RNA Memory Model: A RNA-Mediated Transcriptional Activation Mechanism Involved in Cell Identity

Abstract

I propose a new model, called the “RNA memory” model, for the possible role of RNAs in the maintenance and establishment of cell identity. This is cytoplasmic memory obtained by the transmission of mother noncoding (nc) RNAs to daughter cells. These RNAs are able to activate transcription via sequence homology in daughter cells. Regulation of RNA memory is strictly linked to the regulation of ncRNAs with repressive features, such as the RNAs involved in RNA interference (RNAi). Misregulation of this system could lead to misidentity, and thus it could be involved in cancer transformation, progression of viral or genetic diseases, and progression of senescence.

Introduction

I t is estimated that about 98% of the transcriptional output of the human genome represents RNAs that do not encode protein. This suggests that the genome is either full of largely useless transcription or that these noncoding (nc) RNAs fulfill a wide range of functions in eukaryotic biology. Recent observations strongly suggest that ncRNAs contribute to the complex networks needed to regulate cell function.^1,2

Different models have been developed to explain either the establishment or the maintenance of cell identity, but actual models focus on explaining how an on/off gene status is inherited and not the exact transcriptional profile. In these models, cell identity can be maintained or established mainly by Polycomb and trithorax group proteins, histone variants, DNA methylation, and epigenetic modifications.³

In symmetric cell division, after mitosis, daughter cells are able to activate the same set of genes of the mother cell at the same transcriptional level for each gene, starting from the high condensed and overall silenced state of metaphase chromosomes, thus leading to a cell lineage. We define the maintenance of cell identity as the continued restoring of chromatin and transcriptional profiles through generations.^4
–6 In asymmetric cell division or during commitment, daughter cells can acquire new functions, and their chromatin and transcriptional profile can be different from the maternal one.^4
–6

Even if actual models can explain several events related to the control of cell identity, some phenomena still remain unexplained or poorly explained. An “RNA memory” model could partially fill the gap between actual models and unexplained or partially explained experimental observations.

RNA Memory Model

The RNA memory theory is a son of Occam's razor. The simplest way for daughter cells to restore the transcriptional profile of a mother cell in a cell line is for the daughter cells to have a direct method of reading the transcriptional profile of the mother cell. During its lifetime, the mother cell produces messenger (m) RNAs that represent the key point of its cell identity. The quantity and quality of mRNAs produced are the sum of all the cascades of regulation borne by the cell. These cascades are driven by intracellular, intercellular, humoral, or environmental stimuli and are integrated in the developmental design. The loss of this information after cell division would represent a severe lack of efficiency. For example, enzymatic induction after exposure to an environmental toxin must be retained after cell division, and it would be gradually lost only if the exposure is not reiterated.^7
–9

In the RNA memory model, there is cytoplasmic memory for cell identity via trans-acting RNA diffusible elements (from now on called memRNAs). These RNAs are able to activate transcription via sequence homology in daughter cells either in a gene- or locus-specific manner. In the cytoplasm of the mother cell, memRNAs are functional RNA remnants that mirror the mother cell's own transcriptional profile. After mitosis, the transcriptional profile can be restored through the reading of memRNAs, which reflect the transcriptional profile of the mother cell. This could be considered a sort of transcriptional inertia.

Development programs could take advantage of this effect. The regulated expression of novel memRNAs during development could change the cell fate in the next generation. In this scenario, daughter cells would be different from mother cells. Asymmetric distribution of memRNAs could easily lead to a different cell fate in daughter cells. In this scenario, daughter cells are different one from the other.

It seems possible that memRNAs could lead to a euchromatic state of chromatin moreso than directly activated transcription. Moreover, it seems highly probable that memRNAs are ncRNAs. memRNAs could arise either from introns, mRNAs untranslated regions (UTRs), or noncoding transcripts from promoters and enhancers.

Regulation of RNA memory is strictly linked to the regulation of ncRNAs with repressive features, such as RNAs involved in RNA interference (RNAi). Double-stranded (ds) RNA-driven degradation could be a system used by the cell to modulate RNA memory. RNAi could destroy memRNAs in a gene-specific manner, thus leading to a lack of activation of the target gene, and thus leading to the silencing of that gene. This mechanism is compatible with the observation that the RNAi pathway is required for the establishment of a silenced state but not for its maintenance.^3,10 Several scenarios could be hypothesized, for example, alternative splicing could be an event of regulation for the production of short- or long-lasting memRNAs.

memRNAs could be important regulators in cell fate, and they could be involved in several biological events. Their misregulation could lead to events such as cancer transformation or senescence. Chronic activation of an oncogene via RNA memory could contribute to cancerogenesis; the only evidence for this would be a change in the epigenetic pattern of that gene.^11
–13 From one generation to the other, the abundance of memRNAs for that gene would induce its own transcription greatly, thus leading the cell to enter the cell cycle, and so on.

In senescence, from one generation to the next, transcriptional noise could increase due to a stochastic floating of the RNA memory system, and this could lead to aging-related diseases.^14

–18 Possible applications both in research and in therapies could be significant. These topics will be discussed in detail in this paper.

RNA and Chromatin Structure

Much evidence suggests that RNA-directed processes help to orchestrate chromatin architecture and epigenetic memory.^19,20 RNA has the capacity for a high degree of sequence and locus specificity and could be an important source of information during development and adaptation. The vast majority of genomes of all metazoans are transcribed in a developmentally regulated manner, mainly into complex patterns of ncRNAs.^21,22 It is known that RNA is an integral component of chromatin²³ and that many of the proteins involved in chromatin modifications as well as transcription factors have the capacity to bind RNA or complexes containing RNA or RNA-binding proteins.^21,24,25 There is evidence that many ncRNAs can act locally to regulate the epigenetic state of the nearby chromatin, often recruiting either repressing or activating chromatin remodeling complexes.^{26

–35} These data suggest that RNA can be involved in chromatin remodeling and epigenetic modification in a sequence-specific manner, a key feature of RNA memory model.

RNA and Transcription

RNAs are able to physically contact components of the transcriptional machinery. This is known for the TATA box–binding protein (TBP)³⁶ and for the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Moreover, the CTD can also bind RNA-binding proteins,^37
–39 such as TFIIH, which interacts with U1 small nuclear (sn) RNA, greatly enhancing its kinase and helicase activities.^40,41 These examples show that RNAs have the intrinsic ability to contact components of the transcriptional machinery.

In 1999 it was discovered that some RNA sequences are able to trans-activate transcription,⁴² and that these RNAs seem to bear a consensus sequence.³⁶ Interestingly, a study using longer molecules identified RNAs with a high degree of complexity able to activate transcription even more strongly than in previous experiments. These RNAs do not have a clear consensus sequence.^39,43 These data suggest that RNAs have the ability to trans-activate transcription, a key feature of RNA memory model.

It is also known that in some cases RNAs are able to activate transcription in a highly regulated spatial-temporal way, for example as in the case of trans-activation responsive region (TAR) RNA for the TAT protein in the human immunodeficiency virus type 1 (HIV-1).^44
–46 Other examples are Evf-2 ncRNA, which cooperates with the Dlx-2 protein in a targeted and homeodomain-specific manner, probably the first of a large family to be identified,³² or the steroid receptor RNA activator (SRA) with the SRC-1 protein, which also seems to confer specificity for steroid receptors.^47
–49 Another example is the short noncoding RNA duplex NRSE, which is able to bind to the neuron-restrictive silencing factor NRSF/REST and able to convert it to a transcriptional activator. Interestingly, NRSE has a sequence homology to DNA sites bound by NRSF and does not disrupt the NRSF–DNA complex.⁵⁰ These data suggest that RNAs have in some cases the ability to trans-activate transcription in a regulated way, also by sequence homology. This is another key feature of RNA memory model.

RNA Duplex

Recently, it has been discovered that RNA duplexes with sequence homology with the promoter can promote transcription from that promoter using the same cellular machinery needed for RNAi. This phenomenon is strictly linked to genome-wide antisense transcription.^51
–53 It is interesting to note that there is a great deal of evidence that enhancers and other regulatory sequences are transcribed in the cell in which they are active,^54

–58 and their transcription is often needed to activate the transcription on their target gene.⁵⁹ These data suggest a possible mechanism of action of RNA memory. memRNAs could form RNA duplexes with sequence homology to target promoters or other regulatory sequences, thus driving the transcription of the target gene.

Moreover, there is the possibility that the regulation of RNA memory is strictly linked to the regulation of ncRNAs with repressive features, such as RNAs involved in RNAi. dsRNA-driven degradation could be a system used by the cell to modulate RNA memory. RNAi could destroy memRNAs in a gene-specific manner, thus leading to a lack of activation of the target gene and the silencing of that gene. This is compatible with the observation that the RNAi pathway is required for the establishment of a silenced state but not for its maintenance.^3,10

RNA and Polycomb-Mediated Silencing

Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state.³

In Drosophila, experimental data suggest that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE not only during embryogenesis but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an antisilencing mechanism to prevent the access of repressive PcG complexes to the chromatin. All PREs tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that antisilencing by transcription is a fundamental aspect of the cellular memory system.⁶⁰ It seems that an intergenic ncRNA transcribed from the Polycomb group/trithorax response element (PRE/TRE) of the Ubx gene is retained at the TRE through DNA–RNA interactions and plays an important role in providing an RNA scaffold that is recognized by the epigenetic positive regulator Ash1.^2,26

These data suggest a possible mechanism of action for RNA memory. Transcripts from regulatory sequences could target their specific regulatory sequences, recruiting specific nuclear factors, and thus drive the transcription of the target gene. So, through the inheritance of the transcriptional state of regulatory sequences, the transcriptional state of the target gene could be also inherited. Modulation of transcription of the regulatory sequence could change the cell fate of daughter cells. Transcripts from regulatory sequences with these features would be memRNAs.

Introns and UTRs

MicroRNAs (miRNAs) are 20-nucleotide-long to 24-nucleotide-long ncRNAs acting as posttranscriptional regulators of gene expression in animals and plants. Both human and mouse intronic miRNAs tend to be present in large introns with 5′-biased position distribution.⁶¹ This correlates with the previous observation that most long intronic transcripts are expressed within first introns of the host genes.⁶² A few large-scale studies have shown that certain sets of intronic ncRNAs have the same tissue expression pattern as the corresponding protein-coding genes, but this is not a universal rule.^62

–65

It has been reported that long intronic sequences could coordinate waves of gene expression important for particular cellular processes that are functionally related to the protein-coding transcript of the same locus. Note that non-degraded spliced introns can be exported selectively to the cytoplasm^66–67 and be involved in global gene expression regulation.^66,68 These data suggest a possible role of intronic transcript or intron fragments in RNA memory. If sense or antisense, short or long intronic transcripts or fragments could drive the expression of their homolog gene, and these RNA fragments can survive from one generation to the next, these RNAs would be memRNAs.

Moreover there is evidence that many 3′ UTR of mRNAs can act in trans as ncRNAs,^{69

–80} so it is possible that other fragments of mRNAs, if they could drive the expression of their homolog gene and survive from one generation to the next, could act as memRNAs.

The Nuclear Membrane

In the vast majority of eukaryotes, the nuclear envelope is disrupted during mitosis. In some rare cases this does not happen, so it is not compulsory, but it is probably an event of regulation.⁸¹ Is it possible that nuclear membrane disruption permits the merging of cytoplasmic and nuclear factors, allowing memRNAs to reach the nucleus? Moreover it is known that RNAs can change conformation and activity in response to interacting proteins or metabolites, so the disruption of nuclear membrane could lead to an ulterior step of regulation.^39,82

–85

β-Globin Locus and Transinduction

In mouse erythroid cell lines, cell identity heritage is a clonal phenomenon.⁸⁶ In fact, at least for tested genes, stochastic variations in the transcriptional profile are inherited by daughter cells, without genetic mutations. Tested genes were α- and β-globin genes, which are differentiation markers and have no known nuclear effect.⁸⁶ This means that a mechanism should be present in daughter cells to mimic the transcriptional profile of the mother cell. RNA memory could be this system. For example, if at generation 0 there is a random inactivation of the β-globin gene, there will be a lack of memRNAs for that transcript in mother cell cytoplasm. Daughter cells at generation 0 + 1 could not find any β-globin gene memRNAs, thus the inactivation will be inherited. This effect can extend on through generations, producing a clonal phenomenon. Moreover, because the memRNA activation could be related to sequence homology between memRNAs and the noncoding sequence of the gene, the activation/inactivation could spread through cell generations in an allele-specific mode. Also, asymmetric distribution or variation in the half-life of memRNAs could lead in the next generation to a variation of the transcriptional profile, giving a possible mechanicistic explanation both for the clonal inheritance and the stochastic variation of the transcriptional profile. Moreover, analysis of a multicopy transgenic locus for human β-globin gene in mice showed an all-or-none effect in the activation of all the genes in the locus. Each β-globin gene does not act stochastically in an independent way.⁸⁶ Trans-acting memRNAs with sequence homology for all of the copies of human β-globin gene in the locus could give an easy explanation to this phenomenon.

In human cultured cells, a transient transfection of a β-globin gene induces transcription of the locus control region (LCR) and intergenic region at the chromosomal β-globin locus in nonerythroid cell lines. However, the β-globin genes remain transcriptionally silent. This effect is dependent on transcription, but not on protein expression, and therefore is RNA mediated. This phenomenon was called transinduction. In one experiment, transinduction was mediated by the transfection of a γ gene of the β-globin locus not able to produce a detectable full-length γ-globin mRNA. This suggests that the effect is not mediated by a functional mRNA, but instead by ncRNAs.⁵⁵ A possible explanation is that the transfection is able to produce memRNAs for the euchromatinization of the β-globin locus, but the specific globin genes cannot be expressed due to the lack of the right erythroid transcriptional factors. If this phenomenon is mediated by memRNAs, here there is a clear suggestion that RNA memory can drive a euchromatic state of chromatin more than directly activated transcription.

Moreover, in humans, a regulated intergenic transcription is needed for chromatin remodeling at the β-globin locus to set the right temporal development of the erythroid cell line.⁸⁷ This could be a regulated modulation of RNA memory at the β-globin locus to drive the correct expression pattern through development.

Drosophila melanogaster PARP Gene

In Drosophila melanogaster, there is only one PARP gene that encodes two alternative splicing forms: PARP-I, an enzymatically active form, and PARP-e, an enzymatically inactive form. An insertion mutation near an upstream promoter disrupts all PARP expression. The expression of a transgene for PARP-e can restore the presence and the activity of PARP-I. PARP-e expression induces a more physiological pattern in PARP-I expression, leading to fewer deleterious effects than when PARP-I is misexpressed globally.⁸⁸ Even if authors suggest a different explanation, this phenomenon could be explained by a trans-activation via sequence homology. In my model, PARP-e transgene could have produced memRNAs responsible for the activation of genomic PARP. If this would be true, it implies that alternative splicing could also be a step for the regulation of RNA memory.

Transvection

Many Drosophila loci show a phenomenon called transvection, wherby a wild-type promoter linked to a mutant coding sequence is able to trans-activate a wild-type coding sequence linked to a nonfunctional promoter in the homolog gene.⁸⁹ Usually transvection needs a homologous position on chromosomes of the two interacting genes, but it has been reported some cases in which transvetion occurs over large distances, even between different chromosomes.^90

–93 Moreover, many transvecting promoters are transcribed into ncRNAs, and mutants of epigenetic modifiers can modify transvection.^89,94
–96

Taken together, these observations could suggest an involvement of a diffusible trans-activating signal over great distances, able to modulate locally the epigenetic markers, such RNAs or, in my theory, memRNAs.

Pseudogenes

Pseudogenes bear sequence similarities to a specific protein-coding gene, but they are unable to produce functional proteins. The human genome is estimated to contain up to 20,000 pseudogenes. Pseudogenes may represent a means for additional levels of gene regulation.

Recent studies have suggested the importance of pseudogene transcripts in gene regulation. For example, a mouse not expressing the Makorin1-p1 pseudogene shows abnormal expression of the functional protein-coding Makorin1 gene located elsewhere in the genome.^2,97
–99 Again, this phenomenon could be explained by a trans-activation via sequence homology. In my model, the Makorin1 pseudogene could produce memRNAs responsible for the activation of the functional protein-coding Makorin1.

Celll Reprogramming

The possibility of reprogramming somatic cells to pluripotency through nuclear transfer or cell fusion strongly suggests the presence of trans-acting agents able to radically reprogram the chromatin. These cytoplasmic factors are able to totally erase the epigenetic program of a differentiate cell and convert it into a stem cell–like chromatin pattern.⁵ Even if the efficiency of reprogramming is extremely low, these factors are not only able to physiologically drive the first steps of development, but they also have the intrinsic ability to “remember” how the nucleus should behave. I think that some of these factors could be RNAs that can act in a memRNA-like fashion. Even during direct in vivo reprogramming of somatic cells into embryonic stem-like cells, the production of proteic factors is usually obtained by the use of viral vectors. This method raises the production of both proteins and RNAs of the transfected genes in host cells.⁵ Is it possible that the maintenance of production of stem cell-specific factors through the generations could be mediated also by memRNAs?

Conclusion

In recent years, it has been discovered that ncRNAs could have a broad range of functions, absolutely unexpected until few years ago. RNAi is now a well-known, evolutionarily conserved phenomenon with several implications in many aspects of chromatin dynamics and transcriptional regulation, almost the totality of them with repressive features.^100,101 This paper describes a theory regarding the role of ncRNAs in the activation of transcription and their implications in the control of cell identity. If the theory is confirmed, we would have a mechanism and a tool opposite and complementary to RNAi. This model is called RNA memory. In this theory, the mother cell produces together with mRNAs a class of ncRNAs called memRNAs. memRNAs reflect the transcriptional profile of the mother cell and are retained until the next cell division. In this way, daughter cells are able to read the memRNAs and mimic the transcriptional profile of the mother cell. This mechanism permits retention of transcriptional information due to local functional perturbation, such as enzymatic induction. In this theory, the abundance of memRNAs can be read by the nucleus, using an unknown mechanism, and drive the euchromatinization of specific genes or loci due to sequence homology. I have presented some experimental data in support of such a theory. If this theory is confirmed, it is highly probably that RNAi and RNA memory could work in a very entangled network, especially in the regulation of the developmental program.

Two major objections could be raised against this theory. Why is epigenetic inheritance not sufficient to explain the observed phenomena? The answer is that epigenetic phenomena act in cis and cannot explain the observed trans-acting events. Many of the events discussed seem to use sequence homology to target the right gene or locus, and an RNA-mediated mechanism seems more probable than an epigenetic one. Moreover, many histone covalent modifications seem to be transient and are able to set the local environment to perform specific functions. Histone modifications are and must be very dynamic, but it is uncertain how many of them can spread in time in the absence of a reiterated epigenetic writing.^102,103 The model I propose is that RNA memory targets the gene or the locus that has to be activated and that chromatin remodelers and epigenetic modifiers are the physical effectors.

The second major objection could be the following: Why has this mechanism not yet been discovered? I think that full-length RNAs and/or transcription from regulatory sequences is needed to trigger RNA memory. The enormous use of complementary (c) DNAs in the last decades could have produced experimental drift, which that could have obscured this phenomenon. Moreover, until recently, the analysis of cellular components involved in transcriptional activation has focused on proteic factors.

If this theory of memRNA is confirmed, the implications in the development and cure of several diseases will be enormous. Overproduction of oncogenes could be driven by an alteration of an RNA memory system, leading to a chronic activation of these genes, thus leading to carcinogenic transformation. Interference with RNA memory could revert this activation and could be used in therapy. Viral infection could also take advantage of RNA memory. Production of memRNAs for viral products could contribute to the increase in transcription from viral genes and could be a key point of the regulation of virulence. In senescence, from one generation to the next, transcriptional noise could increase due to a stochastical floating of RNA memory system, and this could lead to aging-related diseases. As a tool, memRNA will be of great use, permitting the activation of specific transcriptional patterns just driving the production of memRNAs, and it will have possible applications in therapy whenever a disease could result from the underproduction of specific gene product, as, for example, in the widespread Gilbert syndrome.¹⁰⁴

Of course, this theory is not a substitute for consolidated theories regarding the regulation of transcription and the control of cell identity. However, if this theory is confirmed, it will be just another piece in the amazing network of cellular life.

Footnotes

Acknowledgments

I want to thank Giordano Bruno and Nikola Tesla for their inspiring visions, and my former laboratories for their help. I want also thank Swonild Ilenia Genovese for her aid revising the manuscript.

Author Disclosure Statement

No competing financial interests exist.

References

Mattick

. The functional genomics of noncoding RNA. Science, 2005; 309:1527–1528.

Prasanth

, Spector

. Eukaryotic regulatory RNAs: An answer to the ‘genome complexity’ conundrum. Genes Dev, 2007; 21:11–42.

Orlando

. Polycomb, epigenomes, and control of cell identity. Cell, 2003; 112:599–606.

Morrison

, Kimble

. Asymmetric and symmetric stem-cell divisions in development and cancer. Nature, 2006; 29;441:1068–1074.

Hanna

, Carey

, Jaenisch

. Reprogramming of somatic cell identity. Cold Spring Harb Symp Quant Biol., 2008; 73:147–155.

Chandebois

, Faber

. From DNA transcription to visible structure: What the development of multicellular animals teaches us. Acta Biotheor, 1987; 36:61–120.

Scassellati-Sforzolini

, Moretti

, Villarini

, Monarca

, Fatigoni

, Pasquini

. In vivo studies on enzymatic induction activity of Linuron. J Environ Pathol Toxicol Oncol, 1994; 13:11–17.

Pasquini

, Monarca

, Scassellati Sforzolini

, Savino

, Fatigoni

, Puccetti

. Urine mutagenicity and biochemical parameters as markers of exposure to petroleum pitch using a rat model. Environ Mol Mutagen, 1990; 15:56–66.

Sendama

, de Hemptinne

, Lambotte

. Recovery of liver function in partially hepatectomized rats evaluated by aminopyrine demethylation capacity. Hepatology, 1985; 5:629–633.

10.

Hall

, Shankaranarayana

, Noma

, Ayoub

, Cohen

, Grewal

. Establishment and maintenance of a heterochromatin domain. Science, 2002; 27;297:2232–2237.

11.

Nagarajan

, Costello

. Molecular epigenetics and genetics in neuro-oncology. Neurotherapeutics, 2009; 6:436–446.

12.

Howell

Jr , Liu

, Ren

, Behlen

, Fodstad

, Riker

. Epigenetics in human melanoma. Cancer Control, 2009; 16:200–218.

13.

Mathews

, Crea

, Farrar

. Epigenetic gene regulation in stem cells and correlation to cancer. Differentiation, 2009; 78:1–17.

14.

Busuttil

, Bahar

, Vijg

. Genome dynamics and transcriptional deregulation in aging. Neuroscience, 2007; 145:1341–1347.

15.

Csermely

, Soti

. Cellular networks and the aging process. Arch Physiol Biochem, 2006; 112:60–64.

16.

Pedraza

, Paulsson

. Effects of molecular memory and bursting on fluctuations in gene expression. Science, 2008; 319:339–343.

17.

Bahar

, Hartmann

, Rodriguez

, Denny

, Busuttil

, Dollé

, Calder

, Chisholm

, Pollock

, Klein

, Vijg

. Increased cell-to-cell variation in gene expression in ageing mouse heart. Nature, 2006; 441:1011–1014.

18.

Martin

. The genetics and epigenetics of altered proliferative homeostasis in ageing and cancer. Mech Ageing Dev, 2007; 128:9–12.

19.

Bernstein

, Allis

. RNA meets chromatin. Genes Dev, 2005; 19:1635–1655.

20.

Amaral

, Mattick

. Noncoding RNA in development. Mamm Genome, 2008; 19:454–492.

21.

Mattick

, Makunin

. Non-coding RNA. Hum Mol Genet, 2006; 15:R17–R29.

22.

Kapranov

, Willingham

, Gingeras

. Genome-wide transcription and the implication for genomic organization. Nat Rev Genet, 2007; 8:413–423.

23.

Rodriguez-Campos

, Azorin

. RNA is an integral component of chromatin that contributes to its structural organization. PLoS ONE, 2007; 2:e1182.

24.

Cassiday

, Maher

. III, Having it both ways: Transcription factors that bind DNA and RNA. Nucleic Acids Res, 2002; 30:4118–4126.

25.

Mattick

, Amaral

, Dinger

, Mercer

, Mehler

. RNA regulation of epigenetic processes. Bioessays, 2009; 31:51–59.

26.

Sanchez-Elsner

, Gou

, Kremmer

, Sauer

. Noncoding RNAs of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax. Science, 2006; 311:1118–1123.

27.

Rinn

, Kertesz

, Wang

, Squazzo

, Xu

, Brugmann

, Goodnough

, Helms

, Farnham

, Segal

, Chang

. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell, 2007; 129:1311–1323.

28.

, Gius

, Onyango

, Muldoon-Jacobs

, Karp

, Feinberg

, Cui

. Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature, 2008; 451:202–206.

29.

Martianov

, Ramadass

, Serra Barros

, Chow

, Akoulitchev

. Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript. Nature, 2007; 445:666–670.

30.

Blin-Wakkach

, Lezot

, Ghoul-Mazgar

, Hotton

, Monteiro

, Teillaud

, Pibouin

, Orestes-Cardoso

, Papagerakis

, Macdougall

, Robert

, Berdal

. Endogenous Msx1 antisense transcript: In vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals. Proc Natl Acad Sci USA, 2001; 98:7336–7341.

31.

Mattick

, Amaral

, Dinger

, Mercer

, Mehler

. RNA regulation of epigenetic processes. Bioessays, 2009; 31:51–59.

32.

Feng

, Bi

, Clark

, Mady

, Shah

, Kohtz

. The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes Dev, 2006; 20:1470–1484.

33.

Pandey

, Mondal

, Mohammad

, Enroth

, Redrup

, Komorowski

, Nagano

, Mancini-Dinardo

, Kanduri

. Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell, 2008; 32:232–246.

34.

Nagano

, Mitchell

, Sanz

, Pauler

, Ferguson-Smith

, Feil

, Fraser

. The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science, 2008; 322:1717–1720.

35.

Terranova

, Yokobayashi

, Stadler

, Otte

, van Lohuizen

, Orkin

, Peters

. Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos. Dev Cell, 2008; 15:668–679.

36.

Saha

, Ansari

, Jarrell

, Ptashne

. RNA sequences that work as transcriptional activating regions. Nucleic Acids Res, 2003; 31:1565–1570.

37.

Kaneko

, Manley

. The mammalian RNA polymerase II C-terminal domain interacts with RNA to suppress transcription-coupled 3′ end formation. Mol Cell, 2005; 20:91–103.

38.

Phatnani

, Greenleaf

. Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev, 2006; 20:2922–2936.

39.

Ansari

. Riboactivators: Transcription activation by noncoding RNA. Crit Rev Biochem Mol Biol, 2009; 44:50–61.

40.

Kwek

, Murphy

, Furger

, Thomas

, O'Gorman

, Kimura

, Proudfoot

, Akoulitchev

. U1 snRNA associates with TFIIH and regulates transcriptional initiation. Nat Struct Biol, 2002; 9:800–805.

41.

O'Gorman

, Thomas

, Kwek

, Furger

, Akoulitchev

. Analysis of U1 small nuclear RNA interaction with cyclin H. J Biol Chem, 2005; 280:36920–36925.

42.

Sengupta

, Wickens

, Fields

. Identification of RNAs that bind to a specific protein using the yeast three-hybrid system. RNA, 1999; 5:596–601.

43.

Buskirk

, Kehayova

, Landrigan

, Liu

. In vivo evolution of an RNA-based transcriptional activator. Chem Biol, 2003; 10:533–540.

44.

Selby

, Bain

, Luciw

, Peterlin

. Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat. Genes Dev, 1989; 3:547–558.

45.

Rosen

, Sodroski

, Haseltine

. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell, 1985; 41:813–823.

46.

Bres

, Yoh

, Jones

. The multi-tasking P-TEFb complex. Curr Opin Cell Biol, 2008; 20:334–340.

47.

Shi

, Downes

, Xie

, Kao

, Ordentlich

, Tsai

, Hon

, Evans

. Sharp, an inducible cofactor that integrates nuclear receptor repression and activation. Genes Dev 1, 2001; 5:1140–1151.

48.

Watanabe

, Yanagisawa

, Kitagawa

, Takeyama

, Ogawa

, Arao

, Suzawa

, Kobayashi

, Yano

, Yoshikawa

, Masuhiro

, Kato

. A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor alpha coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA. EMBO J, 2001; 20:1341–1352.

49.

Lanz

, Razani

, Goldberg

, O'Malley

. Distinct RNA motifs are important for coactivation of steroid hormone receptors by steroid receptor RNA activator (SRA) Proc Natl Acad Sci USA, 2002; 99:16081–16086.

50.

Kuwabara

, Hsieh

, Nakashima

, Taira

, Gage

. A small modulatory dsRNA specifies the fate of adult neural stem cells. Cell, 2004; 116:779–793. 31:1565–1570.

51.

, Okino

, Zhao

, Pookot

, Place

, Urakami

, Enokida

, Dahiya

. Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci USA, 2006; 103:17337–17342.

52.

Janowski

, Younger

, Hardy

, Ram

, Huffman

, Corey

. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nat Chem Biol, 2007; 3:166–173.

53.

Schwartz

, Younger

, Nguyen

, Hardy

, Monia

, Corey

, Janowski

. Antisense transcripts are targets for activating small RNAs. Nat Struct Mol Biol, 2008; 15:842–848.

54.

Calin

, Liu

, Ferracin

, Hyslop

, Spizzo

, Sevignani

, Fabbri

, Cimmino

, Lee

, Wojcik

, Shimizu

, Tili

, Rossi

, Taccioli

, Pichiorri

, Liu

, Zupo

, Herlea

, Gramantieri

, Lanza

, Alder

, Rassenti

, Volinia

, Schmittgen

, Kipps

, Negrini

, Croce

. Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. Cancer Cell, 2007; 12:215–229.

55.

Ashe

, Monks

, Wijgerde

, Fraser

, Proudfoot

. Intergenic transcription and transinduction of the human beta-globin locus. Genes Dev, 1997; 11:2494–2509.

56.

Ling

, Baibakov

, Pi

, Emerson

, Tuan

. The HS2 enhancer of the beta-globin locus control region initiates synthesis of non-coding, polyadenylated RNAs independent of a cis-linked globin promoter. J Mol Biol 2005;, 29; 350:883–896.

57.

Lefevre

, Witham

, Lacroix

, Cockerill

, Bonifer

. The LPS- induced transcriptional upregulation of the chicken lysozyme locus involves CTCF eviction and noncoding RNA transcription. Mol Cell, 2008; 32:129–139.

58.

Lempradl

, Ringrose

. How does noncoding transcription regulate Hox genes? Bioessays, 2008; 30:110–121.

59.

, Elefant

, Liebhaber

, Cooke

. Locus control region transcription plays an active role in long-range gene activation. Molec Cell, 2006; 23:365–375.

60.

Schmitt

, Prestel

, Paro

. Intergenic transcription through a polycomb group response element counteracts silencing. Genes Dev, 2005; 19:697–708.

61.

Zhou

, Lin

. Excess of microRNAs in large and very 5′ biased introns. Biochem Biophys Res Commun, 2008; 368:709–715.

62.

Nakaya

, Amaral

, Louro

, Lopes

, Fachel

, Moreira

, El-Jundi

, da Silva

, Reis

, Verjovski-Almeida

. Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription. Genome Biol, 2007; 8:R43.

63.

Mercer

, Dinger

, Sunkin

, Mehler

, Mattick

. Specific expression of long noncoding RNAs in the mouse brain. Proc Natl Acad Sci USA, 2008; 105:716–721.

64.

Dinger

, Amaral

, Mercer

, Pang

, Bruce

, Gardiner

, Askarian-Amiri

, Ru

, Soldà

, Simons

, Sunkin

, Crowe

, Grimmond

, Perkins

, Mattick

. Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation. Genome Res, 2008; 18:1433–1445.

65.

Katayama

, Tomaru

, Kasukawa

, Waki

, Nakanishi

, Nakamura

, Nishida

, Yap

, Suzuki

, Kawai

, Suzuki

, Carninci

, Hayashizaki

, Wells

, Frith

, Ravasi

, Pang

, Hallinan

, Mattick

, Hume

, Lipovich

, Batalov

, Engström

, Mizuno

, Faghihi

, Sandelin

, Chalk

, Mottagui-Tabar

, Liang

, Lenhard

, Wahlestedt

. RIKEN Genome Exploration Research Group, Genome Science Group (Genome Network Project Core Group), FANTOM Consortium. Antisense transcription in the mammalian transcriptome. Science, 2005; 2;309:1564–1566.

66.

Hill

, Hong

, Wen

, Teng

, McPherson

, Levasseur

, Sorscher

. Micro-RNA-like effects of complete intronic sequences. Front Biosci, 2006; 11:1998–2006.

67.

Clement

, Qian

, Kaplinsky

, Wilkinson

. The stability and fate of a spliced intron from vertebrate cells. RNA, 1999; 5:206–220.

68.

Louro

, Smirnova

, Verjovski-Almeida

. Long intronic noncoding RNA transcription: expression noise or expression choice? Genomics, 2009; 93:291–298.

69.

Abelson

, Kwan

, O'Roak

, Baek

, Stillman

, Morgan

, Mathews

, Pauls

, Rasin

, Gunel

, Davis

, Ercan-Sencicek

, Guez

, Spertus

, Leckman

, Dure

4th , Kurlan

, Singer

, Gilbert

, Farhi

, Louvi

, Lifton

, Sestan

, State

. Sequence variants in SLITRK1 are associated with Tourette's syndrome. Science, 2005; 310:317–320.

70.

Clop

, Marcq

, Takeda

, Pirottin

, Tordoir

, Bibé

, Bouix

, Caiment

, Elsen

, Eychenne

, Larzul

, Laville

, Meish

, Milenkovic

, Tobin

, Charlier

, Georges

. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet, 2006; 38:813–818.

71.

Jupe

, Liu

, Kiehlbauch

, McClung

, Dell'Orco

. Prohibitin in breast cancer cell lines: Loss of antiproliferative activity is linked to 39 untranslated region mutations. Cell Growth Differ, 1996; 7:871–878.

72.

Jupe

, Liu

, Kiehlbauch

, McClung

, Dell'Orco

. The 39 untranslated region of prohibitin and cellular immortalization. Exp Cell Res, 1996; 224:128–135.

73.

Jenny

, Hachet

, Závorszky

, Cyrklaff

, Weston

, Johnston

, Erdélyi

, Ephrussi

. A translation-independent role of oskar RNA in early Drosophila oogenesis. Development, 2006; 133:2827–2833.

74.

Candeias

, Malbert-Colas

, Powell

, Daskalogianni

, Maslon

et al. p53 mRNA controls p53 activity by managing Mdm2 functions. Nat Cell Biol, 2008; 10:1098–1105.

75.

Rastinejad

, Blau

. Genetic complementation reveals a novel regulatory role for 39 untranslated regions in growth and differentiation. Cell, 1993; 72:903–917.

76.

Rastinejad

, Conboy

, Rando

, Blau

. Tumor suppression by RNA from the 39 untranslated region of alpha-tropomyosin. Cell, 1993; 75:1107–1117.

77.

Fan

, Villegas

, Huang

, Wright

. Suppression of malignancy by the 39 untranslated regions of ribonucleotide reductase R1 and R2 messenger RNAs. Cancer Res, 1996; 56:4366–4369.

78.

Amack

, Paguio

, Mahadevan

. Cis and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model. Hum Mol Genet, 1999; 8:1975–1984.

79.

Carninci

, Sandelin

, Lenhard

, Katayama

, Shimokawa

, Ponjavic

, Semple

, Taylor

, Engström

, Frith

, Forrest

, Alkema

, Tan

, Plessy

, Kodzius

, Ravasi

, Kasukawa

, Fukuda

, Kanamori-Katayama

, Kitazume

, Kawaji

, Kai

, Nakamura

, Konno

, Nakano

, Mottagui-Tabar

, Arner

, Chesi

, Gustincich

, Persichetti

, Suzuki

, Grimmond

, Wells

, Orlando

, Wahlestedt

, Liu

, Harbers

, Kawai

, Bajic

, Hume

, Hayashizaki

. Genome-wide analysis of mammalian promoter architecture and evolution. Nat Genet, 2006; 38:626–635.

80.

Shirasawa

, Harada

, Furugaki

, Akamizu

, Ishikawa

, Ito

, Tamai

, Kuma

, Kubota

, Hiratani

, Tsuchiya

, Baba

, Ishikawa

, Tanaka

, Sakai

, Aoki

, Yamamoto

, Sasazuki

. SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease. Hum Mol Genet, 2004; 13:2221–2231.

81.

Anderson

, Hetzer

. The life cycle of the metazoan nuclear envelope. Curr Opin Cell Biol, 2008; 20:386–392.

82.

Winkler

, Breaker

. Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol, 2005; 59:487–517.

83.

Henkin

, Grundy

. Sensing metabolic signals with nascent RNA transcripts: the T box and S box riboswitches as paradigms. Cold Spring Harb Symp Quant Biol, 2006; 71:231–237.

84.

Nudler

. Flipping riboswitches. Cell, 2006; 126:19–22.

85.

Mandal

, Breaker

. Adenine riboswitches and gene activation by disruption of a transcription terminator. Nat Struct Mol Biol, 2004; 11:29–35.

86.

de Krom

, van de Corput

, von Lindern

, Grosveld

, Strouboulis

. Stochastic patterns in globin gene expression are established prior to transcriptional activation and are clonally inherited. Molecular Cell, 2002; 9:1319–1326.

87.

Gribnau

, Diderich

, Pruzina

, Calzolari

, Fraser

. Intergenic transcription and developmental remodeling of chromatin subdomains in the human beta-globin locus. Mol Cell, 2000; 5:377–386.

88.

Tulin

, Stewart

, Spradling

. The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development. Genes Dev, 2002; 16:2108–2119.

89.

Duncan

. Transvection effects in Drosophila. Annu Rev Genet, 2002; 36:521–556.

90.

Hendrickson

, Sakonju

. Cis and trans interactions between the iab regulatory regions and abdominal-A and abdominal-B in Drosophila melanogaster. Genetics, 1995; 139:835–848.

91.

Hopmann

, Duncan

. Transvection in the iab-5,6,7 region of the bithorax complex of Drosophila: Homology independent interactions in trans. Genetics, 1995; 139:815–833.

92.

Muller

, Hagstrom

, Gyurkovics

, Pirrotta

, Schedl

. The mcp element from the Drosophila melanogaster bithorax complex mediates long distance regulatory interactions. Genetics, 1999; 153:1333–1356.

93.

Vazquez

, Muller

, Pirrotta

, Sedat

. The Mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila. Mol Biol Cell, 2006; 17:2158–2165.

94.

Akam

, Martinez-Arias

. The distribution of Ultrabithorax transcripts in Drosophila embryos. EMBO J, 1985; 4:1689–1700.

95.

Lipshitz

, Peattie

, Hogness

. Novel transcripts from the Ultrabithorax domain of the bithorax complex. Genes Dev, 1987; 1:307–322.

96.

Petruk

, Sedkov

, Riley

, Hodgson

, Schweisguth

et al. Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference. Cell, 2006; 127:1209–1221.

97.

Hirotsune

, Yoshida

, Chen

, Garrett

, Sugiyama

, Takahashi

, Yagami

, Wynshaw-Boris

, Yoshiki

. An expressed pseudogene regulates the messenger RNA stability of its homologous coding gene. Nature, 2003; 423:91–96.

98.

Lee

. Molecular biology: Complicity of gene and pseudogene. Nature, 2003; 423:26–28.

99.

Yano

, Saito

, Yoshida

, Yoshiki

, Wynshaw-Boris

, Tomita

, Hirotsune

. A new role for expressed pseudogenes as ncRNA: Regulation of mRNA stability of its homologous coding gene. J Mol Med, 2004; 82:414–422.

100.

Grimm

. Small silencing RNAs: State-of-the-art. Adv Drug Deliv Rev, 2009; 61:672–703.

101.

Moazed

, Bühler

, Buker

, Colmenares

, Gerace

, Gerber

, Hong

, Motamedi

, Verdel

, Villén

, Gygi

. Studies on the mechanism of RNAi-dependent heterochromatin assembly. Cold Spring Harb Symp Quant Biol, 2006; 71:461–471.

102.

Delcuve

, Rastegar

, Davie

. Epigenetic control. J Cell Physiol, 2009; 219:243–250.

103.

Nightingale

, O'Neill

, Turner

. Histone modifications: Signalling receptors and potential elements of a heritable epigenetic code. Curr Opin Genet Dev, 2006; 16:125–136.

104.

Maruo

, Iwai

, Mori

, Sato

, Takeuchi

. Polymorphism of UDP-glucuronosyltransferase and drug metabolism. Curr Drug Metab, 2005; 6:91–99.