Jeannette A. Hovsepian ^{1, @} and John H. Frenster ^{2, @}

Departments of  ¹Radiology and of  ² Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

^@ Present Address:  Physicians' Educational Series, Atherton, California, 94027-5446 USA
Phone:  +1 650 367 6483;   Fax:   +1 650 364 1773
e-mail:   frenster@euchromatin.net

* Supported in part by a USPHS Research Career Development Award (CA-17857) from the National Cancer Institute to J.H.F.

Selective transcription of eukaryotic gene DNA into pre-mRNA molecules is a complex process involving activator molecules to initiate the process, localized melting of the DNA helix to form transcription bubbles, and RNA polymerase II (RNAPII) to form the intact pre-mRNA product. Recent data indicate that small non-coding RNA species  function as co-activators of eukaryotic gene transcription (Science 296: 1260 (2002)). Such RNA species are favored over proteins, since these RNA species are confined to the cell nucleus, are able to form RNA-DNA hybrid molecules to stabilize the DNA bubble, and are able to select specific DNA promoter sequences for transcription initiation [10]. They are further favored for gene regulation because of their short half-lives, allowing quick changes to be made in transcription activity following any changes in the nuclear interior or in other RNA-source genes. Further, small non-coding RNA molecules are transmitted intact from the maternal cell nucleus to the daughter cell nuclei through the course of cell mitosis, thus allowing the preservation of specific RNA programming states of chromatin from one cell generation to the next [8, 9]. Finally, small non-coding RNA species are capable of forming RNA-RNA complexes with the 5’ non-coding leader portions of pre-mRNA that are liberated during splicing of pre-mRNA molecules in forming the final mRNA products. The ability of de-repressor RNA species to form RNA-RNA complexes with 5’ non-coding leaders provides a feedback mechanism [11, 30], whereby excessive rates of transcription and splicing result in increased numbers of RNA-RNA complexes, with these complexes competing with DNA for de-repressor RNA molecules, until such time as a new steady-state of gene transcription within the intact cell nucleus is achieved within each cell for each gene. 

Selective DNA transcription during eukaryotic gene regulation is a complex process involving the chromatin remodeling by transcription activator molecules, the localized melting of gene-specific DNA sites to form transcription bubbles, and the recruitment of RNA polymerase II and other transcription factors to synthesize gene-specific pre-mRNA transcription products [1-4]. Recent data indicate that small RNA sequences function as coactivators of eukaryotic gene-specific transcription [1- 5]. Such RNA sequences have a functional advantage over proteins, since these RNA sequences are able to form RNA-DNA hybrid molecules [6], and are able to select gene-specific DNA  sequences for transcription initiation [7]. Small RNA sequences are transmitted intact from the maternal cell nucleus to the daughter cell nuclei through cell mitosis [8], allowing the preservation of specific RNA programming states within chromatin from one cell generation to the next [9]. Also, small RNA sequences can form RNA-RNA duplexes with the 5’ non-coding leader RNA portions of pre-mRNA that are liberated during splicing of pre-mRNA molecules [4, 10]. Such RNA-RNA duplexes can provide a feedback mechanism, whereby excessive rates of  gene-specific transcription and splicing allow capture of specific RNA activator sequences within RNA-RNA duplexes, thus destabilizing specific DNA bubbles and reducing specific gene transcription [11].

Small RNA molecules are involved in diverse activities within the cell [1-5, 11-12]. Recent reports indicate that small RNA molecules play a significant role during DNA transcription in both prokaryotes [13] and eukaryotes [1- 4], accounting for the selectivity of  gene expression found within these organisms [7]. During DNA transcription by RNA polymerase II, the double-helical structure of DNA undergoes limited strand-separations or melting, to allow the DNA coding bases to be exposed as a template for nascent RNA molecules [14], and it is here that small activator or de-repressor RNA molecules play a role in initiating and stabilizing [15] such transcription bubbles (Fig.1).

Fig. 1: RNA-Induced Chromatin Remodeling and DNA Melting during Selective Gene Transcription.

     Small nuclear RNA species function as de-repressors by displacing [16] repressor proteins (dark blocks), and then binding to the anti-template DNA strand at an initiation site [14]. This initiation stage frees the template DNA strand for transcription to gene-specific pre-messenger RNA [15], following the recruitment to that site, of RNA polymerase II and other transcription factors such as TFIIH [1-3].

     A specific de-repressor RNA sequence may interact with complementary DNA sequences at several gene loci, permitting one RNA sequence population to activate multiple genes synchronously [14, 24].

     An excessive synthesis of gene-specific  pre-messenger RNA may result in formation of RNA-RNA duplexes between the de-repressor RNA and the 5’ leader sequences of that pre-messenger RNA, removing the de-repressor RNA from that initiation site, and reducing the pre-messenger RNA synthesis at that site in a feedback switch mechanism for control of selective gene dosage [11, 30].

     Melted DNA initiation sites are targets for early DNA replication, for single- and double-strand radiation breaks, and for the intercalation of polycyclic hydrocarbons, viral oncogenes or RNA [28].

In isolated nuclei from interphase calf thymus lymphocytes, 80 percent of DNA is inactive in RNA synthesis [18]. Active RNA synthesis occurs within 10 nm euchromatin microfibrils [17, 19], which are structurally continuous with the 30 nm heterochromatin fibrils found within dense nuclear masses [17]. The heterochromatin masses are devoid of RNA synthesis [19]. The active euchromatin microfibrils can be separated from the condensed heterochromatin masses under conditions which preserve the differential RNA synthetic activity after isolation [18]. When compared in thermal hyperchromicity studies, the DNA within isolated repressed heterochromatin is significantly more stable to denaturation than is the DNA within isolated active euchromatin [14, 16], although both are more stable than is the naked DNA isolated from either fraction [16]. Although stabilizing histones are present to the same degree within both fractions [16], excessive amounts of nuclear polyanions are present within isolated active euchromatin [16]. These polyanions, such as RNA, acidic proteins, phosphoproteins, and phospholipids, antagonize the histone-DNA interactions, and render the involved DNA more susceptible to denaturation [16], to DNase I action [20], and to binding by other direct ligands such as actinomycin or acridine orange [20].

Advantage has been taken of such sensitivity to DNase I to develop ultrastructural probes of  active DNA sites within intact cells of human bone marrow [21] during cell division and differentiation [22], and within intact Hodgkin’s Lymphoma lymph nodes [21] during neoplastic cell progression and the surrounding T lymphocytic reaction [23]. These electron microscopic ultrastructural probes allow a single-cell analysis of DNase I-sensitive sites as measured by size, number, and location within the cell nuclei during cell activation, cell division and cell differentiation of large populations of intact cells in native human tissues [24]. More recent studies of isolated active euchromatin reveal that re-programming of gene expression is achieved by a displacement and replacement of one species of de-repressor RNA molecules by those of another species or subset of de-repressor RNA molecules [29].

The finding that the initiation and stabilization of melting of DNA templates during selective gene transcription is mediated by small RNA molecules in the eukaryote cell nucleus [1-4] poses many fundamental questions for the activity of gene clusters and operons [12], for functional genomics [10], and for molecular medicine [11]. These questions include the role of small RNA species in c-myc deregulation during the induction of  neoplastic transformation in mice [25], their role in the oncogenesis of Burkitt lymphoma [26], and their role in the therapy of human acute myelogenous leukemia [27]. The fundamental molecular questions raised by these findings also include the specific base sequences in de-repressor RNA species, the synthesis, storage, and distribution of de-repressor RNA species during oogenesis [28], and the dynamics of  displacement and replacement of de-repressor RNA species from melted DNA during gene re-programming [29-31].

#E. Gottesfeld JM,  and Barbas CFIII, "RNA as a Transcriptional Activator",  Chemistry and Biology, vol 10, no.7, pp. 584-585 (July, 2003).

#D: Frenster JH, and Hovsepian JA, "Overshoot in Late Telophase for RNA Re-Programming of Mitotic Chromatin","RNA2003", p. 211 (July 1-6, 2003), The RNA Society, Bethesda, MD, USA.

#C. Buskirk AR, Kehayova PD, Landrigan A, and Liu DR, "In Vivo Evolution of an RNA-Based Transcriptional Activator", Chemistry and Biology, vol 10, no. 6, pp. 533-540 (June, 2003).

#B. Saha S, Ansari AZ, Jarell KA, and Ptashne M, "RNA Sequences that Work as Transcriptional Activating Regions", Nucleic Acids Research, vol. 31, no. 5, pp. 1565-1570 (March 1, 2003).

#A. Percipalle P, Fomproix N, Kylberg K, Miralles F, Bjorkroth B, Daneholt B, and Visa N, “A Specific Actin-Ribonucleoprotein Interaction Required for Transcription by RNA Polymerase II”, Am. Soc. Cell Biology, 42nd Annual Meeting, Dec. 18, 2002, Late Abstract Poster L235.

2.  Li X-Y, Bhaumik  SR, Zhu X, Li L, Shen W-C, Dixit BL, and Green MR, “Selective Recruitment of TAFs by Yeast Upstream Activating Sequences: Implications for Eukaryotic Promoter Structure”, Current Biol 12, 1240-1244 (2002).

3a. Lanz RB, Razani B, Goldberg AD, and O'Malley BW, "Distinct RNA Motifs are Important for Coactivation of Steroid Hormone Receptors by Steroid Receptor RNA Activator (SRA)", Proc. Natl. Acad. Sci. USA, vol. 99, no. 25, pp. 16081-16086 (December 10, 2002).

3b. Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY, Tsai MJ, and O'Malley BW, "A Steroid Receptor Coactivator, SRA, Functions as an RNA and is Present in an SRC-1 Complex", Cell, vol. 97, no.1, pp. 17-27 (April 2, 1999).

3c. Frenster JH, "Nuclear RNA Species Activate DNA Transcription Within Chromatin", FASEB Journal, Vol. 13, No. 7, A1506 (April 23, 1999).

4a. Lin X, Taube R, Fujinaga K, and Peterlin BM, "P-TEFb Containing Cyclin K and Cdk9 Can Activate Transcription via RNA", J. Biol. Chem. vol. 277, no. 19, pp. 16873-16878 (May 10, 2002).

4b. Pesole G, Liuni S, Grillo G, Licciulli F, Mignone F, Gissi C, and Saccone C, "UTRdb and UTRsite: Specialized Databases of Sequences and Functional Elements of 5' and 3' Untranslated Regions of Eukaryotic mRNAs. Update 2002", Nucleic Acid Research, vol. 30, no. 1, pp. 335-340 (January 1, 2002).

5. Storz G, “An Expanding Universe of Noncoding RNAs”, Science 296, 1260-1263 (2002).

6. Chamberlin MJ, “Comparative Properties of DNA, RNA, and Hybrid Homopolymer Pairs”, Fed Proc 24, 1446-1457 (1965).

7. McCarthy BJ, and Hoyer BH, “Identity of DNA and Diversity of  Messenger RNA. Molecules in Normal Mouse Tissues”, Proc Nat  Acad Sci USA 52, 915-922 (1964).

8. Goldstein L, “Stable Nuclear RNA Returns to Post-Division Nuclei Following Release to the Cytoplasm during Mitosis”, Exp Cell Res  89, 421-425 (1974).

9. Goldstein L, Wise GE, and Ko C, “Small Nuclear RNA Localization during Mitosis. An Electron Microscope Study”, J Cell Biol  73, 322-331 (1977).

10. Frenster JH, “Correlation of the Binding to DNA Loops or to DNA Helices with the Effect on RNA Synthesis”, Nature vol. 208, no. 5015, p. 1093 (December 11, 1965).

11a. Herstein PR, and Frenster JH, "Mated Models of Gene Regulation in Eukaryotes", in: "Embryonic and Fetal Antigens in Cancer", vol. 2, pp. 5-7, (Anderson NG, Coggin JH, eds.), National Technical Information Service, U.S. Dept. Commerce, Springfield, VA., 1972:

11b. Frenster JH, and Herstein PR, “Gene De-Repression”, New Eng J Med 288, 1224-1229 (1973).

12. Blumenthal T, Evans D, Link CD, Guffanti A, Lawson D, Thierry-Mieg J, Thierry-Mieg D,  Chiu WL, Duke K, Kiraly M, and Kim SK, "A Global Analysis of Caenorhabditis elegans Operons", Nature vol. 417, no. 6891, pp. 851 - 854 (June 20, 2002).

13. Wassarman KM, and Storz G, “6S RNA Regulates E. coli RNA Polymerase Activity”, Cell 101, 613-623 (2000).

14a. Frenster JH, “Localized Strand Separations within Deoxyribonucleic Acid during Selective Transcription”, Nature vol. 208, no. 5013, pp. 894-896 (November 27, 1965).

14b. Frenster JH, "Mechanisms of Repression and De-Repression within Interphase Chromatin", In-Vitro, vol. 1, pp. 78-101, (1965).

15. Frenster JH, “A Model of Specific De-repression within Interphase Chromatin”, Nature 206, no. 4990, pp. 1269-1270 (June 19, 1965).

16a. Frenster JH, “Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid”, Nature vol. 206, no. 4985, pp. 680-683 (May 15, 1965).

16b. Huang RC, and Bonner J, "Histone-Bound RNA, a Component of Native Nucleohistone", Proc. Natl. Acad. Sci. USA 54, no. 3, pp. 960-967 (September, 1965).

16c. Benjamin W, Levander AD, Gellhorn A, and DeBellis RH, "An RNA-histone complex in
     mammalian cells and the isolation and characterization of a new RNA species", Proc. Natl. Acad. Sci. USA vol. 55, no. 4, pp. 858 (April, 1966).

16d. Mishra MC, Niu MC, and Tatum EL, "Induction by RNA of Inositol Independence in Neurospora crassa". Proc. Natl. Acad. Sci. USA 72: 642 (1975).

16e. Tseng BY, and Goulian M, "Initiator RNA of Discontinuous DNA Synthesis in Human Lymphocytes", Cell, vol. 12, p. 483 (1977).

16f. Jelinek W, and Leinwand L, "Low molecular weight RNAs hydrogen-bonded to nuclear and
     cytoplasmic poly(A)-terminated RNA from cultured Chinese hamster ovary cells", Cell 15, 205 (1978).

16g. Dobrzelewski J, Milewska Z,  and Panusz H, "Effect on Transcription of Low-Molecular-Weight RNA from Calf Thymus Chromatin", Acta Biochim. Pol. vol. 27, no. 2, pp. 75-87 (February, 1980).

16h. Darlix J-L, Khandian EW, and Weil R, "Nature and Origin of the RNA Associated with Simian Virus 40 Large Tumor Antigen", Proc. Natl. Acad. Sci. USA, vol. 81, pp. 5425-5429 (September, 1984).

16i. Young LS, Dunstan HM, Witte PR, Smith TP, Ottonello S, and Sprague KU, "A Class III Transcription Factor Composed of RNA", Science, vol. 252, pp. 542-546 (April 26, 1991).

16j. Hoffman M, "An RNA First: It's Part of the Gene-Copying Machinery", Science vol. 252, pp. 506-507 (April 26, 1991).

16k. Luo Y, Kurz J, MacAfee N, and Krause MO, “C-Myc Deregulation during Transformation Induction: Involvement of 7SK RNA”, J Cellular Biochem 64, 313-327 (1997).

16l. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin", Clinical Cancer Research, vol. 5, suppl. l, p. 3855s, (624), (November, 1999).

16m. Saha S, Ansari AZ, Jarell KA, and Ptashne M, "RNA Sequences that Work as Transcriptional Activating Regions", Nucleic Acids Research, vol. 31, no. 5, pp. 1565-1570 (March 1, 2003).

17. Frenster JH, “Ultrastructural Continuity between Active and Repressed Chromatin”, Nature vol. 205, no. 4978, pp. 1341-1342 (March 27, 1965).

18. Frenster JH, Allfrey VG, and Mirsky AE, “Repressed and Active Chromatin Isolated from Interphase Lymphocytes”, Proc Natl Acad Sci USA 50, 1026-1032 (1963).

19. Littau VC, Allfrey VG, Frenster JH, and Mirsky AE, “Active and Inactive Regions of Nuclear Chromatin as Revealed by Electron Microscope Autoradiography”, Proc Natl Acad Sci USA 52, 93-100 (1964).

20. Frenster JH, “Electron Microscopic Localization of Acridine Orange Binding to DNA within Human Leukemic Bone Marrow Cells", Cancer Res 31, 1128-1133 (1971).

21. Frenster JH, Nakatsu SL, and Masek MA, "Ultrastructural Probes of DNA Templates within Human Bone Marrow and Lymph Node Cells", Adv Cell Molec Biol 3, 1-19 (1974), ed. DuPraw EJ, New York: Academic Press.

22. Nakatsu SL, Masek MA, Landrum S, and Frenster JH, "Activity of DNA Templates During Cell Division and Cell Differentiation", Nature 248, 334-335 (1974).

23a. Frenster JH, Papalian MM, Masek MA, and Frenster JA, "Electron Microscopic Analysis of Lymph Node Cellular Activity in Hodgkin's Disease", J Natl Cancer Inst 63, 331-335 (1979).

23b. Frenster JH, "Uni-Polar Clustering of Lymphocyte DNA Templates Toward Neoplastic Target Cells within Hodgki's Disease Lymph Nodes", Proc. Am. Assoc. Cancer Res. vol. 43, p. 1134 (March, 2002).

24. Frenster JH, "Single-Cell Analysis of DNase I-Sensitive Sites during Neoplastic and Normal Cell Differentiation within Human Bone Marrow", Ann NY Acad Sci 567, 334-336 (1989).

25a. Darlix J-L, Khandian EW, and Weil R, "Nature and Origin of the RNA Associated with Simian Virus 40 Large Tumor Antigen", Proc. Natl. Acad. Sci. USA, vol. 81, pp. 5425-5429 (September, 1984).

25b. Luo Y, Kurz J, MacAfee N, and Krause MO, “C-Myc Deregulation during Transformation Induction: Involvement of 7SK RNA”, J Cellular Biochem 64, 313-327 (1997).

25c. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin", Clinical Cancer Research, vol. 5, suppl. l, p. 3855s, (624), (November, 1999).

26a. Maruo S, Nanbo A, and Takada K, “Replacement of the Epstein-Barr Virus Plasmid with the EBER Plasmid in Burkitt’s Lymphoma Cells”, J Virol 75, 9977-9982 (2001).

26b. Frenster JH, "Model of Single-Stranded Integration of Oncogenic Viral Genomes", Biophys. J. vol. 15: 137a (1975).

27. DeCarvalho S, “Effect of RNA from Normal Human Marrow on Leukaemia Marrow In-Vivo”, Nature 197, 1077-1080 (1963).

28. Frenster JH, “Selective Control of DNA Helix Openings during Gene Regulation”, Cancer Res., vol. 36, pp. 3394-3398 (September, 1976).

29. Frenster JH, "Yeast RNA Re-Programming of Already-Active Mammalian Chromatin", "RNA 2002", p. 592, Bethesda, MD: The RNA Society, May 28th - June 2nd, 2002.

30a. Frenster JH, and Hovsepian JA, "RNA Feedback Mechanisms during Eukaryotic Gene Regulation", "Northwest Symposium on Systems Biology", p. 15, Oct. 17-18, 2002.

30b. Frenster JH, "Nuclear Ribosomes and RNA-RNA Duplexes", Mol. Biol. Cell vol. 11, suppl. p. 20a (December, 2000).
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31. Frenster JH, "Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".