OTUD1 is a deubiquitinating enzyme involved in many cellular processes including cancer and innate, immune signaling pathways. Here, we perform a proximity labeling-based interactome study that identifies OTUD1 largely present in the translation and RNA metabolism protein complexes. Biochemical analysis validates OTUD1 association with ribosome subunits, elongation factors and the E3 ubiquitin ligase ZNF598 but not with the translation initiation machinery. OTUD1 catalytic activity suppresses polyA triggered ribosome stalling through inhibition of ZNF598-mediated RPS10 ubiquitination and stimulates formation of polysomes. Finally, analysis of gene expression suggests that OTUD1 regulates the stability of rare codon rich mRNAs by antagonizing ZNF598.
BACKGROUND: Limited accessibility to intestinal epithelial tissue in wild animals and humans makes it challenging to study patterns of intestinal gene regulation, and hence to monitor physiological status and health in field conditions. To explore solutions to this limitation, we have used a noninvasive approach via fecal RNA-seq, for the quantification of gene expression markers in gastrointestinal cells of free-range primates and a forager human population. Thus, a combination of poly(A) mRNA enrichment and rRNA depletion methods was used in tandem with RNA-seq to quantify and compare gastrointestinal gene expression patterns in fecal samples of wild Gorilla gorilla gorilla (n = 9) and BaAka hunter-gatherers (n = 10) from The Dzanga Sangha Protected Areas, Central African Republic. RESULTS: Although only a small fraction (< 4.9%) of intestinal mRNA signals was recovered, the data was sufficient to detect significant functional differences between gorillas and humans, at the gene and pathway levels. These intestinal gene expression differences were specifically associated with metabolic and immune functions. Additionally, non-host RNA-seq reads were used to gain preliminary insights on the subjects' dietary habits, intestinal microbiomes, and infection prevalence, via identification of fungi, nematode, arthropod and plant RNA. CONCLUSIONS: Overall, the results suggest that fecal RNA-seq, targeting gastrointestinal epithelial cells can be used to evaluate primate intestinal physiology and gut gene regulation, in samples obtained in challenging conditions in situ. The approach used herein may be useful to obtain information on primate intestinal health, while revealing preliminary insights into foraging ecology, microbiome, and diet.
- MeSH
- Feces * MeSH
- Gastrointestinal Tract metabolism MeSH
- Gorilla gorilla genetics MeSH
- Humans MeSH
- RNA, Messenger genetics MeSH
- Poly A genetics MeSH
- RNA-Seq * MeSH
- Gene Expression Profiling * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
In this study, we describe an approach that enables a highly specific, effective and fast detection of polyadenylated RNA sequences in situ at the light and electron microscopy levels. The method developed is based on the incorporation of 5-bromo-2'-deoxyuridine into the growing cDNA strand by means of the reverse transcriptase. We have shown that unlike the previously used deoxyuridine tagged with biotin or digoxigenin, 5-bromo-2'-deoxyuridine is 'invisible' in the DNA-DNA duplex but easily detectable in the DNA-RNA duplex. This feature is an important pre-requisite for the correct interpretation of the data obtained, as our results strongly indicate that reverse transcriptase uses DNA breaks as primers efficiently. We have also shown that the replacement of deoxythymidine by 5-bromo-2'-deoxyuridine considerably stabilizes the growing DNA-RNA duplex, thus enabling the one-step detection of polyadenylated RNA in structurally well-preserved cells. The method developed provides a highly specific signal with the signal/noise ratio higher than 130 for permeabilized cells and 25 for conventional acrylic resin sections under the conditions used. When the high pressure freezing technique followed by the freeze substitution is employed for the cell's preparation, the ratio is higher than 80.
- MeSH
- Acrylic Resins MeSH
- Microscopy, Fluorescence MeSH
- HeLa Cells MeSH
- In Situ Hybridization, Fluorescence methods MeSH
- Humans MeSH
- RNA, Messenger analysis chemistry MeSH
- Cell Membrane Permeability MeSH
- Poly A analysis MeSH
- Reverse Transcription MeSH
- Tissue Embedding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.
- MeSH
- Aurora Kinase A metabolism MeSH
- Cyclin B1 genetics MeSH
- mRNA Cleavage and Polyadenylation Factors chemistry metabolism MeSH
- Phosphorylation MeSH
- Meiosis * MeSH
- RNA, Messenger metabolism MeSH
- Oocytes enzymology metabolism MeSH
- Polyadenylation MeSH
- Sus scrofa metabolism MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
1 svazek : ilustrace ; 30 cm
Transplantation of insulin-producing tissue is the only curative method for treatment of insulin-dependent diabetes mellitus. Currently, the sole source of transplantable insulin-producing tissue is the cadaveric pancreas, whose availability is three orders of magnitude bellow the clinical need. Published works in principle confirmed that readily available autologous and allogeneic tissues can be differentiated into insulin-producing cell grafts, which can cure diabetes in rodents. The fundamental problem is the lack of an effective and safe differentiation procedure. The key transcription factors controlling the natural beta-cell differentiation were characterized. Bioengineering methods allow for preparation of modified recombinant proteins, which can penetrate into target cells. This innovative project will create a set of modified transcription factors in order to effectively differentiate beta cells. We will evaluate in vitro and in vivo function of the obtained insulin-producing cell grafts.
Transplantace inzulín-produkující tkáně je jediným kurativním způsobem léčby diabetu závislého na inzulínu. Jedinou transplantabilní tkání produkující inzulín jsou dnes kadaverosní slinivky, jejichž dostupnost je řádově nižší než klinická potřeba. Zveřejněné práce v principu potvrdily, že ze snadno dostupných autologních a allogenních tkání lze diferencovat inzulín-produkující buňky, které jsou po transplantaci schopny vyléčit diabetes u hlodavců. Zásadním problémem dosavadních postupů je nízká efektivita diferenciace inzulín-produkujících buněk. Jsou známy klíčové transkripční faktory, které řídí přirozenou diferenciaci beta-buněk. Jsou dostupné bioinženýrské postupy, které umožňují přípravu rekombinatních bílkovin modifikovaných tak, aby pronikly do cílových buněk. V našem předkládaném projektu vytvoříme sadu modifikovaných transkripčních faktorů klíčových pro diferenciaci beta-buněk, využijeme je k optimalizaci diferenciačních podmínek a funkci takto zísaných buněk otestujeme in vitro a in vivo.
- MeSH
- Insulin MeSH
- RNA, Messenger MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- diabetologie
- NML Publication type
- závěrečné zprávy o řešení grantu IGA MZ ČR
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
98 l. : il. ; 32 cm
Exprese genu pro ferochelatázu v erytroidních buňkách ve vztahu k syntéze hemu. XXX XXX XXX
- MeSH
- Cell Line MeSH
- Gene Expression MeSH
- Ferrochelatase MeSH
- Cells, Cultured MeSH
- RNA, Messenger MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- hematologie a transfuzní lékařství
- NML Publication type
- závěrečné zprávy o řešení grantu IGA MZ ČR
Modern cell biology ; Vol.17
354 s.
- MeSH
- Cells MeSH
- RNA, Messenger MeSH
- Conspectus
- Buněčná biologie. Cytologie
- NML Fields
- cytologie, klinická cytologie
