptors, Thyroid Hormone; Thyroid Hormones; Anura; Metamorphosis, Biological; Muscles
PubMed: 36817577
DOI: 10.3389/fendo.2023.1099130 -
Heliyon Feb 2023Common vetch is an important leguminous forage for both livestock fodder and green manure and has a tremendous latent capacity in a sustainable agroecosystem. In the...
Common vetch is an important leguminous forage for both livestock fodder and green manure and has a tremendous latent capacity in a sustainable agroecosystem. In the present study, a comprehensive transcriptome analysis of the aboveground leaves and underground roots of common vetch under multiple abiotic stress treatments, including NaCl, drought, cold, and cold drought, was performed using hybrid-sequencing technology, i. e. single-molecule real-time sequencing technology (SMRT) and supplemented by next-generation sequencing (NGS) technology. A total of 485,038 reads of insert (ROIs) with a mean length of 2606 bp and 228,261 full-length nonchimeric (FLNC) reads were generated. After deduplication, 39,709 transcripts were generated. Of these transcripts, we identified 1059 alternative splicing (AS) events, 17,227 simple sequence repeats (SSRs), and 1647 putative transcription factors (TFs). Furthermore, 640 candidates long noncoding RNAs (lncRNAs) and 28,256 complete coding sequences (CDSs) were identified. In gene annotation analyses, a total of 38,826 transcripts (97.78%) were annotated in eight public databases. Finally, seven multiple abiotic stress-responsive candidate genes were obtained through gene expression, annotation information, and protein-protein interaction (PPI) networks. Our research not only enriched the structural information of FL transcripts in common vetch, but also provided useful information for exploring the molecular mechanism of multiple abiotic stress tolerance between aboveground and underground tissues in common vetch and related legumes.
PubMed: 36816321
DOI: 10.1016/j.heliyon.2023.e13536 -
Molecular Oncology Jul 2023Oestrogen receptor-alpha (ERα) positivity is intimately associated with the development of hormone-dependent breast cancers. A major challenge in the treatment of these...
Oestrogen receptor-alpha (ERα) positivity is intimately associated with the development of hormone-dependent breast cancers. A major challenge in the treatment of these cancers is to understand and overcome the mechanisms of endocrine resistance. Recently, two distinct translation programmes using specific transfer RNA (tRNA) repertoires and codon usage frequencies were evidenced during cell proliferation and differentiation. Considering the phenotype switch of cancer cells to more proliferating and less-differentiated states, we can speculate that the changes in the tRNA pool and codon usage that likely occur make the ERα coding sequence no longer adapted, impacting translational rate, co-translational folding and the resulting functional properties of the protein. To verify this hypothesis, we generated an ERα synonymous coding sequence whose codon usage was optimized to the frequencies observed in genes expressed specifically in proliferating cells and then investigated the functional properties of the encoded receptor. We demonstrate that such a codon adaptation restores ERα activities to levels observed in differentiated cells, including: (a) an enhanced contribution exerted by transactivation function 1 (AF1) in ERα transcriptional activity; (b) enhanced interactions with nuclear receptor corepressor 1 and 2 [NCoR1 and NCoR2 (also known as SMRT) respectively], promoting repressive capability; and (c) reduced interactions with SRC proto-oncogene, non-receptor tyrosine kinase (Src) and phosphoinositide 3-kinase (PI3K) p85 kinases, inhibiting MAPK and AKT signalling pathway.
Topics: Receptors, Estrogen; Estrogen Receptor alpha; Phosphatidylinositol 3-Kinases; Silent Mutation; Cell Line, Tumor; Codon; Neoplasms
PubMed: 36808875
DOI: 10.1002/1878-0261.13399 -
BMC Genomics Jan 2023Tripsacum dactyloides (2n = 4x = 72) and Zea perennis (2n = 4x = 40) are tertiary gene pools of Zea mays L. and exhibit many abiotic adaptations absent...
Integrated single-molecule real-time sequencing and RNA sequencing reveal the molecular mechanisms of salt tolerance in a novel synthesized polyploid genetic bridge between maize and its wild relatives.
BACKGROUND
Tripsacum dactyloides (2n = 4x = 72) and Zea perennis (2n = 4x = 40) are tertiary gene pools of Zea mays L. and exhibit many abiotic adaptations absent in modern maize, especially salt tolerance. A previously reported allopolyploid (hereafter referred to as MTP, 2n = 74) synthesized using Zea mays, Tripsacum dactyloides, and Zea perennis has even stronger salt tolerance than Z. perennis and T. dactyloides. This allopolyploid will be a powerful genetic bridge for the genetic improvement of maize. However, the molecular mechanisms underlying its salt tolerance, as well as the key genes involved in regulating its salt tolerance, remain unclear.
RESULTS
Single-molecule real-time sequencing and RNA sequencing were used to identify the genes involved in salt tolerance and reveal the underlying molecular mechanisms. Based on the SMRT-seq results, we obtained 227,375 reference unigenes with an average length of 2300 bp; most of the unigenes were annotated to Z. mays sequences (76.5%) in the NR database. Moreover, a total of 484 and 1053 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Functional enrichment analysis of DEGs revealed that multiple pathways responded to salt stress, including "Flavonoid biosynthesis," "Oxidoreductase activity," and "Plant hormone signal transduction" in the leaves and roots, and "Iron ion binding," "Acetyl-CoA carboxylase activity," and "Serine-type carboxypeptidase activity" in the roots. Transcription factors, such as those in the WRKY, B3-ARF, and bHLH families, and cytokinin negatively regulators negatively regulated the salt stress response. According to the results of the short time series-expression miner analysis, proteins involved in "Spliceosome" and "MAPK signal pathway" dynamically responded to salt stress as salinity changed. Protein-protein interaction analysis revealed that heat shock proteins play a role in the large interaction network regulating salt tolerance.
CONCLUSIONS
Our results reveal the molecular mechanism underlying the regulation of MTP in the response to salt stress and abundant salt-tolerance-related unigenes. These findings will aid the retrieval of lost alleles in modern maize and provide a new approach for using T. dactyloides and Z. perennis to improve maize.
Topics: Gene Expression Regulation, Plant; Poaceae; Polyploidy; Salt Tolerance; Sequence Analysis, RNA; Zea mays
PubMed: 36717785
DOI: 10.1186/s12864-023-09148-0 -
Frontiers in Genetics 2022Dollar spot is one of the most damaging diseases in turfgrass, reducing its quality and playability. Two species, and (formerly ) have been reported so far in the...
Dollar spot is one of the most damaging diseases in turfgrass, reducing its quality and playability. Two species, and (formerly ) have been reported so far in the United States To study the genome, two isolates H2 and H3, sampled from seashore paspalum in Hawaii in 2019 were sequenced Illumina paired-end sequencing by synthesis technology and PacBio SMRT sequencing. Both isolates were identified as . aff. , a novel species in the United States Using short and long reads, . aff. H3 contained 193 contigs with 48.6 Mbp and presented the most completed assembly and annotation among species. Out of the 13,428 protein models from AUGUSTUS, 349 cytoplasmic effectors and 13 apoplastic effectors were identified by EffectorP. To further decipher pathogenicity, . aff. genomes (H2 and H3), as well as available (LWC-10 and HRI11), (DRR09 and RB-19) genomes were screened for fifty-four pathogenesis determinants, previously identified in . Seventeen orthologs of pathogenicity genes have been identified in species involved in oxalic acid production (, ), mitogen-activated protein kinase cascade (, , ), appressorium formation (, , , , ) and glycolytic pathway (). Within these genes, 366 species-specific SNPs were recorded between species; twenty-eight were non-synonymous and non-conservative. The predicted protein structure of six of these genes showed superimposition of the models among spp. The genomic variations revealed here could potentially lead to differences in pathogenesis and other physiological functions among species.
PubMed: 36685867
DOI: 10.3389/fgene.2022.1033437 -
Frontiers in Molecular Biosciences 2022Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with...
Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with each nucleosome consisting of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. Magic angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy can yield unique insights into histone structure and dynamics in condensed nucleosomes and nucleosome arrays representative of chromatin at physiological concentrations. Recently we used J-coupling-based solid-state NMR methods to investigate with residue-specific resolution the conformational dynamics of histone H3 N-terminal tails in 16-mer nucleosome arrays containing 15, 30 or 60 bp DNA linkers. Here, we probe the H3 core domain in the 16-mer arrays as a function of DNA linker length dipolar coupling-based H-detected solid-state NMR techniques. Specifically, we established nearly complete assignments of backbone chemical shifts for H3 core residues in arrays with 15-60 bp DNA linkers reconstituted with H,C,N-labeled H3. Overall, these chemical shifts were similar irrespective of the DNA linker length indicating no major changes in H3 core conformation. Notably, however, multiple residues at the H3-nucleosomal DNA interface in arrays with 15 bp DNA linkers exhibited relatively pronounced differences in chemical shifts and line broadening compared to arrays with 30 and 60 bp linkers. These findings are consistent with increased heterogeneity in nucleosome packing and structural strain within arrays containing short DNA linkers that likely leads to side-chains of these interfacial residues experiencing alternate conformations or shifts in their rotamer populations relative to arrays with the longer DNA linkers.
PubMed: 36660422
DOI: 10.3389/fmolb.2022.1106588 -
BMC Genomics Jan 2023Pagiophloeus tsushimanus (Coleoptera: Curculionidae), an emerging forest pest exclusively infesting camphor trees, has recently caused severe ecological and economic...
Tolerance to dietary linalool primarily involves co-expression of cytochrome P450s and cuticular proteins in Pagiophloeus tsushimanus (Coleoptera: Curculionidae) larvae using SMRT sequencing and RNA-seq.
BACKGROUND
Pagiophloeus tsushimanus (Coleoptera: Curculionidae), an emerging forest pest exclusively infesting camphor trees, has recently caused severe ecological and economic damage in localized areas in China. Its population outbreak depends largely on the capacity to overcome the pressure of terpenoid-derived metabolites (e.g. linalool) from camphor trees. At present, the molecular basis of physiological adaptation of P. tsushimanus to dietary linalool is poorly understood, and there is no available reference genome or transcriptome.
RESULTS
Herein, we constructed the transcriptome profiling of P. tsushimanus larvae reared on linalool-infused diets using RNA sequencing and single-molecule real-time sequencing. A total of 20,325 high-quality full-length transcripts were identified as a reference transcriptome, of which 14,492 protein-coding transcripts including 130 transcription factors (TFs), and 5561 long non-coding RNAs (lncRNAs) were detected. Also, 30 alternative splicing events and 8049 simple sequence repeats were captured. Gene ontology enrichment of differential expressed transcripts revealed that overall up-regulation of both cytochrome P450s (CYP450s) and cuticular proteins (CPs), was the primary response characteristic against dietary linalool. Other physiological effects possibly caused by linalool exposure, such as increase in Reactive Oxygen Species (ROS) and hormetic stimulation, were compensated by a handful of induced genes encoding antioxidases, heat shock proteins (HSPs), juvenile hormone (JH) epoxide hydrolases, and digestive enzymes. Additionally, based on co-expression networks analysis, a diverse array of hub lncRNAs and TFs co-expressed with CYP450s and CPs were screened as the potential gene regulators. Temporal expression of candidate transcripts determined by quantitative real-time PCR also indicated a cooperative relationship between the inductions of CYP450s and CPs upon exposure to linalool.
CONCLUSIONS
Our present study provides an important transcriptome resource of P. tsushimanus, and lays a valuable foundation for understanding how this specialist pest copes with chemical challenges in its specific host environments.
Topics: Animals; RNA-Seq; Coleoptera; Weevils; Larva; RNA, Long Noncoding; Gene Expression Profiling; Transcriptome; Cytochrome P-450 Enzyme System; Diet
PubMed: 36658477
DOI: 10.1186/s12864-023-09117-7 -
International Journal of Molecular... Dec 2022Rickett's big-footed bat, , which belongs to the family Vespertilionida, is the only known piscivorous bat in East Asia. Accurate whole genome and transcriptome...
Rickett's big-footed bat, , which belongs to the family Vespertilionida, is the only known piscivorous bat in East Asia. Accurate whole genome and transcriptome annotations are essential for the study of bat biological evolution. The lack of a whole genome for has limited our understanding of the molecular mechanisms underlying the species' evolution, echolocation, and immune response. In the present work, we sequenced the entire transcriptome using error-corrected PacBio single-molecule real-time (SMRT) data. Then, a total of 40 GB of subreads were generated, including 29,991 full-length non-chimeric (FLNC) sequences. After correction by Illumina short reads and de-redundancy, we obtained 26,717 error-corrected isoforms with an average length of 3018.91 bp and an N50 length of 3447 bp. A total of 1528 alternative splicing (AS) events were detected by transcriptome structural analysis. Furthermore, 1032 putative transcription factors (TFs) were identified, with additional identification of several long non-coding RNAs (lncRNAs) with high confidence. Moreover, several key genes, including , , , and were also identified as being associated with metabolism, immunity, nervous system processes, and auditory perception. A multitude of pattern recognition receptors was identified, including NLR, RLR, SRCR, the antiviral molecule IRF3, and the IFN receptor subunit IFNAR1. High-quality reference genomes at the transcriptome level may be used to quantify gene or transcript expression, evaluate alternative splicing levels, identify novel transcripts, and enhance genome annotation in bats.
Topics: Animals; Transcriptome; Chiroptera; Gene Expression Profiling; Alternative Splicing; Protein Isoforms; RNA, Long Noncoding
PubMed: 36613506
DOI: 10.3390/ijms24010062 -
Nucleic Acids Research Feb 2023The Th2 cytokine interleukin 4 (IL4) promotes macrophage differentiation into alternative subtypes and plays important roles in physiology, in metabolic and inflammatory...
The Th2 cytokine interleukin 4 (IL4) promotes macrophage differentiation into alternative subtypes and plays important roles in physiology, in metabolic and inflammatory diseases, in cancer and in tissue regeneration. While the regulatory transcription factor networks governing IL4 signaling are already well-characterized, it is currently less understood which transcriptional coregulators are involved and how they operate mechanistically. In this study, we discover that G protein pathway suppressor 2 (GPS2), a core subunit of the HDAC3 corepressor complex assembled by SMRT and NCOR, represses IL4-dependent enhancer activation in mouse macrophages. Our genome-wide and gene-specific characterization revealed that, instead of directly repressing STAT6, chromatin-bound GPS2 cooperates with SMRT and NCOR to antagonize enhancer activation by lysine demethylase 1A (KDM1A, LSD1). Mechanistically, corepressor depletion increased KDM1A recruitment to enhancers linked to IL4-induced genes, accompanied by demethylation of the repressive histone marks H3K9me2/3 without affecting H3K4me1/2, the classic KDM1A substrates for demethylation in other cellular contexts. This in turn caused enhancer and gene activation already in the absence of IL4/STAT6 and sensitized the STAT6-dependent IL4 responsiveness of macrophages. Thus, our work identified with the antagonistic action of a GPS2-containing corepressor complex and the lysine demethylase KDM1A a hitherto unknown epigenetic corepressor-coactivator switching mechanism that governs alternative macrophage activation.
Topics: Animals; Mice; Co-Repressor Proteins; Histone Demethylases; Interleukin-4; Intracellular Signaling Peptides and Proteins; Lysine; Macrophage Activation
PubMed: 36610795
DOI: 10.1093/nar/gkac1230