Petr Svoboda

Publications (75)265.35 Total impact

• Article: FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells; plasma membrane change induced by cholesterol depletion.

  Pavel Ostašov, Jan Sýkora, Jana Brejchová, Agnieszka Olzynska, Martin Hof, Petr Svoboda
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  ABSTRACT: HEK293 cells stably expressing δ-opioid receptor were labeled first with fluorescent analog of cholesterol, 22-NBD-cholesterol, exposed to cholesterol-depleting agent β-cyclodextrin (β-CDX) and analyzed by fluorescence lifetime imaging microscopy (FLIM). In accordance with chemical analysis of cholesterol level, the total cellular signal of this probe was decreased to half. Distribution of lifetime (τtot) values of 22-NBD-cholesterol, however, when screened over the whole cell area indicated no significant difference between control (τtot=4.9±0.1ns) and β-CDX-treated (τtot=4.8±0.1ns) cells. On the contrary, comparison of control (τtot=5.1±0.1ns) and β-CDX-treated (τtot=4.4±0.1ns) cells by analysis of 25-NBD-cholesterol fluorescence implied highly significant decrease of lifetime values of this probe. The observation that 22-NBD-cholesterol appears to be indifferent to the changes in the membrane packing in living cells is in agreement with previous studies in model membranes. However, our data indicate that the alternation of plasma membrane structure induced by decrease of cholesterol level by β-CDX makes the membrane environment of NBD moiety of 25-NBD-cholesterol probe a significantly more hydrated. This finding not only encourages using 25-NBD-cholesterol in living cells, but also demonstrates that previously drawn discouraging conclusions on the use of 25-NBD-cholesterol in model membranes are not valid for living cells.
  Chemistry and physics of lipids 03/2013; · 2.15 Impact Factor
• Article: Production and Application of Long dsRNA in Mammalian Cells.

  Katerina Chalupnikova, Jana Nejepinska, Petr Svoboda
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  ABSTRACT: Double-stranded RNA (dsRNA) is involved in different biological processes. At least three different pathways can respond to dsRNA in mammals. One of these pathways is RNA interference (RNAi) where long dsRNA induces sequence-specific degradation of transcripts carrying sequences complementary to dsRNA. Long dsRNA is also a potent trigger of the interferon pathway, a sequence-independent response that leads to global suppression of translation and global RNA degradation. In addition, dsRNA can be edited by adenosine deamination, which may result in nuclear retention and degradation of dsRNA or in alteration of RNA coding potential. Here, we provide a technical review summarizing different strategies of long dsRNA usage. While the review is largely focused on long dsRNA-induced RNAi in mammalian cells, it also provides helpful information on both the in vitro production and in vivo expression of dsRNAs. We present an overview of currently available vectors for dsRNA expression and provide the latest update on oocyte-specific transgenic RNAi approaches.
  Methods in molecular biology (Clifton, N.J.) 01/2013; 942:291-314.
• Article: RNAi-Based Methods for Gene Silencing in Mouse Oocytes.

  Paula Stein, Petr Svoboda, Richard M Schultz
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  ABSTRACT: RNA interference (RNAi) is an evolutionary conserved gene-silencing pathway that can be efficiently utilized as a tool to study gene function. RNAi is initiated by long double-stranded RNAs (dsRNAs), which are processed into small duplexes called small-interfering RNAs (siRNAs). In turn, these duplexes target mRNAs for degradation in a sequence-specific manner. Mouse oocytes, unlike most mammalian cell types, lack an interferon response to long dsRNA. Moreover, they are a rare example of a mammalian cell type with a robust endogenous RNAi pathway. For these reasons microinjection of either long dsRNAs or siRNAs results in efficient, sequence-specific gene silencing. Here, we describe a protocol for preparation and microinjection of long dsRNA into mouse oocytes.
  Methods in molecular biology (Clifton, N.J.) 01/2013; 957:135-51.
• Article: Maternally-Recruited DCP1A and DCP2 Contribute to Messenger RNA Degradation During Oocyte Maturation and Genome Activation in Mouse.

  Jun Ma, Matyas Flemr, Hynek Strnad, Petr Svoboda, Richard M Schultz
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  ABSTRACT: The oocyte-to-zygote transition entails transforming a highly differentiated oocyte into totipotent blastomeres and represents one of the earliest obstacles that must be successfully hurdled for continued development. Degradation of maternal mRNAs, which likely lies at the heart of this transition, is characterized by a transition from mRNA stability to instability during oocyte maturation. Although phosphorylation of the oocyte-specific RNA-binding protein MSY2 during maturation is implicated in making maternal mRNAs more susceptible to degradation, mechanisms underlying mRNA degradation during oocyte maturation remain poorly understood. We report that DCP1A and DCP2, proteins responsible for decapping mRNA, are encoded by maternal mRNAs recruited for translation during maturation via cytoplasmic polyadenylation elements located in their 3' UTRs. Both DCP1A and DCP2 are phosphorylated during maturation, with CDC2A being the kinase likely responsible for both, although MAPK may be involved in DCP1A phosphorylation. Inhibiting accumulation of DCP1A and DCP2 by RNAi or morpholinos not only decreases degradation of mRNAs during meiotic maturation but also transcription of the zygotic genome. The results indicate that maternally-recruited DCP1A and DCP2 are critical players in the transition from mRNA stability to instability during meiotic maturation and that proper degradation maternal mRNA degradation is successful to execute the oocyte-to-zygote transition.
  Biology of Reproduction 11/2012; · 4.01 Impact Factor
• Article: Transgenic RNAi in mouse oocytes: The first decade.

  Radek Malik, Petr Svoboda
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  ABSTRACT: RNA interference (RNAi), a sequence-specific mRNA degradation induced by double-stranded RNA (dsRNA), is a common approach employed to specifically silence genes. Experimental RNAi in plant and invertebrate models is frequently induced by long dsRNA. However, in mammals, short RNA molecules are used preferentially since long dsRNA can provoke sequence-independent type I interferon response. A notable exception are mammalian oocytes where the interferon response is suppressed and long dsRNA is a potent and specific trigger of RNAi. Transgenic RNAi is an adaptation of RNAi allowing for inducing sequence-specific silencing upon expression of dsRNA. A decade ago, we have developed a vector for oocyte-specific expression of dsRNA, which has been used to study gene function in mouse oocytes on numerous occasions. This review provides an overview and discusses benefits and drawbacks encountered by us and our colleagues while working with the oocytes-specific transgenic RNAi system.
  Animal reproduction science 08/2012; 134(1-2):64-8. · 1.56 Impact Factor
• Article: Deep sequencing reveals complex spurious transcription from transiently transfected plasmids.

  Jana Nejepinska, Radek Malik, Martin Moravec, Petr Svoboda
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  ABSTRACT: Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls.
  PLoS ONE 01/2012; 7(8):e43283. · 4.09 Impact Factor
• Article: Control of the interferon response in RNAi experiments.

  Jana Nejepinska, Matyas Flemr, Petr Svoboda
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  ABSTRACT: The RNA interference (RNAi) and interferons have been an uneasy marriage. Ever since the discovery of RNAi in mammals, the interferon response has been a feared problem. While RNAi became an efficient and widespread method for gene silencing in mammals, numerous studies recognized several obstacles, including undesirable activation of the interferon response, which need to be overcome to achieve a specific and robust RNAi effect. The aim of this text is to provide theoretical and practical information for scientists who want to control interferon response and other adverse effects in their RNAi experiments.
  Methods in molecular biology (Clifton, N.J.) 01/2012; 820:133-61.
• Article: Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal.

  Hana Ujcikova, Katerina Dlouha, Lenka Roubalova, Miroslava Vosahlikova, Dmytro Kagan, Petr Svoboda
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  ABSTRACT: Activation of adenylyl cyclase (AC) by prolonged exposure of mammalian organism to morphine was demonstrated in previous studies of mechanism of action of this drug. However, expression level of individual AC isoforms was not analyzed in crucial cell structure, plasma membrane (PM). Rats were adapted to morphine for 10 days and sacrificed 24h (group+M10) or 20 days (+M10/-M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups-M10 and (-M10/-M20)). Percoll®-purified PM were isolated from brain cortex and analyzed by immunoblotting and specific radioligand binding. ACI (ACII) was increased 8× (2.5×) in morphine-adapted rats (+M10) when compared with controls (-M10). Increase of ACI and II by long-term adaptation to increasing doses of morphine represented a specific effect as the amount of ACIII-ACIX, of prototypical PM marker, Na, K-ATPase and of trimeric G protein α and β subunits was unchanged. Increase of ACI and II was not detected in PM isolated from group (+M10/-M20). Thus, the marked increase of ACI and ACII faded away 20 days since the last dose of morphine. We assume that the specific increase in expression level of ACI and ACII in brain cortex of morphine-adapted rats proceeds as a compensatory, homeostatic response to prolonged exposure to inhibitory drug, morphine. Our findings demonstrate that the dramatic and specific change of the crucial component of the opioid receptor cascade in brain cortex, manifested as an increase in PM level of ACI and II, is reversible.
  Biochimica et Biophysica Acta 12/2011; 1810(12):1220-9. · 4.66 Impact Factor
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  Available from: PubMed Central

  Article: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

  Jana Nejepinska, Radek Malik, Jody Filkowski, Matyas Flemr, Witold Filipowicz, Petr Svoboda
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  ABSTRACT: Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference (RNAi), sequence-independent interferon (IFN) response and editing by adenosine deaminases. To study the routing of dsRNA to these pathways in vivo, we used transgenic mice ubiquitously expressing from a strong promoter, an mRNA with a long hairpin in its 3'-UTR. The expressed dsRNA neither caused any developmental defects nor activated the IFN response, which was inducible only at high expression levels in cultured cells. The dsRNA was poorly processed into siRNAs in somatic cells, whereas, robust RNAi effects were found in oocytes, suggesting that somatic cells lack some factor(s) facilitating siRNA biogenesis. Expressed dsRNA did not cause transcriptional silencing in trans. Analysis of RNA editing revealed that a small fraction of long dsRNA is edited. RNA editing neither prevented the cytoplasmic localization nor processing into siRNAs. Thus, a long dsRNA structure is well tolerated in mammalian cells and is mainly causing a robust RNAi response in oocytes.
  Nucleic Acids Research 09/2011; 40(1):399-413. · 8.03 Impact Factor
• Article: Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1α fusion protein; the effect of cholesterol depletion.

  Jana Brejchová, Jan Sýkora, Kateřina Dlouhá, Lenka Roubalová, Pavel Ostašov, Miroslava Vošahlíková, Martin Hof, Petr Svoboda
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  ABSTRACT: Biophysical studies of fluorescence anisotropy of DPH and Laurdan generalized polarization were performed in plasma membranes (PM) isolated from control and cholesterol-depleted HEK293 cells stably expressing pertussis toxin (PTX)-insensitive DOR-Gi1α (Cys351-Ile351) fusion protein. PM isolated from control, PTX-untreated, cells were compared with PM isolated from PTX-treated cells. Results from both types of PM indicated that i) hydrophobic membrane interior was made more accessible to water molecules and more chaotically organized in cholesterol-depleted samples, ii) cholesterol depletion resulted in an overall increase in surface area of membrane, membrane fluidity, and mobility of its constituents. Analysis of DOR-Gi1α coupling in PTX-treated and PTX-untreated cells indicated that cholesterol depletion did not alter the agonist binding site of DOR (Bmax and Kd) but the ability of DOR agonist DADLE to activate G proteins was markedly impaired. In PTX-untreated membranes, EC50 for DADLE-stimulated [35S]GTPγS binding was shifted by one order of magnitude to the right: from 4.3±1.2×10(-9) M to 2.2±1.3×10(-8) M in control and cholesterol-depleted membrane samples, respectively. In PTX-treated membranes, EC50 was shifted from 4.5±1.1×10(-9) M to 2.8±1.4×10(-8) M. In summary, the perturbation of optimum PM organization by cholesterol depletion deteriorates functional coupling of DOR to covalently bound Gi1α as well as endogenously expressed PTX-sensitive G proteins of Gi/Go family while receptor ligand binding site is unchanged. The biophysical state of hydrophobic plasma (cell) membrane interior should be regarded as regulatory factor of DOR-signaling cascade.
  Biochimica et Biophysica Acta 08/2011; 1808(12):2819-29. · 4.66 Impact Factor
• Article: Targeting genes in living mammals by RNA interference.

  Katerina Podolska, Petr Svoboda
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  ABSTRACT: More than a decade has passed since the discovery of RNA interference (RNAi), an eukaryotic sequence-specific degradation of mRNA induced by complementary double-stranded RNA (dsRNA). RNAi became a common tool for controlled down-regulation of gene expression in cultured cells, as well as in various model organisms. This review summarizes RNAi-based tools for silencing genes in living mammals, which include: (i) transgenic RNAi strategies, where RNAi is triggered by a transgene transmitted through the germline and (ii) approaches, where an RNAi trigger is delivered into an adult animal.
  Briefings in functional genomics 07/2011; 10(4):238-47. · 4.13 Impact Factor
• Article: Ribonucleoprotein localization in mouse oocytes.

  Matyas Flemr, Petr Svoboda
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  ABSTRACT: RNA molecules rarely function alone in cells. For most RNAs, their function requires formation of various ribonucleoprotein (RNP) complexes. For example, mRNP composition can determine mRNA localization, translational repression, level of translation or mRNA stability. RNPs are usually studied by biochemical methods. However, biochemical approaches are unsuitable for some model systems, such as mammalian oocytes and early embryos, due to the small amounts that can be obtained for experimental analysis. In such cases, microscopic techniques are often used to learn about RNPs. Here, we present a review of immunostaining, fluorescence in situ hybridization with subcellular resolution and a combination of both, with emphasis on the mouse oocyte and early embryos models. Application of these techniques to whole-mount fixed oocytes and early embryos can provide information about RNP composition and localization with three-dimensional resolution.
  Methods 02/2011; 53(2):136-41. · 4.01 Impact Factor
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  Available from: PubMed Central

  Article: Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes.

  Lenka Sarnova, Radek Malik, Radislav Sedlacek, Petr Svoboda
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  ABSTRACT: RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type. Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals. We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.
  Journal of Negative Results in BioMedicine 10/2010; 9:8. · 1.47 Impact Factor
• Article: Early postnatal development of rat brain is accompanied by generation of lipofuscin-like pigments.

  Jiří Wilhelm, Joško Ivica, Dmytro Kagan, Petr Svoboda
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  ABSTRACT: The increased generation of free radicals results in the formation of fluorescent end-products of lipid peroxidation, lipofuscin-like pigments (LFPs). The authors observed that LFPs are generated in rat brain after a normal birth during 5 postnatal days. The experimental design of the study comprised 10 groups of animals. The authors measured prenatal values 1 day and 7 days before birth, and then the animals were sampled on postnatal day 1, 2, 5, 10, 15, 25, 35, and 90. Maximum LFP concentration is achieved on the postnatal day 2. Starting from postnatal day 10, LFP concentration returns to prenatal values. A new rise in LFP concentration is observed at 3 months of age. This is associated with the beginning of the aging process. LFPs were characterized by fluorescence spectroscopy using tridimensional excitation spectra, synchronous spectra and their derivatives, and HPLC with fluorescence detection. It was possible to discern several tens of fluorescent compounds of unknown structure that are generated and metabolized during early development. The authors suggest that LFPs are formed after respiratory burst of microglia phagocytosing apoptotic cells.
  Molecular and Cellular Biochemistry 10/2010; 347(1-2):157-62. · 2.06 Impact Factor
• Article: Long-term adaptation to high doses of morphine causes desensitization of mu-OR- and delta-OR-stimulated G-protein response in forebrain cortex but does not decrease the amount of G-protein alpha subunits.

  Lenka Bourova, Miroslava Vosahlikova, Dmytro Kagan, Katerina Dlouha, Jiri Novotny, Petr Svoboda
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  ABSTRACT: The functional activity of trimeric guanine-nucleotide-binding proteins (G-proteins) represents an essential step in linking and regulation of the opioid receptor (mu-, delta- and kappa-OR)-initiated signaling pathways. Theoretical basis and/or molecular mechanism(s) of opioid tolerance and addiction proceeding in the central nervous system were not studied in the forebrain cortex of mammals with respect to quantitative analysis of opioid-stimulated trimeric G-protein activity. G-protein activity was measured in PercollR-purified plasma membranes (PM) isolated from the frontal brain cortex of control and morphine-treated rats by both high-affinity [32P]GTPase and [35S]GTPgammaS binding assays. Exposition to morphine was performed by intra-muscular application of this drug. Control animals were injected with sterile PBS. Both mu-OR (DAMGO)- and delta-OR (DADLE)-responses were clearly desensitized in PM isolated from morphine-treated rats; kappa-OR (U-69593)- and baclofen (GABAB-R)-stimulated [35S]GTPgammaS binding was unchanged, indicating the specificity of the morphine effect. Under such conditions, the amount of G-protein alpha subunits was unchanged. The order of efficacy DADLE>DAMGO>U-69593 was the same in control and morphine-treated PM. Behavioral tests indicated that morphine-treated animals were fully drug-dependent and developed tolerance to subsequent drug addition. Prolonged exposure of rats to high doses of morphine results in decrease of the over-all output of OR-stimulated G-protein activity in the forebrain cortex but does not decrease the amount of these regulatory proteins. These data support the view that the mechanism of the long-term adaptation to high doses of morphine is primarily based on desensitization of OR-response preferentially oriented to mu-OR and delta-OR.
  Medical science monitor: international medical journal of experimental and clinical research 08/2010; 16(8):BR260-70. · 1.70 Impact Factor
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  Available from: ucsd.edu

  Article: The role of miRNAs and endogenous siRNAs in maternal-to-zygotic reprogramming and the establishment of pluripotency.

  Petr Svoboda, Matyas Flemr
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  ABSTRACT: RNA silencing is a complex of mechanisms that regulate gene expression through small RNA molecules. The microRNA (miRNA) pathway is the most common of these in mammals. Genome-encoded miRNAs suppress translation in a sequence-specific manner and facilitate shifts in gene expression during developmental transitions. Here, we discuss the role of miRNAs in oocyte-to-zygote transition and in the control of pluripotency. Existing data suggest a common principle involving miRNAs in defining pluripotent and differentiated cells. RNA silencing pathways also rapidly evolve, resulting in many unique features of RNA silencing in different taxonomic groups. This is exemplified in the mouse model of oocyte-to-zygote transition, in which the endogenous RNA interference pathway has acquired a novel role in regulating protein-coding genes, while the miRNA pathway has become transiently suppressed.
  EMBO Reports 08/2010; 11(8):590-7. · 7.36 Impact Factor
• Article: P-body loss is concomitant with formation of a messenger RNA storage domain in mouse oocytes.

  Matyas Flemr, Jun Ma, Richard M Schultz, Petr Svoboda
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  ABSTRACT: 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.
  Biology of Reproduction 05/2010; 82(5):1008-17. · 4.01 Impact Factor
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  Available from: Veronika Obsilova

  Article: 14-3-3 protein interacts with and affects the structure of RGS domain of regulator of G protein signaling 3 (RGS3).

  Lenka Rezabkova, Evzen Boura, Petr Herman, Jaroslav Vecer, Lenka Bourova, Miroslav Sulc, Petr Svoboda, Veronika Obsilova, Tomas Obsil
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  ABSTRACT: Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for the alpha-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 proteins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking their interaction with G(alpha) subunit. Since RGS proteins interact with G(alpha) subunits through their RGS domains, it is reasonable to assume that the 14-3-3 protein can either sterically occlude the G(alpha) interaction surface of RGS domain and/or change its structure. In this work, we investigated whether the 14-3-3 protein binding affects the structure of RGS3 using the time-resolved tryptophan fluorescence spectroscopy. Two single-tryptophan mutants of RGS3 were used to study conformational changes of RGS3 molecule. Our measurements revealed that the 14-3-3 protein binding induces structural changes in both the N-terminal part and the C-terminal RGS domain of phosphorylated RGS3 molecule. Experiments with the isolated RGS domain of RGS3 suggest that this domain alone can, to some extent, interact with the 14-3-3 protein in a phosphorylation-independent manner. In addition, a crystal structure of the RGS domain of RGS3 was solved at 2.3A resolution. The data obtained from the resolution of the structure of the RGS domain suggest that the 14-3-3 protein-induced conformational change affects the region within the G(alpha)-interacting portion of the RGS domain. This can explain the inhibitory effect of the 14-3-3 protein on GAP activity of RGS3.
  Journal of Structural Biology 03/2010; 170(3):451-61. · 3.41 Impact Factor
• Article: MicroRNA activity is suppressed in mouse oocytes.

  Jun Ma, Matyas Flemr, Paula Stein, Philipp Berninger, Radek Malik, Mihaela Zavolan, Petr Svoboda, Richard M Schultz
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  ABSTRACT: MicroRNAs (miRNAs) are small endogenous RNAs that typically imperfectly base pair with 3' untranslated regions (3'UTRs) and mediate translational repression and mRNA degradation. Dicer, which generates small RNAs in the miRNA and RNA interference (RNAi) pathways, is essential for meiotic maturation of mouse oocytes. We found that 3'UTRs of transcripts upregulated in Dicer1(-/-) oocytes are not enriched in miRNA binding sites, implicating a weak impact of miRNAs on the maternal transcriptome. Therefore, we tested the ability of endogenous miRNAs to mediate RNA-like cleavage or translational repression of reporter mRNAs. In contrast to somatic cells, endogenous miRNAs in oocytes poorly repressed translation of mRNA reporters, whereas their RNAi-like activity was much less affected. Reporter mRNA carrying let-7-binding sites failed to localize to P body-like structures in oocytes. Our data suggest that miRNA function is downregulated during oocyte development, an idea supported by normal meiotic maturation of oocytes lacking Dgcr8, which is required for the miRNA but not the RNAi pathway (Suh et al. [1], this issue of Current Biology). Suppressing miRNA function during oocyte growth is likely an early event in reprogramming gene expression during the transition of a differentiated oocyte into pluripotent blastomeres of the embryo.
  Current biology: CB 02/2010; 20(3):265-70. · 10.99 Impact Factor
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  Available from: PubMed Central

  Article: Dicer is associated with ribosomal DNA chromatin in mammalian cells.

  Lasse Sinkkonen, Tabea Hugenschmidt, Witold Filipowicz, Petr Svoboda
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  ABSTRACT: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.
  PLoS ONE 01/2010; 5(8):e12175. · 4.09 Impact Factor