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Lectures L2.1 L2.2 L2.3 Session 2. Nucleic Acids

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Lectures L2.1 L2.2 L2.3 Session 2. Nucleic Acids Session 2. Nucleic acids Lectures lar from yeast to man. Another report demonstrated that Maf1 is a component of TOR signaling pathway which L2.1 mediates polymerase III transcription repression under various conditions including stationary growing phase Epigenetic phenomena in hormonally and rapamycin stress (Upadhya et al., Mol Cell 2002). Our dependent malignancies in women — review current studies focus on understanding the mechanism of Maf1 action. Our goal is to define the complex regu- Wodzimierz Baranowski latory networks involving the role of Maf1 in control of Department of Gynaecology and Gynaecological Oncology, tRNA synthesis, clearly important step in tumor devel- Military Institute of Medicine, Warszawa, Poland; opment in higher organisms. In order to characterize the e-mail: [email protected] biochemical phenomenon allowing Pol III transcription regulation, we tried to determine which proteins inter- Hormonally dependent malignancies in women (en- act mainly with Maf1. Pol III subunits were identified by dometrial tumors, ovarian and mammary glandcancers) mass spectrometry. We identified the N-terminus domain areleading cause of death among women bearing malig- of the largest subunit of Pol III as possible target domain nant tumors.The steroid receptors (estrogen receptor — of Maf1 interaction with Pol III apparatus. Interaction ER, and progesterone receptor — PR) status ohe tumor, of Maf1 with Pol III is complex; depends on Maf1 phos- apart fromhistological grade of neoplasm differentiation phorylation and varies according to growth conditions. and clinical stage, seems to be an important and valuable- Thus, in exponential growing cells only 10 % of the Pol III prognostic marker as far as hormonal treatment efficacy complexes contain Maf1. In such proliferating cells Maf1 and survival rate is concerned. The steroid receptors as exists in, at least, three phosphorylation states but only transcriptionfactors regulatethe methylation paern of the unphosphorylated Maf1 protein interacts with Pol III. genomic DNA and also the methylation of particular An immunofluorescence experiment suggests that phos- genes. The by-products od estrogen catabolic pathway phorylated forms localize in the cytoplasm. By contrast, (e.g. catecholestrogen derivatives) can induce hydro- in starving cells, that are treated with rapamycin or enter phobic DNA adducts formation leading to mutation(s) stationary phase, Maf1 is unphosphorylated, migrates to of important genes (suppressors and oncogenes). This the nucleus and binds pol III in a stoichiometric manner. review described the state of the art in the field of in- In conclusion we suggest that in response to nutrient star- terplay between estrogen derivatives, estrogen receptor vation stress TOR signaling activates Maf1 repressor by and genomic DNAmethylation (global methylation and its dephosphorylation causing import to the nucleus and metylation of particular genes), as well as paern of DNA recruitment on RNA polymerase III transcribed genes. adducts in hormonal dependent tumors in women. References L2.3 McDonnell D, Norris JD (2002) Science 296: 1642-1644. Cavalieri E, Frenkel K, Liehr J, Rogan E, Roy D (2000) J Natl Can- The arginine-riboswitch regulates cer Inst Mon 27: 75-93. alternative splicing of the Aspergillus Esteller M, Herman J (2002) J Pathol 196: 1-7. nidulans arginase mRNA Nilsson S, Makela S, Treuter E et al. (2001) Physiol Rev 81: 1536- 1,2 2 1565. Piotr Borsuk ,Anna Przykorska , Karina Blachnio2, Michal Koper1,2, Jerzy M. Pawlowicz2, L2.2 Malgorzata Pekala1, Piotr Weglenski1,2 1Department of Genetics, University of Warszawa, Warszawa, Negative regulation of RNA polymerase Poland; 2Institute of Biochemistry and Biophysics, PAS, III transcription by Maf1 protein Warszawa, Poland; Magdalena Boguta e-mail: [email protected] Department of Genetics, Institute of Biochemistry and Expression of the arginase structural gene (agaA) in Biophysics, PAS, Warszawa, Poland; Aspergillus nidulans is subject to complex transcrip- e-mail: [email protected] tional and post-transcriptional regulation, dependent on growth conditions, particularly the presence of ar- The yeast protein, Maf1, functions as a repressor of RNA ginine and various nitrogen and carbon sources in the polymerase III (Pol III) (Pluta et al., Mol Cell Biol 2001). The medium. Arginase mRNA has a long 5’UTR sequence. Maf1 protein is conserved through evolution, suggesting Analysis of this sequence in silico revealed its puta- that the regulatory mechanisms involving Maf1 are simi- tive complex secondary structure, the presence of the Vol. 52 40th Meeting of the Polish Biochemical Society 31 arginine binding motifs (arginine aptamers) and the L2.5 presence of a short intron with two potential 3’ splic- ing sites. We present evidence that: (A) L-arginine Translational control mediated by cis-acting binds directly to the arginase 5’UTR (Kd = 10 mM); (B) sequences located at the 3’ and 5’UTRs of the L-arginine (but neither several other L-amino acids nor rat DNA polymerase β and Hax-1 mRNA D-arginine) invokes drastic changes in the secondary structure of the 5’UTR; (C) L-arginine accelerates the Ewa A. Grzybowska, Elżbieta splicing of the intron present in the 5’UTR and forces Sarnowska, Anna Wilczyńska, Ryszard choice of the one of two 3’ splice sites. We postulate Konopiński, Janusz A. Siedlecki that the expression of the eukaryotic structural gene Cancer Center Institute, Warszawa, Poland; coding for arginase in A. nidulans is regulated at the e-mail: [email protected] level of mRNA stability depending on the riboswitch- mediated alternative splicing of the 5’UTR intron. DNA polymerase β, an enzyme involved in base excision repair, has two transcripts in the rat cells, which differ L2.4 only in the length of their 3’UTRs. Analysis of expression performed in two different reporter systems showed that Ribozymes: recent views on their biological the long 3’UTR is responsible for downregulation of ex- functions and possible applications pression, occurring without changes in mRNA stability, which implies translational regulation. This finding is con- Jerzy Ciesiołka, Mariola Dutkiewicz, sistent with the presence of the sequence similar to the bind- Michał Łęgiewicz, Agata Świątkowska, ing site of translational repressor. Short 3’UTR, causing re- Agnieszka Wichłacz, Jan Wrzesinski porter upregulation, was shown to bind Hax-1 protein. The Institute of Bioorganic Chemistry, PAS, Poznań, Poland; importance of this interaction for mRNA stability and/or e-mail: [email protected] regulation of translation was analyzed in the reporter system by the measurement of luciferase expression and For over two decades the ribozyme research has been subsequent estimation of mRNA levels. The possibility carried out in two major directions: the structure and of translational regulation of Hax-1 expression by small function of natural and in vitro evolved ribozymes, and upstream ORFs present in the 5’UTR of some of its tran- the possibility of using ribozymes in biochemical and scripts was assessed separately. biomedical applications. Current status of these both directions of studies will be reviewed. Special empha- L2.6 sis will be put on the delta ribozymes and selected results obtained by our laboratory will be also pre- Role of RNA structure in pathogenesis sented. We have selected several variants of the antig- of triplet repeat expansion diseases enomic delta ribozyme from its combinatorial library with randomized sequences located in the regions par- Włodzimierz J. Krzyżosiak, Mateusz deMezer, ticipating in formation of the ribozyme catalytic core. Marek Napierała, Krzysztof Sobczak Unexpectedly, the variants differed in preferential use Laboratory of Cancer Genetics, Institute of Bioorganic of catalytic divalent metal ions. Moreover, in some Chemistry, PAS, Poznań, Poland; variants the cytosine residue, which is believed to play e-mail: [email protected] a crucial role in the cleavage mechanism, was mutated. These observations suggest structural flexibility of the The simple sequence repeats (SSRs) known also as short ribozyme catalytic core and will be discussed in terms tandem repeats (STRs) or microsatellites are defined as of the ribozyme cleavage mechanism. We have earlier tandemly repeated tracts of DNA composed of 1-6 base proposed the use of antigenomic delta ribozyme as mo- pair long motifs. Among them the trinucleotide repeats lecular scissors for the purpose of correct processing of are of special interest because of their positive selection the 3’ ends of RNA transcripts obtained in vitro with in exons which suggests some biological roles of these T7 RNA polymerase. Recently, in order to evaluate sequences. Our research is focused on finding normal the usefulness of this ribozyme for directed degrada- functions of trinucleotide repeats in transcripts and deter- tion of structured RNAs, we have synthesized several mining roles of the expanded repeats in pathogenesis of model RNA substrates with the ribozyme’s recognition the so called Triplet Repeat Expansion Diseases (TREDs). sequence always single-stranded but embedded into Our work is also related to potential RNA-directed thera- different RNA structural context. It turned out that pies of these diseases. In the first step of our search for the susceptibility of these substrates to ribozyme cleavage biological functions of trinucleotide repeats in transcripts was substantially differentiated. These results shed we performed their systematic structure analysis using
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