Review:

"MicroRNA control of signal transduction".

Masafumi Inui ¹, Graziano Martello ¹, and Stefano Piccolo ¹

¹ Department of Histology, Microbiology and Medical Biotechnologies, Section of Histology and Embryology, University of Padua, viale Colombo 3, 35100 Padua, Italy.

Correspondence to: Stefano Piccolo ¹,  Email: piccolo@civ.bio.unipd.it

Published online 10 March 2010

"In this issue, Stefano Piccolo and colleagues (page 252) propose that, as they are highly dose-sensitive, signalling pathways are ideal targets for the degree of fine-tuning that miRNAs are likely to achieve in nature. They describe how miRNAs might confer signalling robustness, alter the cellular milieu to influence gene expression induced by various signalling cascades and regulate crosstalk between signalling pathways. As miRNAs modulate signalling cascades that are important in disease, understanding their role in cell signalling might also help us to identify therapeutically relevant miRNAs".

MicroRNAs (miRNAs) are integral elements in the post-transcriptional control of gene expression. After the identification of hundreds of miRNAs, the challenge is now to understand their specific biological function. Signalling pathways are ideal candidates for miRNA-mediated regulation owing to the sharp dose-sensitive nature of their effects. Indeed, emerging evidence suggests that miRNAs affect the responsiveness of cells to signalling molecules such as transforming growth factor-b, WNT, Notch and epidermal growth factor. As such, miRNAs serve as nodes of signalling networks that ensure homeostasis and regulate cancer, metastasis, fibrosis and stem cell biology.

    * Signal transduction pathways are prime candidates for microRNA (miRNA)-mediated regulation: the highly dynamic and dose-sensitive signalling complexes are the ideal targets for the degree of quantitative regulation imposed by miRNAs.

    * Each cell type expresses a unique set of miRNAs, allowing it to interpret the extracellular signals according to its history and environment, leading to the activation of genes that better suit its needs: a process called context-dependent gene expression.

    * Some signalling pathways are kept actively repressed in the absence of stimulation. This default repression mechanism ensures that target genes are activated only in the presence of a signal. miRNAs contribute to this control, inhibiting the expression of transcripts that are either leaky from transcriptional control or that must be expressed at low levels.

    * A miRNA can be regulated by a signalling pathway and, in turn, target a component of another pathway: in doing so, the miRNA can serve as a mediator of crosstalk between signalling pathways, coordinating their activity.

    * miRNAs are part of signalling networks. By buffering environmental and genetic fluctuations, they confer robustness to the cellular response to extracellular signals.

    * Signalling pathways can control the biogenesis of specific miRNAs at the level of processing. Signalling mediators have been shown to be part of miRNA-processing complexes, tuning miRNA maturation.

The first glimpse into the new world of small RNAs came with seminal papers from Ambros, Ruvkun and colleagues 1, 2, 3: they reported that lin-4 and let-7, the first microRNA (miRNA) genes identified, control developmental timing in nematodes by modulating the expression of other genes at the post-transcriptional level. Since then, the miRNA field has grown tremendously, becoming an integral component of the way we think gene expression is regulated. We now know that miRNAs are a universal and pervasive feature of animal and plant genomes; current estimates suggest that the human genome contains at least hundreds of distinct miRNAs, which potentially regulate a large fraction of the transcriptome 4, 5.

miRNAs are a class of 20–25 nucleotide-long non-coding RNAs that modulate gene expression through canonical base pairing between the seed sequence of the miRNA (nucleotides 2–8 at its 5' end) and its complementary seed match sequence (which is present in the 3' UTR of target mRNAs) 4. miRNAs have a peculiar biogenesis (Box 1): they are first transcribed as part of longer precursors (primary transcript; pri-miRNA) that fold on themselves to form hairpin structures. pri-miRNAs are then processed in the nucleus by the Drosha complex and transported to the cytoplasm by exportin 5, where they undergo final processing by the Dicer ribonuclease 6. Mature miRNAs are incorporated in the RNA-induced silencing complex (RISC) by associating with Argonaute proteins. Within the RISC, the single-stranded miRNA is unwound by the helicase activity of Dicer and guides target selection, causing inhibition of translation, stability and localization of target mRNA 6, 7, 8 (Box 1).

Box 1 | RNA biogenesis and mechanisms of action:

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II. Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex, which consists of the RNase III type endonuclease Drosha and its partner, DGCR8 (see the figure). The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA), which is actively exported to the cytoplasm by exportin 5.

In the cytoplasm, the pre-miRNA is recognized by Dicer, another RNase III type endonuclease, and TAR RNA-binding protein (TRBP; also known as TARBP2). Dicer cleaves this precursor, generating a 20-nucleotide mature miRNA duplex. Generally, only one strand is selected as the biologically active mature miRNA and the other strand is degraded. The mature miRNA is loaded into the RNA-induced silencing complex (RISC), which contains Argonaute (Ago) proteins and the single-stranded miRNA. Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target. In particular, perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3' UTR are crucial. The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations: removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4–NOT), followed by mRNA degradation; and blockade of translation at the initiation step or at the elongation step; for example, by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments, known as P-bodies, where they are reversibly stored or degraded.

Figure is modified, with permission, from Ref. 104 Nature Reviews Genetics © 2008 Macmillan Publishers Ltd. All rights reserved. m7G, 7-methylguanosine cap; ORF, open reading frame.

MicroRNA control of signal transduction

Despite great advances, the miRNA world remains largely uncharted with regard to the physiological function of these molecules within cells and organisms, and uncovering the function of individual miRNAs is challenging. First, miRNAs are frequently present as families of redundant genes, which complicates genetic dissections. Second, each miRNA has numerous putative targets that have disparate functions, with no means to decide a priori which one is most meaningful and thus worthy of experimental validation. Third, the degree of target downregulation imposed by miRNAs often tends to be quantitatively modest: measured at the protein level, even an overexpressed miRNA typically downregulates most of its endogenous targets by less than 50% (Ref. 9). Therefore, most proteins should remain effective over this degree of inhibition, an argument supported by the paucity of haploinsufficient phenotypes that have been described so far. Conversely, few genes are known to have phenotypes when duplicated, perhaps mimicking the situation when miRNA-mediated regulation of a target is lost 10. These considerations suggest that even though most genes are predicted to be miRNA targets, only a fraction of these interactions will prove instrumental for overt biological responses and phenotypes4, 10, 11, 12, 13, 14, 15, 16. Remarkably, however, inhibition of miRNA biogenesis (for example, through the ablation of Dicer) clearly reveals that miRNAs are essential for a wide array of biological processes, including control of proliferative homeostasis, differentiation or embryonic stemness17, 18, 19.

As a resolution to this conundrum, rather than querying miRNA–target pairs to predict miRNA biological functions, the reverse question — asking which biological processes might be prime candidates for miRNA-mediated regulation — might be more productive. In this Review, we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells. Signalling complexes are indeed highly dynamic, ephemeral and non-stoichiometric molecular ensembles, which translate into well-established dose-dependent responses. As such, they are the ideal targets for the degree of quantitative fluctuations imposed by miRNAs. This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape, facilitating or opposing the transmission of information to downstream effectors in an effective and timely manner 20. Table 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways. In the first part of this Review, we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling.

Table 1 | microRNAs targeting signalling pathway components*

Abbreviations:

ACVR2A, activin receptor type 2A;
Brd, Bearded;
C. elegans, Caenorhabditis elegans;
D. melanogaster, Drosophila melanogaster;
D. rerio, Dario rerio;
E(spl), enhancer of split;
GFP, green fluorescent protein;
GOF, gain of function;
H. sapiens, Homo sapiens;
ISH, in situ hybridization;
KRAS, Kirsten rat sarcoma RAS;
lef1, lymphoid enhancer-binding factor 1;
lATS, large tumour suppressor;
LOF, loss of function;
miRNA, microRNA;
M. musculus, Mus musculus;
NRAS, neuroblastoma RAS;
Pi3KR2, PI3-kinase regulatory subunit-?;
PTEN, phosphatase and tensin homologue;
spred1, Sprouty-related, EVH1 domain-containing protein 1;
Tcf, t cell factor;
WB, western blot;
X. laevis, Xenopus laevis.

* this is a partial list referring to the examples cited in this Review. See Supplementary information S1 (table) for a more extensive description of miRNAs and corresponding signalling cascades.

‡ Indicates whether the miRNA targets a positive (+) or negative (–) modulator of the signalling pathway.

§GOF indicates miRNA overexpression, LOF indicates miRNA treatment with loss of function reagent.

miRNAs may also help to explain a paradox in evolution. The core protein engines of developmental signalling networks are highly conserved devices, which can be traced back to the common ancestor of all Bilateria 21, 22, 23. However, the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways, demanding the evolution of new layers of regulation. Just like transcription factor binding sites, 3' UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNA–target interactions and counter-select against deleterious pairs 24, 25. Nevertheless, although few new transcription factor families have arisen in animal evolution, continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs 24, 26, 27. Thus, miRNAs may represent ductile and fast-evolving tools, which add sophisticated regulatory tiers to signalling pathways. We discuss the logic of these networks in the second part of this Review. In sum, crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology.

miRNA and signalling: common principles

During embryonic development, a handful of signalling pathways — transforming growth factor-b (TGFb), WNT, Hedgehog, Notch, Hippo, and pathways driven by receptor tyrosine kinases (RTKs) such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptors — precisely coordinate tissue induction, patterning, growth and morphogenesis. The same pathways maintain tissue homeostasis in adults, and their perturbations account disproportionally for human diseases 22, 28, 29, 30, 31, 32. The effectiveness of these signalling pathways relies on their capacity to tightly control the expression of target genes in time and space. Two common principles are adopted to achieve this result: context-dependent transcriptional activation and default repression33 (Fig. 1a,b).

Figure 1 | MicroRNAs in context-dependent transcriptional activation and default repression.

Figure 1 : MicroRNAs in context-dependent transcriptional activation and default repression.

a | MicroRNAs (miRNAs) in context-dependent transcriptional activation. Different cell types respond differently to a signal according to their miRNA milieu. In a certain cell type, signalling mediator A (A) activates the transcription of gene 1 in concert with cofactor (C1). However, part of the downstream gene expression programme cannot be expressed in this cell (gene 2), as a cell-specific miRNA (miR-A) repress its corresponding cofactor (C2).

b | Model of transcriptional default repression. In the absence of the signal, the repressor (R) inhibits the activity of the pathway activator. Signalling relieves this inhibition, activating transcription.

c | miRNAs may contribute to default repression. Signalling cues that are too transient or weak to result in full activation may cause leaky transcription of the target gene (left panel). Default repression is restored if a miRNA targeting either the activator or its downstream transcripts is embedded into the system (right panel).

d | The role of miR-125b in the DNA damage response is an example of a miRNA operating as a primary mediator of default repression. In a normal cell (left panel), miR-125b targets residual p53 activity, avoiding apoptosis. Following genotoxic inputs (right panel), p53 is activated and miR-125b is repressed, inducing apoptosis.

e | The role of miR-372 and miR-373 in the default activation of the genes that are activated by the transcription factors Yes-associated protein (YAP) and Tafazzin (TAZ). In cells in which the Hippo signal is off (left panel), transcription of YAP and TAZ target genes is active. Following Hippo signalling (middle panel), large tumour suppressor (LATS) phosphorylates and inhibits YAP and TAZ. This is prevented by high levels of miR-372 and miR-373, which target LATS (right panel).

miRNAs in default repression.

Default repression ensures that target gene expression is turned on exclusively in the presence of signalling but kept actively repressed in its absence. This is primarily attained at the transcriptional level: typically, the same responsive element on a target gene promoterswitches from mediating default repression to signal-dependent activation 33 (Fig. 1b). But is transcriptional control sufficient to explain tight signalling regulation? It would seem unreasonable to assume that in vivo cells will be challenged only by unambiguous on or off situations. Much more frequently, cells will have to distinguish between what is a real signal — one worthy of activating downstream targets — from inputs that are too weak or too transient. In this grey area, miRNAs could be crucial for signal interpretation: by dampening positive mediators of signalling cascades, miRNAs would raise the threshold for pathway activation, restricting it only to appropriate zones of competence (Fig. 1c).

WNT, Notch and Hedgehog are signalling pathways that are under strong default repression. The role of the transcription factor TCF in WNT signalling serves as a paradigm for this type of regulation. In the absence of WNT ligands, the cytosolic pool of b-catenin, which works in conjunction with TCF to activate specific genes, is phosphorylated and targeted for degradation 28. Following WNT signalling, a cascade is initiated that results in the stabilization and translocation of b-catenin to the nucleus, where it forms a transcriptional activating complex with TCF, which outcompetes co-repressors sitting on target genes 28. miR-8, the Drosophila melanogaster orthologue of the vertebrate miR-200 family, has been identified in a gain-of-function screen for negative regulators of Wingless signalling (WNT signalling in vertebrates) 34. In D. melanogaster and mouse cells miR-8 and miR-200c, respectively, contribute to default repression by targeting both TCF and upstream positive modulators of the pathway, including Wntless (also known as evi), which is required for the secretion of WNT ligands 34.

A similar example relates to Hedgehog signalling 29. In mammals, this pathway controls the proliferation of cerebellar granule progenitor cells, and aberrant pathway activity causes medulloblastoma29. Using a miRNA high-throughput profile in human meduloblastomas, miR-324-5p was identified as a suppressor of Hedgehog signalling. miR-324-5p targets the transcription factor GLI1, the mediator of Hedgehog signalling, and its loss upgrades pathway responsiveness, leading to tumour formation 35.

Default repression by miRNAs does not necessarily have to target core pathway components; it may be equally effective when it intercepts their transcriptional targets (Fig. 1c). A classical example of default repression at the level of dowstream targets is the miRNA-mediated regulation of the enhancer of split (E(spl)) and Bearded (Brd) gene clusters, which are downstream effectors of Notch signalling in D. melanogaster. This is a highly redundant system, in which families of related miRNAs (miR-2, miR-4, miR-7, miR-11 and miR-79 ) promiscuously target a family of related mRNAs, preventing aberrant deployment of Notch-mediated developmental programmes 36, 37. Notably, the Notch targets in E(spl) and Brd are among the few studied examples for which specific mutation of miRNA binding sites in a genomic transgene is sufficient to cause mutant phenotypes, indicating that miRNA regulation is essential for normal Notch signalling 38, 39.

miRNAs may themselves be mediators of default repression. For example, during DNA damage, a cascade of kinases activate the p53 tumour suppressor, leading to cell cycle arrest or apoptosis. p53 is normally a latent transcription factor that is inhibited by ubiquitin-mediated degradation 40. The miRNA miR-125b is essential to complete p53 repression by targeting it, and loss of miR-125b causes p53-dependent apoptosis 41 (Fig. 1d). Interestingly, miR-125b is itself part of the DNA damage network, as it is downregulated after genotoxic treatments 41. Thus, by raising the threshold for p53 activation, miR-125b ensures a safe and robust DNA damage response.

miRNAs in default activation.

Although miRNAs repress gene expression, their function is not just repressive. Indeed, their effect on the output of signalling cascades is strictly dependent on pathway topology. For example, in the Hippo tumour suppressor pathway, which controls tissue growth in D. melanogaster and mammals by regulating cell proliferation and apoptosis, active signalling actually leads to the inactivation of the two downstream transcription factors of the pathway, the Yes-associated protein (YAP) and Taffazin (TAZ) proto-oncogenes. Specifically, signalling causes YAP and TAZ phosphorylation, which inhibits their nuclear activities by localizing them to the cytoplasm 22. YAP and TAZ phosphorylation is mediated by the large tumour suppressor (LATS) kinase and its upstream regulator, Expanded. These Hippo pathway components are targeted by two miRNAs: miR-372 and miR-373 in mammals (LATS) and miR-278 in D. melanogaster (Expanded) 42, 43, 44. This results in the absence of YAP and TAZ phosphorylation, nuclear retention and consequently transcriptional activation of YAP and TAZ target genes. Thus, in the case of Hippo, suppression of signalling mediators leads to the transcription of YAP and TAZ target genes, explaining the oncogenic potential of these miRNAs (Fig. 1e). Interestingly, a key transcriptional target of the Hippo pathway in D. melanogaster is the miRNA bantam, which serves as an essential mediator of organismal growth and patterning 45, 46.

miRNAs and context-dependent signalling

The arrival of an extracellular signal does not typically deliver specific instructions. Instead, it is the cell that interprets the signal according to its history and actual environment, sorting out the repertoire of target genes that better suits its needs. The unique miRNA milieu of each cell type is ideally suited to serve in such context-dependent gene expression (Fig. 1a). This would allow great plasticity in biological outputs, and explains the constant recycling of these signalling cascades in different cell types and stages in development and evolution.

A key challenge in biology is explaining how naive cells acquire distinct fates in response to a limited number of signalling cues. An elegant solution to this riddle relies on the cell's ability to perceive extracellular signals quantitatively (that is, by their intensity and duration) and to couple these 'readings' with the activation of distinct gene expression programmes 47. Such dose dependency seems particularly amenable to miRNA regulation. In this section, we provide examples relating to miRNAs as generators of graded responses or as signalling amplifiers.

miRNAs sharpen morphogen gradients in TGFb signalling. The effects of signalling by TGFb superfamily ligands, such as TGFb and bone morphogenetic protein (BMP), are widespread during morphogenesis and adult tissue homeostasis. This is highlighted by a range of hypomorphic and haploinsufficient phenotypes in mutants for TGFb pathway components observed in fly, worm and vertebrate model systems 32. This also suggests that these components must be somehow limiting in vivo and thus could be ideal targets of miRNA regulation.

Nodal is a TGFb superfamily ligand that serves as a potent morphogen during induction of the germ layers and specification of the body axes. Importantly, in early vertebrate embryos, Nodal activity is asymmetric: in Xenopus laevis, the highest Nodal activity is required for the formation of the Spemann's organizer, which defines the embryo's most dorsal and anterior structures 23 (Fig. 2a). Recent work highlighted the role of two miRNAs, miR-15 and miR-16, in this asymmetry. miR-15 and miR-16 are enriched on the ventral side of the embryo, where they attenuate Nodal signalling by targeting the Nodal receptor activin receptor type 2A (ACVR2A) 48 (Fig. 2b,c). miR-15 and miR-16 also provide a first example of integration between distinct signalling pathways. In X. laevis, the development of the body axes can be traced back to the primeval asymmetry; that is, fertilization. Indeed, soon after sperm entry, WNT–b-catenin signalling becomes activated on the future dorsal side of the embryo 21. The WNT–b-catenin asymmetry is translated into a peak of Nodal activity because, at least in part, WNT signalling inhibits miR-15 and miR-16 biogenesis, thus enhancing Nodal–ACVR2A responsiveness on the future dorsal side 48 (Fig. 2c). Interestingly, distinct species have developed distinct miRNA-based strategies to control Nodal signalling: for example, the miR-15 and miR-16–ACVR2A axis is not conserved in zebrafish, which exploit a different miRNA, miR-430, to control the availability of Nodal ligands in the extracellular space 48, 49 (see below).

Figure 2 | MicroRNAs and signalling gradients.

Figure 2 : MicroRNAs and signalling gradients.

a. The conventional view of a morphogenetic signalling gradient, as exemplified by mesoderm induction by Nodal in amphibian embryos. A dorso–ventral gradient of Nodal ligand expression is translated into graded SMAD2 signalling (indicated by arrows), thus inducing mesodermal tissues of different dorso–ventral identities. The highest dose of signalling induces the Spemann's organizer (ORG), the most dorsal structure of the embryo.

b,c | MicroRNAs (miRNAs) can modify the interpretation of signalling gradients.

b | In Xenopus laevis embryos, miR-15 and miR-16 are enriched on the ventral side, where they inhibit the Nodal receptor activin receptor type 2A (ACVR2A) and consequently SMAD2 signalling.

c | The asymmetric expression of miR-15 and miR-16 is regulated by dorsally enriched b-catenin signalling, which reflects the dorsal-to-ventral activity of the Nodal receptor ACVR2A (magenta triangle, lower panel).

d | miRNAs can modify tissue responsiveness over time. let-7 expression increases during differentiation, resulting in progressive inhibition of RAS signalling. This enables self-renewal in progenitor cells but restrains it while cells differentiate.

e | Signalling downstream of receptor tyrosine kinases (RTKs) involves two main branches, namely the phosphoinositide 3-kinase (PI3K)–AKT and the RAF–mitogen-activated protein kinase (MAPK) cascades. Some miRNAs, such as miR-21 and miR-126, target inhibitors of both branches, leading to a general upregulation of RTK signalling. miR-26a specifically upregulates the PI3K–AKT branch by inhibiting phosphatase and tensin homologue (PTEN). PIK3R2, PI3-kinase regulatory subunit-b; SPRY, sprouty-related genes (Sprouty or SPRED1).

Smad proteins are transcription factors that transduce TGFb signals downstream of their receptors, and they can also be targeted by miRNAs. Indeed, within the developing liver, the miR-23b cluster has been shown to target three Smads (SMAD3, SMAD4 and SMAD5), thereby inhibiting the anti-proliferative response mediated by TGFb and fostering hepatocyte proliferation 50. The fact that a single miRNA cluster targets several Smads concomitantly offers an interesting example of how, despite having a weak effect on their own, the simultaneous attack of miRNAs on a common set of regulatory proteins can amplify their effect.

Attenuation of RAS signalling by let-7.

Beyond spatial regulation, miRNAs can modify tissue responsiveness over time. let-7 was the first nematode miRNA for which clear homologues could be identified in diverse metazoan lineages 51. In nematodes, let-7 functions as regulator of the transition from undifferentiated and proliferative stem cells of the late larva to quiescent, differentiated cells of the adult   3. This is strikingly reminiscent of the function of mammalian let-7, which is expressed at low levels by progenitor cells, but at high levels by their differentiated progeny 52. In cancer cells, loss of let-7 seems to be associated with a reverse embryogenesis programme; that is, with the reactivation of genes that positively regulate proliferation and stem cell self-renewal in youth, but the activity of which activity typically declines with age 53. One of the evolutionary conserved targets of let-7 is RAS, and this regulation controls breast cancer cell self-renewal and, likely, the response to chemotherapy (Fig. 2d). Clearly, this explains only part of the complexity of let-7 biology: in the breast cancer example, epithelial de-differentiation occurs through let-7-mediated downregulation of a different target, high mobility group box A2 (HMGA2; also known as HMGIC) 54, 55. By taming proto-oncogenic signalling, let-7 may oppose cancer, but it may also contribute to the decline of normal stem cell function and tissue renewal associated with ageing 56. Recent advances on the mechanisms of let-7 biogenesis by lIN-28, a protein that promotes pluripotency (see below), suggest exciting possibilities for the targeted manipulation of let-7 expression in different contexts.

miRNAs as signalling amplifiers.

Once a miRNA targets an inhibitor of a signalling cascade, it serves as a positive regulator by either amplifying signal strength or duration, or empowering cell responsiveness to otherwise sub-threshold stimuli. The RAS–RAF–mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K)–AKT cascades are two pleoitropic pathways that branch from activated RTKs 31. The characteristic trait of these pathways is potent signal amplification; this is achieved by numerous kinases acting sequentially and activating one another, as well as by second messengers, such as phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃) 31, 57. The intrinsic risk of this design is mounting a potential chain reaction that leads to senescence or cancer initiation. Negative regulators are in place to prevent this risk; these include phosphatase and tensin homologue (PTEN; which reverses PI3K-mediated phosphorylation of PtdIns(3,4,5)P₃ (Ref. 58)), PI3-kinase regulatory subunit-b (PIK3R2; also known as p85b) (which inhibits PI3K through many mechanisms 59) and Sprouty-related, EVH1 domain-containing protein 1 (SPRED1) or Sprouty (two related antagonists of RAS-mediated RAF activation 60). Several miRNAs have been shown to target both positive and negative regulators of these cascades (Table 1; Supplementary information S1 (table)). Here, we highlight two miRNAs, miR-21 and miR-126, as examples of miRNAs that amplify signals through the coordinated and coherent regulation of multiple targets 61, 62, 63, 64.

miR-126 is the most highly enriched miRNA in endothelial cells, where it sustains VEGF signalling by targeting SPRED1 and PIK3R2 mRNAs (Fig. 2e). By inhibiting the production of natural repressors of VEGF signalling, miR-126 promotes angiogenesis and vascular integrity 61, 62, suggesting that it may serve as an effective target for anti-angiogenic therapies.

miR-21 targets both PTEN and Sprouty 63, 64. Thus, miR-21 serves as a general enhancer of RTK signalling, perhaps explaining its frequent upregulation in various human tumours 65. Interestingly, increased levels of miR-21 not only characterize cancer cells but are also present in other pathological growths. In cardiac fibrosis, miR-21 is highly expressed in proliferating cardiac fibroblasts. Fibrosis has been traditionally considered to be a secondary consequence of failing cardiomyocytes. Strikingly, however, RTK signalling attenuation in cardiac fibroblasts following silencing of miR-21 led to significant attenuation of heart disease, suggesting that fibroblasts may have a more direct and causal role in cardiac hypertrophy and dysfunction than previously thought 64 (Fig. 2e).

In the above examples, a miRNA simultaneously targets distinct RTK signalling branches, thus acting as a general amplifier of the signal response. In other cases, miRNAs can impart specificity to the signalling flow by channelling it towards specific branches. miR-26a, for example, targets only PTEN, thus specifically amplifying AKT-driven gliomagenesis following PDGF stimulation 66 (Fig. 2e).

miRNAs in signalling crosstalk

The regulatory capacity of miRNAs may be exploited to connect distinct signalling pathways (Fig. 3). We mentioned above the example of miR-15 and miR-16, linking WNT and TGFb signalling 48. Furthermore, recent evidence suggests that there are several miRNAs that can act as mediators of signalling crosstalk. One particular example is how TGFb signals through AKT during renal fibrosis 67 (Fig. 3a); this link is assured by a cascade of miRNAs. Specifically, TGFb turns on the transcription of miR-192, which inhibits the expression of zinc finger E-box-binding homeobox 2 (ZEB2). Because ZEB2 is a transcriptional repressor of miR-216a and miR-217, its inhibition allows the derepression of miR-216a and miR-217, leading to PTEN inhibition 67 (Fig. 3a). The ensuing AKT activation causes glomerular mesangial cell survival and hypertrophy in diabetic mouse models. Thus, antagonizing either miR-192 or miR-216a and miR-217 could offer new therapeutic potentials for diabetic nephropathy.

Figure 3 | MicroRNAs in signalling crosstalk and coordination.

Figure 3 : MicroRNAs in signalling crosstalk and coordination.

a | microRNAs (miRNAs) can serve as mediators of crosstalk between signalling pathways. On the left, signal A induces the expression of a miRNA to negatively regulate signal B. On the right is a different example, with miRNAs enabling positive crosstalk between transforming growth factor-b (TGFb) and AKT signalling. In glomerular mesangial cells, TGFb induces the expression of miR-192, which represses the transcription factor zinc finger E-box-binding homeobox 2 (ZEB2). This results in the derepression of miR-216a and miR-217, enabling them to inhibit phosphatase and tensin homologue (PTEN), which leads to enhanced AKT activation. In these cells, this pathway triggers cell survival, extracellular matrix deposition and hypertrophy, all classic features of diabetic nephropathy.

b | miRNAs as signalling coordinators. A single miRNA can act simultaneously on two signalling pathways to coordinate their biological effects in a tissue or cell (upper diagram), as exemplified by miR-203-mediated regulation of skin tissue homeostasis (lower diagram). By antagonizing both WNT signalling (at the level of the transcriptional cofactor lymphoid enhancer-binding factor 1 (LEF1)) and p63 activity, miR-203 may have a general role in skin regeneration and self-renewal.

Similarly to embryogenesis, adult tissue regeneration and wound repair also require the strict, coordinated and dynamic control of various signalling pathways (Fig. 3b). Not surprisingly, miRNAs are emerging as key micromanagers of some of these processes. For example, the skin constantly rejuvenates during homeostasis through the self-renewing capacity of the innermost basal layer. As cells move suprabasally, they embark on a terminal differentiation programme 68. Integral to this switch is the downregulation of the p53-related factor p63, which is essential for epithelial stem cell maintenance 69. miR-203 sharpens the border between basal progenitors and their differentiating progeny because it targets residual p63 expression in suprabasal layers 19. Skin appendages, such as hair follicles or fins, can also regenerate through WNT stimulation 68. Remarkably, another conserved target of miR-203 is lymphoid enhancer-binding factor 1 (LEF1), a b-catenin DNA-binding partner. Although the role of miR-203 in hair follicle regeneration has not been investigated, miR-203 inhibits Lef1-mediated fin regeneration in zebrafish 70 (Fig. 3b). It is therefore tempting to speculate that inhibition of miR-203 may sustain the regenerative capacities of the whole epidermis.

miRNAs confer signalling robustness

Robustness is the capacity of biological systems to generate an invariable phenotype, even when facing genetic or environmental perturbations, and miRNAs have been proposed to contribute to robustness by several mechanisms.

miRNAs as signalling balancers and buffers.

In a simple scenario, miRNAs can target both an activator and an inhibitor of the same pathway. A case in point is the regulation of the Nodal pathway by miR-430 in zebrafish, which targets both the Nodal homologue Squint and its inhibitor, Lefty 49. In this scenario, after lowering miR-430 levels, any gain of Nodal is balanced by a concomitant increase of its antagonist, without causing marked consequences. But what is then the function of miR-430 in the Nodal pathway? Answering this question required the disruption of specific miRNA–target pairs using target protectors — that is, antisense morpholino oligonucleotidespaired with the miR-430-binding sites of the 3' UTR of either Nodal or Lefty, which prevent endogenous miR-430 from binding and regulating their translation. Under these conditions, absence of miR-430 sensitizes embryos to minor fluctuations of Nodal ligand. Thus, some miRNAs target agonist–antagonist pairs to reduce their absolute levels (dampening effect), to regulate their relative levels to achieve optimal signalling efficacy (balancing effect) and to limit undesired signalling fluctuations (buffering effect) 49.

miRNAs in signalling networks.

Signalling pathways are highly interconnected, and the flow of information they carry is controlled by many feedback loops. This renders their appearance and functionality more similar to a network rather than to a linear cascade.

Theoretical models predict that miRNAs are crucial elements of these loops 20, 36, 71, 72, 73. First, miRNAs can act as reinforcers and backups of tissue-specific transcription programmes 20, 36. This defines a coherent feedback loop in which the miRNA and its target are oppositely regulated by the same signal (Fig. 4a). This suggests that a miRNA participates in signalling networks to stabilize fine tissue patterning by repressing its target mRNA in cells where it should be not expressed (that is, 'leaky' mRNAs). Pioneering work in the embryo of D. melanogaster provided some vivid examples of this regulation, with a miRNA and its target being detected in mutually exclusive domains of the embryo 74. However, the concept of coherent regulation can equally apply to less extreme situations; that is, those in which the miRNA and its target are also co-expressed 75. Thus, irrespective of the relative ratio between a miRNA and its target, in coherent regulation the miRNA acts in concert with patterning signals for better control over gene expression.

Figure 4 | MicroRNAs in networks and loops.

Figure 4 : MicroRNAs in networks and loops.

a | MicroRNAs (miRNAs) in coherent feed-forward loops. In the absence of signal, the expression of a target (T) is kept repressed by a miRNA. Following signalling activation, the target is activated directly, but also through inhibition of the miRNA, resulting in coherent regulation.

b | miRNAs in incoherent feed-forward loops. In this case the signal directly activates the target, but at the same time promotes its inhibition by the miRNA, resulting in two opposing regulations. This example applies to the regulation of Lefty by OCT4 and miR-290–miR-295 in mouse embryonic stem cells.

c | miRNAs in bistable fate choices. In a cell, a transcription factor (TF) inhibits the expression of a miRNA; in turn, the miRNA inhibits expression of the TF. The cell can adopt two alternative fates: state A is the default fate, in which miRNA expression dominates and the expression of the TF is turned off. In state B, an extrinsic cue (signal) stabilizes the TF, promoting cell differentiation and leading to repression of the miRNA to stabilize the fate choice (upper panel). Reciprocal inhibition between the transcription factors zinc finger E-box-binding homeobox 1 (ZEB1) and ZEB2 and the miR-200 family regulates the switch between epithelial and mesenchymal states. Transforming growth factor-b (TGFb) signalling induces epithelial to mesenchymal transition (EMT) by stabilizing the expression of ZEB1 and ZEB2 at the expense of miR-200. MET, mesenchymal to epithelial transition.

Although the logic of coherent loops is intuitive, this hardly exhausts the regulatory potential of miRNAs. Not only do evolutionary conserved miRNA–target pairs seem to be co-expressed 72, but genome-wide computational and transcriptomic analyses showed that the expression of miRNAs is more positively than negatively correlated with that of their targets. This extends the functional importance of miRNAs to incoherent network topologies, in which the miRNAs and their target are co-activated (or co-repressed) by the same signalling cues 36 (Fig. 4b). There are two main advantages of such a design. First, it prevents undesired pathway activation from stochastic signalling noise, as only bona fide stimuli can surpass inhibition by the co-expressed miRNA. Second, it may also act homeostatically to maintain steady-state levels of the target protein from unwanted signalling fluctuations, as the miRNA would tune the translation of its target in a direction opposite to that of the signal. Of note, this ensures uniform responsiveness in equivalent groups of cells within a range of signal distribution. For example, pluripotent embryonic stem (ES) cells must tightly control Nodal activity because this pathway defines opposing self-renewal and differentiation fates in a narrow window of concentrations and close temporal succession 76 (M.I. and S.P., unpublished observations). Precise control over Nodal availability is mediated by an incoherent regulatory circuit, in which the pluripotency factors, such as OCT4, turn on the Nodal antagonist Lefty, which remains constantly in check by the concomitant OCT4-mediated activation of the miR-290–miR-295 cluster77 (Fig. 4b). It is important to note that the dynamic kinetic properties of miRNAs are ideally suited to serve in loops that confer robustness, as miRNA processing is faster than protein translation, allowing miRNAs to affect gene expression with a shorter delay than transcriptional repressors 20. Thus, miRNAs can confer cells exquisite temporal and quantitative precision over cell signalling.

This also has implications in cancer, in which numerous oncogenic mutations hit several signal transduction elements. If miRNAs buffer the fluctuations of signal transduction elements, then loss of miRNAs should exacerbate the aberrant activity of signalling molecules. Strikingly, expression profiling experiments show a global downregulation of miRNAs in tumour samples compared with normal tissues 78, 79. Similarly, genetic inhibition of miRNA biogenesis greatly accelerates RAS-induced tumorigenesis 80, 81. As cancer has been effectively portrayed as a Darwinian system that is based on the competition between distinct cellular variants 82, one interesting possibility is that escaping miRNA control may allow the exploitation of more aggressive signalling variants, thereby accelerating cancer progression.

miRNA and targets: reciprocal regulation

Another interesting type of positive feedback module is defined by a miRNA that is negatively regulated by its own target. This double negative configuration is similar to bistable electrical circuits — or toggle switches — and can be used to convert a transient signal into a longer-lasting cellular response: once one of two alternative states is established, the signalling cue that induced the transition is no longer necessary and the status is maintained by itself. Recent studies have reported several examples of miRNAs in toggle switches (Fig. 4c).

miR-200 and EMT: cell memory and epithelial plasticity.

Epithelial to mesenchymal transition (EMT) is a complex gene expression programme that is characterized by loss of cell adhesion through repression of E-cadherin and activation of genes associated with motility, invasion and stemness. As such, EMT is activated during embryonic development and adult tissue remodelling. However, in epithelium-derived tumours, EMT is usurped to foster metastasis and gain of cancer stem cell phenotypes 83. The miR-200 family of miRNAs plays a major part in specifying the epithelial phenotype by preventing the expression of the transcriptional repressors of E-cadherin, ZEB1 and ZEB2 (Ref. 84). In turn, the miR-200 primary transcript is repressed by ZEB1 and ZEB2 (Ref. 85), establishing a double-negative feedback loop between ZEB1 and ZEB2 and the miR-200 family. TGFb signalling, a potent inducer of EMT, activates ZEB1 and ZEB2 (Ref. 85), which then repress miR-200 expression. Once miR-200 levels fall below a threshold, the reprogramming from an epithelial to a mesenchymal phenotype is locked in place(Fig. 4c). The embedded stability of this double-negative feedback loop represents a new form of epigenetic memory. For example, this may explain why, at least in vitro, the stem cell characteristics endowed by a transient EMT stimulation continue to be manifested in the distant descendants of a cell long after the EMT-inducing stimulus has been removed 86.

miR-145, pluripotency and differentiation in ES cells.

miR-145 has a crucial role in the transition between stemness and differentiation through a reciprocal negative feedback loop that includes pluripotency factors. miR-145 is expressed at low levels in pluripotent human ES cells because its promoter is directly repressed by OCT4; in turn, miR-145 targets the 3' UTR of OCT4 and those of the other pluripotency-associated genes, such as SOX2 and Krueppel-like factor 4 (KLF4). BMP signalling promotes the differentiation of human ES cells by inhibiting OCT4 and by sustaining the expression of miR-145, thus ensuring a firm inhibition of pluripotency genes 76, 87.

miR-7 in signalling networks.

An intrinsic risk of double-negative feedback loops is that stochastic fluctuations can fuel loop acceleration, rapidly flipping between alternative cell decisions. However, robustness can be imbued in the system by regulating the miRNA toggle switch downstream of a coherent feed-forward loop. This type of network has been characterized in D. melanogaster eye development 13. In this model, miR-7 is downstream of EGF receptor signalling, a pathway that is essential for the differentiation of progenitor cells into photoreceptors. As shown in Fig. 5, miR-7 and its target Yan (also known as pokkuri) are locked in a reciprocal negative feedback loop that keeps Yan expressed in progenitor cells and miR-7 activated as cells begin differentiating into photoreceptors. EGF signalling breaks tissue homeostasis by inciting a coherent feed-forward loop: it upregulates miR-7 and promotes transient Yan degradation, relieving miR-7 from repression. This two-tiered network embodies most of the sophisticated functions of miRNAs in signalling: stable cellular responses through the reciprocal inhibitory loop and noise buffering effects, as signalling must accumulate miR-7 above a threshold to induce stable changes in gene expression. Interestingly, robustness in gene expression attained through the coherent loop downstream of EGF could be visualized only after challenging miR-7-deficient flies with environmental perturbations, such as heat shock 13.

Figure 5 | MicroRNAs in signalling networks.

Figure 5 : MicroRNAs in signalling networks.

a | In the Drosophila melanogaster eye imaginal disc, the transcription factor Yan is expressed by the progenitor cell, where it represses the microRNA (miRNA) miR-7, the target of which is Yan.

b | During differentiation, epidermal growth factor (EGF) signalling is transiently induced, which initiates a coherent feed-forward loop that is mediated by the transcription factor ETS-like protein pointed, isoform P1 (PNTP1): PNTP1 directly inhibits Yan expression and also induces miR-7 expression.

c | Once the photoreceptor cell is terminally differentiated, the PNTP1 loop is maintained and ensures that Yan expression will remain inhibited even in the presence of fluctuations in the levels of miR-7 or PNTP1. This definitively precludes the possibility that the toggle switch will be destabilized into switching on Yan expression.

Signalling regulation of miRNA processing

A major gap in our knowledge on miRNAs relates to the mechanisms of their expression. Clearly, the identification of the genetic and epigenetic elements responsible for this event is essential to dissect the role of miRNAs in signalling networks. Most miRNA genes are located at intergenic regions, suggesting that they are derived from independent transcriptional units that are regulated by RNA polymerase II or, to a lesser extent, RNA polymerase III. Other miRNAs (about 25–30%) are embedded within the introns of known coding genes and might be regulated by the promoter of their host gene. Intriguingly, chromatin immunoprecipitation studies have shown that the body of mature miRNA genomic coding regions is unusually occupied by nucleosomes 88. The functional importance of this repressive epigenetic mark is unclear, but it may serve as a code to assemble factors involved in processing the miRNA precursors, as suggested by the fact that cleavage of primary miRNAs occurs co-transcriptionally 89. Indeed, miRNA maturation, rather than expression, might be the key regulatory step in miRNA generation: during mouse embryogenesis many miRNA precursors are present at high levels but remain unprocessed 90, but it is still unknown how processing by Drosha and Dicer is actually regulated. A similar scenario is observed in cancer, in which most miRNAs are effectively downregulated irrespective of their genomic organization 78.

Some of the connections between signalling networks and miRNA biogenesis are starting to be revealed at the molecular level. The best-understood example for the regulation of miRNA biogenesis is let-7 miRNA. The levels of mature let-7 increase during differentiation, but this is not caused by an increase in its transcription rate 91. Instead, LIN-28 negatively regulates the processing of let-7 by recognizing the terminal loop of the let-7 pri-miRNA and pre-miRNA and blocking its cleavage by Drosha and Dicer, respectively. Following the binding to pre-let-7, lIN-28 induces 3' terminal uridylation and subsequent degradation of let-7 by recruiting a terminal ribonucleotransferase or poly(U) polymerase, such as terminal uridylyltransferase 4 (Ref. 92). It is unknown whether other RNA-binding proteins use a mechanism that is similar to that used by lIN-28 to regulate processing of specific miRNAs. Nevertheless, the evolutionary conservation of the terminal loop in many miRNAs at least suggests a widespread regulatory role of this sequence.

During DNA damage, p53 binds to the Drosha complex and promotes the post-transcriptional maturation of many miRNAs to pre-miRNA 93. Among the miRNAs that depend on p53 are several putative tumour suppressors, so this could be a new mechanism by which DNA damage induces cell cycle arrest. Mutations in p53 are frequent in cancer; these disable DNA recognition but lead to the gain of metastatic properties in response to signalling cues such as those transmitted by TGFb 94. Intriguingly, mutant p53 also interferes with the formation of the Drosha processing complex, attenuating miRNA biogenesis and thus probably contributing to tumour progression 93.

Another example of post-transcriptional regulation of miRNA relates to TGF? and BMP signalling and the Smad-dependent processing of pri-miR-21 (Ref. 95). Smads promote a rapid increase in the expression of mature miR-21 by associating with the Drosha complex. As a consequence, miR-21 mediates the TGFb-induced differentiation of vascular smooth muscle cells into contractile cells 95. Because both p53 and Smads interact with p68 DEAD box RNA helicase (DDX5) of the Drosha complex, a housekeeping factor that is required for processing numerous miRNAs, it is unclear how transcription factors (such as Smads and p53) could control the biogenesis of a limited, and yet diverse set of miRNAs. It is possible that specificity emerges from the recognition of specific Drosha–pri-miRNA complexes; for example, it will be important to determine whether p53 or Smads directly recognize consensus sites in the RNA duplex that are similar to their cognate DNA responsive element.

Conclusions and future challenges:

With the identification of a vast number of miRNAs, each one carrying a long list of putative targets, the challenge is now to understand their biological function. This challenge is further complicated by the apparent subtlety of the effects of the miRNA–target interaction on gene activity. However, in reviewing the emerging role of miRNAs in signal transduction, it becomes apparent how the highly dose-sensitive nature of developmental signalling pathways renders them prime candidates for miRNA regulation.

A future challenge will be to identify systematically all the miRNAs affecting, and regulated by, cell signalling. Although we are far from this goal, the experimental tools are definitively in place, including the capacity to screen for miRNAs that contribute to discrete signalling events using unambiguous and pathway-specific readouts in cultured cells or other model systems 48, 96. Importantly, we can expect that the use of new loss-of-function reagents, such as antagomirs and locked nucleic acids (LNA), will greatly accelerate the discovery of endogenous miRNA functions 97, 98.

As developmental signalling pathways are disproportionally relevant in human diseases in general, and in cancer in particular, relevant hints to decipher miRNA function will emerge from the identification of miRNAs that are consistently dysregulated in various types of tumour. This should pinpoint the miRNAs that are selected for their oncogenic function or the miRNAs that are downstream effectors of aberrantly dysregulated pathways of human cancers 99. In any case, merging activity-based or expression-based screens with new RNA-based therapeutics may offer opportunities for a signalling therapy for cancer. Instead of focusing on protein-coded oncogenes, which are difficult to target therapeutically, one could focus on their target miRNAs. If these are causal for the malignant phenotype, then anti-miRNA therapeutics could represent readily available smart drugs that can potentially inhibit tumour growth or the metastatic burden of a given tumour.

Another clear indication from current studies is that miRNAs participate in signalling networks, both as backups of transcriptional control and as feed-forward or feedback devices that confer robustness to the output of cell signalling. Thus, the effect of a miRNA can be the result of the net effect of opposing regulations or of the activities against mutually inhibiting factors. Therefore, new miRNA functions may be revealed as we refine our understanding of the networks in which they operate. As outlined in this Review, miRNAs and their targets can be wired with upstream signalling cues in such a way that perturbations in network components can be buffered or tolerated by the system. This fascinating link with biological robustness also carries with it numerous questions. If the main function of miRNAs is to serve robustness, then one might expect that the network, reciprocally, may absorb miRNA perturbations without overt or immediate consequences. In so doing, however, the network may lose its robustness and become more sensitive to genetic or environmental fluctuations. Now the challenge will be to experimentally identify these robustness loops. For example, simple inactivation of a miRNA may not suffice, but dual inhibition together with another transducer may cause the collapse of the entire signalling network, with dramatic phenotypic effects. Alternatively, miRNA functions may not be revealed under uniform laboratory conditions but may need more sophisticated experimental assays that take into account the cell's own complexity and the cell's interactions within tissues or organisms 4, 13, 100.

Little is known about the subcellular localization, turnover and dynamics of many of the macromolecular complexes carrying out miRNA functions; so, an emerging issue is how miRNA processing and activity crosstalk with signalling at the cell biology level. For example, a link has been established recently between RNA silencing and endosomal trafficking, with RISC assembly and turnover occurring at multivesicular bodies. This is intriguing because signalling and endocytic pathways are intimately intertwined: by regulating endosomal trafficking, signalling cues may tune RISC function 101, 102. Conversely, miRNAs might be secreted by exosomes for non-cell autonomous regulatory purposes  103.

Finally, facing the potential complexity of the miRNA–signalling network relationship, it is difficult to escape the prediction that any reductionist approach will greatly benefit from the guidance of quantitative mathematical modelling. The involvement of miRNAs in feed-forward and feedback motifs makes miRNAs ideal reagents in the hands of systems biologists to offer insights into the physical properties of signalling pathways, something that could not be reached by intuition alone. Curiously, aspects of miRNA function can already be perceived in virtue of their analogy with human engineering devices, as in the case of electronic switches. In turn, it may not be long before we might wish to look at the cellular miRNA and signalling framework to borrow new operational principles for the management of complex systems. This understanding may have applications in so far enigmatic, and necessarily holistic, aspects of tissue biology, such as regeneration, self-assembly and homeostasis and, perhaps, even on computing and engineering in general.

Links:

DATABASES

    * miRNA database
    *

      bantam
    * let-7
    * miR-7
    * miR-8
    * miR-21
    * miR-26a
    * miR-126
    * miR-145
    * miR-192
    * miR-203
    * miR-217
    * miR-278
    * miR-290
    * miR-430

FURTHER INFORMATION

* Stefano Piccolo's homepage

Acknowledgements:

We thank the members of the S.P. laboratory for thoughtful insights. We apologize to those whose work could not be cited owing to space limitations. We thank O. Wessely for comments and insights on the manuscript. Our miRNA work is supported by grants from Associazione Italiana Ricerca sul Cancro (AIRC), Comitato Promotere Telethon, Cariparo Foundation (excellence grant), University of Padua (strategic grant) to S.P., by the Ministery of Health (Giovani Ricercatori) to G.M. and by a Uehara Foundation and Marie Curie International fellowship to M.I.

Competing interests statement:
The authors declare no competing financial interests.

Supplementary information:
Supplementary information accompanies this paper. See also:

http://www.nature.com/nrm/journal/v11/n4/suppinfo/nrm2868.html

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   1. Department of Histology, Microbiology and Medical Biotechnologies, Section of Histology and Embryology, University of Padua, viale Colombo 3, 35100 Padua, Italy.

Correspondence to: Stefano Piccolo ¹ Email: piccolo@civ.bio.unipd.it

Published online 10 March 2010

http://www.nature.com/nrm/journal/v11/n4/suppinfo/nrm2868.html

Abbreviations are TF: Transcription factor, GOF: Gain of function, LOF: Loss of function, WB: Western blot, luc: Luciferase reporter assay, IF: Immunofluorescence, ISH: In situ hybridization
*Indicates whether the miRNA targets a positive or negative modulator of the signalling pathway.
Note that some reference which also appear in main-text are referred with different number.

Supplemental References:

Signaling    Target gene Effect* miRNA Species   Validation assays used  Biological processes   Ref.

Abbreviations are TF: Transcription factor, GOF: Gain of function, LOF: Loss of function, WB: Western blot, luc: Luciferase reporter assay, IF: Immunofluorescence, ISH: In situ hybridization

*Indicates whether the miRNA targets a positive or negative modulator of the signalling pathway.

Note that some reference which also appear in main-text are referred with different number.

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In this complete and incisive review, Masafumi Inui, Graziano Martello, and Stefano Piccolo have focused on the role that microRNAs might play in the control of signal transduction. Such gene network signals usually impinge on those gene clusters that are mediating transcription for new RNA or replication for new DNA. These gene loci are a strategic destination for signal molecules in which to exert their continuous modulating effects. They are also a strategic destination for microRNAs in which to exert their continuous modulating effects. The 3D mapping of gene transcription envisions a continuous competition between ligands to DNA or ligands to other transcription  factors, allowing almost instantaneous responses in gene transcription and gene replication. All of these ligands ( including signals and microRNAs ) may be competing by means of doses, affinities, and survival times to influence response rates at particular gene sites within particular gene clusters. In addition, distant active genes on other chromosomes may also influence transcription rates by direct apposition within kissing chromosome complexes. Many of these important ligand interactions occur within hydrophobic microenvironments.

3D Mapping of Competing Ligand Factors during Single Gene Transcription.

3D Mapping of Competing Ligand Factors during Single Gene Transcription.

Several layers of transcription factors are present during the selection and initiation phases of individual gene transcription. The DNA coding sequence within open-looped and/or paired euchromatin 10 nm microfibrils is exposed to a growing cluster of primary ligands to DNA . Primary ligands may encounter counter-ligands in their immediate vicinity, thus forming ligand-counter ligand complexes.

Hydrophobic ligands are bound preferably to single-stranded portions of DNA and/or RNA.

1. The first ligand layer consists of small RNA molecules, synthesized in  upstream or downstream cis- or trans- enhancer loci. Such enhancer RNAs find their preferred binding sites in complementary RNA molecules within the promoter regions of the selected gene.

2. The second layer finds the sense-DNA template already covered by the first layer of ligands. This second layer is also antagonized by intruding antisense-DNA kiss sequences on the same or other chromosomes within the cell. As a consequence, second layer ligands, whether they are  histones, RNAs, proteins, lipids, hormones, or drug molecules, may not gain or retain direct contact with the sense-DNA template during active gene transcription.

RNA Polymerase II and other necessary enzymes are recruited between the layers into the growing transcription clusters early in the initiation stage.

3, 4, and 5. The third, fourth and fifth layers include sequences for binding to other euchromatin, heterochromatin, and nuclear membrane sites, respectively.

Frenster JH, and Hovsepian JA,
"Models of successive levels of resolution during individual gene transcription".

1. Li L, Feng T, Lian Y, Zhang G, Garen A, and Song X, (2009).
"Role of human noncoding RNAs in the control of tumorigenesis".

2. Kim HH, Kuwano Y, Srikantan S, Lee EK, Martindale JL, and Gorospe M, (2009).
"HuR recruits let-7/RISC to repress c-Myc expression".

3.  Schoenfelder S, Sexton T, Chakalova L, Cope NF, Horton A, Andrews S, Kurukuti S, Mitchell JA, Umlauf D, Dimitrova DS, Eskiw CH, Luo Y, Wei C-L, Ruan Y, Bieker JJ, and Fraser P,
"Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells."

4. Junier I, Martin O, and  Képès F, (2010).
"Spatial and Topological Organization of DNA Chains Induced by Gene Co-localization".

5. Zhang H, Li Y, and Lai M,
"The microRNA network and tumor metastasis".

6. Frenster JH, and Hovsepian JA,
"Analysis of Intra-Nuclear Entropy Changes during EMT Activation".

7. Frenster JH, and Hovsepian JA,
"Models of successive levels of resolution during individual gene transcription".

1. Each cell retains all of its embryonic genes for a lifetime.

2. Controls for embryonic genes are often absent in adults.

3. Uncontrolled embryonic genes can replicate wildly.

4.  Replicating genes participate in  intra-cellular competition.

5.  The basis for gene competition is selective transcription.

6.  MicroRNAs can reprogram embryomic transcription.

7.  Gene reprogramming can produce normal phenotypes.

8.  Normal phenotypes can by-pass chromosomal lesions.

9.  MicroRNA therapy may need to be permanent.

10. Transplantation of microRNAs could be preferred.

http://www.embryomas.net/

1. Pathways within cell genomes involve a flow of information.

2. Information can flow by direct contact or by third parties.

3. Direct contact within whole genomes is difficult to regulate.

4. DNA-DNA direct contects are influenced by agents.

5. Nuclear agents include hydrophilic ionic and hydrophobic conforming ligands.

6. Third parties within genomes involve RNAs and proteins.

7.  RNAs and proteins are easy to regulate or reverse.

8.  Information can be shared, lost, or transformed.

9. System information can be hidden during system isolation.

10.  Local information can be permanently lost during system entropy.

http://www.embryomas.net/

Links to Current Research in Euchromatin:
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 RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
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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