Tobacco Documents Online

TELRUIU UMIUER$1TV GEORGE $. WISE FACULTY OF UFE SCIENCES DEPARTMEt,IT OF BIOCHEMISTRY Dr. Arthur D. Eisenberg Associate Research Director The Council for Tobacco Researc 900 Third Avenue New York, NY 10022 USA. '3N-b .£1LI'O'ID.'31N February 9, 1995 Ref. 3773 Dear Dr. Eisenberg, Enclosed please find an project entitled "A naturally repressor of cellular radioresJ information. The ~nticipated duration estimate the direct cost for t~ I hope this project will formal application. Sincerely yours, Dan Canaani, Ph.D. Senior Lecturer (tenured) I !arch [ - a rant 'ull, Dr. Dan Canaani Department of Biochemistry Tel Aviv University Ramat Aviv 69978 Israel. Tel: (972)-3-6408985 Fax: (972)-3-6424270 Encs. 03-6415053 'URg ,05-EA09749 '~D .69978 3~3R-~R ~ 3N'~03 ,nO 033 ]IN~ ~ 3P T£t AV|V UNiV£RSiTY, RAE~AT AV|V. TEL AV~V 69978. T£L 03-6409749, FAX. 97Z-~-6415053 40008969

TEL AUIU UHIUER$1TV .£113 O'ID.LIlN GEORGE S. WISE FACULTY OF UFE SCIENCES DEPARTMENT OF BIOCHEMISTRY February 9, 1995 Ref. 3773 Dr. Arthur D. Eisenberg Associate Research Director The Council for Tobacco Research - USA 900 Third Avenue New York, NY 10022 USA. - - Dear Dr. Eisenberg, Enclosed please find an outline of a proposed research project entitled "A naturally occurring human antisense RNA - a repressor of cellular radioresistance", as well as other relevant information. The anticipated duration of the project is three years and I estimate the direct cost for the first budget year to be $85,000. I hope this project will be judged suitable for a full, formal application. Sincerely yours, Dan Canaani, Ph.D. Senior Lecturer (tenured) Dr. Dan Canaani Department of Biochemistry Tel Aviv University Ramat Aviv 69978 Israel. Tel: (972)-3-6408985 Fax: (972)-3-6424270 Encs. 05-6m,15053 "Op9,05-6A09749 '~O .69978 3"3N'~ ,3~3N'~D] ,,~D~DI]~]IN~ ~]R TEL AViV UN£VERStTZ, RA~4AT AViV, TEL AViV 699'78. TEL. 03-6409749. FAX. 972-3-6415053 40008970

Canaan! Dan A naturally occuring human antisense RNA - a repressor of cellular raflioresistance I. Background and significance The presence of natural antisense RNAs in prokaroytes is well established. These RNAs were shown to affect plasmid replication and maintenance, transcription of plasmid and bacterial genes as well as transposition and DNA conjugation. In contrast, the participation of cellular antisense transcripts in gene regulation in eukaroytes is so far confined to few cases. Natural antisense RNAs have been previously found to be involved in the regulation of p53, N-myc and Xenopus FGF gene, as well as in the developmental programs of slime mold and worms. Recently, we have isolated a new human radiation protecting gene (RPG-1), which stimulates cellular radioresistance (UV- and -f-radiation), while having no effect on nucleotide excision repair. We have also identified a short natural antisense RNA spanning a segment of the 3' untranslated region of RPG-1 mRNA. The RPG-1 protein has been extremely conserved during evolution, and its involvement in cell cycle progression/necrotic or apoptotic cell death is currently being examined. The objective of the proposed study is to clone the full-length cDNA for the antisense RNA to test whether and how it regulates RPG-1 gene expression. Additionally, it will be important to determine how the antisense RNA itself is regulated/affected under different physiological conditions. II. Broad goals and specific aims The long-term objectives are to unravel the biological roles of RPG-1 and its regulation. In the course of this project we plan to achieve the following aims and/or answer the following questions. 1. Molecularly clone the full-length cDNA for the antisense RNA and determine its sequence. 2. Test the existence in vivo of an RNA duplex between RPG-t mRNA and the 0.7 kb antisense RNA. 3. Analyze whether the antisense RNA affects the expression of the RPG-1 gene, and if so at what level. 4. Isolate and identify by functional assays the promoter for the antisense RNA. 5. Examine the interspecies conservation of the antisense RNA gene, and its profile of transcription in different human tissues. 6. Initiate the study on the regulation of the antisense RNA. III. Supporting data Previously our laboratory innovated the approach of cloning human DNA repair genes by transduction of expressible human cDNA libraries into DNA repair deficient human mutant cell lines while selecting for transfectants which acquired elevated radiation resistance (Teitz et al., 1987). This experimental approach resulted in the identification of two human genes which affect cellular UV-resistance. The first turned out to encode the regulatory (9) subunit of human casein kinase II (Teitz et al., 1990a). Casein kinase II is a serinelthreonine kinase whose catalytic subunits exhibit close sequence similarity to a key enzyme in cell division, the human cdc2 (CDK1) gene. We have cloned the cDNAs encoding the human CK II catalytic subunits and together with Dr. Yang-Feng of Yale University have chromosomally mapped each one of the three CK II subunit genes (Yang-Feng et al., 1990; 1991; 1994). To study the biological roles of CK II we have developed a conditional autocatalytic cell system for expression of stringently regulated genes such as CK II (Dotan et al., 1995), as well as 40008971

Canaani Dan applied the yeast two hybrid protein interaction screen to identify th~ in vivo substrates of CK II. The second gene (RPG-1) was isolated following the introduction of an expressible human cDNA library into a highly UV-sensitive fibroblast cell line belonging to a patient, from xeroderma pigmentosum complementation group C (XP-C) and previously immortalized by expression of SV40 early region (Canaani et al., 1986). This led to stable complementation to wild-type levels of both the UV-sensitivity and the excision repair deficiency in primary transformants (Teitz et al., 1987; Stark et al., 1989). Secondary transformants displaying a stable partial UVR phenotype were generated by transfection with a partial digest of total chromosomal DNA from one of the primary transformants while selecting for expression of the "endogenous" neo genes (Teitz et al., 1987). Transfected cDNAs were rescued from the celullar DNA of a secondary transformant by their in vitro amplification using expression vector specific oligodeoxyribonucleotides as primers in a PCR reaction. Their expression in XP-C cells identified a single cDNA (cDNA3), which complemented the UV sensitivity of the recipient cell line to the same partial UV resistance levels exhibited by the secondary transformant from which the cDNA was rescued (Teitz et al., 1990b). The phenotype conferred by expression of cDNA3 was cell specific, as it had no effect on the UV survival of xeroderma pigmentosum cells from complementation group D. DNA sequencing has shown that cDNA3 contains an inser~ of 482 bp including a poly(dA) tail, but no initiation codon for translation and at most an open reading frame of 27 amino acids. Northern blots and DNA sequence analysis determined that cDNA3 constitutes part of the 3' nontranslated region of a 4.5 kb long new human mRNA. Extensive analysis by Southern, Northern and direct sequencing of RT-PCR products derived off mRNAs from 12 XP-C families ruled out the possibility that cDNA3 represents the gene which is defective in XP-C patients. In collaboration with Dr. Aziz Sancar of the University of North Carolina at Chapel Hill, we have shown that in contrast to the situation in XP-C primary transformants, cDNA3 transfectants did not acquire the capability of nucleotide excision repair as tested in a cell-free extract system. Since we have ruled out the possibility of complementation by gene conversion/homologous recombination between a normal copy of cDNA3 and a putative defective segment residing in XP-C cells (see above), how can this partial-length cDNA affect UV-survival? The first clue came from a control experiment where Northern blots of total XP- C RNA were probed with cDNA3-sense riboprobe. Surprisingly, the existence of a 0.7 kb long RNA was detected in the nontransfected XP-C cells. The presence of a naturally occurring antisense RNA to the 4.5 kb mRNA in XP-C cells was verified by the use of cDNA3-sense specific oligonucleotides as primers in a reverse transcriptase reaction from poly(A) containing RNA, followed by PCR. These experiments have shown that the 4.5 kb and 0.7 kb RNAs share at least 322 complementary bases, localized to the 3' untranslated region of the former. These 5' terminal 322 bp of the 0.7 kb antisense RNA do not contain any open reading frame. Our working hypothesis is that the transcription/stability/or translatability of the 4.5 kb mRNA is repressed by the formation of an RNA duplex with the 0.7 kb antisense RNA. Transduction followed by transcription of the mRNA encoded by cDNA3 leads through competition for the 0.7 kb RNA to the generation of cDNA3 mRNA - 0.7 kb RNA duplex, while releasing the 4.5 kb mRNA from the duplex. The latter in turn leads to increased expression of the 4.5 kb mRNA manifested in elevated radioresistance. We propose that the 0.7 kb antisense RNA is a natural repressor of cellular radioresistance. cDNA3 expression complements not only UV- but also 1,-ray sensitivity. One of the hallmarks of the yeast checkpoint mutants is sensitivity to both types of radiation. 2 40008972

Experiments are in progress to analyze the possible role of RPGol in cell cycle progression and radiation induced apoptosis. In addition, during the past few months we have cloned most. of the 4.5 kb mRNA transcripts as cDNA clones. DNA sequencing, v;h[ch is currently in progress, indicates that at least part of the identified open reading frame has been extremely conserved during evolution. The corresponding DNA sequences from the mouse exhibit a high degree of homology which is translated into an 88% identify at the amino acid level, and when conserved amino acid changes are taken into account, the similarity at the protein level approaches 98%. IV. Brief description of experimental design and methods The plan of operation is listed in section II. I reviews, here only some of the methods that we intend to use in addressing the specific aims. 1) Basing ourselves on the poly(A) tail of the antisense RNA, we will clone its 3' end by RT- PCR (RACE) reaction with dTn-adaptor primer for the reverse transcription. The adaptor primer, and two antisense primers (one of them nested) known to reside in the region of overlapping with RPG-1 mRNA, will serve in the PCR reaction. The nucleotide sequences of the PCR products will be compared with those of the RPG-1 mRNNgene. We already know from RT-PCR experiments that the 0.7 kb antisense RNA is not contiguous with the RPG-1 mRNA. Obviously, if the complete sequence of the 0.7 kb antisense RNA is confined to overlapping RPG-1 mRNNgene sequences, then it is likely to be encoded by the same locus. 2) In order to test for in vivo duplexes between the RPG-1 mRNA and the 0.7 kb antisense RNA we will employ the double RNase protection assay. A further proof can be obtained if we will find in the sequencing of the RT-PCR products (mentioned above) a transition of A to G residues when compared to the genomic sequence. Such a phenomenon is due to an RNA duplex unwindase activity which modifies by deanimation A to I residues. 3) Transfection and transcription of cDNA3 in XP-C cells led to increased cellular radioresistance. To test for a correlation between cDNA3 transcription and RPG-1 gene expression we will compare RPG-1 stable mRNA (by Northern blots and RNase protection) and protein (by Western blots) in XP-C versus cDNA3-transfected XP-C cells. As we assume that the 0.7 kb mRNA is noncoding (see previous section), we anticipate that it will be found to affect either both RPG-1 mRNA and protein levels or the protein level per se. If the former turns out to occur, we will compare, in the same cell lines, the rate of RPG-1 transcription by nuclear run-on transcription assays to see whether transcription, splicing or stability are affected. We will also transfect the antisense RNA gone by itself as well as the RPG-1 open reading frame (each incorporated in an expression vector) into XP-C, XP-C/cDNA3, and assay for radioresistance. 4) Following cloning and identification of the.putative promoter region for the antisense RNA gene, it will be cloned into pSVO-CAT type vector and tested for CAT expression in transient transfections into human cells. 5) Evolutionary conservation of the antisense RNA gone will be assessed by interspecies {Zoo) Southern blots. Sense riboprobes will be employed for human Northern tissue blots. 6) We have observed a large variation in the level of the 0.7 kb antisense RNA among human cell lines. As the biological roles of the RPG-1 gone start to unravel it will be easier to define the experiments for this section of the proposal. Obviously, we will look at the expression of the antisense RNA under stress conditions such as radiation, treatment by genoto×ic agents, serum starvation, etc. 3 40008973