InteractiveFly: GeneBrief

mir-1 stem loop: Biological Overview | References

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA-binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, i>miR-1, have been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis has been unknown. This study used Drosophila DM1 models to explore the role of miR-1 in cardiac dysfunction in DM1. miR-1 down-regulation in the heart was found to lead to dilated cardiomyopathy (DCM), a DM1-associated phenotype. In silico screening for miR-1 targets was combined with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. Multiplexin (Mp) was identified as a new cardiac miR-1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila. Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 might be critical in DM1-associated DCM (Souidi, 2023).

To investigate the pathophysiology and the molecular mechanisms underlying DM1, several DM1 models, both mouse, have been created The reduction in MBNL1 and stabilization of CELF1 are thought to be involved in most DM1 phenotypes. Indeed, Mbnl1 knockout mice develop muscle myotonia, weakness/wasting, and cardiac defects including dilated cardiomyopathy and heart conduction block. Mice overexpressing CELF1 in the heart show conduction abnormalities and dilated cardiomyopathy thus confirming the contribution of MBNL1 sequestration and CELF1 up-regulation to DM1 pathogenesis. Overall, the mouse models reproduced multiple DM1 features including RNA foci formation and various alternative splice defects (Souidi, 2023).

A series of inducible Drosophila DM1 lines was generated bearing UAS-iCTG constructs with 240, 480, 600, and 960 CTGs. These lines were used to model DM1 in larval somatic muscles showing not only nuclear foci formation and Mbl sequestration but also muscle hypercontraction, splitting of muscle fibers, reduced fiber size, and myoblast fusion defects leading to impaired larva mobility (Picchio, 2013). The severity of phenotypes in these Drosophila models could be correlated with repeat size (Picchio, 2013), as also observed in DM1 patients. Finally, the overexpression of Drosophila CELF1 ortholog Bru3 and attenuation of MBNL1 counterpart mbl offer further valuable models for identifying gene deregulations underlying DM1 (Souidi, 2023).

Among molecular mechanisms associated with DM1, the deregulation of miRNAs and in particular reduced levels of evolutionarily conserved muscle- and heart-specific miRNA, miR-1, has been reported in DM1 patients and in DM1 models including mouse and Drosophila (Fernandez-Costa, 2013). However, the impact of miR-1 down-regulation on DM1-associated phenotypes has not yet been analyzed (Souidi, 2023).

This study made use of Drosophila DM1 models to explore miR-1 involvement in cardiac dysfunction in DM1. It was observed that dmiR-1 level was reduced in the cardiac cells of DM1 flies and that its down-regulation in the heart led to DCM, thus suggesting that reduced dmiR-1 levels contribute to DM1-associated DCM. Among potential dmiR-1 regulated genes from in silico screening, this study identified Multiplexin (Mp)/Collagen15A1 (Col15A1)/ as a new cardiac dmiR-1 target involved in DM1. Both Mp and Col15A1 proteins were highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. Moreover, the heart-targeted overexpression of Mp was sufficient to induce DCM, whereas its attenuation rescues the DCM phenotype in DM1 flies. miR-1 and its target Mp/Col15A1 thus emerge as molecular determinants of DM1-associated DCM (Souidi, 2023).

The reduction in mammalian MBNL1 and stabilization of mammalian CELF1 are thought to be involved in most DM1 phenotypes. Indeed, Mbnl1 knockout mice develop muscle myotonia, weakness/wasting, and cardiac defects including dilated cardiomyopathy and heart conduction block. Mice overexpressing CELF1 in the heart show conduction abnormalities and dilated cardiomyopathy thus confirming the contribution of MBNL1 sequestration and CELF1 up-regulation to DM1 pathogenesis. Overall, the mouse models reproduced multiple DM1 features including RNA foci formation and various alternative splice defects (Souidi, 2023).

Myotonic dystrophy type 1 is the most common muscular dystrophy in adults. Cardiac repercussions including DCM are among the main causes of death in DM1. However, the underlying mechanisms remain poorly understood, impeding the development of adapted treatments. As was previously demonstrated, Drosophila DM1 models recapitulate all the cardiac phenotypes observed in DM1 patients and so could help gain insight into gene deregulations underlying DM1-associated DCM (Souidi, 2023).

In humans, DCM is characterized by left ventricular dilation and systolic dysfunction defined by a depressed ejection fraction. Similarly, in DCM-developing flies, the cardiac tube is enlarged and shows an increased diastolic and systolic diameter with reduced contractility. The loss of cardiac miRNAs and in particular miR-1 has already been correlated to DCM and heart failure in mice. miR-1 sequence is highly conserved between Drosophila and Human, and it is well known that it regulates genes involved in cardiac development and function including Nkx2.5, SRF, and components of WNT and FGF signaling pathways (Kura et al, 2020) and that its level is reduced in the pathological context of DM1. However, it was not known whether the low miR-1 level caused DM1-associated DCM, nor what were the downstream miR-1 targets. This study shows that two heart-targeting Drosophila DM1 models, Hand > mblRNAi and Hand > Bru3 mimicking sequestration of MBNL1 and stabilization of CELF1, respectively, developed DCM and showed a reduced expression of dmiR-1 in cardiac cells including cardiomyocytes and pericardial cells. Regarding the influence of Hand-Gal4 driven expression in pericardial cells on the DM1 heart phenotypes, previously work tested all the DM1 models using cardioblast-specific Tin-GAL4 driver. DM1 cardiac phenotypes such as conduction defects observed in the Hand > Bru3 model and DCM observed in Hand > mblRNAi and Hand > Bru3 models are observed when using Tin-Gal4 driver. These results suggest that the cardiac phenotypes observed in the DM1 Drosophila heart, including DCM, are mainly due to gene deregulations within the cardiomyocytes. Because the overexpression of CELF1 and the loss of MBNL1 also result in DCM in mice, Drosophila appears well-suited to assessing the impact of reduced miR-1 in DM1-associated DCM. One mechanism explaining why miR-1 levels fall in the DM1 context is the sequestration of MBNL1, which can no longer play its physiological role in pre-miR-1 processing into mature miR-1. This study observed reduced dmiR-1 also upon the cardiac overexpression of CELF1 ortholog Bru3. How CELF1/Bru3 impinges on miR-1 levels is not fully understood, but it was demonstrated that CELF1 could bind UG-rich miRNAs (such as miR-1) and mediate their de-adenylation and degradation by recruiting poly(A)-specific ribonuclease (PARN). Given that Drosophila DM1 models developing DCM showed markedly reduced dmiR-1 in cardiac cells, this study sought to determine whether heart-targeted attenuation of dmiR-1 was sufficient to induce DCM: dmiR-1 knockdown in the heart mimics DM1-associated DCM (Souidi, 2023).

To identify candidate dmiR-1 target genes involved in DCM in silico screening was performed for dmiR-1 seed sites in the 3'UTR regions of genes up-regulated in cardiac cells at 5 weeks of age in DM1 models developing DCM. Among 1,189 3'UTR sequences tested, 162 bore potential dmiR-1 seed sites, including the 3'UTR of Multiplexin (Mp). Mp codes for extracellular matrix protein belonging to a conserved collagen XV/XVIII family. Mp was top-ranked because of its known role in setting the size of the cardiac lumen. The embryos overexpressing Mp display an enlarged cardiac tube and conversely, Mp-/- embryos were found to present a narrower lumen with reduced contractility of the heart tube. In parallel, the mouse mutants of Mp ortholog, Col15A1, showed age-related muscular and cardiac deterioration linked to a degraded organization of the collagen matrix. This prompted an examination Mp expression in the adult fly heart and the effect of its overexpression. Using Mp specific antibody Mp was detected on the surface of the cardiac cells and found that Mp accumulated to a high level in both Hand > mblRNAi and Hand >> Bru3 DM1 lines. Whether the in silico identified dmiR-1 seed site was required for the regulation of Mp expression was examined and confirmed that Mp is a direct in vivo target of dmiR-1 in cardiac cells. As the potential binding site for human dmiR-1 is present also in 3'UTR of Col15A1 transcript it was hypothesize that Mp/Col15A1 are evolutionarily conserved dmiR-1 targets. Consistent with its role downstream of dmiR-1, Mp overexpression in the heart mimicked the dmiR-1 knockdown phenotype, leading to a significantly enlarged heart with reduced contractility. Moreover, heart-specific attenuation of Mp expression in the Hand > Bru3 DM1 context reduced heart dilation and rescued DCM phenotype in aged flies, thus demonstrating that increased Mp levels contribute to DCM observed in DM1 flies. Previous reports revealed increased expression levels of different collagens associated with DCM in both animal models and patients. This study reports evidence that Col15A1 is specifically up-regulated at both transcript and protein levels in cardiac samples from DM1 patients and in particular in those with DCM, with down-regulation of miR-1. Altogether, the observations that Col15A1 expression level is abnormally elevated in DCM-developing DM1 patients and that attenuation of its Drosophila ortholog Mp could ameliorate the DCM phenotype suggest that Col15A1 could be a novel therapeutic target in DM1 (Souidi, 2023).

A large number of genes have so far been implicated in DCM, attesting to the complex molecular origin of this cardiac condition. For example, in Drosophila, DCM was observed in mutants of genes encoding contractile and structural muscle proteins such as Troponin I (TpnI), Tropomyosin 2 (Tm2), δ-sarcoglycan and Dystrophin but also associated with deregulations of EGF, Notch, Cdc42 and CCR4-Not signaling pathway components. In humans, DCM-causing mutations were also identified in a large number of genes including those encoding cytoskeletal proteins such as FLNC, nuclear membrane protein LMNA or involved in sarcomere stability (Titin, TNNT2, MYH7, TPM1) but also RNA-binding protein RBM20 (Souidi, 2023).

This study focused on DCM associated with DM1. A previous study on a mouse model overexpressing CELF1 and developing DCM, identified down-regulation of Transcription factor A mitochondrial (Tfam), Apelin (Apln), and Long-chain fatty acid-CoA ligase 1 (Acsl1) as potentially associated with DCM. It was suggested that CELF1 might regulate their mRNA stability by binding to their 3'UTR regions and causing destabilization and degradation of their transcripts. In this DCM-developing mouse DM1 model, Col15a transcripts were elevated, but the role of Col15a in DCM was not analyzed. Using Drosophila DM1 models with a DCM phenotype, this study identified up-regulation of Col15A1 ortholog Mp as a molecular determinant of DM1-associated DCM. Reduced miR-1 levels were detected in DCM-developing DM1 cardiac cells to the up-regulation of Mp, establishing that Mp is an in vivo target of dmiR-1 (Souidi, 2023).

Importantly, these findings show that in DM1 patients, Collagen 15A1 is up-regulated in the hearts of patients with DCM. In DM1 patients, the DCM phenotype appears several years after onset and is less common than the conduction system defects and arrhythmias. However, DCM is frequently associated with poor prognosis and indication for heart transplant (Souidi, 2023).

In summary, this study report2 evidence for the importance of miR-1-dependent gene deregulations in DM1, and Mp was identified as a new miR-1 target involved specifically in DM1-associated DCM. Mbl depletion and Bru3 up-regulation in the heart were shown to have overlapping impacts on DM1 pathogenesis, both leading to reduced miR-1, up-regulation of Mp, and so to DCM (Souidi, 2023).

The conclusion is that in a physiological context, Mp level is moderately triggered by Mbl-dependent regulation of dmiR-1 processing and Bru3-dependent regulation of dmiR-1 stability. However, in the DM1 context, Mbl is sequestered in nuclear foci while Bru3 levels increase, leading to a reduced dmiR-1 and the up-regulation of its target gene Mp. Considering the known role of Mp as a positive regulator of cardiac lumen size, Mp accumulation in the adult heart would also be expected to promote heart tube enlargement, leading to the DCM phenotype. Whether like in embryos this Mp function involves the Slit/Robo signaling pathway remains to be investigated, but the finding that Robo2 is among identified miR-1 targets up-regulated in DCM-developing DM1 flies upports this possibility. Finally, the fact that Mp ortholog Col15A1 is highly elevated in cardiac samples from DM1 patients with DCM and that reducing Mp rescues the DCM phenotype in DM1 fly model suggests that Mp/Col15A1 could be an attractive diagnostic and/or therapeutic target for DM1-associated DCM (Souidi, 2023).

The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1

miR-1> is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of Drosophila miR-1 produces defects in somatic muscle and embryonic heart development, which have been partly attributed to miR-1 directly targeting Delta to decrease Notch signaling. This study shows that overexpression of miR-1 in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential miR-1 targets revealed that miR-1 directly regulates the 3'UTR of the E3 ubiquitin ligase Nedd4. Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in Drosophila. Larval fly hearts overexpressing miR-1 have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that miR-1 and Nedd4 act together in the formation and actin-dependent patterning of the fly heart. Importantly, it was found that the biochemical and genetic relationship between miR-1 and the mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus, miR-1-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart (Zhu, 2017).

Unexpectedly, overexpression of miR-1 in the anterior-posterior (AP) organizer of the wing disc results in a dose-dependent loss of L3 vein structures, consistent with de-repression of Notch or weakening of a regulatory mechanism that dampens the Notch signal. Using genetic techniques, it was determined that the loss of the distal aspect of L3 could be phenocopied by reducing the gene dose of Notch co-repressors or Nedd4; in the case of Nedd4, the regulation by miR-1 was direct. An expanded model is proposed in which miR-1 expression in the AP organizer has complex effects on Notch signaling owing to its regulation of ligand availability and receptor trafficking. As lower levels of miR-1 expression (18°C) caused wing-vein thickening and tortuosity, and higher levels (22°C) caused vein loss, Delta and Nedd4 may be differentially sensitive to miR-1 regulation, although these studies were not designed to address this issue. It is also possible that indirect effects, such as reductions in Nedd4-mediated ubiquitylation of positive effectors of the Notch receptor (e.g. Deltex) or perturbations in Delta-mediated cis-inhibition, contributed to the de-repression of Notch in the wing-based assay system (Zhu, 2017).

The findings in the mammalian heart indicate that the genetic and biochemical interaction between miR-1 and Nedd4l is physiologically relevant and may provide developmental or tissue-specific regulation of Nedd4l in the myocardium. It is speculated that the additional bands observed on western blots of heart lysates using an anti-Nedd4L antibody might result from post-translational modifications, because Nedd4L can autoregulate its stability through ubiquitylation of its HECT domain. Alternatively, they might represent heart-specific splice variants, because tissue-specific isoforms of Nedd4L have been found in the heart and the liver (Zhu, 2017).

Importantly, although miR-1-mediated reductions in Nedd4 activity caused wing-vein phenotypes induced by Notch, miR-1-mediated dysregulation of Nedd4L in the heart likely affects proteins outside the Notch pathway. Indeed, protein microarrays comparing human Nedd4 with human Nedd4L, suggest that Nedd4L (also known as Nedd4-2) preferentially targets ion channels, whereas Nedd4 targets are enriched for signaling pathways. Thus, in the heart, where miR-1 and murine Nedd4L are both expressed, their genetic and biochemical interaction might influence the excitability and connectivity of cardiomyocytes. Indeed, susceptibility to cardiac arrhythmias and sudden death in humans is associated with six genes that encode ion channels (SCN5A, KCNQ1, KCNH2, KCNE1, KCNE2 and RYR2). Murine Nedd4L regulates the cell-surface densities of the sodium channel, the voltage-gated type V alpha subunit (Scn5a), the potassium voltage-gated channel, KQT-like subfamily member 1 (Kcnq1) and the human Ether-a-go-go-related (KCNH2, previously hERG) channel. Furthermore, miR-1 directly regulates human KCNJ2, a channel that maintains cardiac resting potential. These findings suggest that the regulation of murine Nedd4l by miR-1 contributes to some of the electrophysiological abnormalities seen in miR-1 null mice. It would be interesting to determine whether Nedd4L is dysregulated in the heart after an infarction or under ischemic conditions, when miR-1 is upregulated and fatal cardiac dysrhythmias are common (Zhu, 2017).

MicroRNA directly enhances mitochondrial translation during muscle differentiation.

MicroRNAs are well known to mediate translational repression and mRNA degradation in the cytoplasm. Various microRNAs have also been detected in membrane-compartmentalized organelles, but the functional significance has remained elusive. This study reports that miR-1, a microRNA specifically induced during myogenesis, efficiently enters the mitochondria where it unexpectedly stimulates, rather than represses, the translation of specific mitochondrial genome-encoded transcripts. This positive effect requires specific miR:mRNA base-pairing and Ago2, but not its functional partner GW182, which is excluded from the mitochondria. Evidence is provided for the direct action of Ago2 in mitochondrial translation by crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq), functional rescue with mitochondria-targeted Ago2, and selective inhibition of the microRNA machinery in the cytoplasm. These findings unveil a positive function of microRNA in mitochondrial translation and suggest a highly coordinated myogenic program via miR-1-mediated translational stimulation in the mitochondria and repression in the cytoplasm. (Zhang, 2014)

The Gcm/Glide molecular and cellular pathway: new actors and new lineages

In Drosophila, the transcription factor Gcm/Glide plays a key role in cell fate determination and cellular differentiation. In light of its crucial biological impact, major efforts have been put for analyzing its properties as master regulator, from both structural and functional points of view. However, the lack of efficient antibodies specific to the Gcm/Glide protein precluded thorough analyses of its regulation and activity in vivo. In order to relieve such restraints, an epitope-tagging approach was designed to "FLAG"-recognize and analyze the functional protein both in vitro (exogenous Gcm/Glide) and in vivo (endogenous protein). This study (1) revealed a tight interconnection between the small RNA and the Gcm/Glide pathways. AGO1 and miR-1 are Gcm/Glide targets whereas miR-279 negatively controls Gcm/Glide expression (2) identified a novel cell population, peritracheal cells, expressing and requiring Gcm/Glide. Peritracheal cells are non-neuronal neurosecretory cells that are essential in ecdysis. In addition to emphasizing the importance of following the distribution and the activity of endogenous proteins in vivo, this study provides new insights and a novel frame to understand the Gcm/Glide biology (Laneve, 2013).

The pivotal role of Gcm in orchestrating cell fate specification involves a stringent regulation of its pathway, where multi-level control mechanisms converge. The FLAG-tagged Gcm tool constitutes a useful sensor to characterize post-transcriptional regulatory events (Laneve, 2013).

First, since several bands appear on Western Blots revealing the Gcm–FLAG product and the GCMa vertebrate protein is regulated by phosphorylation, which seems to affect its activity and stability, putative Gcm phosphorylation sites were sought in silico, and several were found. Then cell extracts overexpressing Gcm–FLAG (Act-Gal4) were inclubated with calf intestine phosphatase (CIP) prior to SDS-PAGE and observed a diminished band number and density, thus validating the Gcm phosphorylation prediction. Future studies will assess whether phosphorylation contributes to Gcm stability/activity (Laneve, 2013).

Second, miRNAs are endogenously expressed noncoding RNAs that represent key post-transcriptional regulators of gene expression. An online search carried for miRNAs predicted to target the gcm 3'UTR by miRanda, TargetScan and microCosm databases indicated miR-279/286/996 was the main family of putative effector miRNAs. miR-279, the most characterized member in Drosophila, was initially described for suppressing the formation of heterotopic olfactory neurons and as a component of a complex regulatory circuit orchestrated by the pleiotropic transcription factor Prospero (Pros). Finally, it was also shown to regulate the JAK/STAT pathway, driving rest: activity rhythms and modulating the response to morphogen gradients (Laneve, 2013).

To verify a possible role of miR-279, a sensor construct was developed in which the 3'UTR of gcm was inserted downstream to the FLAG-encoding cassette. Such construct (or its site-specific mutant derivative) was co-transfected in S2 cells along with a vector efficiently over-expressing the miRNA by mean of an Act-Gal4 driver (Laneve, 2013).

Western blot analysis demonstrates a specific downregulation exerted by miR-279 on gcm. Such modulation, abolished in a construct carrying a mutant target site, is specifically mediated by the gcm 3'UTR. Interestingly, ectopic expression of miR-286, a second member of the miR-279 family, diverging from mir-279 at the level of its 3′ sequence, failed to silence Gcm–FLAG. Since the 3′ region of microRNAs is known to play a role in the specificity of microRNA-target recognition, this accounts for the selectivity of gcm post-translational control by miR-279 in vitro (Laneve, 2013).

Finally, this study provides in vivo evidence for miRNA-mediated gcm regulation: (1) miR-279 was co-expressed in the embryonic neural territory (sca-Gal4 driver) in the presence of a Luciferase (Luc) reporter carrying its own 3′UTR or the gcm 3′UTR. By qRT-PCR analysis, a specific downregulation mediated by miR-279 on Luc RNA expression was detected only in the latter case, which parallels the in vitro data (2), an analogous gain-of-function strategy was used to analyze the expression of the endogenous gcm (gcm–FLAG^BAC) in the presence or in the absence of overexpressed miR-279 at RNA and protein level by qRT-PCR and Western blot, respectively. Both approaches show a negative effect, thus validating miR-279 for targeting gcm in vivo in neurogenic territories. In sum, this study identified miR-279 as a negative modulator of gcm both in cell cultures and in embryos. The in vivo relevance of miR-279 on the gliogenic Gcm-dependent pathway was probed. Interestingly, when miR-279 was overexpressed (sca-Gal4) in hypomorphic gcm animals that contain reduced number of glia, a further decrease of glial cell number was expected, but the opposite result was found, and this was obtained upon using two allelic combinations. Thus, further regulatory steps compensate for the effects on gcm, thereby highlighting the complex network linked to small RNAs (Laneve, 2013).

Prompted by the above results, it was asked whether the interplay between Gcm and small RNA metabolism is more pervasive than emerged to date, upon establishing the role of Gcm in this pathway miR-1 was identified in an in vivo DAM ID screen and several GBSs were identified in the region upstream to the miR-1 transcription unit. miR-1 expression was evaluated in vivo upon hs-Gal4 driven Gcm activation and an up-regulation was found, where Gcm plays a crucial role for the development of blood cells. The gcm–Gal4 driver was used to overexpress Gcm and, to restrict the effects to the hemocyte precursor anlagen, and embryonic stages 5–9 were analyzed, when gliogenesis still has to start. An increase of miR-1 levels was confirmed and no effect was detected when Gcm expression was triggered by the sca-Gal4 driver. Interestingly, other miRNAs were identified as potential targets in the DAM ID screen; however their expression did not increase upon Gcm forced expression with any of the used drivers. This further validates the data obtained with miR-1 and suggests that the other miRNAs may work at different developmental stages. Finally, constitutive expression of Gcm in S2 cells, where miR-1 is not endogenously expressed, does not induce miR-1expression, likely due to the absence of appropriate co-activators. Overall, these data call for a cell-specific role of Gcm. Future studies will dissect the role of miR-1 in the Gcm pathways (Laneve, 2013).

Finally, the DAM ID screen also identified Argonaute 1 (AGO1) and an in silico inspection revealed the occurrence of several predicted GBSs mapping upstream to the AGO1 transcription unit. AGO1 a member of the Argonaute/PIWI protein family, involved in small RNA-mediated gene regulation. In Drosophila, AGO1 plays a specific role in miRNA biogenesis and function: it directs the unwinding of the intermediate duplex RNA generated during microRNA biosynthetic pathway and it selects one strand as mature microRNA loaded into the RISC (RNA-induced silencing complex) effector complex. AGO1 is broadly expressed in the embryo as well as in the imaginal discs and this, combined with the well known pleiotropic roles exerted by microRNA, accounts for its involvement in multiple developmental pathways. Interestingly, a genetic screen over a sensitized gcm background identified AGO1 as a putative interactor of gcm (Laneve, 2013).

In short, the Drosophila notum carries a fixed number of sensory organs called bristles. gcm^Pyx/+ flies ectopically express gcm in the larval notum, which triggers the differentiation of supernumerary bristles. gcm^Pyx/+ females show, in average, 18,5 bristles instead of the 11/heminotum typical of WT animals. This phenotype constituted the readout to identify putative gcm interacting genes in a dosage sensitive screen. The AGO1 mutation acts as a suppressor of the gcm^Pyx phenotype in double heterozygous conditions (genotype: gcm^Pyx/AGO1⁰⁸¹²¹), showing a positive genetic interaction with gcm (Laneve, 2013).

A microarray profiling also suggested AGO1 as a possible Gcm target in the neurogenic territories; however its upregulation upon Gcm forced expression as well as in gcm loss of function mutations made unclear the role of Gcm. Therefore (Act-Gal4 driver) Gcm or its tagged derivative was expressed in S2 cells, in which AGO1 is endogenously expressed, and this was found to induce AGO1 accumulation, reflecting and following the temporal accumulation of Gcm–FLAG itself. To further corroborate these data, increasing amounts of the Gcm–FLAG-expressing construct were transfected, and the amount of AGO1 was analyzed at the time-points of Gcm–FLAG highest expression (48–72 h after transfection). This revealed a clear correlation between the quantity of Gcm–FLAG and the expression of AGO1 . Furthermore, to exclude any unspecific influence of the FLAG epitope on target recognition, the same assay employing an untagged version of Gcm. Finally, it was verified AGO1 as a Gcm target in vivo: the UAS–gcm transgene was expressed under the control of the heat-shock (hs) inducible driver hs-Gal4 in Drosophila embryos: the Western blot demonstrates a clear up-regulation of AGO1 upon Gcm ectopic expression. In sum, this study provides significant genetic and molecular evidence for a positive interaction between AGO1 and gcm suggesting a mechanism of direct targeting. Interestingly, no modulation of AGO1 was observed upon Gcm forced expression in the nervous system using sca-Gal4. Since Gcm is required in different cell types, more efforts are required to clarify in which functional pathway Gcm controls AGO1. The data provide nevertheless first evidence for a cell-specific factor modulating the expression of AGO1. Indeed, the widespread distribution and function of miRNAs suggest a complex regulatory network controlling AGO1 expression/activity: Gcm can be proposed as a cell-specific component of the small RNA cascade (Laneve, 2013).

It is concluded that the transcriptional activator Gcm constitutes a paradigmatic example of master regulator, acting as a pivotal cell fate determinant and differentiation factor during Drosophila embryogenesis. It is therefore crucial to outline a trustworthy picture of Gcm biology, from expression to function. The present study provides the first characterization of Gcm at the protein level and reports a large set of data on gcm function, regulation and expression, collected both in vitro and in vivo. Specifically, small RNA metabolism was identified as an important element of the Gcm pathway and a novel gcm-dependent cell type essential in development was identified (Laneve, 2013).

Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients

Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG repeats in the 3' untranslated region of the DMPK gene. Several missplicing events and transcriptional alterations have been described in DM1 patients. A large number of these defects have been reproduced in animal models expressing CTG repeats alone. Earlier studies have also reported miRNA dysregulation in DM1 patients. This study uses a Drosophila model to investigate miRNA transcriptome alterations in the muscle, specifically triggered by CTG expansions. Twenty miRNAs were found to be differentially expressed in CTG-expressing flies. Of these, 19 are down-regulated, whereas 1 is up-regulated. This trend was confirmed for those miRNAs conserved between Drosophila and humans (miR-1, miR-7 and miR-10) in muscle biopsies from DM1 patients. Consistently, at least seven target transcripts of these miRNAs are up-regulated in DM1 skeletal muscles. The mechanisms involved in dysregulation of miR-7 include a reduction of its primary precursor both in CTG-expressing flies and in DM1 patients. Additionally, a regulatory role for Muscleblind (Mbl) is also suggested for miR-1 and miR-7, as these miRNAs are down-regulated in flies where Mbl has been silenced. Finally, the physiological relevance of miRNA dysregulation is demonstrated for miR-10, since over-expression of this miRNA in Drosophila extends the lifespan of CTG-expressing flies. Taken together, these results contribute to understanding of the origin and the role of miRNA alterations in DM1 (Fernandez-Costa, 2013).

Circadian rhythm-dependent alterations of gene expression in Drosophila brain lacking fragile X mental retardation protein

Fragile X syndrome is caused by the loss of the FMR1 gene product, fragile X mental retardation protein (FMRP). The loss of FMRP leads to altered circadian rhythm behaviors in both mouse and Drosophila; however, the molecular mechanism behind this phenomenon remains elusive. This study performed a series of gene expression analyses, including of both mRNAs and microRNAs (miRNAs), and identified a number of mRNAs and miRNAs (miRNA-1 and miRNA-281) with circadian rhythm-dependent altered expression in dfmr1 mutant flies. Identification of these RNAs lays the foundation for future investigations of the molecular pathway(s) underlying the altered circadian rhythms associated with loss of dFmr1 (Xu, 2023).

Consistent with the notion that FMRP is a translational regulator, this study did not detect any change of gene expression in a large number of genes at the mRNA level. Interestingly, a third of the mRNAs that are altered either at CT00 or CT12 display the change only at one time point, which reflects a circadian rhythm-dependent alteration. How these changes could contribute to the altered circadian rhythms through the loss of dFmr1 warrants further investigation. It is also possible that the observed changes could be a direct consequence of a defect in regular sleep pattern rather than a direct consequence of the molecular clock, which would need further investigation. It would also be interesting to test whether dFMR1 associates with these mRNAs in a circadian rhythm-dependent manner, which has not been explored before. Finally, the biological functions of most mRNAs identified in this study are still unknown, and it might prove fruitful to examine their roles in circadian rhythms in general, as well (Xu, 2012).

Among the genes identified in this study, Neurochondrin was shown involved in the regulation of MCHR1 signaling, and play a role in modulating melanin-concentrating hormone-mediated functions in vivo, including neuroendocrine, behaviors and circadian output. Neurochondrin could also interact with a subset of group I mGluRs, which has been implicated in fragile X syndrome. It would be interesting to determine whether Fmrp could directly bind to Neurochondrin mRNA and regulate its expression (Xu, 2012).

This study also examine the profiles of all the known microRNAs in the context of circadian rhythms. In particular, the loss of dFmr1 led to the circadian rhythm-dependent alteration of miR-1 and miR-281 expression. This finding is particularly intriguing, since most of the previously published works on dFmr1 did not use entrained flies. These results indicate that dFmr1 could play a role in modulating expression and biogenesis in circadian rhythms. Since dFmr1 expression remains constant throughout the circadian cycle, it would be interesting to identify the protein(s) that could dynamically interact with dFMR1 and be involved in the modulation of the miRNA pathway. More importantly, further investigation is required into whether there are such alterations in terms of miRNA processing in mammals (Xu, 2012).

In summary, systematic profiling of both mRNA and miRNA was performed in both wild-type and dfmr1 mutant fly heads, and a subset of mRNAs and miRNAs was identified that display circadian rhythm-dependent altered expression in dfmr1 mutant flies, which will provide the foundation for future investigations into the molecular pathway(s) underlying the altered circadian rhythms caused by the loss of dFmr1 (Xu, 2012).

Tinman/Nkx2-5 acts via miR-1 and upstream of Cdc42 to regulate heart function across species

Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, this study found that the Rho-GTPase Cdc42/a cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart-specific interference with Cdc42 function is sufficient to cause these same defects. This study also identified K(+) channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42-Tinman interaction. To determine whether a Cdc42-Nkx2-5 interaction is conserved in the mammalian heart, compound heterozygous mutant mice were examined, and conduction system and cardiac output defects were found. In exploring the mechanism of Nkx2-5 interaction with Cdc42, it was demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. It is concluded that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1 (Quab, 2011).

Functions of miR-1 orthologs in other species

Dual role of miR-1 in the development and function of sinoatrial cells

miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. This study investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). mESCs were generated either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166(+) SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating I(f) currents; miR1OE SAN-like cells instead have only fast-activating I(f) with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and I(f) alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, this study demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate (Benzoni, 2021).

Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system

Dysregulation of intracellular trafficking system plays a fundamental role in the progression of cardiovascular disease. Since Up-regulation of miR-1 contributes to arrhythmia, this study sought to elucidate whether intracellular trafficking contributes to miR-1-driven arrhythmia. By performing microarray analyses of the transcriptome in the cardiomyocytes-specific over-expression of microRNA-1 (miR-1 Tg) mice and the WT mice, it was found that these differentially expressed genes in miR-1 Tg mice were significantly enrichment with the trafficking-related biological processes, such as regulation of calcium ion transport. Also, the qRT-PCR and western blot results validated that Stx6, Braf, Ube3a, Mapk8ip3, Ap1s1, Ccz1 and Gja1, which are the trafficking-related genes, were significantly down-regulated in the miR-1 Tg mice. Moreover, Stx6 was decreased in the heart of mice after myocardial infarction and in the hypoxic cardiomyocytes, and Stx6 was further confirmed is a target of miR-1. Meanwhile, knockdown of Stx6 in cardiomyocytes resulted in the impairments of PLM and L-type calcium channel, which leads to the increased resting ([Ca(2+)](i)). On the contrary, overexpression of Stx6 attenuated the impairments of miR-1 or hypoxia on PLM and L-type calcium channel. Thus, this study reveals that trafficking-related gene Stx6 may regulate intracellular calcium and is involved in the occurrence of cardiac arrhythmia, which provides new insights in that miR-1 participates in arrhythmia by regulating the trafficking-related genes and pathway (Su, 2017).

The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation

Understanding the molecular mechanisms that regulate cellular proliferation and differentiation is a central theme of developmental biology. MicroRNAs (miRNAs) are a class of regulatory RNAs of approximately 22 nucleotides that post-transcriptionally regulate gene expression. Increasing evidence points to the potential role of miRNAs in various biological processes. Thiw study showed that miRNA-1 (miR-1) and miRNA-133 (miR-133), which are clustered on the same chromosomal loci, are transcribed together in a tissue-specific manner during development. miR-1 and miR-133 have distinct roles in modulating skeletal muscle proliferation and differentiation in cultured myoblasts in vitro and in Xenopus laevis embryos in vivo. miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), a transcriptional repressor of muscle gene expression. By contrast, miR-133 enhances myoblast proliferation by repressing serum response factor (SRF). These results show that two mature miRNAs, derived from the same miRNA polycistron and transcribed together, can carry out distinct biological functions. Together, these studies suggest a molecular mechanism in which miRNAs participate in transcriptional circuits that control skeletal muscle gene expression and embryonic development (Chen, 2006).

Search PubMed for articles about Drosophila miR-1

Benzoni, P., Nava, L., Giannetti, F., Guerini, G., Gualdoni, A., Bazzini, C., Milanesi, R., Bucchi, A., Baruscotti, M., Barbuti, A. (2021). Dual role of miR-1 in the development and function of sinoatrial cells. J Mol Cell Cardiol, 157:104-112 PubMed ID: 33964276

Chen, J. F., Mandel, E. M., Thomson, J. M., Wu, Q., Callis, T. E., Hammond, S. M., Conlon, F. L., Wang, D. Z. (2006). The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet, 38(2):228-233 PubMed ID: 16380711

Fernandez-Costa, J. M., Garcia-Lopez, A., Zuniga, S., Fernandez-Pedrosa, V., Felipo-Benavent, A., Mata, M., Jaka, O., Aiastui, A., Hernandez-Torres, F., Aguado, B., Perez-Alonso, M., Vilchez, J. J., Lopez de Munain, A., Artero, R. D. (2013). Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients. Hum Mol Genet, 22(4):704-716 PubMed ID: 23139243

Laneve, P., Delaporte, C., Trebuchet, G., Komonyi, O., Flici, H., Popkova, A., D'Agostino, G., Taglini, F., Kerekes, I., Giangrande, A. (2013). The Gcm/Glide molecular and cellular pathway: new actors and new lineages. Dev Biol, 375(1):65-78 PubMed ID: 23276603

Picchio, L., Plantie, E., Renaud, Y., Poovthumkadavil, P., Jagla, K. (2013). Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression. Hum Mol Genet, 22(14):2795-2810 PubMed ID: 23525904

Qian, L., Wythe, J. D., Liu, J., Cartry, J., Vogler, G., Mohapatra, B., Otway, R. T., Huang, Y., King, I. N., Maillet, M., Zheng, Y., Crawley, T., Taghli-Lamallem, O., Semsarian, C., Dunwoodie, S., Winlaw, D., Harvey, R. P., Fatkin, D., Towbin, J. A., Molkentin, J. D., Srivastava, D., Ocorr, K., Bruneau, B. G., Bodmer, R. (2011). Tinman/Nkx2-5 acts via miR-1 and upstream of Cdc42 to regulate heart function across species. J Cell Biol, 193(7):1181-1196 PubMed ID: 21690310

Souidi, A., Nakamori, M., Zmojdzian, M., Jagla, T., Renaud, Y. and Jagla, K. (2023). Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1. EMBO Rep 24(4): e56616. PubMed ID: 36852954

Su, X., Liang, H., Wang, H., Chen, G., Jiang, H., Wu, Q., Liu, T., Liu, Q., Yu, T., Gu, Y., Yang, B., Shan, H. (2017). Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system. Sci Rep, 7:46259 PubMed ID: 28397788

Xu, S., Poidevin, M., Han, E., Bi, J. and Jin, P. (2012). Circadian rhythm-dependent alterations of gene expression in Drosophila brain lacking fragile X mental retardation protein. PLoS One 7: e37937. PubMed ID: 22655085

Zhang, X., Zuo, X., Yang, B., Li, Z., Xue, Y., Zhou, Y., Huang, J., Zhao, X., Zhou, J., Yan, Y., Zhang, H., Guo, P., Sun, H., Guo, L., Zhang, Y., Fu, X. D. (2014). MicroRNA directly enhances mitochondrial translation during muscle differentiation. Cell, 158(3):607-619 PubMed ID: 25083871

Zhu, J. Y., Heidersbach, A., Kathiriya, I. S., Garay, B. I., Ivey, K. N., Srivastava, D., Han, Z., King, I. N. (2017). The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1. Development, 144(5):866-875 PubMed ID: 28246214

date revised: 10 November 2024

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