Zhenxi Zhang and John M. Burke*

Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington, Vermont

* Corresponding author. Mailing address: The University of Vermont, 95 Carrigan Dr., 220 Stafford Hall, Burlington, VT 05405. Phone: (802) 656-8503. Fax: (802) 656-5172.
E-mail:   John.Burke@uvm.edu

A controlled mutational study was used to determine the site and mechanism of the antiviral action of ribozymes that inhibit Sindbis virus replication. A hairpin ribozyme targeting G575 of the Sindbis virus genomic RNA was designed and cloned into a minimized alphavirus amplicon vector. Cells that were stably transfected with this construct expressed low levels of a constitutive transcript containing the ribozyme plus recognition sequences for Sindbis RNA replicase. Upon infection, the ribozyme transcript was amplified to high levels by the viral replicase, resulting in decreased viral production from infected ribozyme-expressing cells. Mutations were then introduced into the viral RNA target sequence to interfere with ribozyme binding, and compensatory changes were generated in the ribozyme recognition sequence. Single mutations in the virus or ribozyme decreased the efficacy of the ribozyme's inhibition of viral replication, and compensatory mutations restored it. To confirm that ribozyme-catalyzed RNA cleavage was actually needed for inhibition, we performed tests with a cell line expressing an inactivated ribozyme and with a virus containing a single nucleotide target mutation that allowed the ribozyme to bind but blocked cleavage at the recognition site. The results show that most of the antiviral activity of ribozymes is due to ribozyme-catalyzed cleavage at the targeted RNA sequence, but some additional inhibition seems to occur through an antisense mechanism. 

Euchromatin Network Editor's Perspective,  "The RNA Helix and the RNA Hairpin":

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.

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 have ribo-regulator functions.
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.

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. 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".

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".
 

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