Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

• Klíčová slova
• RNA-seq, SW55Ti rotor, mouse early embryo, mouse oocyte, mouse zygote, polysome fractionation, polysome profiling, translatome,
• MeSH
• jaderný obal genetika   metabolismus   MeSH
• meióza genetika   MeSH
• myši MeSH
• oocyty růst a vývoj   metabolismus   MeSH
• polyribozomy genetika   MeSH
• proteiny vázající RNA genetika   MeSH
• RNA messenger skladovaná genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• RNA ribozomální 28S genetika   MeSH
• sekvenování transkriptomu MeSH
• vývojová regulace genové exprese genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny vázající RNA MeSH
• RNA messenger skladovaná MeSH
• RNA ribozomální 18S MeSH
• RNA ribozomální 28S MeSH

More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).

• Klíčová slova
• Grow yeast cultures, Polysome profile analysis, Sucrose density gradient centrifugation, Western and Northern blotting, Yeast whole cell extracts (WCEs),
• MeSH
• centrifugace - gradient hustoty metody   MeSH
• northern blotting metody   MeSH
• polyribozomy chemie   genetika   MeSH
• Saccharomyces cerevisiae chemie   cytologie   genetika   MeSH
• sacharosa chemie   MeSH
• western blotting metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• sacharosa MeSH

Velocity separation of translation complexes in linear sucrose gradients is the ultimate method for both analysis of the overall fitness of protein synthesis as well as for detailed investigation of physiological roles played by individual factors of the translational machinery. Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and Western blotting, genome-wide microarray analysis or qRT-PCR. This chapter provides a basic overview of the polysome profile analysis technique and the RNA isolation procedure from sucrose gradients. We also discuss possible experimental pitfalls of data normalization, describe main alternatives of the basic protocol and outline a novel application of denaturing RNA electrophoresis in several steps of polysome profile analysis.

• MeSH
• centrifugace - gradient hustoty metody   MeSH
• elektroforéza metody   MeSH
• interpretace statistických dat MeSH
• kvasinky MeSH
• polyribozomy chemie   MeSH
• proteosyntéza genetika   MeSH
• regulace genové exprese genetika   MeSH
• RNA izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA MeSH

High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.

• Klíčová slova
• Bovine, Preimplantation embryo development, Ribosome profiling, Transcription, Translation, Translational selectivity,
• MeSH
• blastocysta * metabolismus   MeSH
• embryonální vývoj * genetika   MeSH
• morula metabolismus   MeSH
• oocyty metabolismus   MeSH
• ribozomy genetika   MeSH
• skot MeSH
• těhotenství MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis-in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before-and provide some representative results.

• MeSH
• messenger RNA genetika   MeSH
• mikro RNA metabolismus   MeSH
• myši MeSH
• polyribozomy metabolismus   MeSH
• proteomika metody   MeSH
• srdce růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• messenger RNA MeSH
• mikro RNA MeSH

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 recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue-specific and essential for some tissue development, such as the liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by using a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in the transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays a more relevant role in small RNA metabolism and ribosome biogenesis.

• Klíčová slova
• ADAR1, RNA editing, Y RNA, hepatocyte, miRNA, snoRNA,
• MeSH
• adenosindeaminasa * genetika   metabolismus   MeSH
• buněčné linie MeSH
• editace RNA * MeSH
• genový knockout MeSH
• hepatocyty * metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• polyribozomy metabolismus   genetika   MeSH
• proteiny vázající RNA * genetika   metabolismus   MeSH
• proteosyntéza MeSH
• transkriptom MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ADAR protein, human MeSH Prohlížeč
• adenosindeaminasa * MeSH
• messenger RNA MeSH
• proteiny vázající RNA * MeSH

BACKGROUND: Little is known about the impact of trans-acting genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate the influence of such distant genetic loci on the efficiency of mRNA translation and define their contribution to the development of complex disease phenotypes within a panel of rat recombinant inbred lines. RESULTS: We identify several tissue-specific master regulatory hotspots that each control the translation rates of multiple proteins. One of these loci is restricted to hypertrophic hearts, where it drives a translatome-wide and protein length-dependent change in translational efficiency, altering the stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus across multiple congenic lines points to a translation machinery defect, characterized by marked differences in polysome profiles and misregulation of the small nucleolar RNA SNORA48. Strikingly, from yeast to humans, we observe reproducible protein length-dependent shifts in translational efficiency as a conserved hallmark of translation machinery mutants, including those that cause ribosomopathies. Depending on the factor mutated, a pre-existing negative correlation between protein length and translation rates could either be enhanced or reduced, which we propose to result from mRNA-specific imbalances in canonical translation initiation and reinitiation rates. CONCLUSIONS: We show that distant genetic control of mRNA translation is abundant in mammalian tissues, exemplified by a single genomic locus that triggers a translation-driven molecular mechanism. Our work illustrates the complexity through which genetic variation can drive phenotypic variability between individuals and thereby contribute to complex disease.

• Klíčová slova
• Cardiac hypertrophy, Complex disease, Genetic variation, HXB/BXH rat recombinant inbred panel, Ribosome biogenesis, Ribosome profiling, Ribosomopathy, Spontaneously hypertensive rats (SHR), Translational efficiency, trans QTL mapping,
• MeSH
• biogeneze organel MeSH
• genetická variace MeSH
• iniciace translace peptidového řetězce * MeSH
• kardiomegalie genetika   metabolismus   patologie   MeSH
• krysa rodu Rattus MeSH
• lokus kvantitativního znaku * MeSH
• malá jadérková RNA genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• myokard metabolismus   patologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• potkani inbrední SHR MeSH
• potkani transgenní MeSH
• regulace genové exprese MeSH
• ribozomální proteiny genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   patologie   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sarkomery metabolismus   patologie   MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá jadérková RNA MeSH
• messenger RNA MeSH
• ribozomální proteiny MeSH

Translation initiation starts with the formation of the 43S preinitiation complex (PIC) consisting of several soluble factors, including the ternary complex (TC; elF2-GTP-Met-tRNA(i)(Met)), which associate with the small ribosomal subunit. In the next step, mRNA is recruited to form the 48S PIC and the entire machinery starts scanning the 5' untranslated region of the mRNA until the AUG start codon is encountered. The most widely used method to separate 40S and 60S ribosomal subunits from soluble factors, monosomes and polysomes, is sucrose density centrifugation (SDC). Since PICs are intrinsically unstable complexes that cannot withstand the forces imposed by SDC, a stabilization agent must be employed to detect the association of factors with the 40S subunit after SDC. This was initially achieved by adding heparin (a highly sulfated glycosaminoglycan) directly to the breaking buffer of cells treated with cycloheximide (a translation elongation inhibitor). However, the mechanism of stabilization is not understood and, moreover, there are indications that the use of heparin may lead to artifactual factor associations that do not reflect the factor occupancy of the 43S/48S PICs in the cell at the time of lysis. Therefore, we developed an alternative method for PIC stabilization using formaldehyde (HCHO) to cross-link factors associated with 40S ribosomal subunits in vivo before the disruption of the yeast cells. Results obtained using HCHO stabilization strongly indicate that the factors detected on the 43S/48S PIC after SDC approximate a real-time in vivo "snapshot" of the 43S/48S PIC composition. In this chapter, we will present the protocol for HCHO cross-linking in detail and demonstrate the difference between heparin and HCHO stabilization procedures. In addition, different conditions for displaying the polysome profile or PIC analysis by SDC, used to address different questions, will be outlined.

• MeSH
• buněčné extrakty MeSH
• eukaryotické iniciační faktory chemie   MeSH
• formaldehyd chemie   MeSH
• frakcionace buněk metody   MeSH
• heparin chemie   MeSH
• iniciace translace peptidového řetězce fyziologie   MeSH
• northern blotting MeSH
• polyribozomy fyziologie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• western blotting MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• buněčné extrakty MeSH
• eukaryotické iniciační faktory MeSH
• formaldehyd MeSH
• heparin MeSH
• reagencia zkříženě vázaná MeSH

Translational regulation is pivotal during preimplantation development. However, how mRNAs are selected for temporal regulation and their dynamic utilization and fate during this period are still unknown. Using a high-resolution ribosome profiling approach, we analyzed the transcriptome, as well as monosome- and polysome-bound RNAs of mouse oocytes and embryos, defining an unprecedented extent of spatiotemporal translational landscapes during this rapid developmental phase. We observed previously unknown mechanisms of translational selectivity, i.e., stage-wise deferral of loading monosome-bound mRNAs to polysome for active translation, continuous translation of both monosome and polysome-bound mRNAs at the same developmental stage, and priming to monosomes after initial activation. We showed that a eukaryotic initiation factor Eif1ad3, which is exclusively translated in the 2-Cell embryo, is required for ribosome biogenesis post embryonic genome activation. Our study thus provides genome-wide datasets and analyses of spatiotemporal translational dynamics accompanying mammalian germ cell and embryonic development and reveals the contribution of a novel translation initiation factor to mammalian pre-implantation development.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH