John H. Frenster, M.D., Department of Medicine, Stanford University, Stanford, California 94305, USA. 

1. Introduction

2. Gene De-Repression
a. Heterochromatin and Euchromatin
b. Selective Transcription
c. DNA Helix Openings

3. De-Repressor RNA
a. Assays of Gene De-Repression
b. Isolation and Characterization
c. Interaction with DNA and RNA
     Fig. 1: Gene-Specific Feedback Control of Gene De-repression.

4. Mechanisms of Action
     Fig. 2: Interactions between DNA, De-repressor RNA and Immediate Transcription-product RNA.

5. Addendum
6. Summary
7. References

8. Additional References for RNA as a Therapeutic Agent:
9. Links to Other Sites:
10. Information and Feedback:

Recent experiments utilizing endogenous RNA polymerase (1) have confirmed earlier studies (2-4) which indicated that certain species of low molecular weight RNA can function as de-repressors of gene transcription within higher organisms. Such gene de-repression results in the synthesis of messenger RNA molecules which are characteristic of the particular cells studied (5) and which may account for the positive effects of added nuclear RNA during embryogenesis (6, 7), bacterial (8) or fungal (9) transformation, and the immune response (10). These species of nuclear RNA have been variously referred to as de-repressor RNA (11), chromosomal RNA (3, 4), or activator RNA (12), and are thought to constitute the effector molecules of those complex control systems in higher organisms in which the product of one gene can specifically increase the activity of other genes in the same (12) or adjoining cells (13-15).

Gene regulation in lower organisms, especially bacteria, is mediated largely by protein molecules acting as repressors or inducers of gene activity (16). In these prokaryote organisms, criteria of fitness involve maximum growth when the environment permits it, and minimum growth when the environment demands it. By contrast, in higher organisms the internal environment of the organism is more stable, and criteria of fitness involve restraint of growth in a balanced manner and specialization of function of each cell. Although some protein mechanisms for gene regulation are retained in higher organisms, it is not surprising that the special needs for balanced growth and specialized function in higher organisms should also utilize the higher degree of gene selectivity offered by RNA molecules.

Regulation of gene activity in a positve mechanism by de-repressor RNA provides a unique informational system that maximizes :
1. selectivity for particular gene loci;
2. localization and stability within the cell nucleus;
3. transmission at the time of cell division to the daughter nuclei; and
4. stability within cell nuclei through many cell generations,
all of which reflect the long-term stability of cell differentiation in higher organisms.

2. Gene De-repression:

a. Heterochromatin and Euchromatin:

Higher organisms are composed of a myriad of tissues, each composed of cells differentiated to a specific state and function (5). Such cell differentiation is mediated by selective gene de-repression of an otherwise completely repressed genome (17), with the de-repressed genes being transcribed for specific RNA and protein synthesis characteristic of the differentiated state of that cell (18). The microscopic appearance of such differentiation in the cell nucleus (19) is revealed in the partition of interphase chromatin into at least two phase states within the nucleus:
1. large masses of condensed heterochromatin, and
2. fine microfibrils of extended euchromatin (20).

Detailed studies of the molecular biophysics, biochemistry, and ultrastucture of heterochromatin and euchromatin have revealed a striking partition of function between the two states of chromatin (Table 1), with the most significant being that all active RNA synthesis is confined to the extended euchromatin portion of the cell nucleus (21, 22). Preparative methods have been developed to fractionate the cell nucleus, allowing the physical isolation of either heterochromatin or euchromatin from one tissue sample (21), and these isolated fractions have yielded biochemical and biophysical data which have helped to elucidate the mechanism of gene de-repression (2) and selective transcription (31) within the cells of higher organisms.

b. Selective Transcription:

Each normal diploid cell of an individual animal contains a full and identical complement of all DNA molecular species characterizing that animal (5, 32). Of these DNA molecules, only a fraction are transcribed to RNA molecules within any one cell or tissue (21), with such RNA products being characteristic of the particular tissue type (32, 33).

Stable epigenetic mechanisms select specific portions of the genome for transcription (34), and these mechanisms are stable through cell divisions for up to 50 generations (35). Such selective transcription involves both the choice of a particular gene locus and the choice of a particular DNA strand within the locus on which to effect RNA synthesis (31, 36). The rate of transcription at a particular gene locus appears more influenced by such factors as the local cellular environment and the occurrence of hormonal stimulation (17). Those portions of the genome not selected for gene transcription are usually found in a condensed state, manifested ultrastructurally as masses of inactive heterochromatin (20, 21, 22). When the cell is specifically activated, these masses of condensed heterochromatin may be rapidly converted to extended euchromatin active in gene transcription (30).

c. DNA Helix Openings:

The DNA helix must open in localized areas before the interior base-coded genetic information can be used for new RNA or DNA synthesis (21, 37, 38). These DNA helix openings can be visualized by a high-resolution electron microscopic technique (24, 39), which has been applied to individual cells undergoing normal cell division and differentiation (40), embryogenesis (41), and neoplasia (42, 43). These DNA helix openings are sensitive to digestion with pancreatic DNase I (18, 24) which is relatively specific for single-strand cuts in DNA (44). Such DNA helix openings are found exclusively within transcription-active euchromatin (24), and range in size from 25 to 700 nm in length, corresponding to 70 to 2000 base pairs in DNA helix length (25).

A great variety of molecules entering the cell nucleus preferentially bind to those portions of DNA molecules which are in a single-stranded state (45). The consequences of such ligand binding to single-stranded DNA are to stabilize the particular bound DNA helix openings in the open position (Table 2), thus increasing the rates of RNA synthesis within the DNA helix opening (38, 45).

Carcinogenic chemicals, oncogenic viruses, and steroid hormones all prefer to bind to such single-stranded portions of the host cell's DNA molecules (Table 2), and each of these results in an increase in the rate of RNA synthesis after such binding (38, 45). By contrast, other ligand molecules to DNA bind preferentially to DNA in the double-stranded state (Table 2), and the effect of such ligand binding is to stabilize the closed DNA helix in the inactive state, with a resultant decrease in the rate of RNA synthesis following such binding (38, 45).

Modified from: Frenster JH, "Selective Control of DNA Helix Openings During Gene Regulation", Cancer Research, vol. 36, pp. 3394-3398 (1976).

3. De-Repressor RNA:

a. Assays of Gene De-Repression:

Assays for de-repressor activity range between two major constraints:
1. assays reflecting the biological role of the particular gene de-repression, and
2. assays reflecting the molecular uniqueness of the particular gene de-repression.
Assays relecting the biological role might best be observed in intact animals or intact cells in which the de-repressor molecule produces a specific and crucial effect. These assays are often blurred by the multiple interactions available to the de-repressor molecule in such large and complex systems.

By contrast, assays reflecting the molecular uniqueness of the particular gene de-repression might best be observed in cell-free systems of isolated nuclei, isolated chromatin, or even isolated gene loci, in which the de-repressor molecule interacts with a specific portion of the DNA genome. These assays are often blurred in cell-free systems by the lack of a characteristic biological effect reflecting the significance of the particular gene de-repression.

Some of the more recent assays for de-repressor RNA involve comparisons between nuclear RNA molecules extracted from comparable normal or neoplastic cells (1), comparisons of 5.0 S RNA with 4.5 S RNA (47), comparisons of nuclear RNA with cytoplasmic RNA (2), comparisons of nuclear RNA extracted from immunized or non-immunized subjects (10), comparisons of RNA extracted from embryonic or non-embryonic tissues (6, 48), and comparisons of RNA extracted from cells resistant or sensitive to various drugs (8, 9).

In terms of target systems as assays, cell lysates have been used as targets in assays for initiator RNA activity (49), but isolated DNA sequences have not as yet been used as targets for such assays. By contrast, isolated chromatin is the most frequently used target in assays for de-repressor RNA activity (1, 2, 3, 4, 33, 47), but these are most accurate when they employ endogenous RNA polymerase (1, 2), since exogenous RNA polymerase may copy input RNA artifactually (50).

De-repressor RNA has also been assayed using intact cells (8-10), tissue explants (6, 7, 48), or intact animals (51) as targets, but these complex systems do not permit an unequivocal assessment of the direct effect of RNA upon particular DNA sequences.

b. Isolation and Characterization:

Early studies indicated that 10 S RNA could be separated from messenger RNA after extraction from rat liver (52). Later studies have revealed that such RNA was often found in association with histones after extraction from cells (53, 54), and some of these molecules were found to be restricted to the cell nucleus (55). Chromosomal RNA was originally isolated by a quite complicated procedure (56), but more recently this has been greatly simplified (57). Current studies have indicated that de-repressor activity is found in 4.5 S RNA (47), and can be separated from an accompanying protein which displays a direct stabilizing effect on homologous RNA polymerase (1). The sedimentation and size characteristics of such de-repressor RNA suggests it may be a member of the H class of low molecular weight nuclear RNA molecules synthesized by RNA polymerase I (58).

c. Interaction with DNA and RNA:

When chromosomal RNA is hybridized to native double-stranded DNA, the hybridization is greatly favored by the presence of 5 M urea, a condition which indicates the necessity for some opening of the DNA helix before hybridization with RNA can occur (59). Chromosomal RNA has been found to hybridize to a minimum of 16% of isolated middle-repetitive DNA sequences and a minimum of 1% of isolated single-copy rat DNA (57), or a sum of 4% of total nuclear rat DNA (57). The middle-repetitive DNA sequences to which chromosomal RNA hybridizes are found to be interspersed with single-copy DNA sequences (60) in a manner in which such middle-repetitive DNA sequences are thought to function as control elements in the transcription of the adjacent single-copy DNA sequences (12, 61). In fact, the immediate transcription-product RNA consists of chromosomal RNA sequences on the 5' end of the messenger RNA molecule (60), a position also found to be occupied by double-stranded RNA sequences of the immediate transcription-product RNA (62). These data suggest that de-repressor RNA is often found in a double-stranded state (36, 46), possibly forming a duplex with operator RNA as a complementary sequence (Figure 1). Several studies have recently shown the occurrence of such double-stranded RNA molecules within animal cells (63-65). These double-stranded RNA are quite stable, and may provide the mechanism whereby de-repression can be transported through cell division (66, 67), from the maternal cell nucleus to the nuclei of the daughter cells (68, 69).

Figure 1. Gene-specific feedback control of gene de-repression. The immediate transcription-product RNA molecule consists of both repetitive and single-copy sequences (60), corresponding to operator gene (o) and structural gene (sg) sequences (12, 17, 38). Operator RNA (oRNA) is complementary in base composition to de-repressor RNA (dRNA), and is capable of forming heterometric or homometric RNA-RNA duplexes with de-repressor RNA after excessive rates of gene transcription, thereby inducing the removal of de-repressor RNA from the DNA helix opening, and providing a gene-specific mechanism for feedback inhibition of RNA synthesis.

Since the DNA helix must open in localized areas before the interior base-coded genetic information can be used for new RNA synthesis during gene transcription (31, 38), it is possible that all of the positive effects of added de-repressor RNA are due to the effect of such RNA on promoting such DNA helix openings within particular gene loci (11).

De-repressor RNA offers several unique properties which favor its role in such a selective control mechanism (Figure 1).

• First, it contains sufficient base-sequence complexity (2) to be able to select specific middle-repetitive DNA sequences scattered throuout the genome (57, 60).
• Second, these middle-repetitive DNA sequences are positioned on the 5' end of those single-copy DNA sequences (60, 61) which are coding for structural proteins (12, 61).
• Third, the quantities of such organ-specific middle-repetitive RNA are known to be charcteristic of each organ studied (5).
• Fourth, the formation of double-stranded RNA-DNA duplexes is favored thermodynamically over the DNA-DNA duplex (70,71), especially in the 5 M urea solutions used for chromatin reconstitution assays (4, 59), allowing added RNA to open the DNA helix.
• Fifth, the formation of double-stranded RNA-RNA duplexes (63-65) between de-repressor RNA and its complement (62) on the 5' end of the immediate transcription-product RNA (Figure 1) protects de-repressor RNA against the action of endogenous RNase and allows its transport to the cytoplasm and back to the nucleus during cell division (66-69), thus preserving the particular pattern of gene expression found in the maternal cell on into the daughter cells (34, 35).
• Sixth, such nuclear RNA is found in excess within transcription-active isolated euchromatin fractions (2) as compared to inactive heterochromatin fractions (Table 3).
• Finally, such nuclear RNA is the most capable of all nuclear polyanions (2) in increasing the rate of RNA synthesis when added to previously repressed heterochromatin (Table 4), while having little or no effect upon addition to already active isolated euchromatin fractions (2). 

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  Table 3. Relation of Nuclear Polycations and Polyanions to DNA within Isolated Active Euchromatin and Isolated Repressed Heterochromatin.

Nuclear ligand.	Active Euchromatin	Repressed Heterochromatin
.	(mg/100mg DNA)	(mg/100mg DNA)
Total Nuclear Histones	90.7 +/- 7.7	101.1 +/- 9.1
Non-histone Residual Proteins	109.0 +/- 6.1	54.9 +/- 5.7
Total Nuclear RNA	9.0 +/- 4.7	1.8 +/- 0.7

Modified from: Frenster JH, "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid", Nature Vol. 206, pp. 680-683, May 15, 1965.

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Table 4. Effect of Added Nuclear Polycations or Polyanions on UTP-2-¹⁴C Incorporation into RNA within Isolated Euchromatin and Isolated Heterochromatin.

Nuclear ligand added	Active Euchromatin	Repressed Heterochromatin
.	(cpm/mg DNA)	(cpm/mg DNA)
None	222.2 +/- 13.8	76.2 +/- 4.4
Total Nuclear Histones	97.6 +/- 4.8	59.9 +/- 4.8
Non-histone Residual Proteins	286.0 +/- 1.0	118.4 +/- 7.2
Saline-soluble Proteins	262.0 +/- 1.0	121.7 +/- 0.3
Total Nuclear RNA	249.5 +/- 8.5	185.2 +/- 11.8

It is not yet apparent whether de-repressor RNA functions, in addition, as a primer for new RNA synthesis (72, 73), by being incorporated in the immediate transcription-product RNA molecule in a manner analogous to initiator RNA incorporation into newly synthesized DNA molecules during gene replication (49). The occurrence of double-stranded RNA-RNA duplexes within the cell nucleus (63-65) suggests that these molecules are formed as a type of feedback inhibition of RNA synthesis (Figure 2), with excessive rates of RNA synthesis at any particular gene locus resulting in the removal of de-repressor RNA from that gene locus by formation of a stable RNA-RNA duplex with de-repressor RNA. These complex interactions (Figure 2) between duplex DNA, de-repressor RNA, and immediate transcription-product RNA thus illustrate a complex control system which permits both a positive stimulus and a feedback inhibition for gene transcription at particular gene loci (17, 38).

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Figure 2. Interactions between DNA, de-repressor RNA, and immediate transcription-product RNA during cell division and gene regulation.



Figure 2. Interactions between DNA, de-repressor RNA, and immediate transcription-product RNA during cell division and gene regulation.

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• 1. The DNA helix is stabilized in the double-stranded comformation by closely applied polycationic histone proteins (45).
• 2. The DNA helix is de-stabilized by the displacement of histones from DNA by nuclear polyanions (2), forming transient DNA helix openings (11, 38).
• 3. These transient DNA helix openings are then stabilized in the open conformation at particular gene loci by the binding of de-repressor RNA (D-RNA) containing base sequences complementary to the anti-coding strand of the DNA helix opening (11).
• 4. The coding strand of the stabilized DNA helix opening is then used as a template for messenger RNA (M-RNA) synthesis at the particular gene locus (11).
• 5. Excessive production of M-RNA at the particular gene locus results in the formation of more- stable duplexes between M-RNA and D-RNA (36), removing D-RNA from the DNA helix opening and destabilizing the opening, resulting in a decreased rate of M-RNA synthesis at the particular gene locus and completing a negative feedback inhibition of transcription at that locus.
• 6. The M-RNA D-RNA duplex is transported to the cytoplasm during cell division (68, 69), where it is partitioned between the daughter cells following cell division (68, 69) and allows transmission of the particular pattern of gene transcription in the daughter nuclei (34, 35).
• 7. Random or directed degradation of D-RNA results in a progressive restriction in the range of gene transcription with aging of the particular cell clone (38).

5. Addendum:

More recent data (up to 1980) indicate that although embryonic-inducer RNA does effect transcription of DNA to m-RNA (1), it does not affect translation of such m-RNA by cytoplasmic ribosomes (74).

Nuclear RNA isolated from the livers of mice, rats, chickens, and calves and then administered to the uterine lining cells within living mice was found to induce the synthesis of alien albumin characteristic of the species donating the RNA (75), but was also found to induce the synthesis of host mouse albumin (75), the latter indicating gene de-repression and re-programming of the mouse uterine cells by the donated liver RNA (75). This gene de-repression was inhibited by actinomycin, indicating the need for the synthesis of new RNA rather than the utilization of pre-existing RNA molecules for such induced host albumin synthesis (75). Such re-programming of gene transcription can also be observed in cell hybrids after the activation of repressed hemoglobin genes following the hybridization to cells with active hemoglobin genes (76). Such gene de-repression appears to be gene-specific rather than species-specific in its induction.

Unique DNA sequences adjacent to middle-repetitive DNA sequences are preferentially transcribed and transported as m-RNA to the cytoplasm of mouse cells (77). Such middle-repetitive sequences are found at the 5' end of the unique sequences (60, 61), and provide the targets for complementary sequences in small nuclear RNA species which hybridize with the 5' ends of primary gene transcript DNA (78, 79). These small nuclear RNA species are 4 S in size (90 to 220 nucleotides in length (78, 79)), and are often associated with nuclear proteins (79) in a 30 S ribonucleoprotein particle (80, 81).

During gene transcription (82) and gene replication (83), DNA within active chromatin undergoes a characteristic conformational change (84) consisting of local DNA strand separations with DNA helix openings (31, 36-38) which can be visualized within intact eukaryotic cells by acridine orange ultrastructural probes (24, 25, 39-43, 85) and within isolated DNA molecules by RNA-loop formations (86). Such DNA helix openings may serve as targets for viral, hormonal, and chemical carcinogens (38, 45, 87). DNA helix openings have been found to be asymmetrically located into that half of the immune lymphocyte nucleus closest to the neoplastic cells within Hodgkin's Disease lymph nodes (88), in a manner compatible with the stimulatory effect of immune de-repressor RNA transferred from the neoplastic cell to the nucleus of the immune T-lymphocyte (10, 89).

6. Summary:

Certain species of low molecular weight nuclear RNA have been increasingly implicated as agents of specific gene de-repression in the cells of higher organisms, and may play an important role in controlling gene expression during embryogenesis, cell differentiation, and the immune response.

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76. McBurney MW, Featherstone MS, and Kaplan H, "Activation of teratocarcinoma-derived hemoglobin genes in teratocarcinoma-Friend cell hybrids", Cell 15, 1323 (1978).

77. Kuroiwa A, and Natori S, "Preferential expression of unique sequences adjacent to middle-repetitive sequences in mouse cytoplasmic RNA", Nucleic Acid Res. 7, 751 (1979).

78. 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).

79. Lerner MR, Boyle JA, Mount SM, Wolin SL, and Steitz JA, "Are snRNPs involved in splicing?", Nature 283, 220 (1980).

80. Frenster JH, Allfrey VG, and Mirsky AE, "Metabolism and morphology of ribonucleoprotein particles from the cell nucleus of lymphocytes", Proc. Natl. Acad. Sci. U.S. 46, 432 (1960).

81. Frenster JH, Allfrey VG, and Mirsky AE, "In-vitro incorporation of amino acids into the proteins of isolated nuclear ribosomes", Biochim. Biophys. Acta 47, 130 (1961).

82. Lilley DMJ, Jacobs MF, and Houghton M, "The nature of the interaction of nucleosomes with a eukaryotic RNA polymerase II", Nucleic Acid Res. 7, 377 (1979).

83. Weintraub H, "Assembly of an active chromatin structure during replication", Nucleic Acid Res. 7, 781 (1979).

84. Klevan L, Armitage IM, and Crothers DM, "³²P NMR studies of the solution structure and dynamics of nucleosomes and DNA", Nucleic Acid Res. 6, 1607 (1979).

85. Wang AHJ, Quigley GJ, and Rich A, "Atomic resolution analysis of a 2:1 complex of CpG and acridine orange", Nucleic Acid Res. 6, 3879 (1979).

86. Kaback DB, Angerer LM, and Davidson N, "Improved methods for the formation and stabilization of R-loops", Nucleic Acid Res. 6, 2499 (1979).

87. Frenster JH, Landrum SR, Masek MA, and Nakatsu SL, "DNA targets for carcinogens within living human bone marrow cells", Clin. Res. 26, 434A (1978).

88. 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 (1979).

89. Frenster JH, Nakatsu SL, Masek MA, and Frenster DA, "Active and repressed chromatin within nuclei of normal and neoplastic human cells", Fed. Proc. 39, 2009 (1980). 

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Additional References for RNA as a Therapeutic Agent:

1. DeCarvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo", Nature, vol. 197, no. 4872, pp. 1077-1080 (March 16, 1963).

1. Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY, Tsai M-J, and O'Malley BW, "A Steroid Receptor Coactivator, SRA, Functions as an RNA and is Present in an SRC-1 Complex", Cell 97: 17-27 (April 2, 1999).

2. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin", Clin. Cancer Res. 5: (Suppl.) 3855a (November, 1999).

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euchromation: "the most active portion of the genome within the cell nucleus."