Function of microrna-375 and microrna-124a in pancreas and brain

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1 REVIEW ARTICLE Function of microrna-375 and microrna-124a in pancreas and brain Nadine N. Baroukh 1 and Emmanuel Van Obberghen 1,2 1 INSERM U907, Faculté de Médecine, Institut de Génétique et Signalisation Moléculaire (IFR50), Université de Nice Sophia-Antipolis, Nice, France 2 Laboratoire de Biochimie, Hôpital Pasteur, CHU de Nice, France Keywords development; diabetes; gene regulation; metabolism; microrna; neurons; pancreatic b-cell lines Correspondence N. Baroukh, INSERM U907, IFR50, Faculté de Médecine, Université de Nice Sophia- Antipolis, 28 avenue de Valombrose, Nice Cedex 2, France Fax: Tel: nadine.baroukh@unice.fr (Received 25 March 2009, revised 7 July 2009, accepted 3 September 2009) doi: /j x In recent years, our understanding of how gene regulatory networks control cell physiology has improved dramatically. Studies have demonstrated that transcription is regulated not only by protein factors, but also by small RNA molecules, micrornas (mirnas). The first mirna was discovered in 1993 as a result of a genetic screen for mutations in Caenorhabditis elegans. Since then, the use of sophisticated techniques and screening tools has promoted a more definitive understanding of the role of mirnas in mammalian development and diseases. mirnas have emerged as important regulators of genes involved in many biological processes, including development, cell proliferation and differentiation, apoptosis and metabolism. Over the last few years, the number of reviews dealing with mirnas has increased at an impressive pace. In this review, we present general information on mirna biology and focus more closely on comparing the expression, regulation and molecular functions of the two mirnas, mir- 375 and mir-124a. mir-375 and mir-124a share similar features; they are both specifically expressed in the pancreas and brain and directly bind a common target gene transcript encoding myotrophin, which regulates exocytosis and hormone release. Here, we summarize the available data obtained by our group and other laboratories and provide an overview of the specific molecular function of mir-375 and mir-124a in the pancreas and the brain, revealing a potential functional overlap for these two mirnas and the emerging therapeutic potential of mirnas in the treatment of human metabolic diseases. Introduction Completion of the sequencing of the human genome has led to the identification and mapping of protein-coding genes, which represent only 2 3% of human genomic DNA. Approximately 45% of the remaining DNA consists of repetitive sequences, whereas the rest of the human genome harbours noncoding functional elements and nonfunctional sequences that have been referred to as junk DNA. Increasing evidence supports the notion that the majority of functional elements in the genome do not Abbreviations DGCR8, DiGeorge syndrome critical region gene 8; Foxa2, Forkhead box a2; mirna, microrna; PDK-1, 3 -phosphoinositide-dependent protein kinase-1; Pdx-1, pancreas duodenum homeobox protein 1; pre-mirna, precursor mirna; pri-mirna, primary mirna; REST, response element silencing transcription factor; SCP1, C-terminal domain phosphatase 1. FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works 6509

2 Function of mir-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen code for proteins [1,2]. A major advance in understanding the regulation of genetic information came with the discovery of microrna (mirna) molecules. mirnas are nonprotein-coding small RNAs, nucleotides in length, that are implicated in the posttranscriptional fine tuning of gene regulation. The first mirnas discovered were lin-4 and let-7, which are crucial for regulating developmental timing in the nematode, Caenorhabditis elegans [3,4]. Since these initial reports, several hundred mirnas have been identified in various species. Many mirnas are evolutionarily maintained, suggesting a conservation of function. An interesting study in zebrafish embryos showed that most mirnas are expressed during specific developmental stages and in particular cell types, although some are expressed ubiquitously [5]. These data support the notion of spatiotemporal- and cell type-specific mirna expression [5,6]. In addition, microarray analyses have shown that transient mirna overexpression in cells leads to the downregulation of a large number of transcripts [7]. Theoretically, one mirna could co-ordinate the regulation of hundreds of genes. Comparative genomics has indeed predicted that onethird of human genes could be mirna targets [8]. Once identified, these mirna molecules were deposited for annotation in the mirna catalogue established by the Sanger Institute [9]. mirnas are named using the mir prefix and a unique identifying number [10]. Computational methods have been developed and employed for the prediction of target genes for invertebrate and mammalian mirnas, becoming an important resource for the functional investigation of individual mirnas [11,12]. Our current knowledge indicates that mirnas govern a wide range of physiological and developmental processes. They play an important role in the control of cell survival, proliferation, differentiation and metabolism, whose dysfunction is a potential cause of disease [13 18]. For example, single nucleotide polymorphisms that modify mirna-binding sites have been shown to alter phenotype [19] or cause disease [20]. We and others have focused on the functions of mir-375 and mir-124a and their respective target genes. Biogenesis of mirnas and their mode of action on gene regulation mirnas are generated by a two step processing pathway to yield RNA molecules of 22 nucleotides that regulate target gene expression at the post-transcriptional level [21]. Biogenesis of mirnas starts with the transcription of a long primary precursor product, pri-mirna, synthesized by RNA polymerase II. Like other transcripts, pri-mirna presents a 5 cap structure and a 3 poly(a) tail (Fig. 1). The pri-mirna is processed by a nuclear protein complex, Microprocessor, containing the RNaseIII-type protein Drosha and its double-stranded RNA-binding partner protein Pasha DGCR8 (DiGeorge syndrome critical region gene 8). The Microprocessor complex cleaves pri-mir- NA to precursor mirna (pre-mirna), a nucleotide RNA with a typical stem loop structure [22]. Pasha DGCR8 acts together with the endonuclease Drosha and plays a critical role in the biogenesis and processing of mirnas [23]. Pre-miRNAs are exported into the cytoplasm by the nuclear exportin-5 transporter [24,25]. Once in the cytoplasm, the pre-mirna is processed by another RNaseIII-type protein, Dicer, which acts in concert with another double-stranded RNA-binding protein (the HIV transactivating response RNA-binding protein) and Argonaute proteins to liberate the mature mirna duplex (20 22 nucleotides) [26 29]. Processing by Dicer results in the production of a small double-stranded mirna duplex containing two nucleotide-long 3 overhangs [30]. The mature duplex mirna is incorporated into an effector complex referred to as the RNA-induced silencing complex. On the basis of thermodynamic properties, one strand is eliminated, whereas the other remains integrated in the complex [31,32]. mirnas mediate their effect on gene expression by annealing to the 3 -UTR of target genes. Functional mirna-binding sites in the coding region or 5 -UTR of endogenous mrnas have not been clearly identified, because they are less frequent and appear less effective than those in the 3 -UTR [7,8,33]. However, Lytle et al. [34] demonstrated that introducing a target site for let-7a mirna into the 5 -UTR of a luciferase reporter represses gene expression by let-7a. In many cases, target recognition by a mirna only requires a continuous 6 bp seed match between the 5 end of the mirna and its target. By binding to complementary sequences located at the 3 -UTR of target mrnas and depending on partial or complete sequence homology, mirnas can downregulate transcript levels in addition to suppressing protein translation [35] (Fig. 1). It seems that mirnas might repress protein expression by multiple means, although the exact mechanisms remain unclear. mirnas may interfere with translation at both the initiation and elongation stages, or translation may be unaffected, with nascent polypeptides being degraded. Alternatively, target mrnas may be repressed translationally, because they are sequestered physically from ribosomes and accumulate in P-bodies [36 38]. P-bodies are cytoplasmic subcompartments involved in mrna metabolism, degradation and translation 6510 FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works

3 N. N. Baroukh and E. Van Obberghen Function of mir-375 and 124a in pancreas and brain Pol II mirna gene Nucleus Cytoplasm 5 pri-mirna AAAAA-3 Microprocessor Drosha Pasha- DGCR8 pre-mirna Ran+GTP Exportin 5 Dicer Dicer mirna duplex Fig. 1. Overview of the mirna biogenesis pathway. mirnas are generated as primary transcripts termed pri-mirna. After two ribonuclease cleavage steps, the mature mirna of 22 nucleotides is produced. Mature mirna is incorporated into the RNA interference (RNAi) effector complex RISC (RNA-induced silencing complex), which drives mature mirna to homologous mrnas for direct translational suppression and mrna degradation. For simplicity, not all cellular factors involved in mirna processing are shown. STOP ORF Partial homology mirna RISC Translational repression RISC/target silencing AAAAA mrna target mrna binding RISC High homology ORF mirna degradation P-bodies mirna RISC AAAAA mrna target mrna degradation AAAAA control. These trafficking components are an essential feature of the pathway [39]. Initially, mirnas were only thought to suppress gene expression, but recently it has been shown that they can also have the opposite effect of inducing gene expression by activating transcription [40,41] or upregulating translation [42,43]. Given the known modes of action of mirnas, the temporal and spatial expression profiles of mirnas and their specificity for protein targets, mirnas have opened up research on their potential role in the development and maintenance of cell phenotypes. Specific genomic features for mir-375 and mir-124a Several hundred mirnas have been identified and sequenced in mammalian species, with 700 in human, 500 in mouse and macaque and 300 in rat (from Rfam database, [9]). Generally, most mirna genes are located far away from any annotated gene, implying independent transcription with their own promoters. However, some mirnas lie within predicted introns of genes encoding proteins. In 80% of these cases, the introns have the same orientation as the mirnas, indicating that the protein-coding genes serve as host genes for coexpressed mirnas. Some mirnas are located in close genomic proximity to each other and others are transcribed as polycistronic units [21]. To date, little is known about the transcriptional regulation of mirna genes and studies have mostly concentrated on mirnas located within the intergenic region of the genome. However, a sequence motif GANNNNGA has been found to display a conserved distribution in nematodes. It was observed to be most FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works 6511

4 Function of mir-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen abundant in the upstream sequences of two important mirnas, mir-1 and mir-124 [44]. The mir-375 gene is found on chromosome 2 in humans and chromosome 1 in mice (Table 1). mir-375 is located in an intergenic region between the cryba2 (b-a2 crystallin, an eye lens component) and Ccdc108 (coiled-coil domain-containing protein 108) genes; a genomic region conserving the synteny between humans and mice (see Ensembl, which provides genome sequences for vertebrates). Moreover, the sequences of pre-mir-375 in both species present a 100% homology (Fig. 2A), highlighting the high degree of conservation for this specific mirna. Recently, a study revealed that pancreas duodenum homeobox protein 1 (Pdx-1) and neurogenic differentiation factor 1, two critical components of pancreatic endocrine cell functions, control gene expression of mir-375 in a combinatorial manner [45]. Two regulatory modules have been described in the vicinity of mir-375; the first is located 500 bp upstream of the mirna 5 end and the second 1700 bp downstream. The first domain may correspond to the proximal promoter, whereas the second domain may correspond to a distal enhancer [45]. Taken together, these sequence features indicate that the mir-375 gene is transcribed from its own promoter. mir-124 was first identified by cloning studies in mice [6]. There are three precursor hairpin sequences; mir-124a1 on chromosome 14, mir-124a2 on chromosome 3 and mir-124a3 on chromosome 2 (Table 1). Each mir-124a locus is associated with either expressed sequence tags or annotated mrnas. However, these mrnas do not code for any known proteins, suggesting that they may be part of the primirna transcript. All three mir-124a genes have closely related predicted human homologues (Fig. 2B). Lagos-Quintana et al. [6] also reported a mature mirna sequence, mir-124b, with a G insertion at Table 1. Identification and chromosome (chr) localization of human mouse mir-375 and mir-124a (adapted from Rfam mir registry at hsa, Homo sapiens (human); mmu, Mus musculus (mouse). mir-id Accession Chr Start End hsa-mir-375 MI mmu-mir-375 MI hsa-mir-124a1 MI hsa-mir-124a2 MI hsa-mir-124a3 MI mmu-mir-124a1 MI mmu-mir-124a2 MI mmu-mir-124a3 MI position 12. However, mir-124b has not been found in either the mouse or human genome. mir-124a expression is negatively regulated by the transcriptional repressor, response element silencing transcription factor (REST), in non-neuronal cells and neural progenitors. Indeed, REST functions as a negative regulator of mir-124a via response element (RE1) sites in three mir-124a genomic loci [46]. Additionally, comparative sequence analysis indicates the presence of evolutionary conserved camp response elements recognized by camp response element-binding protein, a basic leucine zipper transcription factor, within the proximal regulatory region of mir-124a, implicating the role of camp response element-binding protein in the positive regulation of this mirna [47]. Despite the importance of characterizing functional DNA activity, few specific transcription elements have been described as regulating mirna gene expression. However, the increasing amount of sequence information from multiple organisms has enabled biologists to use sequence comparisons in gene regulation studies [48 50]. The rationale for using interspecies sequence comparisons in identifying noncoding regulatory elements is based on the observation that sequences that perform fundamental functions are frequently conserved between species. Thus, one possible alternative is to use these available tools for multiple sequence alignments among species to identify conserved regulatory elements regulating mirna genes. Using software for sequence comparisons (i.e. evolutionary conserved region browser) [51], we examined the sequence homology among animal species to search for conserved regions near the mir-124a2 gene that may affect its gene regulation. Our preliminary interspecies analysis of the mir-124a2 gene revealed the presence of a 177 bp sequence with 75% identity between human and zebrafish, 1.8 kbp upstream of mir-124a2 (Fig. 3). On the basis of its high level of sequence conservation (and lacking the characteristics of coding regions), one may propose that this element plays a role in regulating the expression of the mir-124a2 gene. It is crucial to verify this prediction by characterizing this element through in vitro studies and to explore its effect on mir-124a expression. Tissue expression of mir-375 and mir-124a The mir-375 sequence was first cloned from a mouse insulinoma pancreatic b-cell line (MIN6 cells) and identified as the most abundant, evolutionarily conserved, islet-specific mirna [52]. mir-375 is expressed in islet b-cells as well as in non-b-cells of the pancreas [53,54] FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works

5 N. N. Baroukh and E. Van Obberghen Function of mir-375 and 124a in pancreas and brain A B Fig. 2. Human (hsa) and mouse (mmu) mir-375 (A) and mir-124a isoform (B) CLUSTALW stem loop precursor sequence alignments. Mature mirna sequences are underlined. Asterisks indicate conserved nucleotides. Other identified islet-specific mirnas are mir-7, mir-9 and mir-376 [54 56]. Overall, data show that mirnas are necessary for islet cell genesis in mice [57]. Inhibition of mir-375 in zebrafish has a profound deleterious effect on pancreatic development, particularly in endocrine cells [58]. mir-375 was first thought to be restricted to pancreatic cells, but evidence shows that it is also expressed within the brain, exclusively in the pituitary and at a lower level in hypothalamic cells [59]. Several mirnas identified during the mouse pancreatic b-cell line MIN6 cloning were also identified in the brain, indicating an overlap in function of these particular mirna sequences [52]. Furthermore, the pituitary gland and pancreatic cells share similarities in terms of specialized biological functions, such as exocytosis, the final step in the secretory pathway. At this point, it is tempting to speculate that mir-375 has a common function in both tissues and may regulate exocytosis through similar target genes. mir-124a is preferentially expressed in the brain (the most abundant mirna in embryonic and adult central nervous systems) and the retina. The brain is an organ with complex cell type composition, among which neurons and glial cells are predominant. mirna expression analysis in human, mouse and rat brain demonstrates that mir-124, mir-9, mir-128a and mir-128b are highly and specifically expressed in all brain regions, except for the pituitary gland, which shows abundant expression of mir-7, mir-375 and clusters of mir-141 and mir-200a [54,60,61]. During neurogenesis, mir-124a is present at very low levels in neural progenitors, but is highly expressed in differentiating and mature neurons [62]. Because of its absence from proliferative cells and its wide expression in differentiated neurons, mir-124a is not assumed to be associated with a transition in the differentiation states. In addition, this expression pattern is highly specific and consistent with the hypothesis that mir-124a targets genes expressed at differentiation phases [59]. Furthermore, mir-124a overexpression in cultured HeLa cells leads to a decrease in transcript levels of a brain-specific set of genes, and shifts HeLa gene expression towards that of cerebral cortex-like gene expression [7]. Initially described as a brain-specific mirna in mammals, mir-124a, like mir-375, is also well represented in the mouse pancreatic MIN6 b-cell line [52]. Further data from our laboratory have recently demonstrated that the mir-124a expression level is increased in mouse pancreas at embryonic (e) stage e18.5 compared with stage e14.5, indicating a FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works 6513

6 Function of mir-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen Fig. 3. An adapted representation showing the human mir-124a2 genomic region (human localization; chromosome 8: ) compared with fugu (fr2), zebrafish (danrer5), chicken (galgal3), opossum (mondom4), mouse (mm9), rat (rn4) and rhesus macaque (rhemac2) orthologous sequences. Using the EVOLUTIONARY CONSERVED REGION BROWSER the 5 3 region adjacent to the human mir-124a2 gene was compared with their orthologous interval sequences in vertebrate species. Human and rat or mouse sequence comparisons showed a similar genomic structure within this region (high degree of conservation). To identify ECRs (red) with a greater likelihood of containing potential biological activity, we determined which conserved sequences were also present in distant vertebrates, including opossum, chicken, zebrafish and fugu. The multiple alignments revealed the presence of a conserved sequence (177 bp in length, indicated by an arrow), with 75.1% identity between human and zebrafish (Danio rerio). Sequence conservation between human (chromosome 8: ) and zebrafish (chromosome 24: ) is shown in sequence alignment FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works

7 N. N. Baroukh and E. Van Obberghen Function of mir-375 and 124a in pancreas and brain role in development [63]. mir-124a expression in both tissues (pancreas and brain) may play a role in the acquisition and maintenance of tissue identity, which is assumed to be a general function of mirna in development [5]. Organ development is a highly orchestrated process that entails precise control of gene expression (coding or noncoding genes). Interestingly, all tissues maintain a unique mirna expression profile, indicating their contribution to regulating a unique set of target genes that is specific for an organ s development and function. Functional studies implicating mir-375 and mir-124a The biological functions of most mirnas need to be defined and one challenge is to experimentally identify and validate their mrna targets. Some mirnas, including mir-375 and mir-124a, have been characterized for their functional effects. Focusing on mir-375, Poy et al. [52] elucidated the role of this pancreatic islet-specific mirna in cell lines. Overexpression of mir-375 in pancreatic cells impaired glucose-stimulated secretion of insulin with no alteration in glucose-mediated production of ATP or rise in intracellular calcium. In addition, a loss of function of mir-375 revealed an increase in glucosestimulated insulin secretion. These results show that mir-375 is implicated in the regulation of insulin secretion, which is a key determinant of blood glucose homeostasis. The authors demonstrated that myotrophin, a gene described originally in neuronal vesicle transport, is a direct target of mir-375. An interaction between mir-375 and the 3 -UTR of myotrophin mrna was shown to repress myotrophin translation and result in the inhibition of insulin secretion. In addition to its role in exocytosis control, myotrophin is also known as a transcription factor, regulating nuclear factor-kappa B in cardiomyocytes [64]. Nuclear factor-kappa B activity was shown to improve cytoskeleton organization and regulate glucose-induced insulin secretion [65,66]. These findings represent another interesting aspect of the action of myotrophin in cells and may explain the mechanism by which mir-375 also mediates insulin exocytosis. Of course, more work needs to be carried out to confirm this hypothesis. mir-375 target gene regulation is not limited to its action on mytrophin, as described by El Ouaamari et al. [67], who demonstrated that mir-375 negatively regulates 3 -phosphoinositide-dependent protein kinase-1 (PDK-1) [67]. PDK-1 is a key molecule in the phosphatidylinositol-3-kinase cascade stimulated by insulin and it is known to activate, by phosphorylation, a series of substrates involved in cell physiology [68]. Consequently, in response to insulin, mir-375 regulates phosphorylation states of proteins functioning downstream of PDK-1, such as protein kinase B and glycogen synthase kinase. Moreover, our group has shown that mir-375, through its action on phosphatidylinositol-3-kinase PDK-1 protein kinase B signalling reduces the glucose stimulatory effect on insulin gene expression and attenuates the viability and the proliferation of pancreatic b-cells [67]. Similar to our observations, others have demonstrated a downregulation of mir-375 in pancreatic cancer, pointing to an antiproliferative effect of mir-375 [69 71]. Recently, mice lacking mir-375 (375KO) were generated. Using these mice, Poy et al. [53] demonstrated that mir-375 is required for normal glucose homeostasis and influences pancreatic a- and b-cell mass by regulating a cluster of genes controlling cellular growth and proliferation. Taken together, these data demonstrate multiple implications of mir-375 on various cell functions. This is in agreement with the concept that one mirna may target many transcripts, which may confer just as many cell functions [72]. Another example is mir-124a, which was shown to knockdown transcript levels for over 174 genes in HeLa cells, and its introduction in cells promotes a neuronal-like transcript profile [7]. Blocking mir-124a activity in mature neurons selectively increases levels of some non-neuronal transcripts. Thus, it has been proposed that mir-124a suppresses non-neural genes in mammalian neurons and contributes to the acquisition and maintenance of neuronal identity [46]. Specifically, one mir-124a target is the mrna of the antineural function protein small C-terminal domain phosphatase 1 (SCP1), a protein expressed in non-neural tissues during central nervous system development and whose downregulation induces neurogenesis [73]. Interestingly, SCP1 was found among the 174 downregulated genes by mir-124a in HeLa cells [7] and among upregulated genes in mir-124a-depleted cortical neurons [46]. Computational approaches also uncovered mir-124-binding sites in the 3 -UTRs of MeCP2 and CoREST, encoding two components of the REST complex [47]. Together, these data indicate that neurogenesis requires the functions of the REST SCP1 system as well as the post-transcriptional downregulation of non-neuronal transcripts by mir-124a (also under REST control) [46]. REST and mirna are repressor components that participate in a double-negative feedback loop resulting in the stabilization and maintenance of neuronal gene expression [46,47]. More recently, Cheng et al. [74] found that mir-124 is an important regulator of the temporal FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works 6515

8 Function of mir-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen progression of neurogenesis in the subventricular zone in brains of adult mice. Consistent with another study [73], their observations provide evidence that mir-124 promotes neuronal differentiation and cell cycle exit in the subventricular zone stem cell lineage by targeting the mrna of Sox9, whose extinction abolishes the production of neurons in this system [74]. In addition, mir-124a plays an important role in the differentiation of progenitor cells to mature neurons by directly regulating polypyrimidine tract-binding protein 1, which is involved in alternative pre-mrna splicing in nonneural cells [75]. For this mirna the scenario may be even more complex, as investigations carried out on chick neural tubes have identified two other endogenous targets of mir-124a, laminin c1 and integrin b1, both highly expressed by neural progenitors, but repressed upon neural differentiation [76]. The observation that mir-124a is expressed by mature neurons throughout the brain strongly suggests that mir-124a has, in addition to its described role in neurogenesis, other physiological functions in mature neurons. In the retina, mir-124a regulates the retinol dehydrogenase 10 gene, which is known to be relevant to retinal disease [77]. Several predicted targets of mir-124a are genes involved in organ development and may act in a similar manner during retinal development. One may hypothesize that mir-124a or mutations affecting its expression would probably be detrimental for the brain and the retina and contribute to organ abnormalities. mir-124a, abundantly expressed in the pancreas, also represses the myotrophin gene, demonstrating, together with mir-375, a converging translational control of a single protein. In fact, multiple targeting of a transcript may ensure sequential mirna actions and fine tuning of gene expression [72,78]. Recently, we identified the Forkhead box a2 (Foxa2) gene product as a direct mir-124a target. Our work revealed that increasing the level of mir-124a reduced the level of the Foxa2 protein. This subsequently decreased the level of Foxa2 downstream target genes, including Pdx-1, inward rectifier potassium channel member 6.2 (Kir6.2) and sulfonylurea receptor 1 (Sur1). These changes were associated with an increase in basal free calcium, but did not change glucose- or potassiumstimulated hormone secretion [63]. Another group showed that mir-124a modulates the expression of proteins involved in the insulin exocytosis machinery [mir-124a increases the levels of synaptosomalassociated protein 25 (SNAP25), Ras-related protein Rab-3A (Rab3A) and synapsin-1a and decreases those of Rab27A and nuclear complex protein 2 homolog (Noc2)], affecting b-cell secretion [79]. These results demonstrate once again that changes in expression of a single mirna can have an impact on the expression of many genes by direct and or indirect mechanisms and can lead to alterations in cell functions [63,79]. Similar to mir-375, mir-124a is a key regulator of a transcriptional protein network in b-cells. Changes in mir-124a levels may complement the previously described actions of mir-375 by modulating the apparent sensitivity of the exocytotic machinery. mir-124a and mir-375, and other pancreas-specific mirnas, seem to downregulate a greater number of targets than previously appreciated, thereby helping to define pancreas-specific functions. Assigning a function to a mirna might only reveal the tip of the iceberg, as mir-124a overexpression in the HepG2 cell line led to a significant downregulation of many genes in categories related to cell cycle proliferation, indicating that mir-124a is also involved in cell growth control [80]. An increasing number of functions is associated with mir-124a and one of the most recently identified demonstrates its involvement in glucocorticoid responsiveness in the brain [81]. The functional roles of mir-375 and mir-124a in the pancreas and the brain are summarized in Fig. 4. Concluding remarks mirnas are a fascinating new class of molecules that are powerful regulators of gene expression and control many biological processes. Although our knowledge of these tiny molecules is growing each day, their particular characteristics (size, temporal and tissue-specific expression, mode of action) pose a real challenge to studying and elucidating mirnas functions. On the one hand, hundreds of genes are predicted to be regulated by a single mirna. On the other hand, the binding of multiple mirnas to one target gene increases the complexity of predictions [72,82]. However, scientists have widely used computational target predictions to orient lines of investigations and experimental data tend to validate such orientation. mir-375 and mir-124a share similar features; they are both specifically expressed in the pancreas and the brain, albeit at different levels. mir-375 is more abundant in islets and mir-124a is more represented in the brain. This tissue-specific coexpression suggests an overlap of function (redundancy effect or co-ordinate action). mir-375 inhibition has a dramatic effect on pancreas development [58], whereas mir-124a is upregulated during pancreas development [63] and neurogenesis [46]. Together, these findings highlight the involvement of mir-375 and mir-124a in development and their role in the establishment of organ identity. In addition, several studies have demonstrated that 6516 FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works

9 N. N. Baroukh and E. Van Obberghen Function of mir-375 and 124a in pancreas and brain Fig. 4. Schematic representation of the functional and common implications of mir-375 and mir-124a in pancreas and brain. pancreatic b-cells display patterns of gene expression overlapping with those of neuronal cells [83,84]. Moreover, it has been shown that mir-375 and mir-124a directly bind a common target, the myotrophin gene transcript, which encodes a cytoplasmic protein that induces exocytosis and hormone secretion [52,72]. The regulation of myotrophin protein by multiple mirnas provides evidence of a co-ordinated regulation. Both mirnas show an important role in endocrine function and highlight the consequences of their dysregulation on hormone release. Another interesting observation of the action of mirnas is that mirna tissue-specific expression is regulated by tissue-specific transcription factors. The islet-specific mir-375 is controlled by multiple transcription factors, such as Pdx-1 and neurogenic differentiation factor 1, both critical for b-cell development. On the basis of this observation, it is tempting to speculate that mir-375 is involved in b-cell development and that it is temporally controlled during embryogenesis by these two transcription factors. In a similar manner, the brain-specific mir-124a is under the control of REST factor, a neuronal repressor and a regulator of glucose-induced insulin secretion [85], suggesting that a balance between endocrine- and neuron-specific components needs to be reached to exhibit adequate secretory cell functions. Furthermore, like other genes, mirnas are regulated by effectors at a transcriptional level. mir-375 gene expression is negatively regulated by glucose in INS-1E cells and freshly isolated pancreatic islets of Goto-Kakizaki diabetic rats (model of type 2 diabetes); whereas mir-124a expression is increased in freshly isolated diabetic Goto-Kakizaki islets [67]. It is interesting to note that mir-375 and mir-124a regulate insulin gene expression in pancreatic b-cell lines [63,67], probably affecting a final retro-control loop of regulation. mir-375 and mir-124a are expressed in the same tissues, target a common protein, both show glucose sensitivity; yet, they are regulated differentially. They are both involved in pancreatic b-cell development and in the regulation of insulin production and secretion. It seems that mirna acts at multiple hierarchical levels of gene regulatory networks affecting cell functions, and that they are themselves regulated by environmental and or genetic factors. This multilevel regulation may allow individual mirnas to affect the gene expression programme of cells profoundly. It is clear that mirna is involved in organ development, but also in the whole process of an organism s development. Growing evidence demonstrates the vast roles played by mirnas in biological systems and how the alterations of their expression participate in the pathogenesis of human diseases. In the pancreas, b-cells are highly specialized and characterized by the exclusive ability to synthesize and release insulin according to fluctuations in circulating glucose levels. The important roles of mir-375, together with mir-124a, in regulating glucose-stimulated insulin production and secretion, and cell growth proliferation, highlight mirnas as targets for developing novel strategies to correct defective insulin secretion in some forms of type 2 diabetes. The identification of a role for mirna molecules in controlling b-cell gene expression and or b-cell functions may lead to the identification of novel pharma- FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works 6517

10 Function of mir-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen cological targets for the treatment of b-cell failure observed in diabetes. Given the increasing number of mirna sequences identified, it is interesting to investigate their implication and functional roles in metabolic disorders in vivo. A more precise picture should be given with the generation of genetically engineered animal models. Disrupting or overexpressing an mirna gene will allow roles in mammalian physiology to be assigned to each sequence [53,86 88]. Moreover, an interesting report has underlined the possible unintentional deletion of mirna during conventional gene disruption in mouse models [89]. The authors found approximately 200 cases in which mirnas may have been disturbed in mouse gene targeting models. These observations should be used to re-examine gene knockout interpretation and to investigate whether an mirna may contribute to or be responsible for the phenotype observed in vivo. Acknowledgements The authors would like to acknowledge J. Neels, I. Mothe-Satney and P. Grimaldi for their critical reading of the manuscript, suggestions and advice. There is no conflict of interest. References 1 Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, Agarwala R, Ainscough R, Alexandersson M, An P et al. (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420, Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W et al. (2001) Initial sequencing and analysis of the human genome. Nature 409, Lee RC, Feinbaum RL & Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR & Ruvkun G (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, Wienholds E, Kloosterman WP, Miska E, Alvarez- Saavedra E, Berezikov E, de Bruijn E, Horvitz HR, Kauppinen S & Plasterk RH (2005) MicroRNA expression in zebrafish embryonic development. Science 309, Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W & Tuschl T (2002) Identification of tissue-specific micrornas from mouse. Curr Biol 12, Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS & Johnson JM (2005) Microarray analysis shows that some micro RNAs downregulate large numbers of target mrnas. Nature 433, Lewis BP, Burge CB & Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets. Cell 120, Griffiths-Jones S (2004) The microrna registry. Nucleic Acids Res 32, D109 D Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M et al. (2003) A uniform system for micro RNA annotation. RNA 9, Enright AJ, John B, Gaul U, Tuschl T, Sander C & Marks DS (2003) MicroRNA targets in Drosophila. Genome Biol 5, R1. 12 John B, Enright AJ, Aravin A, Tuschl T, Sander C & Marks DS (2004) Human microrna targets. PLoS Biol 2, e Kim KS, Kim JS, Lee MR, Jeong HS & Kim J (2009) A study of micrornas in silico and in vivo: emerging regulators of embryonic stem cells. FEBS J 276, Kim S (2009) A study of micrornas in silico and in vivo. FEBS J 276, Kim S, Hwang do W & Lee DS (2009) A study of micrornas in silico and in vivo: bioimaging of micro RNA biogenesis and regulation. FEBS J 276, Lin Q, Gao Z, Alarcon RM, Ye J & Yun Z (2009) A role of mir-27 in the regulation of adipogenesis. FEBS J 276, Waldman SA & Terzic A (2009) A study of micrornas in silico and in vivo: diagnostic and therapeutic applications in cancer. FEBS J 276, Yousef M, Showe L & Showe M (2009) A study of micrornas in silico and in vivo: bioinformatics approaches to microrna discovery and target identification. FEBS J 276, Clop A, Marcq F, Takeda H, Pirottin D, Tordoir X, Bibe B, Bouix J, Caiment F, Elsen JM, Eychenne F et al. (2006) A mutation creating a potential illegitimate microrna target site in the myostatin gene affects muscularity in sheep. Nat Genet 38, Abelson JF, Kwan KY, O Roak BJ, Baek DY, Stillman AA, Morgan TM, Mathews CA, Pauls DL, Rasin MR, Gunel M et al. (2005) Sequence variants in SLITRK1 are associated with Tourette s syndrome. Science 310, Lee Y, Jeon K, Lee JT, Kim S & Kim VN (2002) MicroRNA maturation: stepwise processing 6518 FEBS Journal 276 (2009) Journal compilation ª 2009 FEBS. No claim to original French government works

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