In biology, most causality is influenced by context. And for RNA, expression precedes function. Within these constraints, RNA is becoming very interesting.

RNA is a molecular family of almost infinite variety: a variety of  base sequences, of molecular configurations and domains, of  ionic charges and molecular topologies and motifs, of molecular dynamics and locations, of molecular synthesis  and  turnovers, and of molecular associations, ligands, and complexes.  In addition, all RNA molecules can be influenced by those universal epigenetic base modifications consisting of  methylation, acetylation,  phosphorylation and other types of covalent bonds.

These new  molecular discoveries of the functions and the futures of RNA are open. But already, each day, we are startled by another RNA surprise,  another RNA discovery.

Storz G, "An Expanding Universe of Noncoding RNAs".

It is necessary to integrate these surprises, each day, lest we suffer intellectual indigestion. And so, we offer here, a short listing of RNA surprises for our consideration, our analysis, and our cognitive integration.

53. RNA genes of noncoding type are ultraconserved and altered within human neoplasms.
52. RNA of the embryonic, non-coding, micro-  type produces in vivo reductions of NSCLC lung cancer.
51. RNAs of Embryonic Gene Re-expression may be unaccompanied by appropriate ribo-regulators.
50. RNAs of diverse types are synthesized by interleaved pervasive transcription.
49. RNAs of long, noncoding macroRNAs average 4.2 kb in length, and are conserved in evolution.
48. RNAs, short, and double-stranded, use antisense to activate specific genes for transcription.
47. RNA selectively represses specific embryonic oncogenes. Are all human neoplasms embryonic ?
46. RNA species of non-coding type function as enhancers of human genes.
45. RNA molecules may have information content in their untranslated 3' terminal base sequences.
44. RNA polymerase catalyzes synthesis of secondary siRNAs from primary siRNAs' mRNA.
43. RNA noncoding gene has undergone rapid evolution for the embryonic human brain.
42. RNA immune vaccine mediates regression of human metastatic malignant melanoma.
41. RNA synthesis of specific allele type is mediated by kissing chromosomes in neurons.
40. RNA mediates the association between splicing factors and RNA Polymerase in human cells.
39. RNA species are required for chromosomal DNA replication in humans cells.
38. RNA and DNA Interactions in the DNA Strand-Separation Model of eukaryotic gene regulation.
37. RNA of antisense type serves as an enhancer and a gene regulator within embryonic stem cells.
36. RNA synthesis occurs in DNase1-sensitive chromatin sites.
35. RNA antisense sequences can be stabilized for in vivo activity in mouse gene regulation.
34. RNA sense and anti-sense  from kissing chromosomes mediate X-chromosome inactivation.
33. RNA synthesis favored as paired sense-antisense type by a kissing DNA-DNA tetraplex.
32. RNA aptamers approved for therapy of human optical macular degeneration.
31. RNA vs. RNA: AntiOncogenes neutralize Oncogenes within human neoplastic cells.
30. RNA antisense is involved in embryogenesis and in human cancer control.
29. RNAs serve as enhancers of specific gene transcription in the vertebrates.
28. RNA de-repressor activates human genes.
27. RNA species in bacteria cooperate to regulate metabolism.
26. RNA antisense alters chromatin ultrastructure.
25. RNA reprogramming corrects defect in alternative splicing.
24. RNA reigns in neurons.
23. RNA meets chromatin.
22. RNA synthesized at the enhancer locus controls transcription at the distant promoter site.
21. RNA antisense synthesis represses the simultaneous promotion of sense RNA.
20. RNA sense and antisense may combine to regulate specific gene locus transcription.
19. RNAs of 6S type (100-200 nt length) are necessary for regulation of DNA transcription.
18. RNAs transcribed in mammalian cells largely remain within the cell nucleus.
17. RNA short 22 nt sequences are deficient in , and a reversible cause of,  human lung neoplasms.
16. RNA from long-term non-progressor AIDS patients downregulates HIV-1 transcription.
15. RNA-like stronger  polyanion is required for spindle assembly and structure during cell mitosis.
14. RNA synthesized within the enhancer activates DNA transcription in the promoter.
13. RNA  5' leader introns of microRNA primary transcripts are long and may be ribo-regulators.
12. RNA may reprogram human neoplastic cells to normal cells.
11. RNA binds tightly to both single-stranded and double-stranded DNA.
10. RNA initiates DNA Synthesis in Eukaryotes.
9. RNA directs reciprocal transcription AND splicing in eukaryotes.
8. RNA forms the Transcription Bubble.
7. RNA as an active agent of genotypic innovation in primates.
6. RNA as an inducer of changed differentiation within living cells and organs.
5. RNA Activator of DNA Transcription can be Engineered into a Chemical Riboswitch.
4. RNA products of SINEs may direct other transcription by RNA polymerase II.
3. RNA species are involved with Prions in Bovine Spongiform Encephalopathy.
2. RNA as an Instructing Agent in the Tumor Immune Response.
1. RNA as a Chromosome-Specific Repressing Agent in X-chromosome inactivation.

Noncoding RNAs may play a role in both initiation and reversion of human neoplasms.

a. Calin GA, Liu C-G, Ferracin M, Hyslop T, Spizzo R, Sevignani C, Fabbri M, Cimmino A, Lee EJ, Wojcik SE, Shimizu M, Tili E, Rossi S, Taccioli C, Pichiorri F, Liu X, Zupo S, Herlea V, Gramantieri L, Lanza G, Alder H, Rassenti L, Volinia S, Schmittgen TD, Kipps TJ, Negrini M, and Croce CM,
"Ultraconserved Regions Encoding ncRNAs Are Altered in Human Leukemias and Carcinomas",
Cancer Cell, Vol 12, 215-229, 11 September 2007.

b. Frenster JH, and Hovsepian JA, “Models of Embryonic Gene-Induced Initiation and Reversion of Adult Neoplasms”.

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death. In 2004, Takamizawa J., et al demonstrated a deficiency of let-7 microRNA within resected human NSCLC tumors, with the severity of the deficiency predicting a worse survival for the patient. They then added let-7 RNA via plasmid to human NSCLC cell cultures, and found that the addition of let-7 RNA resulted in a decreased in vitro growth rate of the culture NSCLC cells. Johnson SM., et al in 2005 then demonstrated that the embryonic oncogene RAS is regulated by the let-7 microRNA family. Kumar MS., et al  have now demonstrated that let-7g, a particular member of the let-7 family, has the greatest effect, both cell cycle arrest and cell death, and that the addition of let-7g to NSCLC tumor-bearing mice results in significant in vivo reductions in NSCLC tumor loads.

a. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endo H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, and Takahashi T, “Reduced Expression of the Let-7 MicroRNAs in Human Lung Cancers in Association with Shortened Postoperative Survival”, Cancer Research 64: 3753-3756 (2004).

b. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, and Slack FJ, “RAS is Regulated by the Let-7 MicroRNA Family”, Cell 120: 635-647 (2005).

c. Kumar MS, Erkeland SJ, Pester RE, Chen CY, Ebert MS, Phillip A. Sharp PA, and Jacks T,
"Suppression of non-small cell lung tumor development by the let-7 microRNA family",
Proc. Natl. Acad. Sci. USA, vol. 105, no. 10 pp. 3903-3908 (March 11, 2008).

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

e. DeCarvalho S, “Effect of RNA from Normal Human Marrow on Leukemic Marrow In-Vivo”, Nature 197:  1077-1080 (March 16, 1963.

f. Frenster JH, and Hovsepian JA, “Models of Embryonic Gene-Induced Initiation and Reversion of Adult Neoplasms”.

Embryonic genes are often re-expressed within adult cells, sometimes resulting in either the initiation and/or  the reversion of adult neoplasms. The molecular products of such gene re-expressions are embryonic RNAs, often unaccompanied by appropriate embryonic ribo-regulators. Such inappropriate embryonic RNA molecules, which are without their usual controls, can be the source of progressive disruption and pathology within the affected adult cells.

a. Frenster JH, and Hovsepian JA, “Models of Embryonic Gene-Induced Initiation and Reversion of Adult Neoplasms”.

b. Okito K, Ichisaki I, and Yamanaka S, “Generation of germline-competent induced pluripotent stem cells”, Nature 448: 313-317 (July19, 2007).

c. Mayr C, Hemann MT, and Bartel DP, "Disrupting the Pairing Betweenlet-7 and Hmga2 Enhances Oncogenic Transformation", Science, 315: 1576-1579 (2007).

d. Koslowski M, Sahin U, Mitnach-Kraus R, Seitz G, Huber C, and Tureci O, “A Placenta-Specific Gene Ectopically Activated in Many Human Cancers is Essentially Involved in Malignant Cell Processes”,
Cancer Research 67: (19) pp. 9528-9534 (October 1, 2007).

e. Eastham AM, Spencer H, Soncin F, Ritson S, Merry CLR, Stern PL, and Ward CM,
"Epithelial-Mesenchymal Transition (EMT) Events during Human Embryonic Stem Cell Differentiation",
Cancer Research 67, 11254-11262, December 1, 2007.

f. Sarrió D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, and Palacios J, "Epithelial-Mesenchymal Transition  (EMT)  in Breast Cancer Relates to the Basal-like Phenotype",
Cancer Research 68, 989-997, February 15, 2008.

g. Wang J, Day R, Dong Y, Weintraub SJ, and Michel L,
"Identification of Trop-2 as an Oncogene and an Attractive Therapeutic Target in Colon Cancers",
Molec. Cancer Therap. 7: 280-285 (February 1, 2008).)

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

In high-resolution studies of transcription of the humn genome, most areas of euchromatin are undergoing interleaved RNA synthesis under multiple layers of apposed control elements.

a. Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT, Stadler PE, Hertel J,  Hackermüller J, Hofacker IL, Bell I, Cheung E, Drenkow J, Dumais E, Patel S, Helt G, Ganesh M, Ghosh S,  Piccolboni A, Sementchenko V, Tammana H, and Gingeras TR,
"RNA Maps Reveal New RNA Classes and a Possible Function for Pervasive Transcription".

b. Rossi JJ, "Transcriptional activation by small RNA duplexes".

c. 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.

New data by Ponjavic J, et al., and by Furuno M, et al., reveal the presence of long noncoding macroRNAs,
averaging 4.2kb in length, tightly conserved during evolution among mammals,  and possibly playing the role of long-range, persistent enhancers of distant genes.

a. Ponjavic J, Ponting CP, and Lunter G,
"Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs".

b. Furuno M, Pang KC, Ninomiya N, Fukuda S, Frith MC, Bult C, Kai C, Kawai J, Carninci P, Hayashizaki Y, Mattick JS, and Suzuki H,
"Clusters of Internally Primed Transcripts Reveal Novel Long Noncoding RNAs".

c. Jones EA, and Flavell RA,
"Distal Enhancer Elements Transcribe Intergenic RNA in the IL-10 Family Gene Cluster".

d. Shin JT, Priest JR, Ovcharenko I, Ronco A, Moore RK, C. Burns CG, and MacRae CA,
"Human-zebrafish non-coding conserved elements act in vivo to regulate transcription".

e. Chen Q, Lin L, Smith S, Lin Q, and Zhou J, "Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation".

f. Ling J, Pi W, Yu X, Bengra C, Long Q, Jin H, Seyfang A, and Tuan D, "The ERV-9 LTR Enhancer is Not Blocked by the HS5 Insulator and Synthesizes Through the HS5 Site Non-Coding, Long RNAs that Regulate LTR Enhancer Function".

MicroRNAs, 21-29 nt in length, have diverse effects on their target genes. New studies now reveal that among such short RNAs, some of those that are double-stranded are capable , through their antisense strands, of acting as activators of gene transcription at specific gene loci.

a. Li L-C, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, and Dahiya R,
"Small dsRNAs induce transcriptional activation in human cells".

b. Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, and Corey DR,
"Activating gene expression in mammalian cells with promoter-targeted duplex RNAs".

c. Rossi JJ, "Transcriptional activation by small RNA duplexes".

d. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

e. Geiss G, Jin G, Guo J, Bumgarner R, Katze MG, and Sen GC, "A Comprehensive View of Regulation of Gene Expression by Double-Stranded RNA-Mediated Cell Signaling",  J. Biol. Chem. vol. 276, no. 32, pp. 30178-30182 (August 10, 2001).

f. Persengiev SP, Zhu X and Green MR, "Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs)",  RNA, vol. 10, no. 1, pp. 12-18 (January, 2004).

g. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH, "A Small Modulatory dsRNA Specifies the Fate of Adult Neural Stem Cells", Cell, vol. 116, no. 6, pp.779-793  (March  19, 2004).

h. A brief history of Activator RNAs:

Embryonic let-7 microRNA selectively represses the RAS and the Hmga2 embryonic oncogenes. This action correlates with the deficiency of let-7 RNA found within non-small cell lung cancer (NSCLC) cells in humans, and with the restoration to normal growth rates in-vitro following the plasmid-induced increase of let-7 RNA within these cells. This may be an example of an embryonic RNA therapy directed successfully against an embryonic oncogene. Are all human neoplasms embryonic ?

a. Lee YS,  and Dutta A, "The tumor suppressor microRNA let-7 represses the HMGA2 oncogene".

b. Mayr C, Hemann MT, and Bartel DP,
"Disrupting the Pairing Between let-7 and Hmga2 Enhances Oncogenic Transformation", 2007.

c. Takamizawa, J., Konishi, H., Yanagisawa, K., Tomida, S., Osada, H., Endoh, H., Harano, T., Yatabe, Y., Nagino, M., Nimura, Y., Mitsudomi T,  and Takahashi T,  (2004). Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 64, 3753–3756, (2004).

d. Johnson, S.M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., Cheng, A., Labourier, E., Reinert, K.L., Brown, D., and Slack, F.J. RAS is regulated by the let-7 microRNA family. Cell 120, 635–647, (2005).

e. Grosshans H, Johnson T, Reinert KL, Gerstein M, and Slack FJ, "The Temporal Patterning MicroRNA let-7 Regulates Several Transcription Factors at the Larval to Adult Transition in C. elegans".

f. Hayes GD, and  Ruvkun G,  "Misexpression of the Caenorhabditis elegans miRNA let-7 is Sufficient to Drive Developmental Programs". (2006).

g. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin". (1999).

h. DeCarvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo", (1963).
 

RNA species of the non-coding RNA type function as enhancers and initiators of downstrean transcription initiation of human genes.

a. Pennacchio LA, Ahituv N, Moses AM, Prabhakar S, Nobrega MA, Shoukry M, Minovitsky S, Dubchak I, Holt A, Lewis KD, Plajzer-Frick I, Akiyama J, De Val S, Afzal V, Black BL, Couronne O, Eisen MB, Visel A, and Rubin EM, "In vivo enhancer analysis of human conserved non-coding sequences".

b. Shin JT, Priest JR, Ovcharenko I, Ronco A, Moore RK, C. Burns CG, and MacRae CA,
"Human-zebrafish non-coding conserved elements act in vivo to regulate transcription".

c. Li L-C, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, and Dahiya R,
"Small dsRNAs induce transcriptional activation in human cells".

d. O’Gorman W, Kwek KY, Thomas B,  and Akoulitchev A,
"Non-coding RNA in transcription initiation".

e. Ling J, Baibakov B, Pi W, Emerson BM, and Tuan D, "The HS2 Enhancer of the b-globin Locus Control Region Initiates Synthesis of Non-coding, Polyadenylated RNAs Independent of a cis-linked Globin Promoter".

f. Pennacchio LA, Loots GG, Nobrega MA, and Ovcharenko I, "Predicting tissue-specific enhancers in the human genome", Genome Research vol. 17: no. 2, pp. 201-211, (January 8, 2007).

 Enhancer Browser:      http://enhancer.lbl.gov/
 Enhancer Identification:  http://www.dcode.org/EI

Messenger RNAs (and also non-coding RNAs) often have poly-A 3' terminals which are not translated in protein synthesis. Now, increasing data suggests that other RNA molecules contain specific RNA sequences (AGUGUU) in their 3' terminals which permit and determine the importation of  such RNA molecules into the cell nucleus. This allows specific cytoplasmic molecules an entrance into affecting the activity of the genome.

a. Hwang H-W,  Wentze EA, and Mendell JT,
"A Hexanucleotide Element Directs MicroRNA Nuclear Import".

b. Jenny A, Hachet O, Závorszky P, Cyrklaff A, Weston MDJ, St Johnston D, Erdélyi M, and Ephrussi A,
"A translation-independent role of oskar RNA in early Drosophila oogenesis".

c. Rastinejad F, and Blau HM, "Genetic Complementation Reveals a Novel Regulatory Role for 3' Untranslated Regions in Growth and Differentiation", Cell, volume 72, pp. 903-917 (1993).

d. Rastinejad F, Conboy MJ, Rando TA, and Blau HM, "Tumor Suppression by RNA from the 3' Untranslated Region of Alpha-Tropomyosin", Cell, volume 75, pp. 1107-1117 (1993).

Two new stdies report that RNA-directed RNA polymerase catalyzes the synthesis of secondary siRNAs from primary siRNAs mRNA. Secondary siRNAs are only of antisense polarity, and carry 5' di- or triphosphates. Analyzing small RNAs associated with ongoing RNAi in C. elegans, it was found that  secondary siRNAs comprise the vast majority.

a. Pak J, and Fire A,
"Distinct Populations of Primary and Secondary Effectors During RNAi in C. elegans".

b. Sijen T, Steiner FA, Thijssen KL, and Plasterk RHA,
"Secondary siRNAs Result from Unprimed RNA Synthesis and Form a Distinct Class".

A 180 bp segment of the HAR1F noncoding gene is transcribed to a 118 nt RNA that has evolved since the human divergence from chimpanzees, and which has vital activity in the embryogenesis of the human cerebral cortex.

a. Pollard KS, Salama SR, Lambert N, Lambot M-A, Coppens S, Pedersen JS, Katzman S, King B, Onodera C, Siepel A, Kern AD, Dehay C, Igel H, Ares M Jr, Vanderhaeghen P, and Haussler D,
"An RNA gene expressed during cortical development evolved rapidly in humans".

b. Lipovich L, Vanisri RR, Kong SL, Lin C-Y, and Liu ET,
"Primate-Specific Endogenous Cis-Antisense Transcription in the Human 5q31 Protocadherin Gene Cluster".

c. Zapala MA, Hovatta I, Ellison JA, Wodicka L, Del Rio JA, Tennant R, Tynan W, Broide RS, Helton R, Stoveken BS, Winrow C, Lockhart DJ, Reilly JF, Young WG, Bloom FE, Lockhart DJ, and Barlow C,  "Adult mouse brain gene expression patterns bear an embryologic imprint".

d. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH, "A Small Modulatory dsRNA Specifies the Fate of Adult Neural Stem Cells". (2004) Cell, 116: 779-793.

Recent studies of human cancer vaccines reveal responses to RNA tumor antigen-derived transfection in patients with metastatic malignant melanoma resistant to other forms of immunotherapy.

a. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, and Rosenberg SA.
"Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes".

b. Liao X, Li Y, Bonini C, Nair S, Gilboa E, Greenberg PD, and Yee C, "Transfection of RNA Encoding Tumor Antigens Following Maturation of Dendritic Cells Leads to Prolonged Presentation of Antigen and the Generation of High-Affinity Tumor-Reactive Cytotoxic T Lymphocytes".

c. Coughlin CM, Vance BA, Grupp SA, and Vonderheide RH, "RNA-transfected CD40-activated B cells induce functional T cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy".
 

During the devlopment of olfactory neurons, only one of the 1,300 odorant receptor genes is expressed, yielding a diverse population of specific cells, each of which is stable for the life of the cell. The enhancer for such allele-specific RNA synthesis is found on one chromosome, and and the promoter for such RNA synthesis is found on a second chromosome, with the two chromosomes forming a kissing DNA tetraplex at the point of RNA synthesis.

a. Lomvardas S, Barnea G, Pisapia DJ, Mendelsohn M, Kirkland J, and Axel R,
"Interchromosomal Interactions and Olfactory Receptor Choice".

b. Frenster JH, and Hovsepian JA,
"Kissing Chromosomes and Paired Sense-Antisense RNA Synthesis".

c. Frenster JH, and Hovsepian JA,
"DNA-DNA Tetraplex Model of Paired Sense-Antisense RNA Synthesis".
 

During human gene transcription, RNA species mediate the association of splicing factors with activated RNA polymerase II on the DNA transcription bubble.

a. Listerman I, Sapra AK,  and Neugebauer KM,
"Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells".

hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G(1)-phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G(1)-phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system.

a. Christov CP, Gardiner TJ, Szüts D, and Krude T,
"Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication".
Molecular and Cellular Biology, vol. 26, no. 18, p. 6993-7004 (September, 2006).

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

c. Frenster JH, Allfrey VG, and Mirsky AE, "Repressed and Active Chromatin Isolated from Interphase Lymphocytes", Proc. Natl. Acad. Sciences, U.S.A., vol. 50, no. 6, pp. 1026-1032 (Dec. 1963).

d. Frenster JH, "Localized Strand Separations within Deoxyribonucleic Acid during Selective Transcription", Nature, vol. 208: no. 5013, pp. 894-896 (November 27, 1965).

38. RNA and DNA  interactions in the DNA Strand-Separation Model of eukaryotic gene regulation.

Control of DNA strand separations at a discrete gene locus is required for selective gene transcription at that locus.

Ligands which prevent DNA strand separation prevent gene transcription, and ligands which favor DNA strand separation at a particular gene locus favor gene transcription at that locus.

Activator RNA species are capable of binding one DNA strand at select gene loci, thereby initiating a DNA transcription bubble for RNA synthesis on the complementary DNA strand of the DNA bubble at that locus.

Open DNA transcription bubbles can bind to complementary second open DNA transcription bubbles at homologous gene loci, thereby forming a DNA-DNA tetraplex for paired sense-antisense RNA synthesis at that gene locus.

The comparative thermodynamic stabilities of RNA-DNA vs. DNA-DNA hydrogen bondings determines the stability state of competitive DNA templates during selective gene transcription.

a. Frenster JH, and Hovsepian JA,
"Activator RNA Initiation of the DNA Transcription Bubble".

b. Hovsepian JA, and Frenster JH,
"Chromosome-Chromosome Contact Points and Paired Sense-Antisense RNA Synthesis".

c. Binnie A, Castelo-Branco P, Monks J, and Proudfoot NJ,
"Homologous gene sequences mediate transcription-domain formation".
 

Synthesis of noncoding antisense RNA species is now recognized as universal throughout the vertebrates, and is often paired with the simultaneous synthesis of complementary coding sense RNA species. Such antisense RNAs have now been found to function as enhancers of other RNA synthesis, and to be present in the transcription products of important key genes within mouse and human embryonic stem cells.

a. Mollica LR, Crawley JTB, Liu K, Rance JB, Cockerill PN, Follows GA, Landry J-R, Wells DJ, and Lane DA, "Role of a 5'-enhancer in the transcriptional regulation of the human endothelial cell protein C receptor gene".

b. Richards M, Tan S-P, Chan W-K, and Bongso A,
"Reverse Serial Analysis of Gene Expression (SAGE) Characterization of Orphan SAGE Tags from Human Embryonic Stem Cells Identifies the Presence of Novel Transcripts and Antisense Transcription of Key Pluripotency Genes".

c. Frenster JH, and Hovsepian JA, "Kissing Chromosomes and Paired Sense-Antisense RNA Synthesis".

DNase1-sensitive chromatin sites contain the promoters, enhancers and transcribers of human RNA synthesis.

a. Crawford GE, Davis S, Scacheri PC, Renaud G, Halawi MJ, Erdos MR, Green R, Meltzer PS, Wolfsberg TG, and Collins FS,
"DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays".

b. Crawford GE, Holt IE, Whittle J, Webb BD, Tai D, Davis S, Margulies EH, Chen YD, Bernat JA, Ginsburg D, Zhou D, Luo S, Vasicek TJ, Daly MJ, Wolfsberg TG, and Collins FS, "Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS)".

c. Sabo PJ, Kuehn MS, Thurman R, Johnson BE, Johnson EM, Cao H, Yu M, Rosenzweig E, Goldy J, Haydock A, Weaver M, Shafer A, Lee K, Neri F, Humbert R, Singer MA, Richmond TA, Dorschner MO, McArthur M, Hawrylycz M, Green RD, Navas PA, Noble WS, and Stamatoyannopoulos JA,
"Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays".
 

RNA sense and antisense molecules have profound effects on mammalian gene regulation, but such molecules are unstable in vivo. New studies now reveal that modifying such RNA antisense molecules chemically while retaining the original RNA linear sequence permits these molecules to be given to living mice for potent long-lasting effects on specific genes and specific metabolic pathways within specific living mouse organs.

a. Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, and Stoffel M,
"Silencing of microRNAs in vivo with 'antagomirs'".

b. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

c. Czech MP, "MicroRNAs as Therapeutic Targets", New Eng. J. Med. vol. 354, no. 11, pp. 1194-1195 (March 16, 2006).

Xist sense RNA repressess all but one X chromosome in mammalian cells, and is controlled by its paired antisense complement Tsix RNA. The entire process is initiated by close pairing (kissing) of two or more X chromosomes, a contact  which favors the synthesis of paired sense-antisense RNA sequences.

a. Xu N, Tsai C-L, Lee JT,
"Transient Homologous Chromosome Pairing Marks the Onset of X Inactivation".

b. Bacher CP, Guggiari M, Brors B, Augui S, Clerc P, Avner P, Eils R, and Heard E,
"Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation".

c. Frenster JH, and Hovsepian JA, "Ultrastructure of Euchromatin Contact Points between the Closed Loops of Adjacent Interphase Chromosomes":

d. Kioussis D, "Gene Regulation: Kissing Chromosomes",  Nature vol. 435, no. 7042, pp. 579-580 (June 2,  2005). http://www.nature.com/nature/journal/v435/n7042/full/435579a.html

e. Frenster JH, and Hovsepian JA, "Kissing Chromosomes and Paired Sense-Antisense RNA Synthesis".
71st Cold Spring Harbor Symposium on Quantitative Biology", Program page 62, May 31-June 5, 2006.
 

Contact points between adjacent non-homologous interphase chromosomes favor the synthesis of paired sense-antisense RNA species. The resulting DNA-DNA kissing tetraplex may be stabilized by interactions within the newly-discovered MER121 family of highly conserved noncoding DNA sequences for enhancer effects on selected sites of DNA transcription.

a. Frenster JH, and Hovsepian JA, "Ultrastructure of Euchromatin Contact Points between the Closed Loops of Adjacent Interphase Chromosomes".

b. Kamal M, Xie X, and Lander ES, "A large family of ancient repeat elements in the human genome is under strong selection".

.c. Shin JT, Priest JR, Ovcharenko I, Ronco A, Moore RK, C. Burns CG, and MacRae CA,
"Human-zebrafish non-coding conserved elements act in vivo to regulate transcription".
 

After a long in vitro selection process, RNA aptamers now demonstrate in clinical trials and a 2-year follow-up that they can effect increased visual acuity in patients with optical macular degeneration. This is the first FDA approval of an RNA therapeutic agent for human disease, and recalls the first demonstrated therapeutic effect of added normal RNA to human patients with acute myelogenous leukemia during pilot studies in 1963.

a. Ng EWM, Shima DT, Calias P, Cunningham, ET Jr. , Guyer DR, and Adamis AP,
"Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease".

b. 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).

c. Ross PJ, George M, Cunningham D, DiStefano F, H. Andreyev JN, Workman P, and Clarke PA, "Inhibition of Kirsten-ras Expression in Human Colorectal Cancer Using Rationally Selected Kirsten-ras Antisense Oligonucleotides", Molecular Cancer Therapeutics vol. 1, no. 1, pp. 29-41 (November,  2001).

New studies of Oncogene activity within the neoplastic cells of Hodgkin lymphoma reveal  a specific Oncogene is unique to a specific lymphoma among other lymphomas, and this Oncogene can be surpressed by a specific AntiOncogene RNA within the neoplastic Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.

a. Janz M, Hummel M, Truss M, Wollert-Wulf B, Mathas S, Johrens K, Hagemeier C, Bommert K, Stein H, Dorken B, and Bargou RC, "Classical Hodgkin lymphoma is characterized by high constitutive expression of activating transcription factor 3 (ATF3) which promotes viability of Hodgkin/Reed-Sternberg cells".

b. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin".

c. Frenster JH, "Single-Cell Analysis of DNase I-Sensitive Sites During Neoplastic Cell Differentiation within Hodgkin's Disease Lymph Nodes".

Long RNA sequences of the cis antisense type play a role in embryonic development in mice and in cancer control in humans, perhaps by antagonizing paired sense RNA activity and expression.

a. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

b. Coudert AE, Pibouin L, Vi-Fane B, Thomas BL, Macdougall M, Choudhury A, Robert B, Sharpe PT, Berda A, and Lezot F, "Expression and regulation of the Msx1 natural antisense transcript during development".

c. Barclay C, Li AW, Geldenhuys L, Baguma-Nibasheka M, Porter GA, Veugelers PJ, Murphy PR, and Casson AG, "Basic Fibroblast Growth Factor (FGF-2) Overexpression Is a Risk Factor for Esophageal Cancer Recurrence and Reduced Survival, which Is Ameliorated by Coexpression of the FGF-2 Antisense Gene".

d. Lipovich L, Vanisri RR, Kong SL, Lin C-Y, and Liu ET,
"Primate-Specific Endogenous Cis-Antisense Transcription in the Human 5q31 Protocadherin Gene Cluster".

e. Frenster JH, and Hovsepian JA, "Ultrastructure of Euchromatin Contact Points between the Closed Loops of Adjacent Interphase Chromosomes".

Many non-coding RNAs are highly conserved from Zebrafish to Humans, and serve as  enhancers for specific gene transcription in the vertebrates.

a. Shin JT, Priest JR, Ovcharenko I, Ronco A, Moore RK, C. Burns CG, and MacRae CA,
"Human-zebrafish non-coding conserved elements act in vivo to regulate transcription".

b. Woolfe A, Goodson M, Goode DK, Snell P, McEwen GK, Vavouri T, Smith SF, North P, Callaway H, Kelly K, Walter K, Abnizova I, Gilks W, Edwards YJK, Cooke JE, and Elgar G,
"Highly Conserved Non-Coding Sequences Are Associated with Vertebrate Development".

c. Bejerano, G., Pheasant, M., Makunin, I., Stephen, S., Kent, W.J., Mattick, J.S., Haussler, D. (2004) "Ultraconserved elements in the human genome", Science, 304, 1321–1325.

d. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

Analysis of the effects of a non-coding mouse RNA on previously-repressed genes within human cells in culture reveals 14 human genes that were activated more than 16-fold.

a. Song X, Sun Y, and Garen A,
"Roles of PSF protein and VL30 RNA in reversible gene regulation".

b. Kuwabara T, Hsieh J, Nakashima K, Warashina M, Taira K, and Gage FH,
"The NRSE smRNA specifies the fate of adult hippocampal neural stem cells".

c. Hovsepian JA, and Frenster JH,
"Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

Bacteria are now found to use small (119nt) RNA species in the control of their metabolism.

a. Kay E, Dubuis C, and Haas D,
"Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0".

The transcription of antisense RNA alters the local heterochromatin methylation and ultrastructure.

a. Cho DH, Thienes CP, Mahoney SE, Analau E, Filippova GN, and Tapscott SJ,
"Antisense Transcription and Heterochromatin at the DM1 CTG Repeats Are Constrained by CTCF".
 

Administration of a trans-splicing RNA corrects defect in alternative splicing in human neurons.

a. Rodriguez-Martin T, Garcia-Blanco MA, Mansfield SG, Grover AC, Hutton M, Yu Q, Zhou J, Anderton BH, and Gallo J-M, "Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies".

b. Frenster JH, and Hovsepian JA, "Activator RNA Exchange during Interphase Chromatin Reprogramming", RNA2004: 305 (2004).

c. Frenster JH, and Hovsepian JA,  "Overshoot in Late Telophase for RNA Re-Programming of Mitotic Chromatin",  RNA 2003, 211 (2003).

d. Frenster JH, "Yeast  RNA  Re-Programming  of  Already-Active  Mammalian Chromatin".  RNA 2002, 592 (2002).

e. Hovsepian JA, and Frenster JH, "Reprogramming as an Approach to Neoplasms".

f. Eder M, and Scherr M, "MicroRNA and Lung Cancer".

g. DeCarvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo".
 

The nervous system and its brain now show the mobility as well as the diversity of RNA species. Its most active region, the cerebral cortex, is alive with embryonic expression. And neural gene expression is often dependent upon RNA de-repressors of repressed genes.

a. Tiedge H, "RNA Reigns in Neurons".

b.  Kuwabara T, Hsieh J, Nakashima K, Warashina M, Taira K, and Gage FH,
"The NRSE smRNA specifies the fate of adult hippocampal neural stem cells".

c. Zapala MA, Hovatta I, Ellison JA, Wodicka L, Del Rio JA, Tennant R, Tynan W, Broide RS, Helton R, Stoveken BS, Winrow C, Lockhart DJ, Reilly JF, Young WG, Bloom FE, Lockhart DJ, and Barlow C,
"Adult mouse brain gene expression patterns bear an embryologic imprint".

d. Song X, Sun Y, and Garen A,
"Roles of PSF protein and VL30 RNA in reversible gene regulation".

e. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH,
"A Small Modulatory dsRNA Specifies the Fate of Adult Neural Stem Cells".

f. Hovsepian JA, and Frenster JH,
"Sense and antisense during RNA initiation of the DNA transcription bubble".

Recent reviews emphasize the close relationship of active euchromatin and repressed heterochromatin to the activator and repressor activities of non-coding RNA species.

a. Bernstein E,  and Allis CD, "RNA meets chromatin".

b. Frenster JH, "Mechanisms of Repression and De-Repression within Interphase Chromatin".

Analysis of the control of b-globin synthesis in human cells reveals that noncoding RNA synthesized at the enhancer locus controls premessenger RNA synthesis at the promoter site 10-55 kb downstream. The RNA may diffuse or be carried by chromatin looping to the promoter site.

a. Ling J, Baibakov B, Pi W, Emerson BM, and Tuan D, "The HS2 Enhancer of the b-globin Locus Control Region Initiates Synthesis of Non-coding, Polyadenylated RNAs Independent of a cis-linked Globin Promoter",  J. Mol. Biol. vol. 350, no. 5, pp. 883-896 (July 29, 2005).

b. Chen Q, Lin L, Smith S, Lin Q, and Zhou J, "Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation".

c. Hovsepian JA, and Frenster JH, "Bioassays of Isolated Nuclear RNA Species as Activators of DNA Transcription".

d. Frenster JH, and Hovsepian JA, "Ultrastructure of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei".
 

During X-chromosome inactivation in mammals, the synthesis of senseXist RNA is required, but  Xist  promotion is repressed by the concurrent synthesis of antisense Tsix RNA.

a. Navarro P, Pichard S, Ciaudo C, Avner P, and Rougeulle C, "Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation".

b. Kelley RL, and Kuroda MI, "The Role of Chromosomal RNAs in Marking the X for Dosage Compensation".

c. Grumbach MM, Morishima A, and Taylor JH, "Human Sex Chromosome Abnormalities in Relation to DNA Replication and Heterochromatinization".

d. Luikenhuis S, Wutz A, and Jaenisch R, "Antisense Transcription Through the Xist Locus Mediates Tsix Function in Embryonic Stem Cells".

The 5' leader sense sequences of premessenger RNA may combine with the antisense sequences of activator RNA to regulate specific gene locus transcription by a feedback mechanism.

a. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

b. Herstein PR, and Frenster JH, "Mated Models of Gene Regulation in Eukaryotes", in: "Embryonic and Fetal Antigens in Cancer", vol. 2, pp. 5-7, Oak Ridge National Lab., Oak Ridge, Tenn., 1972.

Noncoding 6S RNAs in diverse bacterial species are conserved and necessary for regulation of DNA transcription.

a. Trotochaud AE,  and Wassarman KM, "A highly conserved 6S RNA structure is required for regulation of transcription".

b. Barrick JE, Sudarsan N, Weinberg Z, Ruzzo WL, and Breaker RR, "6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter".
 

In an increasing awareness of RNAs that do not play a role in cytoplasmic protein synthesis, two new papers confirm earlier suggestions that a large number of RNA species remain within the mammalian cell nucleus, and play a role in Gene-Gene interactions and in intra-nuclear protein synthesis.

a. Kiyosawa H, Mis N, Iwase S, Hayashizaki Y,  and Abe K, "Disclosing hidden transcripts: Mouse natural sense-antisense transcripts tend to be poly(A) negative and nuclear localized".

b. Cheng J, Kapranov P, Drenkow J, Dike S, Brubaker S, Patel S, Long J, Stern D, Tammana H, Helt G, Sementchenko V, Piccolboni A, Bekiranov S, Bailey DK, Ganesh M, Ghosh S, Bell I, Gerhard DS, and Gingeras TR, "Transcriptional Maps of 10 Human Chromosomes at 5-Nucleotide Resolution".

c. Frenster JH, Allfrey VG, and Mirsky AE, "Metabolism and Morphology of Ribonucleoprotein Particles from the Cell Nucleus of Lymphocytes".
 

Two independent studies have now revealed a deficiency of let-7 RNA within human non-small cell lung carcinomas (NSCLC) (a, b). The greater the deficiency, the greater the mortality of the observed patients after surgery (a). The deficiency of let-7 RNA is most evident in NSCLC  cells compared to other human neoplasms (b). The addition of let-7 RNA to in-vitro cultures of NSCLC neoplastic cells results  in a significantly reduced growth rate of such cells (a). let-7 RNA is found in worms, flies, and humans (c), and plays a decisive role during embryogenesis, inducing embryonic stem cell differentiation and maturation (c). These findings support previous indications of the return of neoplastic cells to an embryonic state (d), and of the role of normal RNA in reversing the human neoplastic state (e, f ).

a. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, and Takahashi T, "Reduced Expression of the let-7 MicroRNAs in Human Lung Cancers in Association with Shortened Postoperative Survival", Cancer Res. 64 (2004), pp. 3753–3756.

b. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, and Slack FJ, "RAS Is Regulated by the let-7 MicroRNA Family".

c. Grosshans H, Johnson T, Reinert KL, Gerstein M, and Slack FJ, "The Temporal Patterning MicroRNA let-7 Regulates Several Transcription Factors at the Larval to Adult Transition in C. elegans".

d. 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.

e. De Carvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo".,

f. Hovsepian JA, and Frenster JH, "Reprogramming as an Approach to Neoplasms ".

g. Eder M, and Scherr M, "MicroRNA and Lung Cancer".

HIV-1 nef dsRNA isolated from AIDS patients who are long-term non-progressors inhibits HIV-1 transcription. A nef-derived miRNA (miR-N367) reduces HIV-1 LTR promoter in human T cells through the negative responsive element of the U3 region in the 5'-LTR, utilizing a transcriptional neo-pathway.

a. Omoto S, and Fujii YR, " Regulation of human immunodeficiency virus 1 transcription by nef microRNA",
J Gen Virol,  vol. 86, no. 3, pp. 751-755 (January 19, 2005).

b. Storz G, Altuvia S, and Wassarman KM, "An Abundance of RNA Regulators".

Poly(ADP-ribose) (PAR) is a large, branched nuclear polyanion that has twice the negative charge of RNA, due to its use of two phosphate groups per polymer unit compared to one such phosphate group per polymer unit in RNA. PAR is indirectly synthesized from ATP by a family of PAR polymerases (PARPs).

PAR is widely distributed in the cell nucleus, and has striking multitasking effects after combining with histones within chromatin and chromosomes. It is now shown to be required for spindle assembly and structure during mitotic anaphase, and for the de-condensation  of chromosomes to chromatin late in mitotic telophase.

a. Chang P, Jacobson MK, and Mitchison TJ, "Poly(ADP-ribose) is required for spindle assembly and structure", Nature vol. 432, no. 7017, pp. 645 - 649 (02 December 2004).

b. Karsenti E, "Cytoskeleton: Spindle saga", Nature, vol. 432, no. 7017, pp. 563 - 564 (02 December 2004),

c. Blower MD, Nachury M, Heald R, and Weis K, "A Rae1-Containing Ribonucleoprotein Complex Is Required for Mitotic Spindle Assembly", Cell, vol. 121, no. 2, pp. 223-234 (22 April 2005).

d. Frenster JH, "Nuclear polyanions as de-repressors of synthesis of Ribonucleic Acid", Nature, volume 206, number 4985, pp. 680 - 683 (15 May 1965).

Intergenic RNA synthesized within the e-globin enhancer activates DNA transcription within the e-globin promoter. The mechanism may be mediated by cis DNA tracking, chromatin looping, and/or activator RNA diffusion in cis or trans.

a. Ling J, Ainol L, Zhang L, Yu X, Pi W, and Tuan D, "HS2 Enhancer Function Is Blocked by a Transcriptional Terminator Inserted between the Enhancer and the Promoter".

b. Ling J, Pi W, Yu X, Bengra C, Long Q, Jin H, Seyfang A, and Tuan D, "The ERV-9 LTR Enhancer is Not Blocked by the HS5 Insulator and Synthesizes Through the HS5 Site Non-Coding, Long RNAs that Regulate LTR Enhancer Function".

c. Hovsepian JA, and Frenster JH, "Bioassays of Isolated Nuclear RNA Species as Activators of DNA Transcription".

microRNAs are ~22 nt long, and function as inhibitors of translation of mRNA s. The primary transcripts yielding  microRNAs are considerably longer (890-1731 nt), and the 5' leader introns after splicing may have independent ribo-regulator activity.

a. Bracht J, Hunter S, Eachus R, Weeks P,  and Pasquinelli AE, "Trans-splicing and polyadenylation of let-7 microRNA primary transcripts".

b.  Mansfield JH, Harfe BD, Nissen R, Obenauer J, Srineel J, Chaudhuri A, Farzan-Kashani R, Zuker M, Pasquinelli AE, Ruvkun G, Sharp PA, Tabin CJ, and McManus MT, "MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression", Nature Genetics  vol. 36, no. 10, pp. 1079-1083 (October, 2004).

c. Persengiev SP, Zhu X, and Green MR, "Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs)",   RNA, vol. 10, no. 1, pp. 12-18 (January, 2004).

d. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription".

e. Herstein PR, and Frenster JH,  "Mated Models of Gene Regulation in Eukaryotes".

f. Cai X, Hagedorn CH, and Cullen BR, "Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs".

RNA is necessary for the formation of epigenetic marks on mammalian cells, and can be used to reprogram mammalian cells in interphase as well as in the more usual time of late telophase.

The administration of normal human bone marrow RNA to patients with acute myelogenous leukemia in relapse has resulted in the conversion of the leukemic cell population in-vivo to a normal granulocyte stage.

a. Frenster JH, and Hovsepian JA, "Activator RNA Exchange during Interphase Chromatin Reprogramming", RNA2004: 305 (2004).

b. Hovsepian JA, and Frenster JH, "Reprogramming as an Approach to Neoplasms".

c. De Carvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo".

d. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, and Takahashi T, "Reduced Expression of the let-7 MicroRNAs in Human Lung Cancers in Association with Shortened Postoperative Survival".

RNA is capable of forming many configurations, but single-stranded portions of these RNA configurations are capable of tight binding to either single-stranded or double-stranded DNA molecules. These interactions take advantage of the greater thermal stability of RNA-DNA duplexes over those of DNA-DNA duplexes, and can be specific for short gene sequences.

The effect of these RNA-DNA interactions within the mammalian cell nucleus is to open the DNA double helix, and then to stabilize the open DNA "bubble" for DNA activity in selective gene transcription and replication.

a. 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).

b. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription", Molec. Biol. Cell, vol. 13, supp. p. 239a (November, 2002).
 

Mammalian DNA is synthesized asynchronously, with the lagging DNA strand primed by a short noncoding RNA initiator molecule.

At any given gene locus, genes active in transcription to RNA in G₁ are also earliest in replication of their DNA in S phase. Short noncoding RNA molecules are involved as activators of DNA transcription, and may also be involved at the same gene site in initating DNA replication.

a. Kao H-I, Campbell JL, and Bambara RA, "Dna2p helicase/nuclease is a tracking protein, like FEN1, for Flap cleavage during Okazaki fragment maturation", J. Biol. Chem, 10.1074/jbc.M409231200

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

c. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription", Molec. Biol. Cell, vol. 13, supp. p. 239a (November, 2002).
 

Recent reviews have re-examined the relationship of splicing after transcription. Although the two processes were thought to to be distinct and not simultaneous, new evidence indicates that the time gap between the two processes may become increasingly small, if not simultaneous, and that each process may also affect the other process reciprocally in specificity and throughput.

In these processes, species of small noncoding RNA are found to play a directing role, perhaps by the same RNA molecule.

a. Kornblihtt AR, de la Mata M, Fededa JP, Munoz MJ, and Nogues G, "Multiple links between transcription and splicing", RNA, vol. 10, no. 10, pp. 1489-1498 (October, 2004).

b. Jiang M, Ma N, Vassylyev DG, and McAllister WT, "RNA Displacement and Resolution of the Transcription Bubble during Transcription by T7 RNA Polymerase", Molecular Cell, vol. 15, no. 5, pp.777-788 (September, 10,  2004).

c. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription", Molec. Biol. Cell, vol. 13, supp. p. 239a (November, 2002).

d. Kwek KY, Murphy S, Furger A, Thomas B, O'Gorman W, Kimura H, Proudfoot NJ, and  Akoulitchev A,
"U1 snRNA Associates with TFIIH and Regulates Transcriptional Initiation", Nature Structural Biology, vol. 9, no. 11, pp. 800-805 (November, 2002).

Is thermodynamics the ultimate arbiter of chemical reactions ? Only in closed systems at equilibrium. In life, with a constant flow of ATP mediating unlikely reactions, literally anything is possible if it enhances survival. And yet, thermodynamics must be considered. The melting temperature of a DNA-DNA helix is lower than that of a DNA-RNA helix, which, in turn, is lower than that of a RNA-RNA helix. So, double-stranded RNA, even if only partially helical, must be respected on thermodynamic grounds, if no other.

New studies on the origins and the mechanics of the transcription bubble indicate that the anti-template DNA strand of the gene being transcribed may be almost as important as the template DNA strand, and that product RNA, and perhaps transactive RNA, are playing important roles in selective gene transcription.

a. Jiang M, Ma N, Vassylyev DG, and McAllister WT, "RNA Displacement and Resolution of the Transcription Bubble during Transcription by T7 RNA Polymerase", Molecular Cell, vol. 15, no. 5, pp.777-788 (September, 10,  2004).

b. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription", Molec. Biol. Cell, vol. 13, supp. p. 239a (November, 2002).
 

7. RNA as an active agent of genotypic innovation in primates.

Alu DNA sequences are SINES (short interspersed elements) of about 300 base pairs in length, which first appeared in primates about 65 million years ago. They are found exclusively in primates, and also help to distinguish the human genome from the chimpanzee genome. Alu DNA is transcribed by RNA polymerase III, forming a noncoding Alu RNA product of uncertain function.  Alu DNA is integrated into several human chromosomes, and is replicated with the chromosomal DNA, but because Alu is an ancient retrotransposon, formed from an original  invading RNA virus, its RNA  may retain the ability for reverse transcription to new Alu DNA sequences. A widespread editing of Alu RNA has been demonstrated in a large number of different human organ systems, especially in the human thymus and in the human brain.

a. Kim DDY, Kim TTY, Walsh T, Kobayashi Y, Matise TC, Buyske S, and Gabriel A, "Widespread RNA Editing of Embedded Alu Elements in the Human Transcriptome", Genome Research, vol. 14, no. 9, pp. 1719-1725 (September 2, 2004).

b. Sorek R, Lev-Maor G, Reznick M, Dagan T, Belinky F, Graur D, and Ast G, "Minimal conditions for
exonization of intronic sequences: 5' splice site formation in Alu exons", Mol. Cell vol. 14, no. 2, pp. 221-231
(April, 2004).

c. Otieno AC, Carter AB, Hedges DJ, Walker JA, Ray DA, Garber RK, Anders BA, Stoilova N, Laborde ME,
Fowlkes JD, Huang CH, Perodeau B, and Batzer MA, "Analysis of the Human Alu Ya-lineage", J. Mol. Biol.,
vol. 342, no. 1: pp. 109-18, (September 3, 2004).

RNA molecules are capable of inducing changes in the degree and type of cell differentiation within living organ systems. The changes occur when the RNA species are presented to the cells, and do not occur when the same RNA species are absent.

a. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH, "A Small Modulatory dsRNA Specifies the Fate of Adult Neural Stem Cells".

b. Czihak G, "Evidence for inductive properties of the micromere RNA in sea urchin embryos".

c. De Carvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo".

RNA molecular sequences can act as activators of DNA transcription. Such specific Activator RNAs can be reacted covalently to other RNA sequences acting as aptamers (binders) of a third species of small molecules of diverse types and concentrations. The aptamer portion of the triplex then reacts more or less strongly to the simple sensor molecule's concentration, transmitting a more or less strong intramolecular signal to the activator domain for control of DNA transcription at a particular gene.

a. 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. Buskirk AR, Landrigan A, and Liu DR, "Engineering a Ligand-Dependent RNA Transcriptional Activator", Chemistry and Biology, Vol 11, 1157-1163, August 2004.

SINEs ( short interspersed elements ) are ancient retrotransposons that have lost their mechanisms for replication, but may retain profound effects on transcription of other genes. SINE families include Alu and B2, and recently studies of B2 transcription by RNA polymerase III and its B2 noncoding RNA product have revealed interesting effects on the activity and mechanisms of other RNA polymerase II molecules.

a. Allen TA, Von Kaenel S, Goodrich JA, and Kugel JF, "The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock", Nature Structural and Molecular Biology, Published online: 08 August 2004; | doi:10.1038/nsmb813

b. Espinoza CA, Allen TA, Hieb AR, Kugel JF, and Goodrich JA, "B2 RNA binds directly to RNA polymerase II to repress transcript synthesis",  Nature Structural and Molecular Biology, Published online: 08 August 2004; | doi:10.1038/nsmb812

c. Ferrigno O, Virolle T, Djabari Z, Ortonne J-P, White RJ, and Aberdam D, "Transposable B2 SINE elements can provide mobile RNA polymerase II promoters", Nature Genetics, vol. 28,  no. 1, pp. 77-81 (May, 2001).
 

Prions are the infectious agents of BSE in cows and CJD in humans. The disease may have had its origin in infected sheep, whose infected nervous tissue was fed to cattle in the 1980s. The disease can now be transmitted to humans by eating infected beef, and can be transmitted between humans by transfusing infected blood or using infected surgical instruments. It has been difficult or impossible to disinfect infected surgical instruments.

Prions are protein particles, with no demonstrated DNA or RNA components. The following two papers suggest a role for RNA species in animal BSE and human CJD infections:

a. Adler V, Zeiler B, Kryukov V, Kascsak R, Rubenstein R, and Grossman A, "Small, Highly Structured RNAs Participate in the Conversion of Human Recombinant PrP^Sen to PrP^Resin vitro".

b. Deleault NR, Lucassen RW, and Supattapone S, "RNA Molecules Stimulate Prion Protein Conversion".

c. Nandi PK, and Nicole J-C, "Nucleic Acid and Prion Protein Interaction Produces Spherical Amyloids which can Function in vivo as Coats of Spongiform Encephalopathy Agent", Journal of Molecular Biology vol. 344, no. 3 , pp.  827-837 (November 26, 2004).
 

The human immune response is initiated by exposure to foreign antigens, usually thought to be of protein or complex polysaccharide composition. It has recently been demonstrated that total tumor RNA extracted from neoplasms of a tumor-bearing individual can induce in specific T-lymphocytes and B-lymphocytes an immune response against the tumor of that individual.

a. Liao X, Li Y, Bonini C, Nair S, Gilboa E, Greenberg PD, and Yee C, "Transfection of RNA Encoding Tumor Antigens Following Maturation of Dendritic Cells Leads to Prolonged Presentation of Antigen and the Generation of High-Affinity Tumor-Reactive Cytotoxic T Lymphocytes".

b. Coughlin CM, Vance BA, Grupp SA, and Vonderheide RH, "RNA-transfected CD40-activated B cells induce functional T cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy".

c. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, and Rosenberg SA.
"Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes".
 

The Barr sex-chromatin body is found in the cell nucleus of all diploid mammalian females, and represents a X-chromosome completely repressed for RNA synthesis and gene expression. The repression is first induced during embryonic life, apparently on a randomized basis in all tissues and cells, resulting in a chimeric phenotype for each adult female with respect to X-chromosome gene expression in each tissue.

If a female has only one X-chromosome (XO, Turner's Syndrome), this chromsome will be actively expressed, and no Barr sex-chromatin body will be observed in the cell nuclei of that individual. If a female has two X-chromosomes (XX, the normal state), one Barr sex-chromatin body is observed: if three X-chromosomes are present (XXX, Trisomy X), two Barr sex-chromatin bodies are observed; and if four X-chromosomes are present, three Barr sex-chromatin bodies are observed within the cell nuclei of each cell.

The rule holds that (n-1) Barr sex-chromatin bodies are observed when (n) X-chromosomes are present.

Recent evidence suggests that Xist RNA is responsible for the repression seen in the entirety of the repressed X-chromosomes, and that antisense Tsix RNA opposes such inactivation. Such repression appears to be sensitive to the number of X-chromosomes within each cell, and is transmitted to the daughter somatic cells of an individual for a lifetime, but not to the germ cells or the born progeny of the individual.

a. Kelley RL, and Kuroda MI, "The Role of Chromosomal RNAs in Marking the X for Dosage Compensation".

b. Grumbach MM, Morishima A, and Taylor JH, "Human Sex Chromosome Abnormalities in Relation to DNA Replication and Heterochromatinization".

c. Luikenhuis S, Wutz A, and Jaenisch R, "Antisense Transcription Through the Xist Locus Mediates Tsix Function in Embryonic Stem Cells".
 

Further Topics in:  Euchromatin,  active DNA, and  RNA  ribo-regulators:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to Reprogramming and Neoplasia:

A Brief History of  Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).

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