InteractiveFly: GeneBrief

mir-7 stem loop: Biological Overview | References

BIOLOGICAL OVERVIEW

The 40,000 neurons of the medulla, the largest visual processing center of the Drosophila brain, derive from a sheet of neuroepithelial cells. During larval development, a wave of differentiation sweeps across the neuroepithelium, converting neuroepithelial cells into neuroblasts that sequentially express transcription factors specifying different neuronal cell fates. The switch from neuroepithelial cells to neuroblasts is controlled by a complex gene regulatory network and is marked by the expression of the proneural gene l'sc. This study discovered that microRNA miR-7 is expressed at the transition between neuroepithelial cells and neuroblasts. miR-7 promotes neuroepithelial cell-to-neuroblast transition by targeting downstream Notch effectors to limit Notch signaling. miR-7 acts as a buffer to ensure that a precise and stereotypical pattern of transition is maintained, even under conditions of environmental stress, echoing the role that miR-7 plays in the eye imaginal disc. This common mechanism reflects the importance of robust visual system development (Caygill, 2017).

Drosophila vision requires the accurate specification of over 80 different types of optic lobe neurons and the establishment of precise visual circuits between the neurons of the optic lobe and the photoreceptors of the eye. The medulla is the largest visual ganglion of the brain. Medulla neurons play roles in motion detection, through input from the R1-R6 photoreceptors via the lamina, and in the perception of color, via direct input from the R7 and R8 photoreceptors. The 40,000 medulla neurons originate from a pseudostratified neuroepithelium. During early development, symmetric division expands the stem cell pool. As development progresses, the medial edge of the neuroepithelium is progressively converted into asymmetrically dividing neuroblasts. Medulla neuroblasts sequentially express a series of transcription factors that specify the differentiation of the medulla neurons (Caygill, 2017).

The transition from neuroepithelial cells into neuroblasts occurs in a highly ordered, sequential manner in response to expression of the proneural gene, lethal of scute (l'sc). Expression of L'sc marks a two- to three-cell-wide boundary between the neuroepithelial cells and the neuroblasts, the so-called transition zone, which moves medially across the neuroepithelium, forming a proneural wave. Within the transition zone, L'sc transiently suppresses Notch activity, triggering the switch from the symmetric, proliferative division of neuroepithelial cells to the asymmetric, differentiative division of neuroblasts. Progress of the wave is regulated by the orchestrated action of the Notch, epidermal growth factor receptor (EGFR), Fat-Hippo, and JAK/STAT signaling pathways (Caygill, 2017).

This study presents evidence here that the transition from neuroepithelial cells to neuroblasts in the developing optic lobe is buffered by the microRNA miR-7. miR-7 is expressed at the transition zone in response to epidermal growth factor (EGF) signaling and is sufficient to promote transition. miR-7 acts via repression of downstream Notch effectors to limit Notch signaling and promote timely transition. In the absence of miR-7, proneural wave progression is disrupted. This disruption becomes more severe under conditions of temperature stress, suggesting that the role of miR-7 is to act as a buffer to ensure the timely and precise transition from neuroepithelial cells to neuroblasts in the developing optic lobe (Caygill, 2017).

This study has shown that miR-7 targets members of the E(spl) family of bHLH transcription factors to define the boundaries of the transition zone, buffering the transition from neuroepithelial cell to neuroblast in the developing optic lobe. Downregulation of Notch signaling is essential for the neuroepithelial-to-neuroblast transition. L'sc provides one mechanism for Notch downregulation. miR-7, expressed in a pattern similar to that of l'sc at the transition zone, provides another, emphasizing the importance of Notch regulation at the transition zone (Caygill, 2017).

The transition zone of the proneural wave mediates the specification of the neuroblasts of the medulla, the largest ganglion of the adult Drosophila visual system. The medulla receives information directly from the R7 and R8 photoreceptors of the ommatidia. Similar to the specification of neuroblasts by the proneural wave in the optic lobe, photoreceptor differentiation is triggered by the movement of the morphogenetic furrow across the epithelium of the eye imaginal disc. As the furrow passes, expression of the proneural gene atonal (ato) is induced in a stripe that is later refined to the R8 cells by Notch-mediated lateral inhibition (Caygill, 2017).

Similar to the observations in the optic lobe, miR-7 has been shown to play a role in photoreceptor differentiation (Li, 2009). Misexpression of miR-7 results in an increase in Ato expression and R8 cell specification, while a loss of miR-7 results in a decrease in Ato expression under conditions of temperature stress. These results show that miR-7 acts to buffer the development of both the medulla and the eye, two tissues that will directly communicate in the adult brain (Caygill, 2017).

miR-7 has been shown to target anterior open (aop; also known as yan) in the eye imaginal disc (Li, 2005). Within the neuroepithelium, aop activity helps to repress the neuroepithelial-to-neuroblast transition. This raises the possibility that miR-7 targeting of aop could also contribute to its function in buffering the neuroepithelial-to-neuroblast transition and that aop could represent a common target during the progression of the proneural wave and the morphogenetic furrow (Caygill, 2017).

In the adult brain, each ommatidium maps to a columnar unit within the lamina and medulla, providing a retinotopic map of the visual field. Signaling from innervating photoreceptors induces the differentiation of lamina neurons. This direct communication provides a strict control of the mapping of photoreceptor and lamina neuron numbers. In contrast, while final numbers of ommatidia and medulla neurons show some co-ordination based on nutrient availability, there is no evidence for direct communication between the eye disc and the developing medulla. The presence of miR-7 in both the eye imaginal disc and the optic lobe represents an independent but conserved buffer that operates to coordinate appropriate developmental progression in each system, in spite of external environmental fluctuations. The presence of this common buffer provides robustness within each system that may contribute to ensuring the eventual connectivity required for retinotopic mapping of the visual system (Caygill, 2017).

MicroRNA miR-7 contributes to the control of Drosophila wing growth

The control of organ growth is critical for correct animal development. From flies to mammals, the mechanisms regulating growth are conserved and the role of microRNAs in this process is emerging. The conserved miR-7 has been described to control several aspects of development. This study has analyzed the function of miR-7 during Drosophila wing development. Loss of miR-7 function results in a reduction of wing size and produces wing cells that are smaller than wild type cells. It was also found that loss of miR-7 function interferes with the cell cycle by affecting the G1 to S phase transition. Further, evidence is presented that miR-7 is expressed in the wing imaginal discs and that the inactivation of miR-7 increases the expression of Cut and Senseless proteins in wing discs. Finally, these results show that the simultaneous inactivation of miR-7 and either cut, Notch, or dacapo rescues miR-7 loss of function wing size reduction phenotype. The results from this work reveal that miR-7 functions to regulate Drosophila wing growth by controlling cell cycle phasing and cell mass through its regulation of the expression of dacapo and the Notch signaling pathway (Aparicio, 2015).

The function of miR-7 in eye morphogenesis and ovariole development has been analyzed using the miR-7^Δ1 and Df(2R)exu¹ deletions and the genetic allelic combination Df(2R)exu¹/miR-7^Δ1 (Li, 2005; Yu, 2009; Tokusumi, 2011; Huang, 2013). In contrast, the function of miR-7 in wing development has only been studied analyzing the phenotypes of miR-7 over-expression in the wing disc (Stark, 2003; Lai, 2005). These wing studies indicated that miR-7 regulates Notch signaling pathway targets like Enhancer of split to control sensory organ precursor determination. The analysis of the loss of miR-7 function using the Df(2R)exu¹/miR-7^Δ1 allelic combination has not been performed. Furthermore, the use of miR-7 sponges has been uninformative. As the Df(2R)exu¹/miR-7^Δ1 allelic combination, used by this study, might also partially inactivate the function of the bancal gene where miR-7 is located, attempts were made to analyze the contribution of miR-7 to the Df(2R)exu¹/miR-7^Δ1 wing phenotype. This study shows that the over-expression of miR-7 and the constitutive and ubiquitous expression of miR-7 rescue, at least partially, the wing phenotype of Df(2R)exu¹/miR-7^Δ1, thereby demonstrating that lack of miR-7 function contributes to the wing phenotype observed in Df(2R)exu¹/miR-7^Δ1 flies and indicating that miR-7 is involved in the control of wing growth. This is consistent with the findings that miR-7 is expressed in the wing imaginal discs and that the G1 to S cell cycle transition is arrested in Df(2R)exu¹/miR-7^Δ1 wing imaginal discs. Expression of mature miR-7 in the wing imaginal discs has been previously reported (Herranz, 2010). The current results show that in situ miR-7 expression in the wing disc is weak but still significant compared to Df (2R) exu¹/miR-7^Δ1 wing discs. Moreover, miR-7 expression in the 'non-boundary cells' along with the absence of its expression in the 'boundary cells' suggests that miR-7 is involved in the regulation of the factors that control the cell interactions regulating growth at the D/V boundary (Herranz, 2008). Although these results strongly suggest that miR-7 has a function in wing growth control, definitive proof will require either a precise miR-7 deletion or an effective miR-7 sponge (Aparicio, 2015).

Bioinformatic analyses have predicted at least 150 miR-7 putative target genes in Drosophila genome. Among these, only a few have been validated using in vivo experiments. To date, miR-7 has been shown to promote photoreceptor differentiation by regulating the expression of yan (Li, 2005; Li, 2009) as well as to control chordotonal organ differentiation through the control of the Enhancer of split-complex genes (Stark, 2003; Li, 2009). Moreover, miR-7 has been shown to regulate dacapo expression to maintain the GSCs proliferation (Yu, 2009) as well as to regulate bag of marbles expression to control the GSCs testis differentiation (Pek, 2009). The current results show that the Df (2R) exu¹/miR-7^Δ1 wing phenotype is rescued by reducing the levels of expression of the genes Notch, cut, and dacapo. These results indicate that miR-7 functions together with the Notch pathway and with dacapo to control wing growth. Curiously, the shape of the Df (2R) exu¹/miR-7^Δ1 wings appears slightly different from wild type wings perhaps due the Insulin-like receptor signaling control of dacapo expression. Neither the Notch nor cut genes have been predicted to be targets of miR-7. However, effectors of the Notch pathway such as components of the Enhancer of split complex genes have been shown to be regulated by high levels of miR-7 to control chordotonal organ differentiation (Aparicio, 2015).

How could miR-7 be involved in the control of wing growth and the regulation of the Notch pathway? This study has shown that the G1-S cell cycle transition is delayed in miR-7 loss of function mutants perhaps as a consequence of the reduced cell size to compensate for the loss of cell mass. Also, this study showed that miR-7 regulates Notch activity. Considering that Notch has been shown to negatively regulate the G1-S transition, a mechanism consistent with all these results is that miR-7 regulates Notch activity, which in turn regulates cell cycle progression and ultimately wing growth (Aparicio, 2015).

dacapo is an inhibitor of the cell cycle progression that is expressed ubiquitously in the wing imaginal cells and is required for normal wing growth. The dacapo 3'UTR contains functional miR-7 binding sites that are required for the maintenance of GSC division. Interestingly, reducing the activity in the wing imaginal discs of dicer, the central element in miRNA biogenesis, produces small wings whose size is rescued when dacapo levels of expression are decreased. Thus, wing growth control mediated by dacapo depends on microRNAs activity. The current results show that reducing the levels of dacapo expression rescues the small wing phenotype produced by miR-7 loss of function. Therefore, the results strongly suggest that miR-7 regulates dacapo expression to control wing growth and, furthermore, that this control is direct as it has been shown using luciferase assays that miR-7 directly regulates the expression of dacapo (Aparicio, 2015).

In the current model, miR-7 controls the expression of Notch pathway components as well as dacapo to allow cell cycle progression and regulate wing growth. A role of miR-7 in the direct regulation of dacapo as well as the direct or indirect regulation of Notch activity is supported by these results. Additionally, Notch has also been proposed to regulate dacapo expression in the follicle cells and it is not unreasonable to think that this regulation might also take place in the wing to control organ growth. These investigations add a new component, the microRNA miR-7, to the group of factors modulating proliferation/growth. Thus, the study of their interactions may help to resolve the intricate relationship between cell cycle and cell growth. Further analysis will be necessary to study the mechanisms involved in this regulation and the factors that together with miR-7, dacapo, and Notch control both the initiation and the termination of cell growth of the wing imaginal disc (Aparicio, 2015).

Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis

Fine-tuned Notch and Hedgehog signalling pathways via attenuators and dampers have long been recognized as important mechanisms to ensure the proper size and differentiation of many organs and tissues. This notion is further supported by identification of mutations in these pathways in human cancer cells. However, although it is common that the Notch and Hedgehog pathways influence growth and patterning within the same organ through the establishment of organizing regions, the cross-talk between these two pathways and how the distinct organizing activities are integrated during growth is poorly understood. An unbiased genetic screen in the Drosophila melanogaster eye has found that tumour-like growth was provoked by cooperation between the microRNA miR-7 and the Notch pathway. Surprisingly, the molecular basis of this cooperation between miR-7 and Notch converged on the silencing of Hedgehog signalling. In mechanistic terms, miR-7 silenced the interference hedgehog (ihog) Hedgehog receptor, while Notch repressed expression of the brother of ihog (boi) Hedgehog receptor. Tumourigenesis was induced co-operatively following Notch activation and reduced Hedgehog signalling, either via overexpression of the microRNA or through specific down-regulation of ihog, hedgehog, smoothened, or cubitus interruptus or via overexpression of the cubitus interruptus repressor form. Conversely, increasing Hedgehog signalling prevented eye overgrowth induced by the microRNA and Notch pathway. Further, it was shown that blocking Hh signal transduction in clones of cells mutant for smoothened also enhance the organizing activity and growth by Delta-Notch signalling in the wing primordium. Together, these findings uncover a hitherto unsuspected tumour suppressor role for the Hedgehog signalling and reveal an unanticipated cooperative antagonism between two pathways extensively used in growth control and cancer (Da Ros, 2013).

A challenge to understand oncogenesis produced by pleiotropic signalling pathways, such as Notch, Hh, and Wnts, is to unveil the complex cross-talk, cooperation, and antagonism of these signalling pathways in the appropriate contexts. Studies in flies, mice, and in human cell cultures have provided critical insights into the contribution of Notch to tumourigenesis. These studies highlighted that Notch when acting as an oncogene needs additional mutations or genes to initiate tumourigenesis and for tumour progression, identifying several determinants for such co-operation. The identification of these co-operative events has often been knowledge-driven, although unbiased genetic screens also identified known unanticipated tumour-suppressor functions. In this sense, this study describes a conserved microRNA that cooperates with Notch-induced overproliferation and tumour-like overgrowth in the D. melanogaster eye, miR-7. Alterations in microRNAs have been implicated in the initiation or progression of human cancers, although such roles of microRNAs have rarely been demonstrated in vivo. In addition, by identifying and validating functionally relevant targets of miR-7 in tumourigenesis, this study also exposed a hitherto unsuspected tumour suppressor role for the Hh signalling pathway in the context of the oncogenic Notch pathway. Given the conservation of the Notch and Hh pathways, and the recurrent alteration of microRNAs in human cancers, it is speculated that the genetic configuration of miR-7, Notch, and Hh is likely to participate in the development of certain human tumours (Da Ros, 2013).

In human cancer cells, miR-7 has been postulated to have an oncogene or a tumour suppressor functions that may reflect the participation of the microRNA in distinct pathways, due to the regulation of discrete target genes in different cell types, such as Fos, IRS-2, EGFR, Raf-1, CD98, IGFR1, bcl-2, PI3K/AKT, and YY1 in humans (Da Ros, 2013).

In Drosophila, multiple, cell-specific, targets for miR-7 have been previously validated via luciferase or in vivo eGFP-reporter sensors or less extensively via functional studiest. Although microRNAs are thought to regulate multiple target genes, when tested in vivo it is a subset or a given target that predominates in a given cellular context. Indeed, of the 39 predicted miR-7 target genes tested by direct RNAi, only downregulating ihog with several RNAi transgenes (UAS-ihog-IR) fully mimicked the effect of miR-7 overexpression in the transformation of Dl-induced mild overgrowth into severe overgrowth and even tumour-like growth. Moreover, it was confirmed that endogenous ihog is directly silenced by miR-7 and that this silencing involves direct binding of the microRNA to sequences in the 3'UTR of ihog both in vivo and in vitro (Da Ros, 2013).

Nevertheless, other miR-7 target genes may contribute to the cooperation with Dl-Notch pathway along with ihog, such as hairy and Tom. While miR-7 can directly silence hairy in the wing, this effect has been shown to be very modest, and thus, it is considered that while hairy may contribute to such effects, it is unlikely to be instrumental in this tumour model. Indeed, the loss of hairy is inconsequential in eye development, although retinal differentiation is accelerated by genetic mosaicism of loss of hairy and extramacrochaetae that negatively sets the pace of MF progression. It is unclear how Hairy might contribute to Dl-induced tumourigenesis (Da Ros, 2013).

The RNAi against Tom produced overgrowth with the gain of Dl albeit inconsistently and with weak penetrance, where one RNAi line did not modify the Dl-induced overgrowth and the other RNAi line caused tumours in less than 40% of the progeny. Tom is required to counteract the activity of the ubiquitin ligase Neuralized in regulating the Notch extracellular domain, and Dl in the signal emitting cells. These interactions are normally required to activate Notch signalling in the receiving cells through lateral inhibition and cell fate allocation. However, although it remains to be shown whether similar interactions are active during cell proliferation and growth, the moderate enhancement of Dl that is induced when Tom is downregulated by RNAi suggests that miR-7-mediated repression of Tom may contribute to the oncogenic effects of miR-7 in the context of Dl gain of function, along with other targets such as ihog (Da Ros, 2013).

Conversely, while the target genes of the Notch pathway, E(spl)m3 and E(spl)m4 as well as E(spl)mγ, Bob, E(spl)m5, and E(spl)mδ, have been identified as direct targets of miR-7 in the normal wing disc via analysis of 3'UTR sensors, there was no evidence that HLHm3, HLHm4, HLHm5, Bob, and HLHmγ are biological relevant targets of miR-7 in the Dl overexpression context. HLHmδ RNAi produced inconsistent phenotypes in the two RNAi transgenic lines available, causing tumour-like growth at very low frequency in only one of the lines. No evidence was obtained that miR-7 provoked overgrowth by targeting the ETS transcription factor in the EGFR pathway AOP/Yan, a functionally validated target of the microRNA miR-7 during retinal differentiation. Neither was any evidence obtained that RNAi of atonal provoked eye tumours with Dl overexpression, although a strong inhibition via expression of a fusion protein Atonal::EN that converts Atonal into a transcriptional repressor has been shown to be sufficient to trigger tumorigenesis together with Dl. Thus, it was reasoned that given that microRNA influenced target genes only subtly (even when using ectopic expression), it is possible that downregulation of atonal contributes to the phenotype along with the other targets (Da Ros, 2013).

In conclusion, this study has identified cooperation between the microRNA miR-7 and Notch in the D. melanogaster eye and identified and validated ihog as a direct target of the miR-7 in this context and have identified boi as a target of Notch-mediated activity at the DV eye organizer, although it remains whether this regulation is direct or indirect. A hitherto unanticipated tumour suppressor activity was uncovered of the endogenous Hh signalling pathway in the context of gain of Dl-Notch signalling that is also apparent during wing development (Da Ros, 2013).

Hh tumour suppressor role is revealed when components of the Hh pathway were lost in conjunction with a gain of Dl expression in both the eye and wing discs. Hh and Notch establish signalling centres along the AP and DV axes, respectively, of the disc to organize global growth and patterning. Where the organizer domains meet, the Hh and Notch conjoined activities specify the position of the MF in the eye disc and the proximodistal patterning in the wing disc. This study also unvailed that in addition antagonistic interaction between the Hh and Notch signalling might help to ensure correct disc growth. Thus, it was shown that Hh signalling limits the organizing activity of Dl-Notch signalling. Although it is often confounded whether Dl-Notch signalling instructs overgrowth by autonomous or nonautonomous (i.e., DV organizers) mechanisms, these findings uncover that loss of Hh signalling enhances a non-cell autonomous oncogenic role of Dl-Notch pathway (Da Ros, 2013).

To date, Hh has not yet to be perceived as a tumour suppressor, although it is noteworthy that human homologs of ihog, CDO, and BOC were initially identified as tumour suppressors. Importantly, both CDO and BOC are downregulated by RAS oncogenes in transformed cells and their overexpression can inhibit tumour cell growth in vitro. Since human RAS regulates tumourigenesis in the lung by overexpressing miR-7 in an ERK-dependent manner, it is possible that RAS represses CDO and BOC via this microRNA. Indeed, the 3'UTR of both CDO and BOC like Drosophila ihog contains predicted binding sites for miR-7. There is additional clinical and experimental evidence connecting elements of the Hedgehog pathway with tumour-suppression. The function of Growth arrest specific gene 1 (GAS1), a Hh ligand-binding factor, overlaps that of CDO and BOC, while its overexpression inhibits tumour growth . More speculative is the association of some cancer cells with the absence of cilium, a structure absolutely required for Hh signal transduction in vertebrate cells (Da Ros, 2013).

Given the pleiotropic nature of Notch, Wnts, BMP/TGFβ, Ras, and Hh signalling pathways in normal development in vivo, it is speculated that competitive interplay as that described in this study between Notch and Hh may not be uncommon among core growth control and cancer pathways that act within the same cells at the same or different time to exert multiple outputs (such as growth and cell differentiation). Moreover, context-dependent tumour suppressor roles could explain the recurrent, unexplained, identification of somatic mutations in Hh pathway in human cancer samples. Indeed, the current findings stimulate a re-evaluation of the signalling pathways previously considered to be exclusively oncogenic, such as the Hh pathway (Da Ros, 2013).

The microRNA miR-7 regulates Tramtrack69 in a developmental switch in Drosophila follicle cells

Development in multicellular organisms includes both small incremental changes and major switches of cell differentiation and proliferation status. During Drosophila oogenesis, the follicular epithelial cells undergo two major developmental switches that cause global changes in the cell-cycle program. One, the switch from the endoreplication cycle to a gene-amplification phase, during which special genomic regions undergo repeated site-specific replication, is attributed to Notch downregulation, ecdysone signaling activation and upregulation of the zinc-finger protein Tramtrack69 (Ttk69). This study reports that the microRNA miR-7 exerts an additional layer of regulation in this developmental switch by regulating Ttk69 transcripts. miR-7 recognizes the 3' UTR of ttk69 transcripts and regulates Ttk69 expression in a dose-dependent manner. Overexpression of miR-7 effectively blocks the switch from the endocycle to gene amplification through its regulation of ttk69. miR-7 and Ttk69 also coordinate other cell differentiation events, such as vitelline membrane protein expression, that lead to the formation of the mature egg. These studies reveal the important role miR-7 plays in developmental decision-making in association with signal-transduction pathways (Huang, 2013).

Germ line differentiation factor Bag of Marbles is a regulator of hematopoietic progenitor maintenance during Drosophila hematopoiesis

Bag of Marbles (Bam) is a stem cell differentiation factor in the Drosophila germ line. This study demonstrates that Bam has a crucial function in the lymph gland, the tissue that orchestrates the second phase of Drosophila hematopoiesis. In bam mutant larvae, depletion of hematopoietic progenitors is observed, coupled with prodigious production of differentiated hemocytes. Conversely, forced expression of Bam in the lymph gland results in expansion of prohemocytes and substantial reduction of differentiated blood cells. These findings identify Bam as a regulatory protein that promotes blood cell precursor maintenance and prevents hemocyte differentiation during larval hematopoiesis. Cell-specific knockdown of bam function via RNAi expression revealed that Bam activity is required cell-autonomously in hematopoietic progenitors for their maintenance. microRNA-7 (mir-7) mutant lymph glands present with phenotypes identical to those seen in bam-null animals and mutants double-heterozygous for bam and mir-7 reveal that the two cooperate to maintain the hematopoietic progenitor population. By contrast, analysis of yan mutant lymph glands revealed that this transcriptional regulator promotes blood cell differentiation and the loss of prohemocyte maintenance. Expression of Bam or mir-7 in hematopoietic progenitors leads to a reduction of Yan protein. Together, these results demonstrate that Bam and mir-7 antagonize the differentiation-promoting function of Yan to maintain the stem-like hematopoietic progenitor state during hematopoiesis (Tokusumi, 2011).

The findings on Bam, mir-7 and Yan suggest a mechanism for the interaction of these regulators in the control of blood cell homeostasis. The role of Yan is to direct a quiescent hematopoietic progenitor, through a primed intermediate progenitor state, towards a blood cell differentiation fate as crystal cell, plasmatocyte or lamellocyte. The final differentiation status of these cells would be subject to the function of distinct lineage-determining transcription factors. By contrast, Bam and mir-7 cooperate to negatively modulate yan mRNA translation in the quiescent hematopoietic progenitor, thus maintaining the initial prohemocyte state. Although details of this possible translational repression remain to be parsed out, it is likely to include an as yet unidentified protein partner of Bam that would facilitate mir-7 and yan mRNA packaging within an inhibitory RNA-induced silencing complex in prohemocytes (Tokusumi, 2011).

Drosophila maelstrom ensures proper germline stem cell lineage differentiation by repressing microRNA-7

Nuage is a germline-unique perinuclear structure conserved throughout the animal kingdom. Maelstrom (Mael) is an unusual nuage component, as it is also found in the nucleus. Mael contains a High Mobility Group box, known to mediate DNA binding. This study shows that Mael nuclear function is required for proper differentiation in the Drosophila germline stem cell (GSC) lineage. In mael mutant testes, transit-amplifying cysts fail to differentiate into primary spermatocytes, instead breaking down into ectopic GSCs and smaller cysts, due to a depletion of Bag-of-marbles (Bam) protein. Mael regulates Bam via repression of miR-7. Mael binds the miR-7 promoter and is required for the local accumulation of HP1 and H3K9me3. miR-7 targets bam directly at its 3'UTR, and a reduction in miR-7 expression can rescue germline differentiation defects found in mael mutants by alleviating Bam repression. It is proposed that Mael ensures proper differentiation in the GSC lineage by repressing miR-7 (Pek, 2009).

GSC maintenance and division are controlled by the miRNA pathway; however, little is known about how miRNAs regulate the GSC lineage. This study shows that Mael is required for male GSC lineage differentiation by repressing miR-7, thus alleviating the repression of Bam by miR-7. A previous study had shown that Mei-P26 restricts growth and proliferation in the ovarian cysts by inhibiting the miRNA pathway. Mei-P26 interacts with Ago1 and possibly regulates the RISC complex. Together it appears that miRNAs are tightly controlled during different stages of their biogenesis in the GSC lineage, and hence a complex network of miRNA regulation operates in the germline (Pek, 2009).

miRNAs show extensive expression patterns and can be regulated both at the transcriptional and posttranscriptional levels. In the Drosophila eye, miR-7 transcription is regulated by Yan, which is in turn regulated by the EGFR signaling pathway. The current data reveal regulation of miR-7 by Mael in the germline. Mouse Mael has been shown to interact with Sin3B (a component of the histone deacetylase complex) and SNF5 (a SWI/SNF chromatin-remodeling complex), implying that Mael may regulate germline gene expression via an epigenetic mechanism. Indeed, this study shows a local accumulation of HP1 and H3K9me3 at the promoter region of miR-7 in a mael-dependent manner. Therefore, in the Drosophila germline, mael may function as a transcriptional regulator for tissue-specific regulation of genes and miRNAs including miR-7 (Pek, 2009).

This work sheds light on a possible mechanism by which Drosophila might replenish their GSCs by regulating mael to induce dedifferentiation. In mammals, breakdown of spermatogonia to replenish GSCs has been reported. Breakdown of differentiating cysts to GSCs have also been shown in the Drosophila germline. Unlike in GSCs, where bam is transcriptionally silenced by the BMP signaling pathway, in differentiating cysts, where transcription of bam takes place, bam can be regulated posttranscriptionally via miRNAs. Since depletion of Bam in differentiating cysts can cause dedifferentiation into GSCs, mael regulation of Bam via miR-7 is a potential mechanism by which to induce cyst dedifferentiation into GSCs in times of need. miRNAs can facilitate the process by inhibiting translation of Bam while the system changes its transcriptional output. Although the HMG-binding motif, which showed significant enrichment by Mael ChIP, as well as the miR-7 target site in the bam 3′UTR are conserved only in the melanogaster subgroup, similar mechanisms involving regulation of miRNAs might have evolved independently in other species (Pek, 2009).

It was shown that activation of Stat in somatic cyst cells is sufficient to cause GSCs to self-renew outside their niche. Although an increase was observed in Stat in the somatic cells in mael mutant testes, it is believed that Mael functions primarily in the germline, as the mael mutants can be rescued by driving Flag-mael transgene under the germline-specific driver, NGT40-Gal4. Therefore, the activation of Stat in somatic cells could be an indirect consequence of signaling from dedifferentiating germline cysts to the somatic cells to establish a microenvironment suitable to maintain ectopic GSCs. However, the possibility that Mael has a role in somatic cells could not be completely excluded (Pek, 2009).

In conclusion, this study has shown that Mael ensures proper differentiation of the GSC lineage by repressing miR-7. Understanding the mechanism by which Mael regulates gene expression and the link between Mael and the mechanism of cellular dedifferentiation would be outstanding subjects for future studies (Pek, 2009).

Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells

It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. Intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, this study shows that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. The miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling (Yu, 2009).

Previous studies have shown that miRNAs may regulate Dap, thereby controlling the cell division of GSCs. This study shows that the dap 3'UTR directly responds to miRNA activities in GSCs. Using luciferase assays, miR-7, miR-278 and miR-309 were identified as miRNAs that can directly repress Dap through the dap 3'UTR in vitro. Although miR-278 and miR-7 play a role in regulating GSC division and cell cycle marker expression, respectively, neither of these mutants showed as dramatic a defect in the GSC cell cycle as Dcr-1-deficient GSCs. Thus, the dap 3'UTR may serve to integrate the effect of multiple miRNAs during cell cycle regulation. It remains possible that some miRNAs involved in this process remain to be identified. It was further shown that InR signaling controls the dap 3'UTR in GSCs. This led to an exploration of the interaction between InR signaling and miRNA/Dap cell cycle regulation. GSCs deficient for InR or Dcr-1 show similar cell cycle defects. Using starvation to control InR signaling, it was shown that both Dcr-1 and dap are required for proper InR signaling-dependent regulation of GSC division. Further, reduction of dap partially rescues the cell division defect of the InR mutant GSCs, suggesting that InR signaling regulates the cell cycle via Dap. These results suggest that miRNAs and Dap act downstream of InR signaling to regulate GSC division (Yu, 2009).

The data suggest that multiple miRNAs can regulate the 3'UTR of dap: miR-7, miR-278 and miR-309 can regulate the dap 3'UTR directly, whereas bantam and miR-8 may regulate it indirectly, or through cryptic MREs in the dap 3'UTR. Using GFP sensor assays, it was also shown that the dap 3'UTR may be directly regulated by miRNAs in the GSCs in vivo. However, which specific miRNAs control endogenous Dap levels in Drosophila GSCs remains unknown. Mammalian p21^cip1 has also been shown to be a direct target for specific miRNAs of the miR-106 family, including miR-290s and miR-372. Further, the mouse miR-290 family has recently been identified as regulating the G1-S transition. In addition, miR-221 and miR-222 have been shown to regulate p27^kip1, thereby promoting cell division in different mammalian cancer cell lines. Neither the miR-290 nor miR-220 family is conserved in Drosophila. Together, these results indicate that the CKIs (Dap) might be a common target for miRNAs in regulating the cell cycle in stem cells. However, the specific miRNAs that regulate the CKIs might vary between organisms (Yu, 2009).

This study reveals novel regulatory roles for miR-7 and miR-278 in the GSC cell cycle. miR-7 and miR-278 can directly target Dap. GSCs deficient for miR-278 show a mild but significant reduction in cell proliferation. Ectopic expression of miR-7 in follicle cells reduces the proportion of cells that stain positive for Dap. Furthermore, ablation of miR-7 in GSCs results in a perturbation of the frequency of CycE-positive GSCs. However, the cell division kinetics of miR-7 mutant GSCs is not reduced, by contrast with the dramatic reduction of cell division in Dcr-1-deficient GSCs. It is plausible that miR-7 and miR-278 act in concert with other miRNAs to regulate the level of Dap in GSCs and thereby contribute to cell cycle control in GSCs (Yu, 2009).

The interaction of multiple miRNAs with the dap 3'UTR might integrate information from multiple pathways. Further studies will reveal what regulates miR-7 and miR-278 expression in GSCs and which other miRNAs might act together in Dap regulation. It is known that miR-7 and the transcriptional repressor Yan mutually repress one another in the eye imaginal disk. In this model, Yan prevents transcription of miR-7 until Erk in the Egfr pathway downregulates Yan activity by phosphorylation, thereby permitting expression of miR-7. Conversely, miR-7 can repress the translation of Yan. Thus, a single pulse of Egfr signaling results in stable expression of miR-7 and repression of Yan. Whether similar regulation will be observed between miR-7 and the signaling pathways that regulate GSC division remains to be seen. It has been suggested that miR-7 might regulate downstream targets of Notch, such as Enhancer of split and Bearded. Thus, miR-7 may have a mild repressive effect on multiple targets in GSCs. Further experiments might illuminate this possibility (Yu, 2009).

miR-278, on the other hand, has been implicated in tissue growth and InR signaling. Overexpression of miR-278 promotes tissue growth in eye and wing imaginal discs. Deficiency of miR-278 leads to a reduced fat body, which is similar to the effect of impaired InR signaling in adipose tissue. Interestingly, miR-278 mutants have elevated insulin/Dilp production and a reduction of insulin sensitivity. Furthermore, miR-278 regulates expanded, which may modulate growth factor signaling including InR. Since InR signaling plays important roles in tissue growth and cell cycle control, it will be interesting to further test how miR-278 may regulate InR signaling, and whether InR signaling might regulate miR-278 in a feedback loop in GSCs (Yu, 2009).

Other miRNAs or miRNA-dependent mechanisms might also play roles in Drosophila GSCs. For example, the miRNA bantam is required for GSC maintenance. A recent study has shown that the Trim-NHL-containing protein Mei-P26, which belongs to the same family as Brain tumor (Brat), affects bantam levels and restricts cell growth and proliferation in the GSC lineage. Interestingly, most miRNAs are upregulated in mei-P26 mutant flies. By contrast, overexpression mei-P26 in bag of marbles (bam) mutants broadly reduces miRNA levels. This suggests that Mei-P26 regulates proliferation and maintenance of GSC lineages via miRNA levels. Since InR signaling cell-autonomously regulates GSC division but not maintenance, the possible interaction between Mei-P26 and InR signaling might be complex (Yu, 2009).

The systemic compensatory effect of insulin secretion in mammals with defective InR signaling is well documented. Insulin levels in mice with liver-specific InR (Insr - Mouse Genome Informatics) knockout are ~20-fold higher than those of control animals owing to the compensatory response of the pancreatic β-cells and impairment of insulin clearance by the liver. Knockout of the neuronal InR also leads to a mild hyperinsulinemia, indicating whole-body insulin resistance. Furthermore, the knockout of components in the InR signaling pathway, such as Akt2 and the regulatory and catalytic subunits of PI3 kinase, also leads to hyperinsulinemia and glucose intolerance. Therefore, a systemic decrease in InR signaling may lead to compensatory responses (Yu, 2009).

To understand the roles of InR signaling in the GSCs while avoiding any systemic compensatory effect the phenotypes of GSC clones were analyzed. Using a panel of cell cycle markers, it was found that InR mutant GSCs show cell cycle defects similar to those of Dcr-1 mutant GSCs: a reduction of cell division rate, an increased frequency of cells staining positive for Dap and CycE, and a decreased frequency of cells staining positive for CycB. Using GFP-dap 3'UTR sensors, it was shown that the dap 3'UTR responds to InR signaling in GSCs, suggesting that InR signaling can regulate Dap expression through the dap 3'UTR. This, together with genetic data indicating that InR/starvation-dependent cell cycle regulation requires Dcr-1 and dap, has led to the proposalthat InR signaling regulates the cell cycle through miRNAs that further regulate Dap levels. Since a reduction in dap only partially rescues the cell cycle defects of InR mutant GSCs, it is possible that InR signaling might also regulate GSC division by additional mechanisms (Yu, 2009).

InR signaling regulates the cell cycle through multiple mechanisms, mainly through the G1-S, but also partly through the G2-M, transition. Recent work has shown a delay in the G2-M transition in GSCs during C. elegans dauer formation. Starvation and InR deficiency may also affect the G2-M checkpoint in Drosophila GSCs. This study has dissected one possible molecular pathway that InR signaling utilizes to regulate the Drosophila GSC G1-S transition and show that InR signaling can control the cell cycle through miRNA-based regulation of Dap (Yu, 2009).

Many studies have connected InR and CKIs to Tor (Target of rapamycin) or Foxo pathways downstream of InR signaling. In S. cerevisiae, the yeast homolog of p21/p27 is upregulated when Tor signaling is inhibited. Foxo, a transcription factor that can be repressed by InR signaling, is known to play important roles in nutrition-dependent cell cycle regulation by upregulating p21 and p27. In C. elegans, starvation causes L1 cell cycle arrest mediated by InR (daf-2) and Foxo (daf-16): InR represses the function of Foxo, thereby downregulating the CKI (cki-1) and upregulating the miRNA lin-4. This study has shown that a miRNA-based regulation of Dap can be coordinated by InR in Drosophila GSCs (Yu, 2009).

Insulin and insulin-like growth factors (Igf1 and Igf2) are known to play important roles in regulating metabolic and developmental processes in many stem cells. In mammals, Igf signaling is required by different stem cell types, including human and mouse ES cells for survival and self-renewal, neural stem cells for expediting the G1-S transition and cell cycle re-entry, and skeletal muscle satellite cells for promoting the G1-S transition via p27^kip1 downregulation. This study has dissected the molecular mechanism of the InR pathway in another adult stem cell type, tDrosophila GSCs, showing that InR signaling can regulate stem cell division through miRNA-based downregulation of the G1-S inhibitor Dap. Further studies will reveal whether miRNAs also mediate InR signaling in other stem cell types (Yu, 2009).

Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs

Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'UTRs) that are complementary to the 5'-ends of miRNAs, or 'seed' regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and all three classes of motif are shown to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the 'box:seed' region. Consistent with this, it was found that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, it is demonstrated that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila (Lai, 2005).

The E(spl)-C and Brd-C of Drosophila melanogaster (Dm) contain two large families of direct Notch target genes, including seven bHLH repressor-encoding genes and 10 Bearded family genes. With the exception of E(spl)mbeta and Ocho, all of these genes contain GY-box (GUCUUCC), Brd-box (AGCUUUA), and/or K-box (UGUGAU) motifs in their 3'UTRs, which are propose to be miRNA-binding sites. Nine of these genes contain three or more box sites, a density that is especially remarkable when one considers how short their 3'UTRs are (often <350 nt in length). The conservation of these sites were systematically assessed in their orthologs from Drosophila pseudoobscura (Dp) and Drosophila virilis (Dv), species that are ~30 million and 60 million years diverged from Dm, respectively. 33/51 Brd-boxes, GY-boxes, and K-boxes have been perfectly conserved and reside in syntenic locations among all three species; 11 additional sites are identical in two of the three species. This indicates that all three motifs are under strong selective constraint (Lai, 2005).

Closer examination of nucleotide divergence surrounding these boxes has revealed some unexpected features that are germane to the proposition that these boxes represent miRNA-binding sites. These features are best illustrated by comparing rapidly evolving genes. Notably, Bearded is an unusually rapidly evolving protein, with only 15 residues preserved between Dm and Dv orthologs (out of 81 and 66 amino acids, respectively), and Dv Bearded has a significantly different arrangement of these 3'UTR motifs. The 3'UTR of Dv E(spl)m5 is also quite different from its counterparts in Dm/Dp. Alignment of Dm/Dp orthologs of Bearded and E(spl)m5 reveals that sequences upstream of most GY-boxes are well conserved; these regions include most sequences presumed to pair with miR-7. Similar patterns are seen for many other GY-boxes in other Notch target genes. However, the sequence upstream of many Brd- and K-boxes is strongly diverged, so that only 'box'-pairing is often preserved. In fact, many Brd- and K-boxes generally lack extensive pairing to miRNAs outside of the 'box' sequence. These factors likely preclude their identification by various published computational algorithms for miRNA-binding sites. Indeed, Brd- and K-boxes in Notch target genes have been deemed unlikely to represent miRNA-binding sites. In contrast, rapid divergence of the upstream portion of miRNA-binding sites is consistent with the idea that pairing between the miRNA 'seed' (positions ~2-8) and the 3'UTR 'box' (approximately the last one-third of the miRNA-binding site) is most critical for miRNA-mediated regulation (Lai, 2005).

It is also noted that precise spacing of several motif occurrences that are closely paired is also conserved, even though orthologous 3'UTRs otherwise display significant insertions and deletions. In these cases, one would presume that simultaneous binding of miRNAs to their respective sites would not be possible unless the 3'-end of the downstream miRNA was unpaired, a configuration that unexpectedly proved functional in vitro. Finally, there are a few nonconserved boxes in these 3'UTRs (7/51 total sites). In several cases, the nonconserved site is highly related to a neighboring conserved site [i.e., the first and second GY-boxes of Dp E(spl)m4 are equally similar to the first GY-box in Dm E(spl)m4; the third and fourth Brd-boxes in Dp E(spl)m5 are highly related to the third Brd-box in Dm E(spl)m5], implying that these nonconserved sites may be functional, newly evolved miRNA-binding sites (Lai, 2005).

GY-box-, Brd-box-, and K-box-class miRNAs are highly conserved among diverse insects, and many are, indeed, identical. Therefore Brd-boxes, GY-boxes, and K-boxes were sought in the predicted 3'UTRs of E(spl)bHLH and Brd genes from mosquitoes, bees, and moths; these species cover ~350 million years of divergence from Drosophila. Impressively, homologs of both E(spl)bHLH and Brd genes in these highly diverged species all contain multiple copies and multiple classes of 'box' motifs in their 3'UTRs. This strongly suggests that regulation by all three families of miRNAs is an ancient feature of Notch target gene regulation in insects (Lai, 2005).

To directly test the capacity of miRNAs to regulate the 3'UTRs of these Notch target genes, an in vivo assay was used. The target in this assay is a ubiquitously expressed reporter (tub>GFP or arm>lacZ) fused to an endogenous 3'UTR (a 3'UTR sensor). The reporter transgene is introduced into a genetic background in which a UAS-DsRed-miRNA transgene is activated with dpp-Gal4 or ptc-Gal4. This results in ectopic miRNA production in a stripe of red-fluorescing cells at the anterior–posterior boundary of imaginal discs. Inhibition of the green reporter within the red miRNA-misexpressing domain reflects direct miRNA-mediated negative regulation. Focus was placed on the central wing pouch region of the wing imaginal disc (Lai, 2005).

The ability of sensor transgenes for most Bearded family genes [Bob, Bearded, Tom, Ocho, E(spl)malpha, and E(spl)m4] and most E(spl)bHLH repressor genes [E(spl)mgamma, E(spl)mdelta, E(spl)m3, E(spl)m5, and E(spl)m8] to be regulated by ectopic GY-box-, Brd-box-, and K-box-class miRNAs was extensively analyzed. Sensor expression is influenced by the level to which it is negatively regulated by endogenous factors, including miRNAs. In this assay, the disc sensor must be expressed at sufficient levels before one can observe its knock-down by ectopic miRNAs. 3'UTR sensor constructs for different Notch target genes accumulate to different levels in vivo, consistent with variable amounts of endogenous miRNA-mediated regulation. Nevertheless, it was possible to reliably detect expression of all sensors excepting E(spl)m8. As detailed in the following three sections, these sensors were used to unequivocally demonstrate GY-boxes, Brd-boxes, and K-boxes to be sites of miRNA-mediated negative regulation by corresponding families of complementary miRNAs in vivo (Lai, 2005).

miR-7 is the only known Drosophila miRNA whose 5'-end is complementary to the GY-box (GUCUUCC). miR-7 has been shown to regulate three GY-box targets, including two members of the E(spl)-C, E(spl)m3 and E(spl)m4. While these two genes scored well in a genome-wide prediction of miR-7 targets, many other members of the Brd-C and E(spl)-C also contain between one and three GY-boxes in their 3'UTRs [Bob, Bearded, Tom, E(spl)mgamma, E(spl)m5]. Of these, only Tom was computationally identified as a compelling candidate for miR-7 (Lai, 2005).

The specificity of the disc sensor assay was assayed by showing that neither an empty tub-GFP sensor nor an Ocho sensor were affected by miR-7. The previous experiments done with E(spl)m3 and E(spl)m4 were repeated and it was observed that both were, indeed, inhibited by ectopic miR-7. This assay was used to demonstrate that miR-7 negatively regulates all seven GY-box-containing members of the Brd-C and E(spl)-C, including those with single sites [E(spl)m3, E(spl)mgamma, and Bearded], those with two sites [E(spl)m4, Tom, Bob], and those with three sites [E(spl)m5]. These data convincingly support the hypothesis that GY-boxes are general signatures of miR-7-binding sites in Notch target genes, irrespective of the overall amount of pairing between miR-7 and sequences outside of the GY-box. In order to more definitively demonstrate that miR-7-mediated regulation occurs through identified GY-boxes, mutant sensors bearing point mutations in the GY-boxes were tested. A Bearded sensor carrying five point mutations in its single GY-box no longer responded to miR-7. In a more stringent test, an E(spl)m5 sensor carrying 2-nt mutations in each of its three GY-boxes was generated. These targeted changes also abolished the ability of miR-7 to negatively regulate E(spl)m5. Therefore, ~7 continuous base pairs between the 'box' motif and its cognate miRNA seed are critical for in vivo target regulation. It is also noted that when mutant 3'UTRs are tested, a mild increase in reporter activity in miRNA-misexpressing cells was sometimes observed, the reason for which has not been determined (Lai, 2005).

Previous work has suggested synergism between miRNA-binding sites on the same transcript. Multiple GY-box 3'UTRs were generally subject to greater regulation than single-site 3'UTRs, even though the amount of miR-7 pairing to individual GY-boxes in multiple-site 3'UTRs is often less than its pairing with single GY-box 3'UTRs. Indeed, negative regulation of E(spl)m4, Tom, Bob, and E(spl)m5 by miR-7 was qualitatively indistinguishable from an artificial sensor containing two perfectly miR-7-complementary sites, even though many sites in these genes display relaxed pairing with miR-7 outside of GY-boxes. This suggests that as little as 7–8 nt of complementarity may suffice for miRNA target recognition, especially where multiple sites are present. However, since all three single GY-box-containing 3'UTRs were also regulated by miR-7, synergism is not required for biologically significant regulation by miRNAs (Lai, 2005).

There are two Drosophila miRNAs, miR-4 and miR-79, whose 5'-ends are complementary to the Brd-box (AGCUUUA). Both miRNAs are resident in miRNA clusters, and miR-4 resides in particularly dense clusters containing several unrelated miRNAs. Use was made of a UAS-DsRed-miR-286, miR-4, miR-5 transgene that is referred to as 'UAS-miR-4' and a UAS-DsRed-miR-79 transgene. miR-4 and miR-79 have only limited similarity outside of their Brd-box seed, and there is little indication from pairwise alignments that these miRNAs are specifically 'tuned' to different Brd-box sites in Notch target genes. In fact, all of these Brd-boxes lack the extended complementarity to miRNAs that is typical of miR-7:GY-box pairs, and no Notch target genes were previously predicted computationally as targets of miR-4 or miR-79 (Lai, 2005).

Seven Brd-box-containing Notch target genes were validated as being regulated by Brd-box-family miRNAs, including those with single sites [Tom, E(spl)mdelta, E(spl)mgamma] and those with multiple sites [Bearded, E(spl)malpha, E(spl)m4, and E(spl)m5]. Curiously, the negative regulatory effects of miR-4 on E(spl)mgamma, E(spl)malpha, E(spl)m4, and E(spl)m5 were greater than those of miR-79 on these same 3'UTRs, even though miR-4 is no more complementary to these sites than is miR-79. Nevertheless, the common ability of miR-4 and miR-79 to down-regulate individual sensors indicates that cross-regulation of individual sites by multiple members of a given miRNA family may occur. Notably, both miRNAs are expressed at high levels during embryonic development (Lai, 2005).

The specificity of miR-4 and miR-79 was tested using two mutant Bearded sensors, one bearing several point mutations in each of its three Brd-boxes and another containing mutations in the Brd-boxes and the GY-box. In both cases, the mutant transgenes accumulate to higher levels, consistent with relief from negative regulation by endogenous Brd-box-class miRNAs in the wing disc. In addition, they are no longer responsive to ectopic Brd-box-class miRNAs, indicating that the observed regulation occurs directly via Brd-boxes. As well, this experiment demonstrates that regulation by the miR-4 transgene is not attributable to miR-286 and miR-5 carried on this construct. Nevertheless, this miRNA construct efficiently down-regulates a miR-5 sensor containing two miR-5 sites, indicating that the other miRNAs carried on this construct are functional. As a final test of the specificity of this assay, it was observed that this three-miRNA construct fails to inhibit the expression of an empty tub-GFP sensor (Lai, 2005).

Having demonstrated that Brd-boxes are bona fide miRNA-binding sites, it was asked whether regulation of the Bearded 3'UTR by miR-7 requires the presence of Brd-boxes. This might be the case, for example, if negative regulation of a given 3'UTR required synergism between different types of miRNA-binding sites. A Bearded 3'UTR carrying mutations in each of the three Brd-boxes was observed to be still strongly inhibited by miR-7, indicating that individual types of miRNA-binding sites suffice for regulation in this assay (Lai, 2005).

The largest family of Drosophila miRNAs includes those whose 5'-ends are complementary to the K-box (cUGUGAUa, where the lowercase nucleotides represent positions of strong bias). The K-box is also the most pervasive motif within these Notch target genes; it is present in almost every member of the Brd-C and E(spl)-C [excepting E(spl)mbeta and Ocho, which lack any box motifs]. The maximum overall site complementarity of any given K-box site to any K-box family miRNAs is generally modest, and less than that seen with other demonstrated targets of the K-box family miRNA miR-2, namely, the proapoptotic genes grim, reaper, and sickle. In fact, the sole Notch target gene that was predicted informatically as a target of a K-box family miRNA in any study was E(spl)m8: miR-11, and this pair ranked only 46^th (Lai, 2005).

The ability was tested of two quite distinct K-box family miRNAs, those of the miR-2 cluster (miR-2a-1, miR-2a-2, and miR-2b-2) and miR-11, to regulate K-box-containing 3'UTRs. Given the abundance of K-box complementary miRNAs (as a class, they are among the more frequently cloned fly miRNAs), the occupancy of K-box sites by endogenous K-box-class miRNAs may be near-saturating in some cases. In fact, negative regulation of E(spl)m8, whose K-boxes mediate 10-fold negative regulation and nearly eliminate expression of this sensor, could not be convincingly demonstrated. In spite of this, positive evidence was obtained that four other K-box-containing 3'UTRs, E(spl)m4, Bob, E(spl)malpha, and E(spl)mdelta, are directly regulated by K-box-family miRNAs, although the amount of regulation observed was weaker than that seen with GY-box- or Brd-box-class miRNAs. As was the case with the two Brd-box-class miRNAs, both miR-2 and miR-11 are capable of regulating some of the same K-box-containing targets. This constitutes further evidence for the possibility of cross-regulation of miRNA-binding sites, even where the miRNAs in question display very little similarity outside of their seeds (Lai, 2005).

In performing pairwise tests of these miRNAs with Notch target gene sensors, two instances were observed of miRNA-mediated regulation of sensors lacking canonical boxes. (1) It was observed that the E(spl)mdelta sensor was inhibited by miR-7. Although E(spl)mdelta lacks a canonical GY-box, it does contain a GY-box-like site that would have a single G:U base pair with the miR-7 seed. The nucleotides that are 5' and 3' to the box are also paired with miR-7, and there is a significant region of pairing to the 3'-end of the miRNA. These factors may allow this site to be recognized by miR-7. The 9-mer AGUUUUCCA is found in both Dp and Dv orthologs of E(spl)mdelta, indicating that this site is under selection and therefore is likely important for regulation of E(spl)mdelta. (2) It was observed that the Bob sensor was negatively regulated by both Brd-box-class miRNAs, miR-4 and miR-79. Although Bob lacks a canonical Brd-box, it does contain two matches to positions 2-7 of the Brd-box, which would pair to positions 2-7 of the miR-4/79. In this regard, this type of site is reminiscent of the 6-mer K-box, which pairs to positions 2-7 of K-box miRNAs. One of these Brd-box-like sites is conserved in Dp, and the syntenic site in Dv is, in fact, a canonical Brd-box, further indicating a functional relationship between Bob and miRNAs of the Brd-box family (Lai, 2005).

The apparent functionality of these noncanonical sites led to a search for other such sites in Notch target 3'UTRs. Although one might expect to find many-fold more copies of degenerate sites relative to canonical sites, instead only a few additional examples of relaxed GY-box-like or Brd-box-like sites were found. For comparison, there are 28 canonical sites of these classes in Notch target 3'UTRs (16 Brd-boxes and 12 GY-boxes), but only three additional examples of a 7-mer box-like site with a G:U base-pair to a miRNA seed [all are GY-box-like sites in E(spl)mdelta, E(spl)m3, and E(spl)m7]. In addition, there are only five additional examples of sites that match only positions 2-7 of the GY-box or the Brd-box [all of which are Brd-box-like sites: the two in Bob, one in E(spl)m7, one in E(spl)malpha, and one in E(spl)mdelta]. These considerations strongly suggest that the much more restricted, canonical sites are actively selected for function in these Notch target 3'UTRs, a conclusion that is bolstered by the patterns of evolutionary conservation of these sites (Lai, 2005).

These experiments presented thus far demonstrate that target gene 3'UTRs harboring sequence elements with Watson-Crick complementarity to the 5'-ends of miRNAs are, indeed, regulated by these miRNAs in vivo, and that such sites are necessary for miRNA-mediated regulation. Are these sites sufficient for regulation by complementary miRNAs? Although a variety of studies of model sites in tissue culture assays indicate site sufficiency, tests in animals suggest that miRNA site context can be less forgiving in vivo. For example, certain reporters containing multimers of six lin-4 or three let-7 sites are not appropriately regulated by lin-4 or let-7 in nematodes. In addition, mutation of sequences outside of the let-7-binding sites in lin-41 abolishes regulation by let-7 in vivo. Therefore, it was of interest to test the functionality of GY-boxes, Brd-boxes, and K-boxes when abstracted from endogenous 3'UTR context (Lai, 2005).

To do so, a tandem of isolated GY-box, Brd-box, and K-box elements were cloned from Bob, Bearded, and E(spl)m8, respectively, into tub-GFP transgenes. Also mutant versions were cloned containing single changes in the Brd-box sites or dual changes in the GY-boxes. The ability of these 'box' sensors to respond to exogenously expressed miRNAs was tested. It was found that wild-type GY-box, Brd-box, and K-box sensors are all negatively regulated by corresponding miRNAs. These data directly demonstrate that all three types of box sites are sufficient for miRNA-mediated negative regulation. In contrast, mutant box sensors are nonfunctional in this assay. Since the mutant box sensors contain only one or two changes in each site, these data provide strong in vivo support for the idea that Watson-Crick pairing to the 5'-end of the miRNA (the 'seed') is the key essential feature of miRNA target recognition. As a further test of this idea, the ability of the three different K-box miRNAs, miR-6, miR-2, and miR-11, to down-regulate a miR-6 sensor was tested. All three inhibited miR-6 sensor expression, consistent with the ability of seed-pairing to mediate regulation by miRNAs (Lai, 2005).

With these UAS-miRNA transgenic lines in hand, the consequences of ectopically expressing miRNAs on Drosophila development were tested. It should be noted that Notch target-regulating miRNAs were fully expected to regulate other functionally unrelated targets in vivo. For example, it has been established that K-box-family miRNAs also negatively regulate the proapoptotic genes reaper, sickle, and grim via K-boxes in their 3'UTRs, while Brd-box-family miRNAs target the mesodermal determinant bagpipe via a Brd-box in its 3'UTR. Therefore, even if ectopic miRNAs are able to affect normal development, they would not necessarily be expected to affect Notch signaling exclusively. Nevertheless, it has been previously reported that ectopic miR-7 induces loss of molecular markers of wing margin development, resulting in wing notching. This indicates that phenotypic characterization of miRNA misexpression can be informative (Lai, 2005).

Using an independently derived UAS-miR-7 construct lacking DsRed, it was verified that dpp-Gal4>miR-7 wings display notching and loss of Cut expression at the developing wing margin of wing imaginal discs; the size of the L3-L4 intervein domain was also reduced. It was next observed that ectopic K-box miRNAs of the miR-2a-1, miR-2a-2, miR-2b-2 cluster or miR-6-1, miR-6-2, miR-6-3 cluster had similar effects on wing margin development, although two UAS-transgenes were necessary to produce this effect. Also loss of anterior crossvein and occasional L3 vein breaks was observed, although these are not indicative of loss of N signaling. More generalized expression of miR-7 using bx-Gal4 induced strong thickening of wing veins, which is indicative of compromised Notch signaling during lateral inhibition of wing veins. Expression of K-box miRNAs using bx-Gal4 had severe effects on wing development, resulting in tiny, crumpled wings. It is suspected that this results from misregulation of non-Notch-pathway-related targets. The Brd-box miRNAs miR-4 and miR-79 and the K-box miRNA miR-11 did not affect wing margin development, even when these transgenes were present in two copies, indicating that this phenotype is not generally due to misexpression of miRNAs. However, miR-79 induced strong wing curling at high levels, potentially due to misregulation of non-Notch-pathway-related targets (Lai, 2005).

Next, focus was placed on development of the adult peripheral nervous system, as exemplified by the bristle sensory organs that decorate the body surface. A classic role for Notch signaling is to restrict the number of sensory organ precursors. It was found that misexpression of miR-6 using bx-Gal4 results in a strong increase in microchaete bristle density and clustered dorsocentral macrochaetes, phenotypes that are consistent with loss of Notch signaling during lateral inhibition of sensory organ precursors. Ectopic miR-2 had a similar, but milder, effect and mostly induced clustered dorsocentral and scutellar macrochaetes. Therefore, divergent members of the K-box miRNA family have similar effects on sensory organ development, consistent with data indicating that seed-related miRNAs can regulate overlapping sets of target genes. Ectopic miR-7 also induces macrochaete tufting, which correlates with the differentiation of supernumerary sensory organ precursors in wing imaginal discs. Finally, occasional duplication of bristles was observed upon misexpression of the Brd-box miRNA mir-79, although this construct also induced occasional bristle loss. Ectopic expression of miRNAs does not in itself induce bristle defects per se, since misexpression of miR-4 or miR-11 does not interfere with bristle development (Lai, 2005).

Overall, the ability of different classes of Notch-regulating miRNAs to specifically induce phenotypes that are characteristic of Notch pathway loss of function in multiple developmental settings is a strong indication that Notch pathway targets validated in this study are key endogenous targets of these miRNAs (Lai, 2005).

It appears, therefore, that cells go through a significant amount of trouble to actively inhibit Notch signaling. Core components of the Notch pathway are subject to significant negative regulation at every step in their life cycle, including at the transcriptional, post-transcriptional, and post-translational levels. For example, in the absence of activated nuclear Notch, CSL proteins are transcriptional repressors that actively repress Notch target gene activity. In addition, multiple dedicated ubiquitin ligases promote degradation of Notch pathway components, including the receptor Notch itself. To this list, may be added transcripts of most direct Notch target genes in Drosophila that are negatively regulated by multiple families of miRNAs (Lai, 2005).

The evidence provided in this study to support this conclusion is that (1) three different classes of miRNA-binding sites (GY-boxes, Brd-boxes, and K-boxes) are pervasive among two major classes of Notch target genes; (2) all three classes of motif are selectively constrained in 3'UTRs during evolution; (3) transcripts bearing these box sites are negatively regulated by complementary miRNAs in vivo; (4) all three classes of sites are both necessary and sufficient for miRNA-mediated regulation in vivo; and (5) ectopic expression of Notch target-regulating miRNAs phenocopies Notch pathway loss of function during multiple developmental settings. Perhaps most importantly, it has been shown that genomic transgenes specifically mutated for miRNA-binding sites are sufficiently hyperactive so as to perturb normal development of the peripheral nervous system. This places these Drosophila Notch target genes in a relatively select group of miRNA targets for which miRNA-mediated regulation is phenotypically essential for normal development (Lai, 2005).

While most of the previously characterized in vivo targets of miRNAs are of the 'extensive pairing' variety, many of the validated targets in this study display much more limited 'box:seed'-pairing to miRNAs. In fact, within the context of the set of Notch target gene 3'UTRs, the presence of conserved GY-boxes, Brd-boxes, and K-boxes allowed for highly effective prediction of miRNA:target relationships. This is the case even without first taking into account the extent of miRNA-pairing outside of box motifs. Rapid divergence of sequences upstream of box motifs, particularly those of the Brd-box and K-box classes, further indicates that extensive pairing is not selected for in these bona fide target sites. Consistent with this, multiple lines of evidence are presented that show that divergent seed-related miRNAs can regulate overlapping sets of target in vivo. Conversely, the importance of pairing between 3'UTR boxes to miRNA seeds was demonstrated by endogenous 3'UTR and box sufficiency tests, where even single-nucleotide disruption of seed-pairing abolishes regulation by miRNAs in vivo (Lai, 2005).

Identification and characterization of miRNA-binding sites in these Notch target 3'UTRs mesh well with other recent bioinformatics and experimental studies that together help to define the 'look' of miRNA-binding sites. The concept of using conserved 'boxes' with Watson-Crick complementarity to miRNA seeds to identify miRNA targets is at the heart of the TargetScanS approach. A recent study has identify statistically significant signal not only for conserved 3'UTR sites that match positions 2-8 of the miRNA (as is characteristic of the Brd-box and GY-box), but also for matches to positions 2-7 of the miRNA (as is characteristic of the K-box). In addition, a significant bias was identified for the nucleotide corresponding to position one of the miRNA to be an adenosine in predicted target sites. Interestingly, 27/42 (64%) of GY-boxes, Brd-boxes, and K-boxes in Dm Notch target genes also have an adenosine in this position, consistent with the notion that this feature can help to identify genuine target sites. These results are also consistent with directed tests of model sites using an imaginal disc sensor assay. Together with the recent observation that miRNAs can down-regulate large numbers of transcripts that contain box:seed matches in their 3'UTRs, a current view emerges that conserved 3'UTR boxes that are 6-7 nt in length and complementary to the 5'-ends of miRNAs need to be considered seriously as functional regulatory sites. While seed-pairings with G:U base pairs are evidently not generally selected for, evidence is shown that rare sites of this class are functional. This is consistent with other studies that demonstrate that G:U seed-pairing impairs, but does not necessarily abolish target site function (Lai, 2005).

Finally, the presence of multiple classes of miRNA-binding sites in most Notch target gene 3'UTRs raises the possibility of combinatorial regulation. Although this has been widely suggested as a formal possibility, extensive evidence has been provided that 3'UTRs can bear multiple classes of functional sites. Phylogenetic considerations indicate that 10 different Notch target genes are likely regulated by multiple classes of miRNAs, and direct experimental support of this was provided for six Notch target genes. Multiple Brd-box-, K-box-, and GY-box-class miRNAs are present at high levels in the Drosophila embryo, and the Brd-box miRNA miR-4 is co-transcribed with the K-box miRNAs miR-6-1, miR-2, miR-3, suggesting that combinatorial control of Notch target genes actually occurs during normal development. Future studies are aimed at examining how different miRNA-binding sites collectively contribute to overall regulation of an individual gene (Lai, 2005).

Of the few animal miRNAs whose in vivo functions and targets are well understood, most act as genetic switches that determine binary, on/off states of target gene activity. For example, lin-4 and let-7 are temporal switches that control progression through nematode larval stages by inhibiting their targets at designated times in development. lsy-6 and miR-273 are spatial switches whose extremely restricted cell-type-specific expression patterns control neuronal identity. In these cases, temporally or spatially restricted miRNA expression is central to their control of specific processes, and each of these miRNAs appears to have a small number of key targets (Lai, 2005).

A different rationale is proposed for Brd-box and K-box miRNAs during Drosophila development. Although endogenous territories of GY-box-mediated regulation are not known, negative regulation by Brd-boxes and K-boxes appears spatially and temporally ubiquitous. Thus, Notch target transcripts of the Brd family and E(spl)bHLH families are subject to modes of miRNA-mediated regulation that operate in all cells, even though the genes themselves display highly restricted patterns of spatial expression. This suggests that these miRNAs are not dedicated to regulating Notch signal transduction, but may 'tune' the expression of many target genes. Indeed, the K-box-family miRNAs miR-2, miR-6, and miR-11 also directly regulate K-box-containing proapoptotic genes, and the Brd-box-family miRNAs miR-4 and miR-79 regulate the mesodermal determinant bagpipe. One prediction is that even though mutation of Brd-boxes and K-boxes in individual Notch target genes results in specific defects in Notch-mediated cell fate decisions, mutation of Brd-box and K-box miRNAs would have more general developmental consequences. This is supported by the observation that many, but not all, of the phenotypes induced by ectopic expression of Notch-regulating miRNAs appear to be obviously related to repression of Notch pathway activity (Lai, 2005).

An important advance of this study is the in vivo validation of a large number of biologically relevant miRNA targets that are minimally paired to miRNAs outside of the 'box:seed' region. Since modestly complementary sites are both necessary and sufficient for miRNA-mediated regulation, it might be relatively easy for novel miRNA-binding sites to arise in 'tuning' targets. Indeed, a subset of box sites has apparently newly evolved during Drosophilid radiation. In the greater context of insect Notch target genes, it appears to have been important that they be negatively regulated by miRNAs, although the precise numbers and arrangement of different sites is variable. These features of tuning targets seem to allow for highly customized regulation of individual genes (Lai, 2005).

The experimental validation of many tuning targets may be challenging or impossible to obtain where quantitative regulation is subtle. Nevertheless, minor changes in gene activity, even of a fraction of a percent, could become highly significant when selecting the fitness of individuals at the population level. Deep evolutionary profiling of related species will therefore be key to revealing the full complement of biologically important miRNA-binding sites. The data suggest that multiple classes of miRNA-binding sites can be recognized with confidence as highly conserved 3'UTR 'boxes' complementary to miRNA seeds, and this approach has been applied to the analysis of mammalian genomes. By mid-2005, 12 Drosophila genomes will be completed, which should enable high-confidence identification of miRNA-binding sites on the genome-wide scale -- even in cases in which only 7 nt of the target are paired to a miRNA (Lai, 2005).

Recent computational work pointed to regulation of vertebrate Notch and Delta by miR-34; however, no Notch target genes were similarly singled out in various bioinformatics efforts. miR-34 is conserved in flies; however, inspection of fly Notch or its ligands Delta and Serrate failed to reveal 'boxes' that might indicate similar regulation by miR-34. Brd-box-, GY-box-, and K-box-complementary miRNAs are likewise conserved between flies and vertebrates. Are any vertebrate Notch target genes predicted to be targeted by these miRNAs by virtue of 'boxes'? Although Brd proteins have thus far been found only in insects, E(spl)bHLH proteins are conserved in and are primary effectors of Notch signaling in all vertebrates. No enrichment for Brd-boxes, GY-boxes, and K-boxes is observed across the set of vertebrate E(spl)bHLH 3'UTRs as a whole. However, members of a specific subset of E(spl)-related repressors, named the Hey genes, contain a preponderance of these boxes in their 3'UTRs. This appears to be the case in a variety of mammals (human, mouse, and rat) and fish (fugu and zebrafish). Therefore, miRNA-mediated regulation may be a conserved feature of Notch target genes, a scenario that is under current experimental investigation (Lai, 2005).

Identification of Drosophila microRNA targets

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. This study screened conserved 3' UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA-target heteroduplex structures and energies. This approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. The target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. Three predicted targets each for miR-7 and the miR-2 family were experimentally verified, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. Features shared by all validated targets were identified that can be used to evaluate target predictions for animal miRNAs. Initial evaluation and experimental validation of target predictions suggest functions for two miRNAs. For others, the screen suggests plausible functions, such as a role for miR-277 as a metabolic switch controlling amino acid catabolism. Cross-genome comparison proved essential, as it allows reduction of the sequence search space. Improvements in genome annotation and increased availability of cDNA sequences from other genomes will allow more sensitive screens. An increase in the number of confirmed targets is expected to reveal general structural features that can be used to improve their detection. While the screen is likely to miss some targets, this study shows that valid targets can be identified from sequence alone (Stark, 2003).

Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation

Micro RNAs are a large family of noncoding RNAs of 21-22 nucleotides whose functions are generally unknown. A large subset of Drosophila RNAs has been shown to be perfectly complementary to several classes of sequence motif previously demonstrated to mediate negative post-transcriptional regulation. These findings suggest a more general role for micro RNAs in gene regulation through the formation of RNA duplexes (Lai, 2002).

A new strategy of gene regulation was defined by the activities of Caenorhabditis elegans let-7 and lin-4. These RNA molecules of 21-22 nt are complementary to the 3' untranslated regions (UTRs) of target transcripts and mediate negative post-transcriptional regulation through RNA duplex formation. Several recent reports now reveal that a large family of RNAs of 21-22 nt, collectively termed micro RNAs (miRNAs), exists in organisms as diverse as worms, flies and humans. Although it was presumed that many of these new miRNAs would also act in post-transcriptional gene regulation, initial searches did not reveal obvious targets based on sequence complementarity (Lai, 2002).

In Drosophila, two 3'UTR sequence motifs, the K box (cUGUGAUa) and the Brd box (AGCUUUA) mediate negative post-transcriptional regulation. Although originally identified in the 3' UTRs of Notch pathway target genes encoding basic helix-loop-helix (bHLH) repressors and Bearded family proteins, modes of regulation mediated by both motifs are spatially and temporally ubiquitous. This suggests that at least some of the many other Drosophila transcripts that contain K boxes or Brd boxes in their 3' UTRs are also actively regulated by these motifs. Since RNA-binding proteins typically show relatively relaxed binding specificities, it was hypothesized that an RNA component might be involved in recognition of these highly constrained motifs. This was bolstered by the finding that another motif common to the 3' UTRs of many of the same Notch pathway target genes, the GY box (uGUCUUCC), is complementary to and mediates RNA duplex formation through the proneural box (AUGGAAGACAAU), a motif located in the 3' UTRs of transcripts encoding proneural bHLH activators, (Lai, 2002).

Drosophila miRNAs encoded by 11 of 21 distinct genomic miRNA loci are complementary to the K box at their 5' end, with all but miR-11 having a perfect (8/8) antisense match to the extended K box consensus (UAUCACAG). Notably, the most 5' nucleotide of miR-11 is a cytosine residue, making it complementary to the second most common nucleotide at this position in identified K boxes. In addition, perfect antisense matches to the Brd box and GY box were found at the 5' ends of fly miR-4 and fly miR-7, respectively. The precise complementarity of these miRNAs to K box, Brd box and GY box motifs suggests that they bind these sequences in 3' UTRs and, in the case of the former two motifs, mediate negative post-transcriptional regulation. Complementarity between miRNAs and 3' UTRs extends beyond core sequence motifs in many cases, providing additional support for the existence of the proposed RNA duplexes. Examples exist of extended miRNA complementarity to 3' UTRs containing K boxes, Brd boxes and GY boxes. Complements to all three sequence motifs are located exclusively at the 5' ends of miRNA, suggesting that some aspect of regulation may be shared by these different miRNAs. For example, a common factor might be involved in the recognition or stabilization of these short miRNA-3' UTR duplexes (Lai, 2002).

Several miRNAs complementary to K boxes (miR-11 and the miR-2b and miR-13 subfamilies) are broadly expressed throughout Drosophila development, consistent with their proposed involvement in temporally ubiquitous regulation mediated by K boxes; the GY box-complementary miRNA miR-7 is similarly broadly expressed during development. The expression of the single identified Brd box-complementary miRNA miR-4 is restricted to embryogenesis. However, since the search for miRNAs has not yet been saturating, other miRNAs complementary to Brd boxes that are expressed later in development might yet be found (Lai, 2002).

The regulatory role of the K box and Brd box in other organisms has not yet been tested. Nevertheless, the presence of their complements in worm and human miRNAs suggests that these modes of regulation have potentially been conserved. Notably, the complements to these motifs are also located specifically at the 5' ends of miRNA. The restricted location of complements in these different species further suggests that the regulatory targets of many other miRNAs will be determined by the sequence of their 5' ends. In agreement with this idea, most of the known lin-4 and let-7 target sequences also involve perfect complements with the 5' ends of these miRNAs. Systematic searches for the complements of other 5' miRNA ends in 3' UTRs may therefore identify new post-transcriptional regulatory sequence elements. It should be noted, however, that despite the existence of three conserved sites in the lin-14 3' UTR that include perfect complements to lin-4, normal regulation of lin-14 actually depends on variant lin-4 binding sites containing a bulged nucleotide in the 5' complementary region. Thus, this rule is probably not absolute (Lai, 2002).

Initially, miRNAs are transcribed as RNAs of approximately 70 nt containing a stem-loop structure; these are cleaved by the RNAse III enzyme Dicer to generate the mature miRNA. Curiously, only a single strand of the duplex precursor stem structure is generally stable and is recovered as miRNA. The model proposed here may help to explain this phenomenon, since the strand that is complementary to these identified 3' UTR motifs is nearly exclusively the one that is isolated as miRNA. The single exception is miR-5, whose sequence contains a K box. Notably, miR-5 and the K box-complementary miRNAs miR-6-1,2,3 (whose loci are incidentally located next to each other in the genome) are complementary at 20 of 21 continuous nucleotide positions. This suggests that miR-5 might influence or possibly interfere with the ability of miR-6-1,2,3 to interact with 3' UTRs that contain K boxes (Lai, 2002).

Negative regulation by K box- and Brd box-complementary miRNA must differ from lin-4-mediated regulation, because K boxes and Brd boxes have significant, though distinct, effects on both transcript stability and translational efficiency, whereas lin-4 is thought to act at a step following translational initiation. The GY box does not seem to have a strong effect at the cis-regulatory level. Other miRNAs may show additional regulatory capacities; efforts are underway to understand the different molecular mechanisms of regulation mediated by miRNA-3' UTR RNA duplexes (Lai, 2002).

MicroRNA-7 compromises p53 protein-dependent apoptosis by controlling the expression of the chromatin remodeling factor SMARCD1

The epidermal growth factor receptor (EGFR) up-regulates miR-7 to promote tumor growth during lung cancer oncogenesis. Several lines of evidence have suggested that alterations in chromatin remodeling components contribute to cancer initiation and progression. This study identified SMARCD1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 1) as a novel target gene of miR-7. miR-7 expression reduced SMARCD1 protein expression in lung cancer cell lines. Luciferase reporters carrying wild type or mutated 3'UTR of SMARCD1 were used, and miR-7 was found to block SMARCD1 expression by binding to two seed regions in the 3'UTR of SMARCD1 and down-regulated SMARCD1 mRNA expression. Additionally, upon chemotherapy drug treatment, miR-7 down-regulated p53-dependent apoptosis-related gene BAX (BCL2-associated X protein) and p21 expression by interfering with the interaction between SMARCD1 and p53, thereby reducing caspase3 cleavage and the downstream apoptosis cascades. Although SMARCD1 sensitized lung cancer cells to chemotherapy drug-induced apoptosis, miR-7 enhanced the drug resistance potential of lung cancer cells against chemotherapy drugs. SMARCD1 was down-regulated in patients with non-small cell lung cancer and lung adenocarcinoma cell lines, and SMARCD1 and miR-7 expression levels were negatively correlated in clinical samples. This investigation into the involvement of the EGFR-regulated microRNA pathway in the SWI/SNF chromatin remodeling complex suggests that EGFR-mediated miR-7 suppresses the coupling of the chromatin remodeling factor SMARCD1 with p53, resulting in increased chemo-resistance of lung cancer cells (Hong, 2016).

EGFR promotes lung tumorigenesis by activating miR-7 through a Ras/ERK/Myc pathway that targets the Ets2 transcriptional repressor ERF

MicroRNAs (miRNA) mediate distinct gene regulatory pathways triggered by epidermal growth factor receptor (EGFR) activation, which occurs commonly in lung cancers with poor prognosis. This study reports the discovery and mechanistic characterization of the miRNA miR-7 as an oncogenic 'oncomi' and its role as a key mediator of EGFR signaling in lung cancer cells. EGFR activation or ectopic expression of Ras as well as c-Myc stimulated miR-7 expression in an extracellular signal-regulated kinase (ERK)-dependent manner, suggesting that EGFR induces miR-7 expression through a Ras/ERK/Myc pathway. In support of this likelihood, c-Myc bound to the miR-7 promoter and enhanced its activity. Ectopic miR-7 promoted cell growth and tumor formation in lung cancer cells, significantly increasing the mortality of nude mice hosts, which were orthotopically implanted with lung cancers. Quantitative proteomic analysis revealed that miR-7 decreased levels of the Ets2 transcriptional repression factor ERF, the coding sequence of which was found to contain a miR-7 complementary sequence. Indeed, ectopic miR-7 inhibited production of ERF messages with a wild-type but not a silently mutated coding sequence, and ectopic miR-7 rescued growth arrest produced by wild-type but not mutated ERF. Together, these results identified that ERF is a direct target of miR-7 in lung cancer. These findings suggest that miR-7 may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel prognostic biomarker and therapeutic target in lung cancer (Chou, 2010).

Search PubMed for articles about Drosophila mir-7

Aparicio, R., Simoes Da Silva, C. J. and Busturia, A. (2015). MicroRNA miR-7 contributes to the control of Drosophila wing growth. Dev Dyn 244(1): 21-30. PubMed ID: 25302682

Caygill, E. E. and Brand, A. H. (2017). miR-7 buffers differentiation in the developing Drosophila visual system. Cell Rep 20(6): 1255-1261. PubMed ID: 28793250

Chou, Y. T., Lin, H. H., Lien, Y. C., Wang, Y. H., Hong, C. F., Kao, Y. R., Lin, S. C., Chang, Y. C., Lin, S. Y., Chen, S. J., Chen, H. C., Yeh, S. D. and Wu, C. W. (2010). EGFR promotes lung tumorigenesis by activating miR-7 through a Ras/ERK/Myc pathway that targets the Ets2 transcriptional repressor ERF. Cancer Res 70(21): 8822-8831. PubMed ID: 20978205

Da Ros, V. G., Gutierrez-Perez, I., Ferres-Marco, D. and Dominguez, M. (2013). Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis. PLoS Biol 11(5): e1001554. PubMed ID: 23667323

Herranz, H., Perez, L., Martin, F. A. and Milan, M. (2008). A Wingless and Notch double-repression mechanism regulates G1-S transition in the Drosophila wing. EMBO J 27(11): 1633-1645. PubMed ID: 18451803

Herranz, H., Hong, X., Perez, L., Ferreira, A., Olivieri, D., Cohen, S. M. and Milan, M. (2010). The miRNA machinery targets Mei-P26 and regulates Myc protein levels in the Drosophila wing. EMBO J 29(10): 1688-1698. PubMed ID: 20400939

Hong, C. F., Lin, S. Y., Chou, Y. T. and Wu, C. W. (2016). MicroRNA-7 Compromises p53 Protein-dependent Apoptosis by Controlling the Expression of the Chromatin Remodeling Factor SMARCD1. J Biol Chem 291(4): 1877-1889. PubMed ID: 26542803

Huang, Y. C., Smith, L., Poulton, J. and Deng, W. M. (2013). The microRNA miR-7 regulates Tramtrack69 in a developmental switch in Drosophila follicle cells. Development 140(4): 897-905. PubMed ID: 23325762

Lai, E. C. (2002). Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat. Genet. 30(4): 363-4. 11896390

Lai, E. C., Tam, B. and Rubin, G. M. (2005). Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs. Genes Dev 19(9): 1067-1080. PubMed ID: 15833912

Li, X. and Carthew, R. W. (2005). A microRNA mediates EGF receptor signaling and promotes photoreceptor differentiation in the Drosophila eye. Cell 123(7): 1267-1277. PubMed ID: 16377567

Li, X., Cassidy, J. J., Reinke, C. A., Fischboeck, S. and Carthew, R. W. (2009). A microRNA imparts robustness against environmental fluctuation during development. Cell 137(2): 273-282. PubMed ID: 19379693

Pek, J. W., Lim, A. K. and Kai, T. (2009). Drosophila maelstrom ensures proper germline stem cell lineage differentiation by repressing microRNA-7. Dev Cell 17(3): 417-424. PubMed ID: 19758565

Stark, A., Brennecke, J., Russell, R. B. and Cohen, S. M. (2003). Identification of Drosophila MicroRNA targets. PLoS Biol 1(3): E60. PubMed ID: 14691535

Tokusumi, T., Tokusumi, Y., Hopkins, D. W., Shoue, D. A., Corona, L. and Schulz, R. A. (2011). Germ line differentiation factor Bag of Marbles is a regulator of hematopoietic progenitor maintenance during Drosophila hematopoiesis. Development 138(18): 3879-3884. PubMed ID: 21813570

Yu, J. Y., Reynolds, S. H., Hatfield, S. D., Shcherbata, H. R., Fischer, K. A., Ward, E. J., Long, D., Ding, Y. and Ruohola-Baker, H. (2009). Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells. Development 136(9): 1497-1507. PubMed ID: 19336466

date revised: 20 October 2017

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