Danio rerio, commonly known as zebrafish, is an established model organism for the developmental and cell biology studies. Although significant progress has been made in the analysis of the D. rerio genome, cytogenetic studies face challenges due to the unclear identification of chromosomes. Here, we present a novel approach to the study of the D. rerio karyotype, focusing on the analysis of lampbrush chromosomes isolated from growing oocytes. Lampbrush chromosomes, existing during diplotene, serve as a powerful tool for high-resolution mapping and transcription analysis due to their profound decondensation and remarkable lateral loops decorated by RNA polymerases and ribonucleoprotein (RNP) matrix. In D. rerio, lampbrush chromosomes are about 20 times longer than corresponding metaphase chromosomes. We found that the lampbrush chromosome stage karyotype of D. rerio is generally undifferentiated, except for several bivalents bearing distinct marker structures, including loops with complex RNP matrix and locus-associated nuclear bodies. Locus-associated nuclear bodies were enriched for coilin and snRNAs; the loci where they formed presumably correspond to the histone gene clusters. Further, we observed the accumulation of splicing factors in giant terminal RNP aggregates on one bivalent. DAPI staining of Danio rerio lampbrush chromosomes revealed large and small chromomeres non-uniformly distributed along the axis. For example, D. rerio lampbrush chromosome 4, comprising the sex-determining region, is divided into two halves-with small chromomeres bearing long lateral loops and with large dense chromomeres bearing no or very tiny lateral loops. As centromeres were not distinguishable, we identified centromeric regions in all bivalents by FISH mapping of pericentromeric RFAL1, RFAL2, and RFAM tandem repeats. Through a combination of morphological analysis, immunostaining of marker structures, and centromere mapping, we developed cytological maps of D. rerio lampbrush chromosomes. Finally, by RNA FISH we revealed transcripts of pericentromeric and telomeric tandem repeats at the lampbrush chromosome stage.

• Klíčová slova
• Centromere, FISH-mapping, Germinal vesicle, Histone locus bodies, Karyotype analysis, Lampbrush chromosomes, Non-coding RNA, Oocyte nucleus, Tandem repeats, Telomere, Zebrafish,
• MeSH
• chromozomy * MeSH
• dánio pruhované * genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• karyotyp MeSH
• karyotypizace MeSH
• oocyty metabolismus   MeSH
• ribonukleoproteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ribonukleoproteiny MeSH

Here, we present a protocol for labeling and live visualization of RNA-protein complexes in the form of ribonucleoprotein particles (RNPs) in tobacco pollen tubes. We describe steps for constructing RNA-pp7/MS2 tag and biolistic gene-gun-mediated pollen transformation. We then provide detailed procedures for RNA labeling using PP7 aptamer nascent RNA tagging and a fluorescently labeled Pseudomonas aeruginosa PP7 bacteriophage coat protein (PCP) reporter that binds to PP7 RNA stem loops. This protocol is adaptable to other cell types by employing tissue-specific promoters.

• Klíčová slova
• cell biology, developmental biology, microscopy, plant sciences,
• MeSH
• barvení a značení metody   MeSH
• Pseudomonas aeruginosa genetika   metabolismus   MeSH
• pylová láčka * metabolismus   genetika   MeSH
• ribonukleoproteiny metabolismus   MeSH
• RNA rostlin genetika   metabolismus   MeSH
• tabák * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ribonukleoproteiny MeSH
• RNA rostlin MeSH

BACKGROUND AND HYPOTHESIS: Schizophrenia is associated with altered energy metabolism, but the cause and potential impact of these metabolic changes remain unknown. 22q11.2 deletion syndrome (22q11.2DS) represents a genetic risk factor for schizophrenia, which is associated with the loss of several genes involved in mitochondrial physiology. Here we examine how the haploinsufficiency of these genes could contribute to the emergence of schizophrenia in 22q11.2DS. STUDY DESIGN: We characterize changes in neuronal mitochondrial function caused by haploinsufficiency of mitochondria-associated genes within the 22q11.2 region (PRODH, MRPL40, TANGO2, ZDHHC8, SLC25A1, TXNRD2, UFD1, and DGCR8). For that purpose, we combine data from 22q11.2DS carriers and schizophrenia patients, in vivo (animal models) and in vitro (induced pluripotent stem cells, IPSCs) studies. We also review the current knowledge about seven non-coding microRNA molecules located in the 22q11.2 region that may be indirectly involved in energy metabolism by acting as regulatory factors. STUDY RESULTS: We found that the haploinsufficiency of genes of interest is mainly associated with increased oxidative stress, altered energy metabolism, and calcium homeostasis in animal models. Studies on IPSCs from 22q11.2DS carriers corroborate findings of deficits in the brain energy metabolism, implying a causal role between impaired mitochondrial function and the development of schizophrenia in 22q11.2DS. CONCLUSIONS: The haploinsufficiency of genes within the 22q11.2 region leads to multifaceted mitochondrial dysfunction with consequences to neuronal function, viability, and wiring. Overlap between in vitro and in vivo studies implies a causal role between impaired mitochondrial function and the development of schizophrenia in 22q11.2DS.

• Klíčová slova
• 22q11.2DS, energy metabolism, mitochondria, schizophrenia,
• MeSH
• DiGeorgeův syndrom * genetika   MeSH
• lidé MeSH
• mikro RNA * metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• ribonukleoproteiny metabolismus   MeSH
• ribozomální proteiny metabolismus   MeSH
• schizofrenie * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mikro RNA * MeSH
• MRPL40 protein, human MeSH Prohlížeč
• proteiny vázající RNA MeSH
• ribonukleoproteiny MeSH
• ribozomální proteiny MeSH

Varietal differences within a species with agronomic importance are often based on minor changes in the genomic sequence. For example, fungus-resistant and fungus-susceptible wheat varieties may vary in only one amino acid. The situation is similar with the reporter genes Gfp and Yfp where two base pairs cause a shift in the emission spectrum from green to yellow. Methods of targeted double-strand break induction now allow this exchange precisely with the simultaneous transfer of the desired repair template. However, these changes rarely lead to a selective advantage that can be used in generating such mutant plants. The protocol presented here allows a corresponding allele replacement at the cellular level using ribonucleoprotein complexes in combination with an appropriate repair template. The efficiencies achieved are comparable to other methods with direct DNA transfer or integration of the corresponding building blocks in the host genome. They are in the range of 35 percent, considering one allele in a diploid organism as barley and using Cas9 RNP complexes.

• Klíčová slova
• Biolistic, CRISPR/Cas, Cereals, Homology-directed repair, Tissue culture, Triticeae,
• MeSH
• alely MeSH
• CRISPR-Cas systémy MeSH
• DNA MeSH
• ječmen (rod) * genetika   metabolismus   MeSH
• ribonukleoproteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• ribonukleoproteiny MeSH

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

• Klíčová slova
• RNA chaperones, genome assembly, ribonucleoproteins, rotavirus,
• MeSH
• elektronová kryomikroskopie MeSH
• genom virový genetika   MeSH
• molekulární chaperony metabolismus   MeSH
• molekulární modely MeSH
• proteiny vázající RNA metabolismus   MeSH
• ribonukleoproteiny metabolismus   MeSH
• RNA virová genetika   MeSH
• Rotavirus genetika   růst a vývoj   metabolismus   MeSH
• sbalování RNA genetika   MeSH
• virové nestrukturální proteiny metabolismus   MeSH
• zabalení virového genomu genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• molekulární chaperony MeSH
• proteiny vázající RNA MeSH
• ribonukleoproteiny MeSH
• RNA virová MeSH
• virové nestrukturální proteiny MeSH

Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear shape, and cell cycle progression. We show that fibrillarin has an additional activity as a ribonuclease. The activity is affected by phosphoinositides and phosphatidic acid and insensitive to ribonuclease inhibitors. Furthermore, the presence of phosphatidic acid releases the fibrillarin-U3 snoRNA complex. We show that the ribonuclease activity localizes to the GAR (glycine/arginine-rich) domain conserved in a small group of RNA interacting proteins. The introduction of the GAR domain occurred in evolution in the transition from archaea to eukaryotic cells. The interaction of this domain with phospholipids may allow a phase separation of this protein in nucleoli.

• Klíčová slova
• fibrillarin, nucleolus, phosphoinositides, rRNA, ribonucleolar particle, viral progression,
• MeSH
• chromozomální proteiny, nehistonové chemie   genetika   metabolismus   MeSH
• fosfolipidy metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• malá jadérková RNA metabolismus   MeSH
• mutace genetika   MeSH
• proteinové domény MeSH
• rekombinantní proteiny metabolismus   MeSH
• ribonukleasy chemie   genetika   metabolismus   MeSH
• ribonukleoproteiny metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• fibrillarin MeSH Prohlížeč
• fosfolipidy MeSH
• malá jadérková RNA MeSH
• rekombinantní proteiny MeSH
• ribonukleasy MeSH
• ribonukleoproteiny MeSH
• RNA, U3 small nucleolar MeSH Prohlížeč

Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (Nicotiana tabacum), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes. We demonstrated that the composition of the aforementioned complexes is not rigid and that numerous transcripts were redistributed among these complexes during pollen development, which may represent an important mechanism of translational regulation. Therefore, we defined the pollen sequestrome as a distinct and highly dynamic compartment for the storage of stable, translationally repressed transcripts and demonstrated its dynamics. We propose that EDTA/puromycin-resistant particle complexes represent aggregated nontranslating monosomes as the primary mediators of messenger RNA sequestration. Such organization is extremely useful in fast tip-growing pollen tubes, where rapid and orchestrated protein synthesis must take place in specific regions.

• MeSH
• polyribozomy genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• proteomika metody   MeSH
• pyl genetika   růst a vývoj   metabolismus   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• ribonukleoproteiny genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• stanovení celkové genové exprese metody   MeSH
• tabák genetika   růst a vývoj   metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH
• ribonukleoproteiny MeSH
• rostlinné proteiny MeSH

In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs). Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of S. cerevisiae also contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) as well as translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.

• MeSH
• cytoplazmatická granula metabolismus   MeSH
• elongační faktory chemie   metabolismus   MeSH
• fyziologický stres MeSH
• molekulární sekvence - údaje MeSH
• peptidy - faktory ukončení chemie   metabolismus   MeSH
• reakce na tepelný šok * MeSH
• ribonukleoproteiny metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• elongační faktory MeSH
• peptidy - faktory ukončení MeSH
• ribonukleoproteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH

The mRNA-protein complexes (mRNPs, Messenger ribonucleoprotein particles) are the "couriers" of the modern eukaryotes that process, store and deliver messages (transcripts) from the nucleus to the appropriate subcellular compartments and beyond. Presence of mRNPs arbitrates the posttranscriptional control of gene expression by editing the precursor RNA to maturity, postulate its subcellular localization and/or storage and dictate its fate once in the cytoplasm; either to be translated or dispensed through mRNA degradation. Initiation of transcription is coupled with processing of the transcribed message and the immediate association of the transcript with a set of structural and regulatory proteins. Per se, mRNP complexes sub-optimize transcription by recruiting RNA-binding proteins which are the core component of the RNP activities that culminate overall distribution and abundance of individual proteins. This asymmetric distribution of the mRNA is the determinant of protein gradient and is known to influence cell polarity, cell fate and overall patterning during development. Embryo patterning in Drosophila, polarization of maternal mRNA to daughter cell in budding yeast and directional growth of mammalian neural cell and pollen tubes of flowering plants, are the most prominent examples of mRNP facilitated posttranscriptional control, influencing cell fates and patterns of development.This chapter addresses the current knowledge on the mechanisms of posttranscriptional control reinforced by the formation of RNP particles and reviews differences in the underlying mechanisms. The outline of the chapter encompasses step-wise cellular processes leading to the formation of mRNPs and its implication to cellular activities. A dedicated section is also integrated discussing the recent findings on the unique mechanism of RNP formation in the male gametophyte of Nicotiana tabaccum. A proposed model outlines the network of posttranscriptional control with a focus on the role of RNPs is also presented aiming to stimulate future research with a perspective of advancing our knowledge on the subject and its plausible application in improving food quality.

• MeSH
• biologické modely MeSH
• cytoskelet metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• posttranskripční úpravy RNA * MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• ribonukleoproteiny genetika   metabolismus   MeSH
• stabilita RNA MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• messenger ribonucleoprotein MeSH Prohlížeč
• messenger RNA MeSH
• proteiny vázající RNA MeSH
• ribonukleoproteiny MeSH

In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• cytoplazmatická granula metabolismus   MeSH
• endoribonukleasy MeSH
• intracelulární prostor MeSH
• konformace proteinů MeSH
• multiproteinové komplexy metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• ovarium růst a vývoj   metabolismus   MeSH
• proteiny vázající RNA fyziologie   MeSH
• ribonukleoproteiny metabolismus   MeSH
• RNA čepičky metabolismus   MeSH
• RNA messenger skladovaná metabolismus   MeSH
• trans-aktivátory metabolismus   MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• endoribonukleasy MeSH
• multiproteinové komplexy MeSH
• proteiny vázající RNA MeSH
• ribonukleoproteiny MeSH
• RNA čepičky MeSH
• RNA messenger skladovaná MeSH
• smad4-interacting protein SMIF, mouse MeSH Prohlížeč
• trans-aktivátory MeSH