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19 publications mentioning dme-mir-124

Open access articles that are associated with the species Drosophila melanogaster and mention the gene name mir-124. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

[+] score: 845
Moreover, as well-conserved targets were expressed at overall higher absolute levels than poorly-conserved targets in miR-124+ cells, we conclude that a dominant feature of the miR-124 target network has selected for substantial co -expression of the miRNA and its targets, perhaps to fine-tune their levels. [score:13]
Nevertheless, comparison of gene expression in mir-124 -expressing cells from wildtype and mir-124 mutant animals revealed strong enrichment in miR-124 target sites amongst upregulated transcripts, revealing the impact of miR-124 on neuronal gene expression [9]. [score:12]
Loss of miR-124 broadly upregulated its direct targets but did not support the proposed mutual exclusion mo del, as its functional target genes were relatively highly expressed in neurons. [score:11]
Our studies of Drosophila mir-124 demonstrate that its loss is compatible with grossly normal neural development and differentiation, despite broad changes in gene expression and global upregulation of direct miR-124 targets. [score:10]
In addition, miR-124 provided one of the first illustrations of spatially anticorrelated expression of a miRNA and its targets [15], and was exploited for analysis of Ago-bound target transcripts [16]– [19] and direct identification of Ago-bound target sites [20]. [score:10]
We also did not observe overall enrichment for epidermal genes amongst all upregulated genes (thus including both direct and indirect effects, Table S2), and only a few transcripts with miR-124 targets were absent in wild-type miR-124:DsRed+ cells and now present in mir-124 mutant cells (17/204 putative targets, but only 2 of these bore conserved sites; Table S1). [score:10]
Furthermore, while we could confirm that mutual exclusion with epidermal genes is clearly a feature of the target network selected by Drosophila miR-124, it does not seem to be a major determinant in directing neuronal-specific programs of gene expression, since epidermal genes were not overall substantially upregulated in the absence of the miR-124. [score:9]
Based on the data in Table S1, this series of worksheets summarizes GO term enrichments found in “all” up- and down-regulated genes as well as in just upregulated genes bearing miR-124 target sites; the latter were also separated on the basis of miR-124 site conservation. [score:9]
Moreover, well-conserved miR-124 targets were generally more highly expressed than poorly-conserved targets, even in wild-type miR-124 -expressing cells (Figure 7B). [score:9]
We conclude that evolutionary selection of miR-124 target sites in miR-124 -expressing cells is biased for transcripts that accumulate to above-average levels, even though the presence of miR-124 target sites clearly decreases the endogenous levels of these target transcripts (Figure 6E–6G). [score:9]
There were certainly individual miR-124 targets that are expressed and/or function in epidermal development, but this was not an overall trend amongst derepressed miR-124 targets (Figure 6E–6G). [score:8]
Few of these contain miR-124 target sites, and all of these are lowly-expressed at best, suggesting that these differences do not contribute substantially to direct changes in gene expression in mir-124 mutants. [score:8]
Moreover, derepression of direct targets accounted for a substantial proportion of the most deregulated genes in mir-124 mutants, since 24/59 genes upregulated >2-fold with p-value<0.05 bore miR-124 seed sites (Table S1). [score:8]
The impact of endogenous Drosophila miR-124 on neuronal gene expressionThe Drosophila system has been critical for elucidating fundamental features of miRNA target recognition in animals [3], [15], [46], [61]– [63], and for studying specific miRNA-target interactions that mediate phenotype [64]. [score:7]
Cell-autonomous misregulation of direct targets in mir-124 mutantsHaving established a variety of clear phenotypes in mir-124 mutants, we wished to query changes in gene expression in the mutant cells. [score:7]
Here, miR-124 directly targets the transcription factor Sox9, which maintains TAs and is downregulated during neural differentiation [24]. [score:7]
We infer from these gene expression patterns that the derepression of the miR-124 target network, impedes the normal transition of gene expression from neuroblasts to differentiated neurons in mir-124 mutants. [score:7]
Transcriptome analysis revealed strong enrichment of direct miR-124 targets amongst genes upregulated in mir-124-mutant cells. [score:7]
In any case, these data provide clear evidence that the derepression of direct miR-124 targets is the major determinant causing gene upregulation in mir-124 mutant cells. [score:7]
These gene lists overlapped rather poorly with direct miR-124 targets, and that the number of direct targets in the neuroblast and neuronal clusters was comparable (51 and 74, respectively). [score:7]
For example, studies of miR-124 yielded the first demonstration of the downregulation of hundreds of direct targets detected by transcriptome analysis, and that this activity was driven by the miRNA seed region [14]. [score:7]
Loss of miR-124 further correlated with increased activity of other neural miRNAs and the neural translational regulator Pumilio, and had the net effect of impairing transition from the neuroblast to neuronal gene expression signature. [score:6]
miR-124 targets exhibited a strong trend to be amongst the more highly expressed genes compared to non -targeted transcripts; this was true not only in the mir-124 mutant but also in wildtype (Figure 7B and Figure S7). [score:6]
Cross-regulatory effects of mir-124 loss on other modes of RNA–based regulationOne may speculate that dysfunction of miRNAs, which have large networks of targets, may trigger global changes in other modes of gene regulation. [score:6]
Nevertheless, recently-evolved miR-124 target sites exerted palpable regulatory impact in the intact animal, since transcripts with such sites were detectably shifted in their expression relative to background. [score:6]
We further analyzed the glial marker Repo, which was reported as a direct miR-124 target with anti-correlated expression [15], [33], but its pattern was not substantially altered (Figure 3F, 3G). [score:6]
We could indeed validate many such targets as being upregulated in miR-124:dsRed+ cells isolated from mir-124 mutants by qPCR (Figure 6C). [score:6]
We chose this as a temporal window that was late enough to permit the full pattern of miR-124 expression to be established, but putatively early enough to minimize highly indirect changes in gene expression (i. e., that might arise during the remainder of embryogenesis from 16–22 hrs). [score:6]
To complement these quantitative data with cellular data, we examined the expression of the miR-124 target Repo [33], which we confirmed to be directly responsive to miR-124 (Figure 6H). [score:6]
We analyzed a knockout of Drosophila mir-124, which is conserved in sequence and neuronal expression across the animal kingdom, and predicted to have hundreds of mRNA targets. [score:6]
Although deregulation of other targets likely contributes to the observed mir-124 mutant phenotypes, the similarity in electrophysiological defects upon deletion of miR-124 and overactivity of retrograde BMP signaling suggests that deregulation of this pathway may contribute to aberrant physiology of mir-124 mutant synapses. [score:5]
Amongst motifs associated with decreased gene expression in mir-124 mutants, the top motif corresponds to the Pumilio site; others motifs include the seeds of K box family miRNAs, miR-10-5p, and an orphan motif (AUGCAAA) with several hundred conserved matches (defined by TargetScan). [score:5]
Expression of Drosophila mir-124 was confidently detected only in CNS, but did not exclude potential expression in the PNS. [score:5]
We generated a transcriptional reporter of mir-124 that recapitulated the CNS expression of endogenous pri-mir-124, and used this to purify mir-124 -expressing cells from stage-matched wild-type and mir-124-mutant embryos. [score:5]
We first tested whether we could reproduce the mutual exclusivity principle amongst miR-124 target genes, as defined by an independent set of miRNA target predictions generated using mirSVR [41] and multiZ alignments of twelve Drosophila genomes [42]. [score:5]
Predictions of conserved miRNA binding sites (e. g. TargetScan or mirSVR) did not identify miR-124 target sites in the annotated pumilio 3′ UTR or CDS; however modENCODE data [72] revealed that pumilio transcription extends >2 kb downstream of its annotated 3′ end. [score:5]
Note that the top -upregulated gene, white, is a consequence of genetic background, since the mir-124 knockout is marked by an extra copy of mini-white. [score:5]
Figure S7Absolute expression of miR-124 target genes in miR-124:DsRed+ cells. [score:5]
As has been observed in vertebrate systems, well-conserved targets of fly miR-124 were overall repressed more potently than poorly-conserved targets (Figure 6F). [score:5]
The expression of miR-124 is not mutually exclusive with its functional targets. [score:5]
Cell-autonomous misregulation of direct targets in mir-124 mutants. [score:5]
1002515.g007 Figure 7(A) Consistent with earlier reports [15], transcripts bearing miR-124 target sites predicted by mirSVR are enriched for genes annotated with non-neural expression. [score:5]
Previously, miR-124 was selected as a particularly compelling case in which its Drosophila targets were depleted for in situ terms related to nervous system development, and enriched for terms related to epidermal development [15]. [score:5]
Amongst the broad network of miR-124 targets, we are struck by the coordinate targeting of multiple components of the retrograde BMP signaling pathway [44], including all three receptors (Sax/Tkv/Wit), the downstream transcription factor (Mad) and its cofactor (Medea). [score:5]
In contrast, we observed very few changes in these same transcripts in mir-124 mutant cells that did not express miR-124:DsRed, indicating that their deregulation was likely a direct consequence of miR-124 activity. [score:5]
The broad, but phenotypically-tolerated, misregulation of miR-124 targets in this species is potentially consistent with the “fine-tuning” mo del for miRNA regulation. [score:5]
Representative images of Even-skipped (A,B) and Hunchback (C,D) expression in the neuronal layers, and Miranda (E, F) and Deadpan (G, H) expression in the neuroblast layers of wild type and mir-124[6/6] mutants are shown. [score:5]
Indeed, cross-referencing these target predictions against in situ annotations catalogued from Drosophila embryogenesis [43] confirmed that epidermal genes were enriched amongst miR-124 targets at stages 11–12 and 13–16 (Figure 7A), as reported earlier [15]. [score:5]
For example, neural genes were depleted of miR-124 target sites while epidermal genes were enriched for miR-124 target sites. [score:5]
Given that we failed to observe substantial contribution of mutual exclusion to the functional miR-124 target network, we sought connections between de-repressed miR-124 targets and mutant phenotypes. [score:5]
The group of neural genes is shifted towards lower expression levels in the mir-124 mutant, while the NB cluster is shifted towards higher expression. [score:5]
However, when performing a similar analysis using our data from functional derepression in mir-124 mutant cells, we failed to observe broad derepression of epidermal target genes, either amongst well-conserved or poorly-conserved target sets (Table S2). [score:5]
Overall, these data indicate a substantial trend for co -expression of miR-124 and its targets genomewide, as similarly deduced from studies of miR-124 in zebrafish [35] and C. elegans [9]. [score:5]
Second, miR-124 targets were preferentially amongst the higher-expressed transcripts in miR-124+ cells, even in wild-type. [score:5]
Our detailed in vivo transcriptome-wide analysis of endogenous miR-124 targets sets the stage for future studies of how individual targets might affect different settings of miR-124 function. [score:5]
Multiple transgenic lines exhibited identical expression in the embryonic nervous system that recapitulated endogenous pri-mir-124 expression. [score:5]
Left panel is the same as in main Figure 7B, indicating that both miR-124 well-conserved and poorly-conserved targets are expressed at relatively high levels in both wt and mir-124 mutants. [score:5]
Donor insertions on chromosome X or III were used for mir-124 targeting, and were crossed to flies carrying heat shock-inducible FLP recombinase and I-SceI endonuclease, to mobilize the miRNA targeting element from the donor chromosome and linearize the excised fragment. [score:5]
Amongst neural genes upregulated ∼2-fold in mir-124 mutant cells and contain miR-124 binding sites in their 3′ UTRs were multiple members of the retrograde BMP signaling pathway, including the receptors saxophone (sax) (Figure 6C) and wishful thinking (wit), and the transcription factor Mad (Tables S1 and S2). [score:4]
Pum transcript was only mildly upregulated in mir-124 mutant cells, and available antibodies were not suitable for immunostaining (not shown). [score:4]
Since coordinate regulation of multiple aspects of an entire pathway by an individual miRNA is only rarely observed [1], [46], [47], this property is a distinctive aspect of the miR-124 target network. [score:4]
In the embryonic mammalian brain, miR-124 was reported to direct neural differentiation by targeting polypyrimidine tract binding protein 1 (PTBP1), a global repressor of alternative splicing in non-neural cells [23]. [score:4]
The electrophysiological defects in mir-124 mutants phenocopy activation of BMP signaling at the synapse, and miR-124 directly targets multiple components of this pathway (Figure 8). [score:4]
Because of the specific expression of mir-124 in the CNS, we were interested to see if we could uncover any defects in neural development. [score:4]
Nevertheless, gene deregulation in mir-124 mutant cells could be interpreted as a failure to consolidate the neural gene expression signature. [score:4]
One notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons phenocopies loss of miR-124. [score:4]
Amongst 7-nt motifs associated with transcripts that increased in the mir-124 mutant nervous system, the highest-scoring motif (p-value = 0) corresponded to the miR-124 seed region (positions 2–8), while the next highest-scoring motifs amongst globally upregulated transcripts corresponded to variations of 2–7 miR-124 seeds (Figure 8G). [score:4]
Neural expression of Drosophila mir-124 Northern analysis first detected mature miR-124 at 4–6 hrs of development (Figure 1A), corresponding approximately to embryo stages 9–10. [score:4]
The miREDUCE analysis also revealed several motifs associated with transcripts that were downregulated in the absence of miR-124. [score:4]
Lack of strong defects in neural production or differentiation in mir-124 mutantsBecause of the specific expression of mir-124 in the CNS, we were interested to see if we could uncover any defects in neural development. [score:4]
We used Northern analysis to verify that multiple independent mir-124 knockout alleles did not express mature miR-124 (Figure 2B), demonstrating that these are truly null backgrounds. [score:4]
However, as the three vertebrate mir-124 loci are co-expressed in the nervous system, analysis of the null situation will require a triple knockout. [score:4]
We also observed that Pumilio binding sites were strongly associated with downregulated transcripts in mir-124 mutants. [score:4]
Similarly, C. elegans deleted for mir-124, which is expressed mostly in ciliated sensory neurons, do not reveal obvious defects in neural development [9]. [score:4]
Since these tissues derive from a common developmental progenitor, the neuroectoderm, this led to a mo del in which miR-124 may solidify the neural fate by widespread suppression of epidermal genes that should be absent from neurons. [score:4]
Left is the retrograde BMP signaling pathway, red are miR-124 targets, which are all on the positive direction of BMP signaling. [score:4]
Further inspection showed that another BMP receptor thickveins (tkv) and the co-Smad Medea also contain highly conserved miR-124 binding sites, although tkv mRNA was not upregulated in the microarray and Medea was not detected by this platform (even though it has a critical function in neurons). [score:4]
The second worksheet summarizes the top upregulated genes in mir-124 mutant cells. [score:4]
Shifts to the right reflect overall upregulation of genes in the mir-124 mutant. [score:4]
Having documented both primary and secondary effects of loss of miR-124 on neural gene expression, we asked whether such gene deregulation exerted a coherent overall effect on cell identity. [score:4]
Nevertheless, the strong enrichment of Pumilio binding sites amongst transcripts downregulated in mir-124 mutants suggests its overactivity. [score:4]
However, complicating the picture is the recent report that Xenopus miR-124 represses neurogenesis by directly targeting the proneural bHLH factor NeuroD1 [28]. [score:4]
Therefore, miR-124 does not seem to have an overarching theme in, for example, directly targeting neuroblast genes. [score:4]
In addition to a global effect on neuroblast-to-neural transition, we observed that genes downregulated upon in vivo loss of miR-124 were enriched for seeds of K box miRNAs and miR-10-5p (Figure 8G). [score:4]
We conducted sensor assays to examine the response of these targets to miR-124, and observed that all five targets were indeed repressed by ectopic miR-124, with especially strong repression of the sax and tkv sensors that contained conserved paired sites (Figure 8C). [score:4]
The fourth -highest scoring motif (GCGCGCC) amongst up-regulated transcripts did not match a continuous region of miR-124, but exhibited notable similarity. [score:4]
Derepression of the direct miR-124 target network had many secondary effects, including over-activity of other post-transcriptional repressors and impaired transition from neuroblast to neuronal transcriptome signatures. [score:4]
Analysis of 8 such sensors, bearing single conserved 7mer or 6mer sites, showed that all were significantly repressed upon transfection of ub-Gal4 and UAS-mir-124 expression constructs (Figure 6H). [score:3]
We note that analogous mir-124 promoter fusions in nematode and zebrafish were correctly expressed in the absence of endogenous mir-124 and Dicer, respectively [9], [35]. [score:3]
Table S2GO term enrichments in mir-124 expression data. [score:3]
Lack of evidence for mutual exclusion amongst the functional miR-124 target network. [score:3]
The impact of endogenous Drosophila miR-124 on neuronal gene expression. [score:3]
Post-sort analysis showed that ∼80% of the selected cells were DsRed+, and qPCR analysis of these sorted wild-type cells confirmed that the DsRed+ cells specifically expressed pri-mir-124 (Figure 6B). [score:3]
This viewpoint is consistent with analyses of miR-124 targets in human [50], zebrafish [35] and C. elegans [9], indicating a unifying theme for this particular miRNA across animals. [score:3]
The first worksheet summarizes gene expression between miR-124:DsRed+ cells isolated from wild-type and mir-124−/− 10-16 hr embryos. [score:3]
Other possibilities are that miR-124 regulates a transcriptional regulator of pumilio, or that Pumilio activity is altered in mir-124 mutants. [score:3]
1002515.g005 Figure 5 mir-124 suppresses variation in dendrite numbers on sensory neurons. [score:3]
Studies in chick ascribed miR-124 as a proneural factor that inhibits the anti-neural phosphatase SCP1 [21]. [score:3]
The triplicate wild-type and mir-124-mutant transcriptomes were highly segregated by unsupervised hierarchical clustering (Figure 6D), indicating that major changes in expression profiles were due to genotype and not to technical variation. [score:3]
We were in a position to test this using our gene profiling data from wildtype and mutant miR-124 -expressing cells. [score:3]
Looking more ventrally into the progenitor layer, we observed strong co -expression of miR-124:DsRed with neuroblasts marked by Deadpan (Figure 7D), as noted earlier (Figure 1E–1G). [score:3]
Transcriptome-wide derepression of miR-124 targets. [score:3]
org/) and conserved miR-124 target sites in their 3′ UTRs (Figure 6C). [score:3]
Derepression of a sufficient number of such non-neural transcripts may contribute collectively to the incomplete capacity of mir-124 mutant cells to transition from a neuroblast to neuronal gene expression signature (Figure 8H). [score:3]
We investigated this further by examining the absolute levels of predicted miR-124 targets in miR-124 -expressing cells. [score:3]
Still, a “one size fits all” description of miR-124 activity is not appropriate, since we certainly do observe a number of functional miR-124 targets whose predominant activities are in epidermal or other non-neural derivatives. [score:3]
Coordinate targeting of retrograde BMP signaling components by miR-124. [score:3]
Given that these invertebrate orthologs of miR-124 are identical in sequence to their vertebrate counterparts, and are highly and specifically expressed in their respective nervous systems, there is not strong reason a priori to suspect that miR-124 should not have comparable requirements amongst different animals. [score:3]
1002515.g006 Figure 6Gene expression in miR-124+ cells from wild-type and mir-124 mutants. [score:3]
mir-124 suppresses variation in dendrite numbers on sensory neurons. [score:3]
Thus, the large miR-124 network accommodates a range of target properties [69], [70]. [score:3]
1002515.g001 Figure 1Temporal and spatial expression of Drosophila miR-124. [score:3]
Still, it remains possible that the many other gene expression changes in mir-124 mutant neurons (Figure 6, Figure 7, Figure 8) contribute to its loss of function phenotype. [score:3]
mir-124:DsRed was active in the full complement of neurons in the CNS, but Elav alone was highly expressed in the peripheral nervous system (Figure 1H, 1I). [score:3]
Cross-regulatory effects of mir-124 loss on other modes of RNA–based regulation. [score:3]
Gene expression in miR-124+ cells from wild-type and mir-124 mutants. [score:3]
The miR-124 target network included coordinate repression of multiple components in the retrograde BMP signaling pathway, whose activity controls synaptic release. [score:3]
Neural expression of Drosophila mir-124. [score:3]
Altogether, our analyses reveal a complex set of primary and secondary effects on neuronal gene expression in mir-124 mutants, which are collectively associated with behavioral dysfunction in larval and adult stages. [score:3]
We infer that a phase of coexpression of miR-124 and repo precedes the adoption of their mutually-exclusive state. [score:3]
The third worksheet summarizes expression of genes present in mir-124−/− that were not called present in wild-type. [score:3]
Having established a variety of clear phenotypes in mir-124 mutants, we wished to query changes in gene expression in the mutant cells. [score:3]
Such conservation implies substantial functions of miR-124 in controlling neural gene expression. [score:3]
Although many of these sites in BMP pathway targets were only 6mers (matching positions 2–7 of miR-124), all of them except the Mad site were well-conserved across Drosophilid evolution (Figure 8B and Figure S8), implying their functional constraint. [score:3]
Therefore, direct and indirect consequences may both contribute to electrophysiological defects caused by the absence of miR-124. [score:3]
Since all of these cell types derive from a common progenitor, the neuroectoderm, this led to the mo del that expression of miR-124 helps to repress epidermal potential in neurons [15]. [score:3]
High magnification insets of panels E–F show gradual expression of miR-124:DsRed in all Deadpan+ and Prospero+ positive cells in CNS. [score:3]
To facilitate analysis of mir-124 expression, we generated a transcriptional reporter. [score:3]
Figure S8Conservation of miR-124 target sites amongst components of the retrograde BMP signaling pathway. [score:3]
Pumilio is well-characterized as a neural RNA binding protein and translational regulator, and affects synaptic function and dendrite morphogenesis [53]– [55], which we also observed to be miR-124-regulated settings. [score:3]
We therefore used in situ hybridization to primary miRNA transcripts to analyze expression of Drosophila mir-124 at the cellular level [8]. [score:3]
Altogether, we demonstrate that endogenous miR-124 has substantial impact on CNS gene expression, which underlie its requirement for organismal behavior and physiology. [score:3]
Consistent with the lack of substantial neural specification defects in the mutant, the expression of the mir-124 reporter was similar in the presence or absence of the miRNA. [score:3]
This defect was rescued by the mir-124 genomic transgene, indicating that miR-124 suppresses variability in dendritic branching numbers. [score:3]
About half of these contain miR-124 target sites. [score:3]
Below are the miR-124 targeting of BMP pathway genes, red boxes on the conservation graphs indicate sequences pairing with miR-124 seed region and their extent of conservation. [score:3]
Functional interpretation of direct and indirect consequences of miR-124 loss. [score:3]
We recently showed that misexpression of activated Sax and Tkv receptors in motoneurons increases evoked excitatory junctional potentials without affecting spontaneous activity, very similar to that of mir-124 mutants [48]. [score:3]
Table S1Gene expression changes in mir-124 mutants. [score:3]
One mechanism involves direct repression by miR-124 of Baf53a, a neural progenitor-specific chromatin regulator that must be exchanged for a neural-specific homolog to consolidate neural fate [27]. [score:3]
Figure S1Expression of the miR-124:dsRed reporter in stage 8 embryos. [score:3]
Figure S5 Expression of the miR-124:dsRed reporter in larval CNS. [score:3]
In principle, such a pattern might reflect an active role of miR-124 to suppress the epidermal program in neurons, or might reflect a fail-safe program that is secondary to transcriptional mechanisms. [score:3]
The latter phenotype is perhaps reminiscent of reports that inhibition of Aplysia miR-124 similarly results in an increase in evoked EPSP amplitude [10]. [score:3]
Temporal and spatial expression of Drosophila miR-124. [score:3]
3′ UTRs of predicted miR-124 targets were cloned into the psiCHECK-2 vector (Promega) using cold fusion cloning (System Biosciences). [score:3]
This is consistent with the view that on the transcriptome-wide level, the exclusion of epidermal genes from miR-124 -expressing cells is primarily enforced by transcriptional mechanisms. [score:3]
Analysis of mir-124 overexpression in the mir-124 mutant background showed a strong decrease in branching complexity; still, the variation in dendritic end numbers was rescued. [score:3]
Notably, we recently showed that misexpression of activated Sax and Tkv receptors in motoneurons increases synaptic activity without affecting NMJ structure [48], [49], similar to mir-124 mutants. [score:3]
Instead, we took advantage of the mir-124:DsRed reporter to isolate mir-124 -expressing cells from dissociated embryos using fluorescence activated cell sorting (FACS). [score:3]
Of note, C. elegans mir-124 is predominantly expressed in sensory neurons [9]. [score:3]
miR-124 is strictly conserved in both primary sequence and spatial expression pattern, being restricted to the nervous system of diverse metazoans, including flies [8], nematodes [9], Aplysia [10], and all vertebrates studied [11]– [13]. [score:3]
The predicted miR-124 target sites were partitioned into well-conserved and poorly-conserved based on the Multiz 15 fly species alignment in the UCSC genome browser [42]. [score:3]
This yielded clusters of 1109 and 1415 unique genes that were mostly restricted to neuroblasts and neurons, respectively, of which 1002 and 1269 were expressed in miR-124+ cells. [score:3]
By purifying cognate miRNA -expressing cells from wild-type and miRNA-mutant backgrounds, we were able to assess transcriptome-wide effects of genetic removal of miR-124 with precision. [score:3]
Overall, these observations suggested that mutual exclusion of miR-124 and target accumulation is not a feature actively driven by miRNA activity. [score:3]
The spatial expression of miR-124 and Repo was previously reported to be mutually exclusive [15], and we confirmed exquisite exclusion of their domains in the ventral ectoderm, where miR-124 is active in neurons and Repo in glia (Figure 7C–7C′″). [score:3]
We also tested the effect of misexpressing miR-124 in class I neurons, building on our observation that ectopic miR-124 reduces dendrite numbers in wild-type [31]. [score:3]
To validate the capacity for direct targeting of these transcripts by miR-124, we assayed the response of luciferase-3′ UTR sensors to ectopic miR-124 in S2 cells. [score:3]
Misexpression of miR-124 in mir-124 mutants, using a newly constructed UAS-DsRed-mir-124 transgene, recapitulated this defect (Figure 5C). [score:3]
miR-124:dsRed is mostly active in the central complex, both in neuroblasts and neurons; it is expressed only weakly in the optic lobe. [score:3]
Future studies should address the cross-talk of post-transcriptional regulation in neurons mediated by miR-124, neuronal miRNAs and Pumilio. [score:2]
The analysis of vertebrate mir-124 knockouts is therefore highly anticipated. [score:2]
Like most other miRNA mutants in this species, the loss of miR-124 did not cause obvious developmental, physiological or behavioral phenotypes. [score:2]
For flies in which mini-white mapped to chromosome II, PCR was performed to verify the integration of the targeting construct at the mir-124 locus using primers that bind outside the left homology arm and within unique vector sequence downstream of the left homology arm but upstream of the mini-white gene. [score:2]
Overactivity of other neural post-transcriptional regulators in mir-124 mutants. [score:2]
Overactivity of other neural post-transcriptional regulators in mir-124 mutantsThe bioinformatic analyses presented thus far focused specifically on motifs of interest, e. g. miR-124 seeds. [score:2]
This suggested that transcriptional profiling by this strategy was not likely to be substantially affected by the absence of cell types whose specification might require miR-124, or that might fail to be isolated because of positive autoregulatory feedback of miR-124 onto its own transcription. [score:2]
Here, we analyze a knockout of the sole mir-124 gene in D. melanogaster. [score:2]
We speculate that environmental or genetic stress may reveal additional requirements for miR-124 in development and differentiation of the nervous system. [score:2]
It remains to be seen if synaptic overactivity in the mir-124 mutant can be directly linked to the behavioral defects we observed at the organismal level (Figure 4). [score:2]
In light of the broad roles ascribed to endogenous miR-124 in neurogenesis, neural differentiation, and neural physiology [60], all from antisense strategies, the extensive negative data from our Drosophila mir-124 knockout are equally compelling. [score:2]
Specific behavioral and electrophysiological defects in mir-124 mutantsSince we did not observe substantial defects in neural development, we checked for functional defects in the central nervous system. [score:2]
Neuronal gene expression was globally decreased in miR-124:DsRed cells isolated from mir-124 mutants compared to wild-type (p<2.2E-16). [score:2]
So far, a mir-124 knockout has only been described in C. elegans, which harbors a single copy of this gene [29]. [score:2]
Northern analysis first detected mature miR-124 at 4–6 hrs of development (Figure 1A), corresponding approximately to embryo stages 9–10. [score:2]
Generation of mir-124 knockout and genomic rescue strains. [score:2]
Generation of mir-124 knockout and genomic rescue strainsWe used ends-out homologous recombination to replace the endogenous mir-124 hairpin with a white+ marker flanked by loxP sites (Figure 2A). [score:2]
To do so, we separated miR-124:DsRed+ and DsRed− cells from stage 13–16 embryos (∼10–16 hrs of development) that were wildtype or deleted for mir-124. [score:2]
Therefore, deregulation of BMP signaling may contribute to the electrophysiological defects observed in mir-124 mutants. [score:2]
Since mir-124 is activated in neuroblasts and maintained in differentiated neurons (Figure 1), we hypothesized that the absence of miR-124 might be manifest in the transition from the neuroblast to neural state. [score:1]
While we may not have examined the relevant neural subpopulation, our studies indicate that miR-124 is not required for gross aspects of neurogenesis and differentiation in the embryonic and larval nervous system. [score:1]
miR-124:dsRed is active in the brain and the ventral nerve cord (VNC). [score:1]
Only the Mad miR-124 site is restricted to melanogaster group species, but it is perfectly conserved amongst these five genomes. [score:1]
We introduced mir-124:DsRed into the mir-124 mutant background, so that we could isolate the relevant mutant cells (Figure 6A). [score:1]
However, Deadpan/Elav staining showed relatively normal patterns of neuroblasts and neurons in the mir-124 mutant brain (Figure 3H, 3I). [score:1]
We therefore checked for PNS phenotypes, judging that defects that were rescuable should reflect endogenous requirements for miR-124. [score:1]
We used ends-out homologous recombination to replace the endogenous mir-124 hairpin with a white+ marker flanked by loxP sites (Figure 2A). [score:1]
Figure S3Analysis of CNS markers in mir-124 mutants. [score:1]
Drosophila stocksDeletion alleles of mir-124 were generated using ends-out recombination [73]. [score:1]
Activity of mir-124 initiates in neuroblasts and is maintained in GMCs and CNS neurons. [score:1]
Indeed, transcripts bearing miR-124 sites predicted by mirSVR [41] exhibited a highly statistically significant shift to higher levels in mir-124 mutants (p-value<1.11e-12) (Figure 6E); i. e. shifted to the right in the CDF plot. [score:1]
These observations suggest that miR-124 is detectably required for organismal fitness. [score:1]
We observed that 60–70% of mir-124 deletion embryos of various genotypes failed to hatch, and that embryonic lethality was substantially (although not fully) rescued by the mir-124 genomic transgene (Figure S2). [score:1]
Sensor plasmid and ub-Gal4 were cotransfected with UAS-DsRed-miR-124 or empty pUAST vector into S2-R+ cells using Effectene (Qiagen). [score:1]
In the adult mammalian brain, miR-124 promoted neural differentiation of the immediate progenitors, the transit-amplifying cells (TAs). [score:1]
Functional studies have connected vertebrate miR-124 to various aspects of neural specification or differentiation. [score:1]
However, mir-124 mutant adult males exhibited substantially shortened lifespan, and this defect was completely rescued by introduction of the mir-124 genomic transgene (Figure 2C). [score:1]
The effect was more pronounced in ddaD than ddaE neurons, but both were rescued by the mir-124 genomic transgene. [score:1]
1002515.g004 Figure 4Requirement of mir-124 for larval locomotion and synaptic transmission. [score:1]
Deletion alleles of mir-124 were generated using ends-out recombination [73]. [score:1]
Representative images are shown from mir-124 loss- and gain-of-function backgrounds. [score:1]
Recognizing that substantial manipulation is incurred during embryo dissociation and cell sorting, we were interested to obtain confidence that potential changes in gene expression in our measurements could be specifically attributed to miR-124 activity. [score:1]
Inset of panel (B) highlights the detection of nuclear dots that reflect the chromosomal locations of mir-124 transcription. [score:1]
We recombined the 39N16 rescue with mir-124 deletion alleles, and used Northern analysis to validate that this transgene restored a normal level of miR-124 to mutant adults (Figure 2B). [score:1]
1002515.g003 Figure 3Absence of major defects in specification of the nervous system of mir-124 mutants. [score:1]
Interestingly, miR-124 is not required for normal dendrite formation per se, but its absence caused a broader distribution of dendrite numbers on ddaD and ddaE neurons, i. e. a “robustness” defect. [score:1]
These defects phenocopied the electrophysiological defects of mir-124 mutant synapses (Figure 4E–4K). [score:1]
mEJCs were indistinguishable in the two groups, but mir-124 mutants showed an increase in the average EJC amplitudes, indicating a significant elevation in quantal content at the NMJ (Figure 4K). [score:1]
Therefore, miR-124 serves to limit synaptic activity. [score:1]
The apparent temporal fluctuation in miR-124 levels appeared to be a consequence of its tissue-specificity. [score:1]
However, they were not easily kept as homozygous stocks, potentially reflecting detrimental effects of mir-124 deletion. [score:1]
Nevertheless, two observations suggest that the feature of mutual exclusion in the Drosophila miR-124 network is of subtle consequence. [score:1]
Although the average numbers of dendritic ends for ddaD and ddaE neurons in mir-124[6] or mir-124[7] mutants were not statistically different those in wildtype, the variation in their numbers was significantly increased (by F test). [score:1]
All of these phenotypes were rescued by a single copy of a 19 kilobase (kb) genomic transgene encompassing the mir-124 locus. [score:1]
Specific behavioral and electrophysiological defects in mir-124 mutants. [score:1]
miR-124 has been a popular mo del for genomewide investigations of miRNA targeting principles. [score:1]
We observed that mir-124 mutant neuroblast clones appropriately maintained a single neuroblast and could undergo multiple divisions to generate many neurons (Figure 3J). [score:1]
Another plausible mechanism might be that miR-124 represses a transcriptional repressor of these other miRNAs. [score:1]
The mir-124 mutant alleles were viable and fertile, and exhibited normal external morphology. [score:1]
∼400 bp genomic fragment containing the pre-mir-124 sequence was cloned into the UAS-dsRed [47] to generate the UAS-dsRed-mir-124 transgene. [score:1]
However, no substantial effect of miR-124 on chick neurogenesis was found in a parallel study [22], although miR-124 was observed to repress neural progenitor genes such as laminin gamma1 and integrin beta1. [score:1]
Absence of major defects in specification of the nervous system of mir-124 mutants. [score:1]
Wandering third instar larvae were dissected in cold HL3 solution without Ca [2+] following standard protocol [79], using the mir-124 genotypes described above and BG380-Gal4> UAS-TkvA [80]. [score:1]
Loss of miR-124 impairs neuroblast to neuronal transition. [score:1]
All vertebrate miR-124 loss-of-function studies have relied on antisense strategies and have yet to be validated by bona fide mutant alleles. [score:1]
Within the brain, activity or mir-124:DsRed was highest in the central complex (Figure S5B). [score:1]
Lack of strong defects in neural production or differentiation in mir-124 mutants. [score:1]
We therefore generated a P[acman] insertion of 19 kb of mir-124 genomic DNA (39N16, Figure 2A), a region lacking annotated protein-coding genes; note that it contains mir-287, but this locus has not been confirmed in largescale sequencing [30], [32]. [score:1]
miR-124 reduces the variability of dendritic numbers of sensory neurons. [score:1]
We detected abundant activity of mir-124:DsRed in the larval CNS, including both the brain and ventral nerve cord (Figure S5A). [score:1]
We confirmed these phenotypes to be due to miR-124 loss, as shown by their rescue by a mir-124 genomic transgene. [score:1]
Requirement of mir-124 for larval locomotion and synaptic transmission. [score:1]
However, pan-CNS Drosophila miR-124 does not appear to be required for bulk aspects of neurogenesis or differentiation, as has been concluded for its vertebrate counterparts. [score:1]
Endogenous requirements for the highly conserved neural locus miR-124. [score:1]
Therefore, miR-124 is required for normal locomotion. [score:1]
Close examination showed that its primary transcription, as reflected by nuclear dots of elongating pri-mir-124 transcripts (Figure 1B, inset), was first detected in the ventral nerve cord around stage 8 during germband elongation (Figure S1) and became more prominent in subsequent stages. [score:1]
Other mammalian studies bolster the concept that miR-124 promotes neurogenesis [25] or neural differentiation [26]. [score:1]
We purified three biologically independent samples of miR-124:DsRed+ cells from dissociated wildtype and mir-124 mutant stage 13–16 embryos and profiled them using Affymetrix microarrays. [score:1]
To analyze larval neuroblast clones, we heat-shocked hsflp, tubGal4, UAS-GFP; FRT40A, tubGal80/FRT40A mir-124[6] for 37°C for 90 minutes at 24 hr ALH (after larval hatching) and dissected at 96 hr ALH. [score:1]
Therefore, the loss of the abundant CNS miRNA miR-124 may result in the overactivity of other CNS miRNAs. [score:1]
mir-124 exhibited less movement, and their behavior was restored by inclusion of a 19 kb mir-124 rescue transgene. [score:1]
These results suggest that miR-124 helps maintain the consistency of dendritic branching patterns of specific neurons. [score:1]
While dispensable for gross neural specification and differentiation, deletion of mir-124 caused short lifespan, increased variation in dendrite numbers, impaired larval locomotion, and aberrant synaptic release at the NMJ. [score:1]
In all panels, miR-124:DsRed is at left, the neural markers in the middle, and merged images at right; the signal in the center of panels G is gut autofluorescence. [score:1]
miR-124:dsRed was generated by cloning 4 kb upstream of the hairpin into Red-H-Stinger [75]. [score:1]
The increase in EJCs was fully rescued when we included a mir-124 genomic transgene in the homozygous mutant larvae, indicating that the increase in EJCs and quantal content was attributable to the mir-124 deletion. [score:1]
The mir-124 rescue transgene was generated by injection of P[acman] clone CH322-39N16 into attP16 strain [74] (Genetic Services Inc. [score:1]
However, the variation in dendrite numbers was substantially increased in mir-124 mutants (Figure 5D); this was especially noticeable for ddaD. [score:1]
Mira, Dpn, and Eve wild type images are taken from mir-124 genomic rescue embryos, and the Hb image is taken from yw embryo. [score:1]
In situ hybridization for pri-mir-124 confirms detection of nuclear primary transcripts at stage 8 (arrows, inset). [score:1]
Therefore, endogenous miR-124 strongly influences the transcriptome of the Drosophila nervous system. [score:1]
Figure S2Lethal phase analysis of mir-124 mutants. [score:1]
These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. [score:1]
Nevertheless, we observed strikingly opposite behavior of neuroblast and neuronal genes as a whole, in the absence of mir-124 (Figure 8H). [score:1]
However, this layer also contained strongly Repo -positive cells (Figure 7D′) that colabeled with miR-124:DsRed but were exclusive of Deadpan; we infer these to be glioblasts. [score:1]
Different trans-heterozygous mir-124 mutant combinations exhibited a clear defect in both parameters, and these were fully rescued by the mir-124 genomic transgene (Figure 4A–4C and Figure S6); the differences were highly statistically significant (Figure 4D). [score:1]
Substantial embryonic lethality was observed in mir-124 mutants, which was demonstrably (although not completely) rescued by the mir-124 genomic transgene. [score:1]
Deletion of mir-124 resulted in no differences in spontaneous activity, but caused significant increases in evoked currents and quantal content; these phenotypes were rescuable. [score:1]
Finally, mir-124 mutants exhibited grossly normal axonal architecture in the late embryo, as marked by 22C10 (Figure S4). [score:1]
Following embryogenesis, we did not observe substantial differences in viability between the mir-124 mutant and wildtype, at larval/pupal/adult stages (Figure S2). [score:1]
On the one hand, the average number of dendritic branches in mir-124 mutants did not show a statistical difference from that in wildtype larvae. [score:1]
We plotted the cumulative distribution function (CDF) of various sets of genes, comparing their levels in the mir-124 mutant relative to wildtype. [score:1]
Figure S4Loss of mir-124 does not result in abnormal axonal architecture as labeled by 22C10 in st15 embryos, either in CNS (A,B) or PNS (C,D). [score:1]
Similar to endogenous pri-mir-124, the mir-124:DsRed transgene was faintly active at stage 8 (Figure S1), and exhibited nearly completely colocalization with the pan-neuroblast marker Deadpan in the stage 9 CNS (Figure 1E, 1E′); at this stage mature neurons have not yet been specified. [score:1]
To gain functional insight into the basis of this defect, we first tested for a possible role of miR-124 in synaptic structure. [score:1]
For example, most miR-124 in the adult was present in the head (Figure 1A), consistent with comparison of head and body small RNA data [30]. [score:1]
Wild type (mir-124:dsRed) and mutant (mir-124[del12/12]; mir-124:dsRed) flies were raised in collection cages at 25°C. [score:1]
Figure S6Quantitative analysis of locomotion defects in mir-124 mutant larvae. [score:1]
The bioinformatic analyses presented thus far focused specifically on motifs of interest, e. g. miR-124 seeds. [score:1]
GMCs can be marked by Prospero, and these cells were similarly labeled by mir-124:DsRed (Figure 1E, 1E″). [score:1]
A 19 kb mir-124 rescue transgene lacks known protein-coding genes; it overlaps mir-287 but this locus has not been validated as a miRNA from deep sequencing [32]. [score:1]
We fused 4.2 kb of sequence upstream of the mir-124 hairpin, including ∼1 kb more genomic sequence than the previously studied mir-124:Gal4 transgene [31], to a nuclear DsRed gene in the insulated H-Red-Stinger vector. [score:1]
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[+] score: 37
Three hStau1-specific mRNAs contained predicted miR-124 targets and have been described as related to neuron function: The homeobox-containing gene engrailed2 (en2), which is involved in autism disorder [61], [62], the magnesium transporter 1 gene (magt1), identified by differential expression during epilepsy [63] and most interestingly, synaptic cell-adhesion molecule2/ leucine-rich repeat and fibronectin III domain-containing molecule1 (salm2/lrfn1) gene. [score:5]
Among these, miR-124 stands out as particularly enriched in hStau1-containing complexes and is over-expressed upon differentiation of human neuroblastoma cells in vitro and (ii) Expression of hStau1 is essential for proper dendritic arborisation during neuroblastoma cell differentiation, yet it is not necessary for maintenance of the differentiated state. [score:5]
Previous reports have documented that miR-124 participates in the neural development by modifying several of the regulation layers indicated above, like transcription [24], alternative splicing [48] or specific protein silencing [49]. [score:3]
To verify these results in a more physiological setting we used human neuroblastoma SH-SY5Y cells, since both miR-124 and miR-9 are highly expressed in neural cells [45], [46]. [score:3]
Interestingly, two of the most prominent miRNAs found associated to hStau1 during screening were miR-124 and miR-9, which have been described as highly relevant for neural development [24], [47]– [49]. [score:2]
To analyse the size pattern of miR-124–containing complexes during differentiation, total cell extracts derived from SH-SY5Y cells were prepared at days 0 and 10 in the differentiation process and fractionated by gel filtration on Sephacryl S400 as indicated above. [score:1]
Fraction pools F1 to F4 were generated as indicated in Fig. 4 and the RNA was used for miR-124 determinations using RT-qPCR. [score:1]
Figure S1 Induction of miR-124 upon neuroblast differentiation. [score:1]
In addition, miR-124 showed higher concentration in the hStau1 fractions than in the initial cell extract, whereas the rest of the miRNAs analysed were not enriched in the hStau1 complexes (Fig. 3D). [score:1]
The size pattern of miR-124-containing complexes changes during neuroblast differentiation. [score:1]
Particularly interesting were miR-124 and miR-9, that showed the highest hStau1 vs TAP ratio, using as a control miR-147a, that was not present among those detected in the initial screening (Fig. 3C). [score:1]
Association of miR-124 to hStau1 complexes in undifferentiated and differentiated neuroblastoma cells. [score:1]
Furthermore, the association of both miR-124 and miR-9 to hStau1 complexes was verified in non -transfected SH-SY5Y human neuroblastoma cells (Figs. 4, 5). [score:1]
All miRNAs tested were detected in the hStau1-containing F1 pool and, interestingly, miR-124 and miR-9 were preferentially found in this fraction. [score:1]
Here we identify miR-124 and miR-9 as miRNAs specifically associated to hStau1, one of these proteins, and show that hStau1 is important for the proper differentiation of human neuroblastoma to neuron-like cells. [score:1]
In agreement with the reported role of miR-124 in neuronal cell differentiation in chick and mouse [47]– [49], a large increase in the total miR-124 concentration was observed in human neuroblastoma SH-SY5Y cells upon differentiation in vitro (Fig. S1). [score:1]
Whereas most of the miR-124 co-migrated with the hStau1 complexes in undifferentiated cells, it was mostly present in smaller complexes co-migrating with Ago2 when the cells became differentiated. [score:1]
Here we show that, as expected, the levels of miR-124 increase during the differentiation of neuroblastoma to neuron-like cells in vitro (Fig. S1) and, furthermore, its pattern of association to hStau1 complexes changes along this process (Fig. 5), suggesting a role for hStau1 in neural differentiation. [score:1]
We show the association of hStau1 with the Ago components of the RISC and identify miR-124 and miR-9 as the miRNAs preferentially associated to hStau1 RNA granules. [score:1]
These results were verified for miR-124 and miR-9 in three independent filtration experiments and the data are presented in Fig. 4C. [score:1]
miR-124 accumulates in human Staufen1 complexes. [score:1]
The specific association of miR-124 with hStau1 complexes and the alterations observed in this interaction during human neuroblast differentiation prompted us to address the role of hStau1 in this process. [score:1]
In addition, miR-124 was the only miRNA among those tested that showed higher concentration in the hStau1 -associated RNA than in total cell RNA (Fig. 3D). [score:1]
Total cell RNA was isolated and the concentration of miR-124 was determined by TaqMan RT-qPCR. [score:1]
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[+] score: 14
First miR-124 and miR-iab-4-5p are downregulated in wt animals in response to hyperthermic stress, but their levels are not altered appropriately in Dys and Dg mutants. [score:4]
Here we were able to determine that in the absence of Dys or Dg the levels of miR-124 and miR-iab-4-5p fail to undergo the appropriate downregulation seen in wt animals in response to high temperature stress. [score:4]
We were particularly interested in Dys signaling miRNA regulation, thus we tested the “Dys Dependent” cluster from Figure  2d: miR-956, miR-252, miR-124, miR-970, miR-283, miR-927, miR-iab-4-5p, miR-962, miR-980, miR-959, and miR-975 (Additional file 1). [score:2]
miR-124 has been shown to be specific to the nervous system and can be regulated by fragile X mental retardation protein 1 (dFR1) [45]. [score:2]
Potential Dys-regulated miRNAs are miR-956, miR-252, miR-980, miR-124, miR-970, miR-283, miR-927, miR-iab-4-5p, miR-962, miR-959, and miR-975. [score:2]
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[+] score: 12
The number of predicted sites under selection does not appear to correlate with the breadth of miRNA expression, as among the miRNAs with the largest number of predicted target sites we find some that are highly tissue specific (miR-9 and miR-124 that are expressed in the nervous system [5], and miR-155 that is specific to lymphoid cells [5]) as well some that have broad expression (e. g. the families of miR-29 [9, 10, 41, 42] and miR-30 [41, 42]). [score:9]
As miR-124 is highly expressed in mature neurons, it is possible that its function is to maintain previously established neuronal circuits. [score:3]
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[+] score: 8
Of the 78 miRNAs that we probed by expression profiling only dme-miR263a and -263b displayed strong evidence of circadian regulation (with possible weak cycling for dme-miR-124). [score:4]
Likewise, miR-31a (Fig. 3F) might also exhibit low amplitude cycling similar to that of miR-124. [score:1]
Although the differences in daily levels for miR-124 in wildtype flies do not reach significance even when less stringent criteria was applied (p = 0.0813, ANOVA without FDR), it is possible that miR-124 undergoes low amplitude circadian oscillations in abundance. [score:1]
Finally, the D. melanogaster dme-miR-124 is similar to vertebrate miR-124a (Fig. 4D), which also cycled in the mouse retina [50]. [score:1]
Of these, dme-miR-124 showed a pattern very similar to that of dme-miR-263a and -263b, exhibiting trough levels during the mid-day that were followed by increases during the early to late-night in wildtype flies and constantly elevated levels in the cyc [0 ]mutant (Fig. 3B). [score:1]
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[+] score: 7
Drosophila miR-124 regulates neuroblast proliferation through its target anachronism. [score:4]
miR-124 activity is required to support proliferation of neuroblasts in the larval brain by limiting expression of Anachronism (Weng and Cohen, 2012). [score:3]
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[+] score: 7
Other miRNAs from this paper: dme-mir-92a, dme-mir-92b
A pri-miR-124 expression plasmid was generated by amplification of 500 bp of primary sequence from mouse genomic DNA and cloning into pSuper-GFP vector. [score:3]
HEK293T cells were transfected with wild type or mutant miR-92 sensor plasmids or empty psiCHECK-2, pSuper-GFP-pri-miR-92a, or pSuper-GFP-pri-miR-92b expression vectors (described above) or pSuper-GFp-miR-124 using Fugene6 (Promega). [score:3]
MiR-124 used as a control miRNA. [score:1]
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[+] score: 7
Importantly, profiling of Drosophila microRNA expression in dissected thoracic muscles, had previously demonstrated miR-124, miR-100, miR-277 and miR-304 expression in these muscles 35. [score:5]
We selected dme-miR-92a, dme-miR-100 and dme-miR-124 based on data generated in our laboratory and their orthology with human miRNAs. [score:1]
57. miRNA sponge lines (UAS-miR-SP) for dme-miR-92a, dme-miR-100, dme-miR-124, dme-miR-277, dme-miR-304 and scramble-SP (control) were obtained from Dr. [score:1]
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[+] score: 6
Indeed, decreased mRNA levels (in human HeLa cells) were observed for dozens of genes upon transfection of two distinct miRNAs, miR-1 and miR-124; it was also shown that the 3′ untranslated regions (UTRs) of these down-regulated mRNAs have significant complementarity to the 5′ extremity of the transfected miRNAs. [score:6]
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[+] score: 6
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-27a, hsa-mir-29a, hsa-mir-101-1, dme-mir-1, dme-mir-2a-1, dme-mir-2a-2, dme-mir-2b-1, dme-mir-2b-2, dme-mir-10, mmu-let-7g, mmu-let-7i, mmu-mir-1a-1, mmu-mir-101a, mmu-mir-124-3, mmu-mir-126a, mmu-mir-133a-1, mmu-mir-137, mmu-mir-140, mmu-mir-142a, mmu-mir-155, mmu-mir-10b, mmu-mir-183, mmu-mir-193a, mmu-mir-203, mmu-mir-143, hsa-mir-10a, hsa-mir-10b, hsa-mir-34a, hsa-mir-183, hsa-mir-199b, hsa-mir-203a, hsa-mir-210, hsa-mir-222, hsa-mir-223, dme-mir-133, dme-mir-34, dme-mir-79, dme-mir-210, dme-mir-87, mmu-mir-295, mmu-mir-34c, mmu-mir-34b, mmu-let-7d, dme-let-7, dme-mir-307a, dme-mir-2c, hsa-let-7g, hsa-let-7i, hsa-mir-1-2, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-137, hsa-mir-140, hsa-mir-142, hsa-mir-143, hsa-mir-126, hsa-mir-193a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-29a, mmu-mir-27a, mmu-mir-34a, mmu-mir-101b, hsa-mir-1-1, mmu-mir-1a-2, hsa-mir-155, mmu-mir-10a, mmu-mir-210, mmu-mir-223, mmu-mir-222, mmu-mir-199b, mmu-mir-124-1, mmu-mir-124-2, hsa-mir-101-2, hsa-mir-34b, hsa-mir-34c, hsa-mir-378a, mmu-mir-378a, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, mmu-mir-411, hsa-mir-193b, hsa-mir-411, mmu-mir-193b, hsa-mir-944, dme-mir-193, dme-mir-137, dme-mir-994, mmu-mir-1b, mmu-mir-101c, hsa-mir-203b, mmu-mir-133c, mmu-let-7j, mmu-let-7k, mmu-mir-126b, mmu-mir-142b, mmu-mir-124b
For example, pre-let-7a hairpins in the four species had a consensus structure with a folding energy of –21.1 Kcal/mol, but pre-miR-124 hairpins in the four species had a much higher folding energy of the consensus structure (–10.5 Kcal/mol), indicating that pre-miR-124 hairpins were less conserved across the four species. [score:1]
Besides shifted seeds, many well-conserved 5′-isomiRs with the same or nearly identical seed regions had different arm abundances among the four species, exemplified by miR-124, miR-193, miR-210, miR-2 and miR-87 (Table 3). [score:1]
Beyond mammals, 20 miRNA families were found conserved across fruitfly, mouse and human, 8 were conserved between fruitfly and worm, and 4 families (let-7, miR-1, miR-34 and miR-124) were found conserved among all the 4 species. [score:1]
Similar observations were made for miR-124, miR-137, miR-193, miR-210, miR-2, miR-79 and miR87 across species, with miRNAs following the loop-counting rule having lower arm abundances of 5′-isomiRs. [score:1]
For instance, miR-124-3p family produced 22% and 27% of reads as 5′-isomiRs in human and mouse, respectively, but only the maximum of 0.69% of reads in fruitfly. [score:1]
Interestingly, conserved pre-miRNAs with similar 5′-isomiR arm abundances (e. g. let-7 in Table 3) have more conserved secondary structures than those with large variations of 5′-isomiR arm abundances (e. g. miR-124 in Table 3), inferred by the folding energies of consensus structures (T-test P < 0.05, Figure 3A). [score:1]
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[+] score: 5
We recovered four targets from this list (miR-7/HLHm5, miR-279/SP555, miR-124/Gli, and miR-310/imd) but failed to locate conserved nuclei for the other six targets (see comments in Table 3). [score:5]
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[+] score: 5
Phylogenetic analysis, target gene prediction and pathway analysis showed that, among the 13 conserved miRNAs (miR-1, miR-100, miR-10a, miR-124, miR-125, miR-184, miR-33, miR-34, miR-7, miR-9, miR-92a, miR-92b and miR-let7), several highly conserved miRNAs (miR-1, miR-7 and miR-34) targeted the same or similar genes leading to the same pathways in shrimp, fruit fly and human (Figure 3b). [score:5]
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[+] score: 4
Sun K Westholm JO Tsurudome K Hagen JW Lu Y Kohwi M Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutantsPLoS Genet. [score:2]
AREs and miR124 seed sequences had no significant enrichment or depletion in neural fate or localized mRNAs (Figure  5A) or in the entire set of low or high-stability neural mRNAs. [score:1]
Based on these criteria, we tested for enrichment or depletion of AU-rich elements (AREs) [33], miR-124 seed sequences (miR-124 is a nervous system-specific microRNA [34]), and Pumilio recognition elements (PREs) [35]. [score:1]
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[+] score: 2
Searches of the C. elegans 3′ miRNA motifs in Drosophila and humans identified 3′ relationships of cel-miR-80 and cel-miR-799 with hsa-miR-208a, and interestingly revealed 3′ relationships of hsa-miR-208a with hsa-miR-129-3p and hsa-miR-129* and of hsa-miR-124 with hsa-miR-377* (Figure S3). [score:1]
These groups are: 1) cel-lin-4, cel-miR-87; 2) cel-miR-90, cel-miR-124 (3′ region of identity also conserved to some extent in cel-miR-80, cel-miR-81, cel-miR-82 and cel-miR-234); 3) cel-miR-81, cel-miR-799 (3′ region of identity also conserved to some extent in cel-miR-80 and cel-miR-82); and 4) cel-miR-52, cel-mir-53, cel-miR-70, cel-miR-229 and cel-miR-272. [score:1]
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[+] score: 2
The microinjection of miR-124 was another experiment which further supported the role of RNA -mediated epigenetic inheritance in mice [30]. [score:1]
The miR-124 microinjected mice grew a body size 30–40 percent larger than controls and had a frequent occurrence of twin pregnancies. [score:1]
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[+] score: 1
Indeed, we were able to identify one copy of mir-133, two copies of mir-137, and three copies of mir-124 (supplementary file S4, online) in the genome of P. tepidariorum, further increasing the number of microRNAs that we identified. [score:1]
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[+] score: 1
elegans, Drosophila,Mouse, Humans miR-279 upd Rhythmicity N Insects miR-276a timeless Rhythmicity Peak at ZT10 Trough at ZT18 Insects miR-124 – Phase Peak at ZT19 Trough at ZT7C. [score:1]
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[+] score: 1
Third, miR-124 loss of function increases synaptic release at the NMJ, presumably by coordinating the repression of mRNAs encoding for components in the BMP signaling pathway including the BMP receptors Wit and Saxaphone (Sax), and the downstream transcription factor Mothers against dpp (Mad) [64]. [score:1]
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[+] score: 1
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-17, hsa-mir-25, hsa-mir-92a-1, hsa-mir-92a-2, hsa-mir-105-1, hsa-mir-105-2, dme-mir-1, dme-mir-10, mmu-let-7g, mmu-let-7i, mmu-mir-1a-1, mmu-mir-124-3, mmu-mir-134, mmu-mir-10b, hsa-mir-10a, hsa-mir-10b, dme-mir-92a, dme-mir-92b, mmu-let-7d, dme-let-7, hsa-let-7g, hsa-let-7i, hsa-mir-1-2, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-134, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-92a-2, hsa-mir-1-1, mmu-mir-1a-2, mmu-mir-10a, mmu-mir-17, mmu-mir-25, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-92a-1, hsa-mir-379, mmu-mir-379, mmu-mir-412, gga-let-7i, gga-let-7a-3, gga-let-7b, gga-let-7c, gga-mir-92-1, gga-mir-17, gga-mir-1a-2, gga-mir-124a, gga-mir-10b, gga-let-7g, gga-let-7d, gga-let-7f, gga-let-7a-1, gga-mir-1a-1, gga-mir-124b, gga-mir-1b, gga-let-7a-2, gga-let-7j, gga-let-7k, dre-mir-10a, dre-mir-10b-1, dre-mir-430b-1, hsa-mir-449a, mmu-mir-449a, dre-let-7a-1, dre-let-7a-2, dre-let-7a-3, dre-let-7a-4, dre-let-7a-5, dre-let-7a-6, dre-let-7b, dre-let-7c-1, dre-let-7c-2, dre-let-7d-1, dre-let-7d-2, dre-let-7e, dre-let-7f, dre-let-7g-1, dre-let-7g-2, dre-let-7h, dre-let-7i, dre-mir-1-2, dre-mir-1-1, dre-mir-10b-2, dre-mir-10c, dre-mir-10d, dre-mir-17a-1, dre-mir-17a-2, dre-mir-25, dre-mir-92a-1, dre-mir-92a-2, dre-mir-92b, dre-mir-124-1, dre-mir-124-2, dre-mir-124-3, dre-mir-124-4, dre-mir-124-5, dre-mir-124-6, dre-mir-430b-2, dre-mir-430b-3, dre-mir-430b-4, dre-mir-430b-6, dre-mir-430b-7, dre-mir-430b-8, dre-mir-430b-9, dre-mir-430b-10, dre-mir-430b-11, dre-mir-430b-12, dre-mir-430b-13, dre-mir-430b-14, dre-mir-430b-15, dre-mir-430b-16, dre-mir-430b-17, dre-mir-430b-18, dre-mir-430b-5, dre-mir-430b-19, dre-mir-430b-20, hsa-mir-412, hsa-mir-511, dre-let-7j, hsa-mir-92b, hsa-mir-449b, gga-mir-449a, hsa-mir-758, hsa-mir-767, hsa-mir-449c, hsa-mir-802, mmu-mir-758, mmu-mir-802, mmu-mir-449c, mmu-mir-105, mmu-mir-92b, mmu-mir-449b, mmu-mir-511, mmu-mir-1b, gga-mir-1c, gga-mir-449c, gga-mir-10a, gga-mir-449b, gga-mir-124a-2, mmu-mir-767, mmu-let-7j, mmu-let-7k, gga-mir-124c, gga-mir-92-2, gga-mir-449d, mmu-mir-124b, gga-mir-10c, gga-let-7l-1, gga-let-7l-2
The family defining bootstrap cutoff values are tree-specific, and are set to be the smallest bootstrap value of the reference miRNA families (let7, mir-124, mir-17 and mir-1, See additional file 3: Reference miRNA families) in each input tree. [score:1]
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