Research topics
Our lab explores several research topics related to RNA silencing and repression of mobile elements in mammals. Current research topics include activity and silencing of L1 retrotransposons in mammals, diverse roles of long double-stranded RNA in mammalian cells, stability of maternal mRNAs in the oocyte, and transgenic RNAi.
Activity and silencing of mobile elements in mammalian cells.
The project has two major aims: 1) to understand the differences between active and silent L1 elements by analyzing epigenetic modifications and expression of individual retrotransposition-competent L1 elements; and 2) to delineate the role of RNAi and related silencing pathways in regulation of L1 expression. We use bisulfite sequencing and chromatin immunoprecipitation to study epigenetic modifications of the potentially most active human L1 elements in human samples (biopsies, and stable cell lines). Preliminary data suggest differential regulation of retrotransposition-competent L1 elements in different human cells. Further analysis will also address transcription and its regulation at these loci. We will also analyze epigenetic reprogramming of a transgenic human L1 element and its de novo insertions during the mouse life cycle. Preliminary data suggest silencing of the human transgenic L1 element in murine somatic cells but relief of repression in the germline. Another part of the project studies an L1 retrotransposition model in cell culture to identify the mechanisms involved in the control of L1 silencing. Finally, we want to address in cell culture whether several L1 inverted repeats dispersed throughout the human genome generate double-stranded RNA that contributes to L1 silencing.
Effects of long dsRNA expression in mammalian cells.
We have generated transgenic mice ubiquitously transcribing a long inverted repeat, which should give a rise to dsRNA. These mice do not exhibit any non-specific phenotype except of RNAi effects in the female germline. Characterization of effects of long dsRNA expression in somatic cells of transgenic mice is currently in progress. We are analyzing interferon pathway activation, adenosine deamination of the long dsRNA, RNAi effects, localization of the long dsRNA, etc. Simultaneously, we analyze the same long dsRNA expression system in the cell culture.
Further development of transgenic RNAi approach.
Transgenic RNAi is an excellent tool to inhibit gene expression. The classical transgenic RNAi approach in mouse oocytes is utilizing expression of long dsRNA controlled by the oocyte-specific ZP3 promoter. The recent development of transgenes allowing for expression of short RNA hairpins by a tissue-specific pol II promoter allows one to simplify transgenic RNAi in the oocyte by generating a ZP3-driven expression cassette designed for inserting short hairpins. This would greatly improve transgenic RNAi in the oocyte because of common problems with cloning long inverted repeats. As a proof-of-principle, we use the established Mos model system, which has been extensively used for RNAi experiments previously.
Degradation of mRNAs during oocyte-to-zygote transition in the mouse.
Transformation of a differentiated mouse oocyte into a pluripotent blastomere of the 2-cell embryo is accompanied by extensive degradation of maternal mRNA. Maternal mRNA translation and degradation are key processes for regulating gene expression during a period of transcriptional quiescence between the resumption of meiosis and zygotic gene activation (ZGA). Degradation of maternal mRNAs is not uniform. Degradation of some maternal mRNAs is triggered by resumption of meiosis or by fertilization while other maternal mRNAs are degraded independently upon developmental events. The majority of maternal mRNAs is degraded by the mid 2-cell stage. There are numerous specific examples illustrating the complexity of maternal mRNA degradation but a thorough analysis of the stability of the maternal transcriptome has not been provided yet. In order to understand degradation of the maternal transcriptome during meiotic maturation and after fertilization, we begin with computational analysis of previously published microarray data from GV oocytes, MII eggs, zygotes and other early developmental stages in order to identify sequence motifs enriched in degraded maternal mRNAs. These motifs are subsequently tested for whether they serve as miRNA or protein binding sites. Simultaneously,we analyze the localization and temporal pattern of expression of several key proteins associated with mRNA stability and degradation. The ultimate goal is to develop an experimentally supported model describing degradation of the maternal transcriptome prior to the zygotic genome activation.
Current Grant Support
2007-2009 EMBO Installation Grant, project 1483, Regulation of mRNA stability during oocyte-to-zygote transition in the mouse.
2007-2009 GA AV ČR, IAA501110701, Activity and inhibition of mobile elements in mammalian cells.
2009-2013 GAČR, 204/09/0085, Effects of long dsRNA expression in mammalian cells.
Most important recent papers
(2003-present; names of the lab members underlined)Stein, P., Svoboda P., Anger, M., Schultz, R.,M.: RNAi: Mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 9: 187-192, 2003.
Stein, P., Svoboda P., Schultz ,R. M.: Transgenic RNAi in mouse oocytes: A simple and fast approach to study gene function. Dev. Biol. 256: 188-94, 2003.
Fedoriw, A.,M., Stein, P., Svoboda P., Schultz, R.M., Bartolomei, M.,S.: Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 303: 238-40, 2004.
Svoboda, P., Stein, P., Anger, M., Bernstein, E., Hannon G.J., Schultz R.M.: RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Dev. Biol. Dev. Biol. 269: 276-85, 2004.
Svoboda, P., Stein, P., Filipowicz, W., Schultz, R.M.: Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes. Nucleic Acids Res. 32: 3601-6, 2004.
Bultman, S.J., Gebuhr, T.C., Pan, H., Svoboda, P., Schultz, R.M., Magnuson, T.: Maternal BRG1 regulates zygotic genome activation in the mouse. Genes Dev. 20: 1744-54, 2006
Puschendorf, M., Stein P., Oakeley, E.J., Schultz, R.M., Peters, A.H., Svoboda, P.: Abundant transcripts from retrotransposons are unstable in fully grown mouse oocytes. Biochem Biophys Res Commun.1347: 36-43, 2006.
Schmitter, D., Filkowski, J., Sewer A., Pillai, R.S., Oakeley, E.J., Zavolan, M., Svoboda, P., Filipowicz, W.: Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic Acids Res. 34: 4801-15, 2006.
Sinkkonen, L., Hugenschmidt, T., Berninger, P., Gaidatzis, D., Mohn, F., Artus-Revel, C.G., Zavolan, M., Svoboda, P., Filipowicz W.: MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nat Struct Mol Biol. 15(3):259-67, 2008
Reviews
Stein, P., Svoboda, P.: Guide to RNAi in mouse oocytes and preimplantation embryos, Cold Spring Harbor Press, book chapter, August 2003.
Svoboda, P.: Long dsRNA and silent genes strike back: RNAi in mouse oocytes and early embryos. Cytogenet. Genome Res. 105: 422-34, 2004.
Horman, S. R., Svoboda, P., Luning Prak, E. T.: The potential regulation of L1 mobility by RNA interference. J. Biomed. Biotechnol. Vol. 2006, article ID 32713: 1-8, 2006.
Svoboda, P., Di Cara, A.: Hairpin RNA - a secondary structure of primary importance. Cell. Mol. Life Sci. 63: 901-8, 2006.
Svoboda P. Off-targeting and other non-specific effects of RNAi experiments in mammalian cells. Curr Opin Mol Ther. 9(3):248-57, 2007
Grosshans H., Svoboda P. miRNA, siRNA, piRNA - Kleine Wiener Ribonukleinsauren. Bioessays. 29(9):940-3, 2007
Svoboda P. RNA silencing in mammalian oocytes and early embryos. Curr Top Microbiol Immunol. 320: 225-256, 2008
Kutter, C., Svoboda P.: miRNA, siRNA, piRNA: Knowns of the unknown. RNA Biology 5(4): 181-188, 2008.
Svoboda, P. MicroRNAs in stem cells. Controlling Pluripotency and Differentiation, Bioforum Europe 11/2008: 28-30, 2008.
Svoboda, P., Stein, P.: RNAi Experiments in Mouse Oocytes and Early Embryos. CSH Protocols 4(1): (DOI:10.1101/pdb.top56), 2009.
Teaching, lectures, public presentations
B150P11 Developmental Biology
B150C07 Practical Course in Developmental Biology
B150P85 Epigenetics
Co-organizer of Advances in Molecular Biology and Genetics