scholarly journals Fine Tuning of Phosphorothioate Inclusion in 2′-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yoshitsugu Aoki ◽  
Cristina S. J. Rocha ◽  
Taavi Lehto ◽  
Shouta Miyatake ◽  
Henrik Johansson ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Exon Skipping ◽  
Treatment Method ◽  
Bone Cell ◽  
Fine Tuning ◽  
Nuclear Accumulation ◽  
Specific Cell ◽  
Reporter Cell ◽  
Cell Lineages ◽  
Proteomic Approach

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2′-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.

scholarly journals RALF1-FERONIA complex affects splicing dynamics to modulate stress responses and growth in plants

2020 ◽  
Vol 6 (21) ◽  
pp. eaaz1622 ◽  
Author(s):  
Long Wang ◽  
Tao Yang ◽  
Bingqian Wang ◽  
Qinlu Lin ◽  
Sirui Zhu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Rna Splicing ◽  
Rna Binding ◽  
Fine Tuning ◽  
Nuclear Accumulation ◽  
Nonsense Mediated Decay ◽  
Rapid Alkalinization Factor ◽  
Transcriptomic Changes ◽  
Mrna Binding ◽  
External Stimuli

The environmentally responsive signaling pathways that link global transcriptomic changes through alternative splicing (AS) to plant fitness remain unclear. Here, we found that the interaction of the extracellular rapid alkalinization FACTOR 1 (RALF1) peptide with its receptor FERONIA (FER) triggered a rapid and massive RNA AS response by interacting with and phosphorylating glycine-rich RNA binding protein7 (GRP7) to elevate GRP7 nuclear accumulation in Arabidopsis thaliana. FER-dependent GRP7 phosphorylation enhanced its mRNA binding ability and its association with the spliceosome component U1-70K to enable splice site selection, modulating dynamic AS. Genetic reversal of a RALF1-FER–dependent splicing target partly rescued mutants deficient in GRP7. AS of GRP7 itself induced nonsense-mediated decay feedback to the RALF1-FER-GRP7 module, fine-tuning stress responses, and cell growth. The RALF1-FER-GRP7 module provides a paradigm for regulatory mechanisms of RNA splicing in response to external stimuli.

scholarly journals Deficiency of Lipin2 Results in Enhanced NF-κB Signaling and Osteoclast Formation in RAW-D Murine Macrophages

2021 ◽  
Vol 22 (6) ◽  
pp. 2893
Author(s):  
Asami Watahiki ◽  
Seira Hoshikawa ◽  
Mitsuki Chiba ◽  
Hiroshi Egusa ◽  
Satoshi Fukumoto ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Inflammatory Responses ◽  
Osteoclast Formation ◽  
Fine Tuning ◽  
Nuclear Accumulation ◽  
Innate Immune Responses ◽  
Osteoclastic Resorption ◽  
Bone Disorder ◽  
Proinflammatory Responses ◽  
Potential Therapeutic Intervention

Lipin2 is a phosphatidate phosphatase that plays critical roles in fat homeostasis. Alterations in Lpin2, which encodes lipin2, cause the autoinflammatory bone disorder Majeed syndrome. Lipin2 limits lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. However, little is known about the precise molecular mechanisms underlying its anti-inflammatory function. In this study, we attempted to elucidate the molecular link between the loss of lipin2 function and autoinflammatory bone disorder. Using a Lpin2 knockout murine macrophage cell line, we showed that lipin2 deficiency enhances innate immune responses to LPS stimulation through excessive activation of the NF-κB signaling pathway, partly because of TAK1 signaling upregulation. Lipin2 depletion also enhanced RANKL-mediated osteoclastogenesis and osteoclastic resorption activity accompanied by NFATc1 dephosphorylation and increased nuclear accumulation. These results suggest that lipin2 suppresses the development of autoinflammatory bone disorder by fine-tuning proinflammatory responses and osteoclastogenesis in macrophages. Therefore, this study provides insights into the molecular pathogenesis of monogenic autoinflammatory bone disorders and presents a potential therapeutic intervention.

scholarly journals Physical cues of cell culture materials lead the direction of differentiation lineages of pluripotent stem cells

2015 ◽  
Vol 3 (41) ◽  
pp. 8032-8058 ◽  
Author(s):  
Akon Higuchi ◽  
Qing-Dong Ling ◽  
S. Suresh Kumar ◽  
Yung Chang ◽  
Abdullah A. Alarfaj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Pluripotent Stem Cells ◽  
Specific Cell ◽  
Cell Lineages ◽  
Physical Cues

Differentiation methods of hPSCs into specific cell lineages. Differentiation of hPSCsviaEB formation (types AB, A–D) or without EB formation (types E–H).

scholarly journals The L-type amino acid transporter LAT1 inhibits osteoclastogenesis and maintains bone homeostasis through the mTORC1 pathway

2019 ◽  
Vol 12 (589) ◽  
pp. eaaw3921
Author(s):  
Kakeru Ozaki ◽  
Takanori Yamada ◽  
Tetsuhiro Horie ◽  
Atsushi Ishizaki ◽  
Manami Hiraiwa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bone Loss ◽  
Glycogen Synthase ◽  
Amino Acid Transporter ◽  
Nuclear Accumulation ◽  
Specific Cell ◽  
Nuclear Factor ◽  
Bone Homeostasis ◽  
Mtorc1 Pathway ◽  
Acid Transporter

L-type amino acid transporter 1 (LAT1), which is encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types, contributing to the pathogenesis of cancer and neurological disorders. Amino acid substrates of LAT1 have a beneficial effect on bone health directly and indirectly, suggesting a potential role for LAT1 in bone homeostasis. Here, we identified LAT1 in osteoclasts as important for bone homeostasis. Slc7a5 expression was substantially reduced in osteoclasts in a mouse model of ovariectomy-induced osteoporosis. The osteoclast-specific deletion of Slc7a5 in mice led to osteoclast activation and bone loss in vivo, and Slc7a5 deficiency increased osteoclastogenesis in vitro. Loss of Slc7a5 impaired activation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway in osteoclasts, whereas genetic activation of mTORC1 corrected the enhanced osteoclastogenesis and bone loss in Slc7a5-deficient mice. Last, Slc7a5 deficiency increased the expression of nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1) and the nuclear accumulation of NFATc1, a master regulator of osteoclast function, possibly through the canonical nuclear factor κB pathway and the Akt–glycogen synthase kinase 3β signaling axis, respectively. These findings suggest that the LAT1-mTORC1 axis plays a pivotal role in bone resorption and bone homeostasis by modulating NFATc1 in osteoclasts, thereby providing a molecular connection between amino acid intake and skeletal integrity.

3-Iodothyronamine Induces Diverse Signaling Effects at Different Aminergic and Non-Aminergic G-Protein Coupled Receptors

2019 ◽  
Vol 128 (06/07) ◽  
pp. 395-400 ◽  
Author(s):  
Heike Biebermann ◽  
Gunnar Kleinau
Keyword(s):  
Body Temperature ◽  
G Protein ◽  
Intracellular Signaling ◽  
Serotonin Receptor ◽  
Fine Tuning ◽  
Specific Cell ◽  
Knock Out ◽  
Alpha2 Adrenoceptor ◽  
Knock Out Mice ◽  
G Protein Coupled

AbstractThe thyroid hormone metabolite 3-iodothyronamine (3-T1AM) exerts diverse physiological reactions such as a decrease of body temperature, and negative inotropic and chronotropic effects. This observed pleomorphic effect in physiology can be barely explained by interaction with only one target protein such as the trace-amine receptor 1 (TAAR1), a class A G-protein coupled receptor (GPCR). Moreover, Taar1 knock-out mice still react to 3-T1AM through physiological responses with a rapid decrease in body temperature. These facts propelled our group and others to search for further targets for this molecule.The group of TAARs evolved early in evolution and, according to sequence similarities, they are closely related to adrenoceptors and other aminergic receptors. Therefore, several of these receptors were characterized by their potential to interplay with 3-T1AM. Indeed, 3-T1AM acts as a positive allosteric modulator on the beta2-adrenoceptor (ADRB2) and as a biased agonist on the serotonin receptor 1B (5HT1b) and the alpha2-adrenoceptor (ADRA2A). In addition, 3-T1AM was reported to be a weak antagonist at a non-aminergic muscarinic receptor (M3).These findings impressively reflect that such trace amines can unselectively and simultaneously function at different receptors expressed by one cell or at different tissues. In conclusion, the role of 3-T1AM is hypothesized to concert the fine-tuning of specific cell reactions by the accentuation of certain pathways dependent on distinct receptors. 3-T1AM acts as a regulator of signals by blocking, modulating, or inducing simultaneously distinct intracellular signaling cascades via different GPCRs.

scholarly journals High-resolution transcriptional profiling of Anopheles gambiae spermatogenesis reveals mechanisms of sex chromosome regulation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chrysanthi Taxiarchi ◽  
Nace Kranjc ◽  
Antonios Kriezis ◽  
Kyros Kyrou ◽  
Federica Bernardini ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Dosage Compensation ◽  
Transcriptional Repression ◽  
Sex Chromosome ◽  
Transcriptional Profiling ◽  
Differential Expression Analysis ◽  
Model Organisms ◽  
Specific Cell ◽  
Cell Lineages ◽  
Targeted Analysis

Abstract Although of high priority for the development of genetic tools to control malaria-transmitting mosquitoes, only a few germline-specific regulatory regions have been characterised to date and the presence of global regulatory mechanisms, such as dosage compensation and meiotic sex chromosome inactivation (MSCI), are mostly assumed from transcriptomic analyses of reproductive tissues or whole gonads. In such studies, samples include a significant portion of somatic tissues inevitably complicating the reconstruction of a defined transcriptional map of gametogenesis. By exploiting recent advances in transgenic technologies and gene editing tools, combined with fluorescence-activated cell sorting and RNA sequencing, we have separated four distinct cell lineages from the Anopheles gambiae male gonads: premeiotic, meiotic (primary and secondary spermatocytes) and postmeiotic. By comparing the overall expression levels of X-linked and autosomal genes across the four populations, we revealed a striking transcriptional repression of the X chromosome coincident with the meiotic phase, classifiable as MSCI, and highlighted genes that may evade silencing. In addition, chromosome-wide median expression ratios of the premeiotic population confirmed the absence of dosage compensation in the male germline. Applying differential expression analysis, we highlighted genes and transcript isoforms enriched at specific timepoints and reconstructed the expression dynamics of the main biological processes regulating the key stages of sperm development and maturation. We generated the first transcriptomic atlas of A. gambiae spermatogenesis that will expand the available toolbox for the genetic engineering of vector control technologies. We also describe an innovative and multidimensional approach to isolate specific cell lineages that can be used for the targeted analysis of other A. gambiae organs or transferred to other medically relevant species and model organisms.

scholarly journals Ontogenetic Survey of Histone Modifications in an Annelid

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Glenys Gibson ◽  
Corban Hart ◽  
Robyn Pierce ◽  
Vett Lloyd
Keyword(s):  
Gene Expression ◽  
Histone Modifications ◽  
Marine Invertebrates ◽  
Marine Invertebrate ◽  
Specific Cell ◽  
Early Cleavage ◽  
Cell Lineages ◽  
Common Time ◽  
Wide Range ◽  
Development And Differentiation

Histone modifications are widely recognized for their fundamental importance in regulating gene expression in embryonic development in a wide range of eukaryotes, but they have received relatively little attention in the development of marine invertebrates. We surveyed histone modifications throughout the development of a marine annelid, Polydora cornuta, to determine if modifications could be detected immunohistochemically and if there were characteristic changes in modifications throughout ontogeny (surveyed at representative stages from oocyte to adult). We found a common time of onset for three histone modifications in early cleavage (H3K14ac, H3K9me, and H3K4me2), some differences in the distribution of modifications among germ layers, differences in epifluorescence intensity in specific cell lineages suggesting that hyperacetylation (H3K14ac) and hypermethylation (H3K9me) occur during differentiation, and an overall decrease in the distribution of modifications from larvae to adults. Although preliminary, these results suggest that histone modifications are involved in activating early development and differentiation in a marine invertebrate.

scholarly journals Adaptation of Bacillus subtilis to growth at low temperature: a combined transcriptomic and proteomic appraisal

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 831-853 ◽  
Author(s):  
Ina Budde ◽  
Leif Steil ◽  
Christian Scharf ◽  
Uwe Völker ◽  
Erhard Bremer
Keyword(s):  
Bacillus Subtilis ◽  
Low Temperature ◽  
Response Regulator ◽  
Proteome Analysis ◽  
Atp Synthesis ◽  
Fine Tuning ◽  
Transcriptional Responses ◽  
Natural Habitats ◽  
Regulatory Circuit ◽  
Proteomic Approach

The soil bacterium Bacillus subtilis frequently encounters a reduction in temperature in its natural habitats. Here, a combined transcriptomic and proteomic approach has been used to analyse the adaptational responses of B. subtilis to low temperature. Propagation of B. subtilis in minimal medium at 15 °C triggered the induction of 279 genes and the repression of 301 genes in comparison to cells grown at 37 °C. The analysis thus revealed profound adjustments in the overall gene expression profile in chill-adapted cells. Important transcriptional changes in low-temperature-grown cells comprise the induction of the SigB-controlled general stress regulon, the induction of parts of the early sporulation regulons (SigF, SigE and SigG) and the induction of a regulatory circuit (RapA/PhrA and Opp) that is involved in the fine-tuning of the phosphorylation status of the Spo0A response regulator. The analysis of chill-stress-repressed genes revealed reductions in major catabolic (glycolysis, oxidative phosphorylation, ATP synthesis) and anabolic routes (biosynthesis of purines, pyrimidines, haem and fatty acids) that likely reflect the slower growth rates at low temperature. Low-temperature repression of part of the SigW regulon and of many genes with predicted functions in chemotaxis and motility was also noted. The proteome analysis of chill-adapted cells indicates a major contribution of post-transcriptional regulation phenomena in adaptation to low temperature. Comparative analysis of the previously reported transcriptional responses of cold-shocked B. subtilis cells with this data revealed that cold shock and growth in the cold constitute physiologically distinct phases of the adaptation of B. subtilis to low temperature.

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