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33 publications mentioning dme-mir-9a

Open access articles that are associated with the species Drosophila melanogaster and mention the gene name mir-9a. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

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[+] score: 333
Other miRNAs from this paper: dme-mir-9c, dme-mir-9b, dme-let-7
Since N-cad is probably not the only mRNA target of miR-9a, it will be interesting to study whether these or other mRNA targets should be downregulated by miR-9a in order to regulate spermatogenesis during ageing. [score:9]
A comparison of this list to the 194 in silico predicted miR-9a targets (Targetscan Fly) yielded six potential direct targets, one of which was senseless, a previously characterized target of miR-9a, confirming library reliability (Fig.   3a and Table  1) 14, 17. [score:8]
Scale bars, 10 µm miR-9a is expressed in GSCs and progenitor germ cellsTo identify the cells that express miR-9a in the testis we used a green fluorescent protein (GFP) sensor that utilizes the unique property of miRNAs to silence protein expression [18]. [score:7]
Moreover, ectopic expression of miR-9a-DsRed in hub cells, which do not express miR-9a in wild-type, was partial as only some of the testes expressed DsRed. [score:7]
miR-9a overexpression in GSCs and spermatogonia cells that normally express miR-9a resulted in fragmented N-cad expression in the adherent junctions between GSCs and hub cells (Fig.   4a, b). [score:7]
These data confirm that miR-9a directly inhibits N-cad protein expression and destabilizes its mRNA through canonical sequences within the 3′UTR. [score:6]
Furthermore, our data suggest that at the apical tip of the testis miR-9a serves to disconnect GSCs from the hub by downregulating the expression of N-cad. [score:6]
Alternatively, it is possible that N-cad expression in the hub is non-autonomously regulated by N-cad levels or by other miR-9a targets in the GSCs. [score:6]
Scale bars, 10 µm To identify the cells that express miR-9a in the testis we used a green fluorescent protein (GFP) sensor that utilizes the unique property of miRNAs to silence protein expression [18]. [score:5]
Biryukova I Asmar J Abdesselem H Heitzler P Drosophila mir-9a regulates wing development via fine-tuning expression of the LIM only factor, dLMODev. [score:5]
Consistent with these observations, flow cytometry (FACS) analysis of the GFP -expressing cells population revealed a marked 83% decrease in the mean fluorescent intensity (MFI) when miR-9a was co-expressed with gfp- N-cad-3′ UTR [WT] reporter (Fig.   3g). [score:5]
We used UAS-miR-9a-DsRed [15] to overexpress miR-9a either in GSCs and spermatogonia cells (nos-GAL4; UAS-miR-9a-DsRed) or in the hub (upd-GAL4; UAS-miR-9a-DsRed), whereas the DsRed fluorescent signal marks the positive -expressing cells. [score:5]
Ectopic expression of DsRed-miR-9a in GSCs and progenitor germ cells (a, b) or the hub (c– e) or in both the hub and GSCs (f, g) reduces N-cad expression. [score:5]
Two miR-9a binding consensus sites are located at the 3′UTR of N-cad to mediate translation inhibition and mRNA destabilization in vitro and in vivo. [score:5]
Identification of miR-9a targets in the testismiRNAs repress mRNA translation, which is often followed by the mRNA deadenylation and decay [19]. [score:5]
Expressing miR-9a sensor in a miR-9a null mutant background of miR-9a[E39] [14] resulted in GFP expression in GSCs and spermatogonia cells, indicating that in the sensor flies GFP is specifically repressed by miR-9a in these cells (Supplementary Fig.   1a, b). [score:5]
Furthermore, miR-9a directly downregulates Neural-Cadherin (N-cad) to control the adhesion between GSCs and the hub, thus promoting GSCs detachment from the niche to allow differentiation and functional spermatogenesis. [score:5]
Therefore, cells that endogenously express miR-9a create a silencing mechanism that prevents the expression of GFP. [score:5]
Misregulation of miR-9a in the stem cell nicheGiven that miR-9a is expressed in GSCs and not in the hub, we tested the possibility that it regulates the adherent junctions between GSCs and hub cells via N-cad levels. [score:5]
Simultaneous expression of UAS-miR-9a-DsRed in both the hub and GSCs (upd-GAL4; nosGAL4, UAS-miR-9a-DsRed) also caused a dramatic reduction in N-Cad expression (Fig.   4f). [score:5]
Given that miR-9a is expressed in GSCs and not in the hub, we tested the possibility that it regulates the adherent junctions between GSCs and hub cells via N-cad levels. [score:4]
We present several lines of evidence to support that N-cad is a direct target of miR-9a. [score:4]
Thus, the mRNA levels of direct miR-9a targets in the testis are expected to be elevated in miR-9a[E39] mutants. [score:4]
Fig. 3 N-cad is a direct target of miR-9a. [score:4]
control in both young and aged testis was compared to a list of in silico predicted mir-9a targets (Targetscan Fly, http://www. [score:4]
Note that N-cad [RNAi] rescues miR-9a[E39] sterility Lastly, to directly address the possibility that the miR-9a[E39] mutant phenotypes of increased GSCs and decreased fertility are due to the presence of elevated N-cad at the hub-GSC junctions, we reduced its expression in GSCs of the mutants (nos-GAL4, UAS-N-cad [RNAi]; miR-9a[E39], Fig.   5g–l). [score:4]
Note that N-cad [RNAi] rescues miR-9a[E39] sterility N-cad [RNAi] in GSCs rescues the miR-9a[E39] phenotypeLastly, to directly address the possibility that the miR-9a[E39] mutant phenotypes of increased GSCs and decreased fertility are due to the presence of elevated N-cad at the hub-GSC junctions, we reduced its expression in GSCs of the mutants (nos-GAL4, UAS-N-cad [RNAi]; miR-9a[E39], Fig.   5g–l). [score:4]
Together, these data suggest that miR-9a expression in GSCs regulates the dynamic adhesion between these cells and the hub. [score:4]
Note the significant increase in N-cad expression in GSCs-hub adherent junction of miR-9a[E39] mutants. [score:3]
Fig. 1 miR-9a increases during ageing and is expressed in GSCs and spermatogonia. [score:3]
We propose that the differential expression of miR-9a in GSCs and progenitors, but not in hub cells, enables dynamic adherence of GSCs to the hub without affecting the hub cells themselves. [score:3]
Moreover, miR-9a is not expressed in terminally differentiated spermatocytes, mature sperms, somatic cyst, and, notably, not in hub cells (Fig.   1f, g). [score:3]
Moreover, overexpression of miR-9a in miR-9a[E39] mutant regained GSCs division frequency to 3% (n = 102) and rescued fertility of the aged mutant males (Fig.   2n). [score:3]
Fig. 4 miR-9a overexpression reduces N-cad. [score:3]
In support of these data, miR-9a fluorescence in situ hybridization (FISH) shows that miR-9a is expressed in GSCs and spermatogonia and absent from the hub. [score:3]
Our transcriptome analysis revealed two additional adhesion proteins, Sticks and Stones and lame duck as potential targets of miR-9a (Table  1). [score:3]
To determine whether these mutant phenotypes are due to lack of miR-9a in GSCs and spermatogonia cells, we ectopically expressed UAS-DsRed-miR-9a [15] in these cells of miR-9a[E39] mutants (nos-GAL4, UAS-miR-9a-DsRed; miR-9a[E39]). [score:3]
Identification of miR-9a targets in the testis. [score:3]
In accordance with this observation, reducing N-cad levels in GSCs of the mir-9a[E39] mutant flies resulted in an overall reduction of N-cad expression (Supplementary Fig.   3c, d). [score:3]
In support of miR-9a as a negative regulator of adhesion are the findings that it represses two functionally orthologues of N-cad: in Drosophila the cadherin protein Flamingo (Fmi) and in mammals E-cad 22, 23, suggesting that miR-9 plays a broad role in regulating cell–cell adhesion. [score:3]
Note that miR-9a is not expressed in spermatocytes or the somatic niche (hub and cyst cells). [score:3]
We used this method to compare the expression pattern of GFP-control and GFP -miR-9a sensor both driven under a tubulin promoter. [score:3]
Western analysis in Schneider 2 (S2R+) cells showed that miR-9a causes a 90% reduction in the expression of a GFP reporter containing the N-cad 3′UTR (gfp- N-cad-3′ UTR [WT]). [score:3]
miR-9a is expressed in GSCs and progenitor germ cells. [score:3]
Quantification of gfp by qRT-PCR revealed a 90% reduction in mRNA levels of the wild-type reporter only when miR-9a was co-expressed, with no effect on the mutant (Fig.   3i). [score:3]
Validation of N-cad as a miR-9a targetConsistent with the transcriptome analysis, immunofluorescence staining of controls and mir-9a[E39] mutants with anti-N-cad revealed higher levels in adherent junction of mutants, among hub cells and between hub and GCSs (Fig.   3b). [score:3]
Of these, 231 genes showed higher expression in miR-9a[E39] mutant vs. [score:3]
Note decreased fertility of aged miR-9a[E39] mutants and rescue in DsRed-miR-9a ectopic expression in GSCs and spermatogonia of the mutant background To our surprise, the marked increase in GSCs number in miR-9a[E39] mutants did not result in improvement of the ageing phenotype. [score:3]
i– l Rescue of miR-9a[E39] mutants by ectopic expression of DsRed-miR-9a in GSCs and progenitor germ cells (nos-GAL4, UAS-DsRed-miR-9a; miR-9a[E39]), Con. [score:3]
Note decreased fertility of aged miR-9a[E39] mutants and rescue in DsRed-miR-9a ectopic expression in GSCs and spermatogonia of the mutant background To our surprise, the marked increase in GSCs number in miR-9a[E39] mutants did not result in improvement of the ageing phenotype. [score:3]
However, in testes of both young and aged flies, the miR-9a sensor revealed that miR-9a is expressed in GSCs and spermatogonia progenitor germ cells. [score:3]
A group of genes that showed higher expression in mir-9a[E39] mutant vs. [score:3]
Two miR-9a canonical recognition sites are located within 3′UTR of N-cad (Targetscan Fly, Table  1 and Fig.   3d). [score:3]
In contrast, the levels of Epithelial-cadherin (E-cad), an N-cad family member that is not a predicted miR-9 target, were unchanged (Table  1). [score:3]
a, b Control (nos-GAL4 outcrossed to w [1118]) and miR-9a overexpression in the GSCs and progenitor germ cells (nos-GAL4,UAS-DsRed-miR-9a). [score:3]
a Venn diagram of genes increased in testis from young (green) or aged (yellow) miR-9a[E39] mutants relative to age-matched controls transcriptome analysis in compare to computationally predicted miR-9a targets (red; http://www. [score:3]
Comparison of this list to the predicted miR-9a targets resulted in the presented six candidates. [score:3]
After filtration based on log Fold Change (logFC ≥ 0.9), significance cutoff (P-value ≤ 0.05), and minimal CPM per each gene (≥1), we obtained a group of 450 genes that showed higher expression in young mir-9a[E39] mutant vs. [score:3]
To facilitate miR-9a target identification we analyzed the transcriptome of cDNA libraries (Illumina) of four RNA samples (each in at least two biological repeats) prepared from testis of young (1-day), aged (30-days), control (w [1118]), and miR-9a[E39] mutants. [score:3]
The GFP -miR-9a sensor contains two repeats of the complementary sequence of miR-9a in an artificial 3′ untranslated region (3′UTR) following a reporter GFP sequence [15]. [score:3]
However, here too, clear detection of the DsRed signal in both hub and GSCs was only apparent in a few samples (14/39), while the rest of the samples expressed miR-9a-DsRed only in the germline (25/39, Fig.   4g). [score:3]
f, g Simultaneous expression of miR-9a in both the hub and GSCs (upd-GAL4;nos-GAL4,UAS-DsRed-miR-9a). [score:3]
The transfection efficiency of miR-9a co -expression with gfp- N-cad-3′ UTR [WT] or gfp- N-cad-3′ UTR [Mut] reporters was measured by qRT-PCR for mature miR-9a, and confirmed similar expression levels (Fig.   3h). [score:3]
c– e Control (upd-GAL4 outcrossed to w [1118]) and miR-9a overexpression in the hub (upd-GAL4;UAS-DsRed-miR-9a). [score:3]
Validation of N-cad as a miR-9a target. [score:3]
Taken together, these findings indicate that the phenotype of miR-9a mutants origins at least partially from N-cad overexpression in stem cells. [score:3]
d– i miR-9a targets a gfp-N-cad-3′UTR reporter in S2R+ cells. [score:3]
Moreover, miR-9a overexpression did not affect cell viability. [score:3]
Fig. 5N-cad overexpression mimics miR-9a[E39] phenotype and N-cad [RNAi] rescues miR-9a[E39] mutants. [score:3]
Misregulation of miR-9a in the stem cell niche. [score:2]
Ma L miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasisNat. [score:2]
The appearance of high levels of N-cad in the niche of the miR-9a null mutants may be explained by the fact that GSCs are directly connected to the majority of the hub cells. [score:2]
Scale bars 10 µm To determine whether N-cad overexpression presents a similar phenotype to that of miR-9a mutants, we measured the stem cell number, division frequency, and fertility of N-cad overexpressing flies in stem and spermatogonia germ cells (UAS-N-cad; nos-GAL4) compared to controls (nos-GAL4 outcrossed to w [1118]). [score:2]
Wang Y Wang H Li X Li YEpithelial microRNA-9a regulates dendrite growth through Fmi-Gq signaling in Drosophila sensory neuronsDev. [score:2]
Therefore, what may appear as an increase in N-cad among hub cells following miR-9a knockdown is in fact an increase that occurs mainly between hub and GSCs. [score:2]
rescue (nos-GAL4, UAS-DsRed-miR-9a; miR-9a[E39]/TM6), young (i) and aged (k); Vasa, Fas3, and pHH3 (green) DsRed (red) and DAPI (blue). [score:1]
Fig. 2 miR-9a mutants increase GSCs maintenance but reduce spermatogenesis. [score:1]
rescue 1-day (n = 45), 30-days (n = 24); miR-9a[E39] rescue 1-day (n = 33), 30-days (n = 16); Statistical significance was determined as in Fig.   1d. [score:1]
While both young and aged miR-9a mutants hold a much higher number of stem cells in their niche, this does not improve spermatogenesis. [score:1]
Note a 10-fold increase of miR-9a in 30-days. [score:1]
d Schematic representation of GFP reporter constructs of N-cad 3′UTR WT or Mut (mutated bases to disrupt miR-9a seed pairing are indicated). [score:1]
miR-9a levels increase in testis of aged malesTo determine whether the levels of specific miRNAs are age-altered, we analyzed the miRNAome of testes dissected from 1-day (young), 15-days (mid-aged), and 30-days (aged) wild-type (w [1118]) flies by NanoString technology (Fig.   1b, c). [score:1]
In conclusion, we propose that the increase in miR-9a during ageing in stem and progenitor germ cells modulates degeneration in spermatogenesis and promotes detachment toward sperm maturation. [score:1]
To obtain identical genetic backgrounds that are critical for ageing experiments [27], miR-9a[E39] flies were first outcrossed in our lab to w [1118]. [score:1]
Similar to miR-9a[E39], a second miR-9a null allele, miR-9a[j22] [14], also maintains a high average number of GSCs in the niche of young and aged males (Supplementary Fig.   1f). [score:1]
miR-9a levels increase in testis of aged males. [score:1]
rescue: nos-GAL4, UAS-N-cad [RNAi]; miR-9a[E39]/TM6). [score:1]
Fly strains used in this research were w [1118], control GFP sensor (S. M. Cohen [18]), miR-9a-GFP sensor (E. C. Lai) [15], upd-GAL4 (T. Xie), sco/cyo; nos-Gal4:VP16/MKRS (M. Van Doren [24]), nos-GAL4/Cyo;Sb/Tm6b (E. Arama), Imp [CB04573] (A. Spradling [25]), UAS-DsRed-miR-9a (E. C. Lai) [15], UAS-DN-cadherin on 2nd (T. Uemura [26]), UAS-N-Cad [RNAi] (2 [nd] Chr. [score:1]
e– f Western blot analysis of cells transfected with 50 nM miR-9a mimic or negative control miRNA (miR-CN) with GFP reporters of N-cad 3′UTR WT or Mut as indicated. [score:1]
DsRed fluorescent signal was used to mark the miR-9a -positive cells (Fig.   2i–l). [score:1]
rescue 1-day (n = 27), 30 days (n = 29) ; miR-9a rescue 1-day (n = 30), 30-days (n = 31). [score:1]
Illumina cDNA libraries were prepared from 1 µg total RNA extracted from testes of young and aged control w [1118] and miR-9a[E39] backcrossed mutants with TruSeq RNA V2/Illumina kit. [score:1]
Moreover, no miR-9a FISH signal was obtained in testis of miR-9a[E39] null mutants (Supplementary Fig.   1c–e). [score:1]
Consistent with the transcriptome analysis, immunofluorescence staining of controls and mir-9a[E39] mutants with anti-N-cad revealed higher levels in adherent junction of mutants, among hub cells and between hub and GCSs (Fig.   3b). [score:1]
b Representative images of testes taken at the same time exposure from control (w [1118]; n = 45) and miR-9a[E39] mutants (n = 45) immunostained for N-cad (red) and Vasa (green; scale bars, 10 µm). [score:1]
d qRT-PCR for mature miR-9a relative to control (2S rRNA) in the testes of 1-day, 15-days, or 30-days-old wild-type (w [1118]) males. [score:1]
36% of the testes showed DsRed-miR-9a both in the hub and germline (f, n = 39) and 64% showed DsRed-miR-9a only in the germline (g). [score:1]
g– l N-cad [RNAi] in GSCs and progenitor germ cells of miR-9a[E39] mutants rescue mutant phenotype (rescue: nos-GAL4, UAS-N-cad [RNAi]; miR-9a[E39], Con. [score:1]
a– h Testes from 1-day (a, c) or 30-days (e, g)-old control (w [1118]) or 1-day (b, d) or 30-days (f, h)-old miR-9a[E39] mutants immunostained for Fas3 (blue, hub), Vasa (green, germ cells), and with pHH3 (green, c– d). [score:1]
This restored normal GSC number in both young and aged adults (Fig.   5k), regained normal division rate of GSCs in aged males (n = 178), and importantly rescued the age-related sterility of miR-9a[E39] mutants (Fig.   5l). [score:1]
However, GSCs of miR-9a[E39] and miR-9a[J22] mutant alleles completely arrested division in aged flies (n = 233 and n = 178, respectively). [score:1]
Thus, although the niche of miR-9a null mutants consists of a higher number of GSCs, these cells fail to maintain spermatogenesis in aged males due to reduced division frequency. [score:1]
miR-9a null present higher GSCs number with reduced division. [score:1]
To determine why the increase in GSCs of miR-9a[E39] mutants does not improve spermatogenesis, we immunostained testes with anti-Thr 3-phosphorylated histoneH3 (pHH3) to mark mitotic cells (Fig.   2c, d) and counted pHH3 -positive GSCs. [score:1]
The miR-9a sensor detect endogenous levels of miR-9a in GSCs and spermatogonia. [score:1]
e– g’ Testes of control GFP sensor (e, e’) and miR-9a sensor 1-day (f, f’) and 30-days (g, g’) stained for Fas3 (blue) to mark the hub (asterisks), Vasa to mark the germ cells (red) and GFP (green). [score:1]
miR-9a null mutant alleles (miR-9a[E39] (a backcrossed line) and miR-9a[J22]) were generated in Prof. [score:1]
Gao’s lab by ends-out homologous recombination that replaced the 78 nt-long miR-9a precursor with the white gene [14]. [score:1]
Samples were then washed in Exiqon hybridization buffer and incubated for 1 h at 55 °C with 40 mM xtr- mir-9a-5p 5′ and 3′Dig labeled LNA probe (619314-360, Exiqon). [score:1]
pAc-GFP reporter plasmids (0.5 µg DNA per well) and dm-miR-9a mimic or negative control miR (10 nM; Applied Biosystems) were co -transfected according to the manufacturer’s recommendations (LipoJet™). [score:1]
miR-9a FISHWhole-mount testes were dissected in PBS diethyl pyrocarbonate (DEPC) and placed in Terasaki plates in 10 µl fix solution of 4% PFA in PBT buffer (PBS DEPC, 0.1% Tween-20) for 20 min at room temperature, rinsed and washed twice in PBTH (PBT, 50 µg/ml Heparin, and 250 µg/ml tRNA). [score:1]
rescue, and miR-9a [E39] rescue) of the same age. [score:1]
The abnormal increase in stem cell maintenance in miR-9a mutant testis and the age-related loss of fertility illustrate the severe consequences of failure to restrain stem cell adherence to their niche. [score:1]
miR-9a FISH. [score:1]
N-cad [RNAi] in GSCs rescues the miR-9a[E39] phenotype. [score:1]
On the contrary, GSC division frequency is reduced and spermatogenesis of miR-9a mutants is gradually decreased leading to premature sterility in aged males. [score:1]
P < 0.005 between N-cad [RNAi] miR-9a[E39] rescue and miR-9a[E39] in young and aged males. [score:1]
We also suggest that the age-related increase in miR-9a levels correlates with the increase in dedifferentiation events of spermatogonia progenitors and possibly prevents accumulation of stem cells at the expense of spermatogenesis. [score:1]
Interestingly, one of the top candidates identified was the evolutionary conserved miR-9a 14– 17. miR-9a was also included in the top four most abundant miRNAs in the testis and in aged flies represents ~1% of the entire miRNAome. [score:1]
Furthermore, these differences were maintained also in aged males, where miR-9a[E39] mutants showed 45% more GSCs (Fig.   2e, f, m). [score:1]
rescue (n = 29), miR-9a[E39] (n = 21), and N-cad [RNAi] miR-9a[E39] rescue (n = 24). [score:1]
In summary, our results suggest that miR-9a levels increase significantly in the testis during ageing to promote stem cell differentiation and detachment from their niche. [score:1]
We found that miR-9a levels increase in the GSCs during ageing. [score:1]
The siblings obtained (miR-9a[E39]/+) were then crossed again to obtain the miR-9a[E39]. [score:1]
h, i qRT-PCR, average of n = 3, biological repeats (each in three replicas) for (h) mature miR-9a levels relative to miR-CN in N-cad 3′UTR WT. [score:1]
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[+] score: 285
These results support the hypothesis that the sNPFR1 and NPY2R mRNAs are legitimate targets of miR-9a/miR-9. To further support our hypothesis that miR-9a inhibits growth and suppresses insulin signalling via its target sNPFR1, we simultaneously overexpressed both miR-9a and sNPFR1 in the Drosophila IPCs. [score:11]
These results support the hypothesis that the sNPFR1 and NPY2R mRNAs are legitimate targets of miR-9a/miR-9. To further support our hypothesis that miR-9a inhibits growth and suppresses insulin signalling via its target sNPFR1, we simultaneously overexpressed both miR-9a and sNPFR1 in the Drosophila IPCs. [score:11]
To further verify the expression of miR-9a in larval and adult IPCs, we examined Tubulin–GFP–miR-9a sensor lines 9 (Fig. 2d) whose ubiquitous expression of GFP is suppressed in miR-9a -expressing cells. [score:9]
In the IPCs, miR-9a is likely mediating its effects by targeting the known positive regulator of insulin expression sNPFR1 as miR-9a [−/−] null mutants express elevated levels of sNPFR1 (Supplementary Fig. 7). [score:8]
Dilp2 expression is restored in miR-9a [E39/J22] null mutants by simultaneous IPC-specific miR-9a overexpression (miR9a [−/−] +Dilp2>miR-9a), but Dilp3 expression is further reduced. [score:7]
IPC-specific overexpression of miR-9a also reduces body size, while knockdown of miR-9a via expression of a miR-9a sponge increases body size. [score:6]
These results indicate that miR-9a modulates body growth and metabolism by regulating IPC insulin expression and consequently insulin signalling in peripheral target tissues. [score:6]
This effect is likely caused by modulation of insulin expression, as miR-9a overexpression reduces Dilp2 levels, while miR-9a knockdown enhances Dilp2 levels (Fig. 3). [score:6]
miR-9 in mammals is expressed in both the brain and the pancreatic beta cells where it has been shown to regulate glucose levels via its targets onecut2 and sirt1 (refs 18, 19). [score:6]
miR-9a regulates growth and insulin signallingWe next increased miR-9a expression in the IPCs using UAS-miR-9a (Dilp2>miR-9a) and reduced miR-9a expression in the IPCs using UAS-miR-9a sponge (Dilp2>miR-9a sponge). [score:6]
When miR-9a expression is suppressed outside the IPCs (Dilp2-G80 [ts] ; Tub>miR-9a sponge, 18 °C), body length is significantly reduced compared with the Dilp2-G80 [ts] ; Tub-Gal4 control but Dilp2 expression is unchanged (Fig. 5b,c). [score:6]
Simultaneous sNPFR1 overexpression rescues the phenotypes induced by miR-9a overexpression. [score:5]
Of the candidate miR-9a targets predicted by Targetscan (www. [score:5]
In addition, we show that IPC-specific miR-9a overexpression rescues the Dilp2 expression of the null mutant (Fig. 4c–e; Supplementary Fig. 4b,d,f). [score:5]
We next increased miR-9a expression in the IPCs using UAS-miR-9a (Dilp2>miR-9a) and reduced miR-9a expression in the IPCs using UAS-miR-9a sponge (Dilp2>miR-9a sponge). [score:5]
When miR-9a is suppressed everywhere by shifting the Dilp2-G80 [ts] ; Tub>miR-9a sponge flies to the restrictive temperature, body length is also reduced and Dilp2 expression is increased like the phenotype we observed in miR-9a [−/−] null mutant flies (compare Fig. 5b,c with Fig. 4a,c). [score:5]
sNPFR1 and its orthologue NPY2R are targets of miR-9aSince the sequence of mature miR-9a is well-conserved from flies to humans (Fig. 1c), we reasoned that the miR-9a target responsible for the growth control phenotype may also be conserved. [score:5]
Although manipulation of miR-9a does not alter larval Dilp5 expression, IPC overexpression of miR-9a (Dilp2>miR-9a) reduces larval Dilp2 and Dilp3 mRNA levels, while reduction of miR-9a (Dilp2>miR-9a sponge) significantly increases larval Dilp2 mRNA levels (Fig. 3d–f). [score:5]
In further confirmation of our findings, Dilp2 expression in miR-9a [−/−] null mutant flies is increased like in flies whose IPCs express the miR-9a sponge (Fig. 4c). [score:5]
IPC-specific miR-9aoverexpression rescues Dlip2 expression of the miR-9a null mutant. [score:5]
When Dilp2-Gal80 [ts] is inactivated at 29 °C and miR-9a is ubiquitously suppressed (Dilp2-G80 [ts] ; Tub>miR-9a sponge), body length is less significantly reduced but Dilp2 expression is increased. [score:5]
As expected, the changes in wing cell size and number induced by miR-9a overexpression in the IPCs are rescued by simultaneous overexpression of sNPFR1 (Dilp2>miR-9a+sNPFR1) (Fig. 7f,g). [score:5]
The elevated concentration and development time required for the adult in situ may indicate either lower expression or some degree of probe degradation, but the staining was clearly specific as the miR-9a null mutant brains stained at the same time and under the same conditions were blank. [score:4]
We have uncovered a novel function for miR-9a in the regulation of insulin signalling and body growth through its target sNPFR1 in the Drosophila brain IPCs. [score:4]
Thus, miR-9a in the IPCs seems to regulate body growth via modulation of insulin expression. [score:4]
We next asked whether reduced sNPFR1 dosage (sNPFR1 [minos−/+] heterozygote) can restore body length and Dilp2 expression in the background of miR-9a knockdown via miR-9a sponge. [score:4]
In addition, the levels of haemolymph glucose and trehalose rise with miR-9a overexpression and fall with miR-9a knockdown (Supplementary Fig. 6a). [score:4]
Contrary to expectations, we found that while sponge -mediated knockdown of miR-9a in the IPCs increases body size, miR-9a [−/−] null mutants and flies in which miR-9a is suppressed in non-IPCs show reduced body size. [score:4]
sNPFR1 heterozygous rescues the body length and Dilp2 expression by IPC-specific knockdown of miR-9a. [score:4]
These results confirm that miR-9a in IPCs regulates insulin signalling and body growth via its target sNPFR1 (Fig. 9). [score:4]
How to cite this article: Suh, Y. S. et al. Genome-wide microRNA screening reveals that the evolutionary conserved miR-9a regulates body growth by targeting sNPFR1/NPYR. [score:4]
Larval IPC-specific miR-9a overexpression (Dilp2>miR-9a) reduces activated pAKT, while miR-9a knockdown (Dilp2>miR-9a sponge) increases activated pAKT. [score:4]
We found miR-9a overexpression in the IPCs (Dilp2>miR-9a) reduces body length, wing length and pupal volume, while miR-9a knockdown in the IPCs (Dilp2>miR-9a sponge) increases body length, wing length and pupal volume (Fig. 3a–c; Supplementary Fig. 3a). [score:4]
Similar to our findings in Dilp2>miR-9a sponge flies, pAKT levels are increased and 4E-BP levels are decreased in the insulin target tissues of miR-9a [−/−] null mutants (Fig. 4f–h; Supplementary Fig. 5b). [score:3]
To further support the differential effects of miR-9a in the IPCs and in non-IPCs on body growth, we generated a Dilp2-Gal80 [ts] line that suppresses Gal4 activity in the IPCs at the 18 °C permissive temperature but not at the 29 °C restrictive temperature. [score:3]
sNPFR1 and its orthologue NPY2R are targets of miR-9a. [score:3]
This allowed us to visualize miR-9a expression in larval and adult brains. [score:3]
The sNPFR1 heterozygote also restores Dilp2 expression in the miR-9a sponge and miR-9a [−/−] null mutant backgrounds (Dilp2>miR-9a sponge+sNPFR1 [minos−/+]or miR-9a [−/−] +sNPFR1 [minos−/+]) (Fig. 8b,d). [score:3]
We were able to verify that miR-9a is endogenously expressed in the larval and adult IPCs via LNA in situ hybridization against the mature miRNA and via miR-9a sensor lines (Fig. 2; Supplementary Fig. 2). [score:3]
miR-9a is expressed in the insulin-producing cells. [score:3]
This defect can be rescued by simultaneous sNPFR1 overexpression (Dilp2>miR-9a+sNPFR1). [score:3]
Consistently, IPC overexpression of miR-9a (Dilp2>miR-9a) also reduces Dilp2, 3 and 5 mRNA levels, while reduction of miR-9a (Dilp2>miR-9a sponge) significantly increases Dilp2 mRNA levels (Supplementary Fig. 4a,c,e) in adult heads. [score:3]
Similar to the known miR-9a/miR-9 targets senseless 13 and Foxg1 (ref. [score:3]
In a miRNA overexpression screen using the IPC driver Dilp2-Gal4, we found that miR-9a and the closely related miR-9b dramatically reduce wing size (Fig. 1). [score:3]
The largest and most significant reductions in wing length are induced by overexpression of miR-9a, miR-9b and miR-79, which are all members of the conserved miR-9 miRNA family (Fig. 1c). [score:3]
miR-9a is expressed in the insulin-producing cells of brains. [score:3]
Thus, both our in situ hybridization and Tubulin–GFP–miR-9a sensor results demonstrate miR-9a expression in both larval and adult IPCs. [score:3]
Since UAS-miR-9a significantly reduces wing length via overexpression of a single miRNA, simplifying subsequent analyses, we focused the rest of our efforts on miR-9a. [score:3]
As expected, overexpression of miR-9a in the IPCs (Dilp2>miR-9a) reduces the level of pAKT in both larvae (Fig. 3g,h) and adults (Supplementary Fig. 5a) and increases the level of 4E-BP in adults (Fig. 3i). [score:3]
Although this staining seems to be widespread, its absence in the miR-9a mutants is consistent with some level of endogenous miR-9a expression in the IPCs of both larvae and adults (Fig. 2; Supplementary Fig. 2). [score:3]
Since the sequence of mature miR-9a is well-conserved from flies to humans (Fig. 1c), we reasoned that the miR-9a target responsible for the growth control phenotype may also be conserved. [score:3]
Transfection of miR-9a leads to enrichment of sNPFR1 and NPY2R mRNAs just like known target mRNAs senseless and foxg1 when normalized to rp49 and GAPDH levels. [score:3]
Indeed, overexpression of sNPFR1 rescues the reduced body and wing length phenotypes of Dilp2>miR-9a flies (Fig. 7a,b). [score:3]
Compared with the control (Dilp2-Gal4/+), overexpression of miR-9a in the IPCs (Dilp2>miR-9a) reduces both wing cell size and number, while knockdown of miR-9a (Dilp2>miR-9a sponge) increases wing cell size and number. [score:3]
miR-9a regulates growth and insulin signalling. [score:2]
Here we show that both fly sNPFR1 and mammalian NPY2R are targets of miR-9a/miR-9 by demonstrating direct binding of miR-9a/miR-9 to the sNPFR1 and NPY2R 3′-UTRs using an in vitro binding assay and a CLIP assay (Fig. 6). [score:2]
miR-9a regulates growth and insulin signalling. [score:2]
miR-9a regulates the insulin modulator sNPFR1 and its mammalian orthologue NPY2R. [score:2]
The body length, wing length and pupal volume of miR-9a [−/−] null mutants, however, are all reduced (Fig. 4a,b; Supplementary Fig. 3b), in seeming contrast with the results we observed in the Dilp2>miR-9a sponge flies. [score:1]
Dilp5 levels remain unchanged by miR-9a manipulation. [score:1]
To determine whether miR-9a directly binds to sNPFR1 and NPY2R mRNA, we designed primers to amplify fragments of their 3′-UTRs that include the predicted miR-9a seed sequence matches (Supplementary Table 4). [score:1]
In addition, miR-9a [−/−] null mutant flies produce significantly more sNPFR1 protein than the wild-type control strain (Supplementary Fig. 7). [score:1]
We performed a cross-linking immunoprecipitation (CLIP) assay to determine whether miR-9a binds directly to the sNPFR1 and NPY2R 3′-UTRs. [score:1]
An in situ hybridization with an LNA probe specific to the mature miR-9a sequence stains the IPCs in the w [1118] control genotype (b), but not in miR-9a [E39/J22] null mutant (miR-9a [−/−]) brains (c). [score:1]
We transfected the cells with either synthetic scrambled miR-9a or miR-9a duplex (Bioneer, Korea) (final concentration 80 μM) using Xtremegene HP (Roche, USA). [score:1]
In other words, loss of miR-9a in the IPCs alone increases growth while ubiquitous or non-IPC loss of miR-9a reduces growth (Figs 3a, 4a and 5b). [score:1]
The sNPFR1 heterozygote restores body length significantly in the miR-9a sponge (Dilp2>miR-9a sponge+sNPFR1 [minos−/+]) and slightly in the miR-9a [−/−] null mutants (miR-9a [−/−] +sNPFR1 [minos−/+]) (Fig. 8a,c). [score:1]
Compare with the Dilp2>miR-9a sponge, Dilp2-Gal4/+ and Dilp2-Gal4; sNPFR1 [minos−/+] controls. [score:1]
After transfection with either biotinylated miR-9a or a scrambled miRNA, we isolated the biotin–miRNA–RISC–mRNA complex with streptavidin beads. [score:1]
It will thus be interesting to see if the relationship we have uncovered between the miR-9, NPY, insulin signalling and body growth extends to in vivo mammalian mo dels despite the differences in anatomical location and embryonic origin of the IPCs. [score:1]
We found, however, that the level of Dilp2 mRNA is increased in both Dilp2>miR-9a sponge and miR-9a [−/−] null mutant flies and the level of the downstream signalling protein pAKT is significantly increased in the miR-9a mutants (Figs 3d,g and 4c,f). [score:1]
WT, sNPFR1 [minos−/+] and miR9a [−/+] +sNPFR1 [minos−/+] are the controls. [score:1]
This activation of insulin signalling in both the miR-9a sponge flies and the miR-9a [−/−] null mutants suggests that miR-9a has positive effects on growth in non-IPCs that are able to mask its negative effect on growth in the IPCs. [score:1]
We used a DIG -labelled miR-9a-specific LNA probe purchased from Exiqon (#88078-15) at 50 nM for larval brains and 250 nM for adult brains. [score:1]
Glucose and trehalose are also reduced in miR-9a [−/−] null mutant haemolymph as in the Dilp2>miR-9a sponge flies (Supplementary Fig. 6b). [score:1]
14), we found that miR-9a binds to and enriches sNPFR1 mRNA over RP49 control RNA in Drosophila S2 cells and NPY2R mRNA over GAPDH control RNA in rat insulinoma INS-1 cells (Fig. 6b,e). [score:1]
miR-9a inside and outside the IPCs differentially affects body growth. [score:1]
The larval and adult IPCs in the Tubulin–GFP–miR-9a sensor flies, here identified by a Dilp2-specific antibody, exhibit less GFP staining than surrounding cells (Fig. 2e,f). [score:1]
miR-9a sponges were provided by D. Van Vactor (Havard medical school, USA). [score:1]
These data reveal that miR-9a in the IPCs and in non-IPCs affect body growth differently. [score:1]
This suggests that miR-9a in non-IPCs may affect body growth differently than miR-9a in the IPCs. [score:1]
The UAS-miR-9a sponge contains 10 × miR-9a binding sites and tissue specifically reduces miR-9a levels by soaking up endogenous miR-9a molecules 10. [score:1]
Reduction of miR-9a in the IPCs (Dilp2>miR-9a sponge) increases the level of pAKT in larvae (Fig. 3g,h). [score:1]
Dilp2-Gal4 was provided by E. Rulifson (University of California San Francisco, USA), and miR-9a [J22] and miR-9a [E39] mutants were provided by F. B. Gao (University of Massachusetts Medical School, USA). [score:1]
We observed staining associated with miR-9a probe binding in both the larval (Supplementary Fig. 2) and adult PI regions in a control genotype (w [1118]) (Fig. 2b; Supplementary Fig. 2b), but not in homozygous miR-9a [−/−] null mutant brains stained under identical conditions (Fig. 2c; Supplementary Fig. 2c). [score:1]
Transfection with a scrambled version of miR-9a (Supplementary Table 3) causes no such enrichment. [score:1]
We were able to observe several two to six nucleotide deletions in the miR-9a seed sequence matches of random sNPFR1 and NPY2R 3′-UTR clones from both cell types transfected with miR-9a (Fig. 6a,d; Supplementary Table 5). [score:1]
Tubulin–GFP–miR-9a sensor was provided by E. Lai (Sloan-Kettering Institute, USA). [score:1]
This confirms that miR-9a binds to the 3′-UTRs of sNPFR1 and NPY2R. [score:1]
miR-9a is expressed in the insulin-producing cells of brainsTo investigate the mechanism by which miR-9a alters growth, we used a locked nucleic acid (LNA) probe specific to the mature form of miR-9a in an in situ hybridization experiment. [score:1]
Our results suggest an evolutionarily conserved relationship between the miR-9 family and the sNPF/NPY receptors (Fig. 9). [score:1]
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3
[+] score: 273
Other miRNAs from this paper: dme-mir-9c, dme-mir-9b
To confirm that the muscle defects observed resulted directly from miR-9a overexpression in the IFMs during myofibrillogenesis, we studied the effect of suppressing miR-9a expression in the overexpression background. [score:10]
Since several putative miR-9a targets are important during muscle development, we asked whether the hypercontraction phenotype resulted directly from the downregulation of TnT by miR-9a or was due to the repression of other targets. [score:10]
Given that up mutations have been implicated in muscle hypercontraction (Nongthomba et al. 2003, 2007), we investigated whether miR-9a can cause the downregulation of TnT, possibly through binding to its target sequence at the 3′-UTR of TnT mRNA and suppressing the translational process as represented in Figure 2D. [score:9]
Flies carrying Mhc [P401S] mutation in the miR-9a overexpression background showed suppression of the muscle structural defects associated with the miR-9a overexpression (Figure 1, F–H). [score:8]
To determine whether the inherently low expression of miR-9a during myofibrillogenesis (Figure S1A) is important for the critical roles of its targets during this stage of muscle development, we overexpressed miR-9a in the IFMs throughout myofibrillogenesis. [score:8]
While, miR-9a is strongly expressed in all developmental stages, its expression is reduced in adult flies, including the adult IFMs where its expression is much reduced compared to earlier developmental stages (Sempere et al. 2003). [score:8]
To further confirm that that the muscle hypercontraction resulting from overexpressing miR-9a is a direct result of knockdown of TnT alone, we carried out a rescue of the muscle phenotype by overexpressing TnT devoid of the miR-9a binding site in flies with elevated levels of miR-9a in their IFMs. [score:7]
Thus, our finding that miR-9a can alter levels of thin filament components via translational control of TnT demonstrates that miRNAs are not just “regulators of regulators,” but can act as direct regulators in coordinating a complex process such as myofibrillar assembly. [score:7]
Clearly, downregulation of TnT by miR-9a phenocopies the mutation in up via the same mechanism of stoichiometric imbalance that drives the mis-regulation of the acto-myosin interaction. [score:6]
Overexpression of miR-9a results in downregulation of TnT. [score:6]
These data suggest that the expression of some miR-9a targets could be required for IFM development and function. [score:6]
Of these miR-9a targets in muscles, 4 genes are reported to be involved in larval muscle development, 19 in the development of pupal and adult muscles (Figure 2A) (Schnorrer et al. 2010), and 6 genes are associated with muscle development (Madan et al. 2017; https://www. [score:6]
Figure 3Transgenic lines with overexpression of TnT (10a or 10b isoform) restore Troponin-T levels and rescue the muscle hypercontraction phenotype resulting from overexpression of miR-9a. [score:5]
miR-9a is expressed in developing IFMs and the muscle attachment sites (Yatsenko and Shcherbata 2014), and expression is highly reduced in adult IFMs (Supplemental Material, Figure S1A). [score:5]
miR-9a target prediction was done using five different target prediction software suites. [score:5]
Compared to flies with overexpression of miR-9a alone (Figure 1, H and H’), the hypercontraction suppressed flies possessed six DLMs (asterisks) (Figure 1G) with close to normal sarcomeric structures (Figure 1G’, white arrow shows a Z-disc), confirming that the miR-9a muscle phenotype results from unregulated acto-myosin interactions. [score:5]
We identified TnT, a structural component of the thin filament of the sarcomere, as a major target for miR-9a in muscles, and have shown that miR-9a overexpression leads to repression of TnT and that this is sufficient to explain the IFM hypercontraction phenotype. [score:5]
The upheld gene that encodes TnT is highlighted in red (within blue box) (B) Expression profile of the putative miR-9a targets in the IFMs obtained from the microarray data from IFM of wild-type flies. [score:5]
While miR-9a expression was barely detectable in wild-type IFMs, UH3-Gal4 -mediated (Singh et al. 2014) miR-9a overexpression clearly increased miR-9a levels (Figure S1A) and adversely affected both wing posture and flight performance (Figure 1, A–B’ and E). [score:5]
Taurine -upregulated gene-1 (TUG1) negatively regulates miR-9 in a human cancer cell line (Zhao and Ren 2016). [score:5]
All the putative miR-9a targets associated with muscle development proved to be involved in muscle function. [score:4]
Knockdown of TnT resulted in the disruption of muscle structures (Figure S3, E and E’), which was comparable to the muscle defects seen in overexpression of miR-9a (Figure S3, F and F’). [score:4]
IFM, indirect flight muscles; miR-9a, microRna-9a; PCR, polymerase chain reaction; TnT, Troponin-T; UTR, untranslated region; NS, non-significant. [score:4]
First, we observed that the knockdown of TnT in IFMs during myofibrillogenesis generated a phenotype very similar to the overexpression of miR-9a. [score:4]
Overexpression of transgenic TnT, either the TnT 10a or 10b isoform, devoid of the miR-9a binding sequence, restored TnT protein levels in these flies compared to flies overexpressing miR-9a alone (Figure 3A). [score:4]
Among the putative targets of miR-9a genes involved in muscle development and function is the up gene, which codes for all the TnT isoforms in Drosophila melanogaster. [score:4]
We have now shown that miR-9a is involved in the translational regulation of TnT levels during sarcomeric assembly. [score:4]
Knockdown of miR-9a in the overexpression background rescued the flight ability to levels similar to wild-type flies (Figure S1B). [score:4]
We further tested if the knockdown of some other predicted miR-9a targets can lead to similar defects in muscle structure and function. [score:4]
UAS-miR-9a [third chromosome, Bloomington Drosophila Stock Center (BDSC) #41138] and UAS-miR-SP-9a (third chromosome, kind gift from David Van Vactor, Harvard Medical School) were used for overexpression and knocking-down of miR-9a, respectively. [score:4]
Figure 2Putative target genes of miR-9a that are involved in muscle development. [score:4]
TnT is a major target of miR-9a during myofibrillogenesis in the IFMs. [score:3]
Both of these transgenic lines (TnT 10a and TnT 10b) showed almost complete restoration of muscle integrity and sarcomeric structure (Figure 4, B, B’, D, and D’) comparable to that of wild-type controls (Figure 4, A and A’), in stark contrast to the muscles in the flies overexpressing miR-9a (Figure 4, C and C’). [score:3]
Since miR-9a is capable of regulating TnT levels in Drosophila, it is possible that the human miR-9 may also play a role in regulating TNNT levels. [score:3]
There was also a concomitant decrease in the levels of other structural proteins (which are not targets of miR-9a) that are part of the thin filament (Actin), including the Tn complex (TnI) (Figure S3A). [score:3]
The loss of muscle integrity and sarcomeric structure caused by miR-9a overexpression (Figure 1, D–D’’) is very similar to the hypercontraction phenotype reported earlier (Nongthomba et al. 2003). [score:3]
However, overexpression of miR-9a resulted in abnormal muscle and loss of myofibril integrity in the IFMs, with defects in sarcomeric organization and no organized Z-discs (Figure 1, D–D’’’). [score:3]
Flies of both sexes, overexpressing miR-9a, also exhibited a complete loss of flight ability [100% flightless (n = 35)] unlike their control counterparts (+; +; UAS miR-9a/+) [90% up-flighted and 10% horizontally-flighted (n = 31)] (Figure 1E). [score:3]
Thus, the hypercontraction was rescued and the IFMs showed ordered sarcomeres (Figure S2, D–E’) in contrast to those with the phenotype from overexpression of miR-9a alone (Figure 1, D and D’). [score:3]
Importantly, the restoration of TnT levels in the background of miR-9a overexpression completely rescued the hypercontraction phenotype. [score:3]
Whereas flies overexpressing miR-9a exhibited broken muscle fibers that appeared to be hypercontracted and pulled toward attachment sites (highlighted in Figure 1, D–D’’’), control wild-type adult hemithoraces showed the typical six well-organized DLM fascicles (Figure 1C) and, at higher magnification, individuals showed well-arranged fibers (Figure 1C’). [score:3]
The miR-9a targets thus obtained were then functionally annotated using DAVID (http://david. [score:3]
Figure 1 IFM-specific overexpression of miR-9a causes muscle hypercontraction. [score:3]
However, reduction in Sls, which is a structural component of the Z-disc, did result in some tearing of the sarcomeres (rectangle, Figure S3D’), but this was not comparable to the damage following miR-9a overexpression. [score:3]
We report here that overexpression of miR-9a causes a hypercontraction phenotype in the IFMs. [score:3]
These data argue that TnT is the major target of the miR-9a responsible for the muscle hypercontraction phenotype. [score:3]
Incidentally, bioinformatic analysis failed to find a miR-9 target site in the mRNA sequence of mouse TNNT. [score:3]
identified 135 potential miR-9a targets that were then functionally annotated. [score:3]
Overexpression of miR-9a in the IFMs during myofibrillogenesis causes hypercontraction. [score:3]
Unlike those of the wild-type flies (Figure S2, A and A’), flies overexpressing miR-9a had severe muscle disorganization (Figure S2B), with whole fascicles missing (black arrowheads, Figure S2B’). [score:3]
On the other hand, Flightin, a thick filament component, was not reduced in flies overexpressing miR-9a compared to wild-type flies (Figure S3A). [score:2]
DLMs, dorsal longitudinal muscles; GFP, green fluorescent protein; IFM, indirect flight muscles; miR-9a, microRna-9a; SEM, scanning electron microscope; TRITC, tetramethylrhodamine. [score:2]
Knockdown of miR-9a in the IFMs during myofibrillogenesis does not affect muscle structure and function. [score:2]
The knockdown of miR-9a during myofibril assembly had no detrimental effect on flight (Figure S1B). [score:2]
Indeed, there was a significant reduction in TnT protein levels (P value < 0.002) in the DLMs of flies overexpressing miR-9a compared to wild-type (Figure 2E). [score:2]
The TnT transcripts (both 10a and 10b) were amplified using cDNA extracted from wild-type thoraces, using primers designed to target the 5′- and 3′-UTRs but to exclude the miR-9a binding site. [score:2]
Further, the flight ability of these flies was also partially rescued: control flies [(UH3/+; +; +) Gal4 flies] were 100% up-flighted (n = 31); for TnT 10a, 19.6% of flies were horizontally-flighted, 28.2% down-flighted, and 52.2% flightless (n = 46); and for TnT 10b: 4.8% flies were up-flighted, 37.1% horizontally-flighted, 22.6% down-flighted, and 35.5% were flightless (n = 62); as compared to the flies with overexpression of miR-9a alone, of which were all flightless (n = 60) (Figure 3D). [score:2]
Our study suggests that miR-9a might be involved in specifically regulating the levels of cardiac TnT in humans. [score:2]
Flies with knockdown of miR-9a (UH3 > miR-SP-9a) exhibited close to normal flight ability, with 74.2% of flies capable of upward flight and 25.8% exhibiting horizontal flight (n = 31), which is similar to wild-type, where 80.6% of flies were up-flighted and 19.4% were horizontally-flighted (n = 31) (Figure S1B). [score:2]
It would be interesting to know if the increase in cardiac TNNT that occurs in response to hypertrophic stimulus is mediated by miR-9. Many mutations of the human cardiac TnT give rise to the hypertrophic condition (Di Pasquale et al. 2012). [score:2]
The present study throws light on a new role played by miR-9a during muscle development and function. [score:2]
Further, both wild-type (Figure S1C) and miR-9a knocked-down flies (Figure S1D) had six normal dorsal longitudinal muscles (DLM) fibers in each hemithorax with normal sarcomeric structures (Figure S1, C’–D’’). [score:2]
We have confirmed that miR-9a is barely detectable in adult IFMs (Figure S1A). [score:1]
It would be interesting to determine what represses miR-9 during myofibril assembly. [score:1]
Therefore, we investigated if any such proteins are targets of miR-9a. [score:1]
** signifies P value < 0.005, DLMs, dorsal longitudinal muscles; miR-9a, microRna-9a; NS, non-significant; PCR, polymerase chain reaction; TnT, Troponin- T. Figure 4 Rescue of the loss of muscle integrity. [score:1]
miR-9 is required for maintaining protein stoichiometry and may have implications in the etiology of myopathies. [score:1]
Interestingly, sequence analysis of the human skeletal and cardiac isoforms of TNNT reveals that only the cardiac TNNT possesses the miR-9 binding site (Figure S4C) while the skeletal isoforms lack it. [score:1]
When UH3-Gal4 was used to drive both UAS-miR-9a and UAS-GFP, the progeny still exhibited hypercontracted muscles in hemithoraces with loss of sarcomeric structure (Figure S4, B and B’), similar to the phenotype that results from driving UAS-miR-9a alone using UH3-Gal4 (Figure 1, D and D’). [score:1]
The 3′-UTR of the up gene has the miR-9 binding site sequence (Figure 2D). [score:1]
Drosophila miR-9a is identical to human miR-9 and the human TnT (TNNT) has significant homology to Drosophila TnT (Lagos-Quintana et al. 2001). [score:1]
Locked nucleic acid (LNA) Probe for miR-9a (5′-TCATACAGCTAGATAACCAAAGA-3′) and control probe for U6snoRNA (5′-GTCATCCTTGCGCAGGGGCCATGC-3′) was labeled using 1 µl T4 polynucleotide kinase (PNK), 1 µl [γ- [32]P] ATP, and 2 µl PNK buffer, and the final volume was made up to 20 µl. [score:1]
It is important to note that the initial report of miR-9’s role in muscle hypertrophy were from studies on mice (Wang et al. 2010), so miR-9a could be playing varied roles in different organisms. [score:1]
Previously, Drosophila miR-9a has only been shown to be involved in neuronal differentiation, wing margin patterning, and myotendinous junction formation (Biryukova et al. 2009; Bejarano et al. 2010; Yatsenko and Shcherbata 2014). [score:1]
DLMs, dorsal longitudinal muscles; GFP, green fluorescent protein; miR-9a, microRna-9a. [score:1]
Generation of UAS-TnT lacking the miR-9 binding site. [score:1]
Repression of TnT by miR-9a is responsible for the hypercontraction phenotype. [score:1]
** signifies P value < 0.005, DLMs, dorsal longitudinal muscles; miR-9a, microRna-9a; NS, non-significant; PCR, polymerase chain reaction; TnT, Troponin- T. Figure 4 Rescue of the loss of muscle integrity. [score:1]
The present study provides a plausible candidate in the form of miR-9 to explore in the etiology of idiopathic cardiomyopathies. [score:1]
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4
[+] score: 50
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]
Additionally, let-7 is predicted to target several kinases and phosphatases in this pathway, and, importantly for the postulated involvement of let-7 in malignancy, the Fas ligand, TGF β receptor I, nerve growth factor and fibroblast growth factor 11. miR-9 has been described as a brain-specific miRNA [5], and recent evidence suggests that its expression is highest in fetal brain and oligodendrogliomas [54]. [score:5]
This is because this conservation pattern corresponds better to the inferred selection pattern of miR-544 than the inferred selection pattern of miR-9. One of the issues that has been extensively discussed in the miRNA literature is the question of the typical number of functional targets per miRNA, and the related question of what fraction of seed matches in 3' UTRs corresponds to functional target sites. [score:5]
This suggests that, whereas the target repertoires of miR-9 and miR-124a have been largely conserved since the common ancestor of the mammals, significant changes have occurred in the target repertoires of miR-544 and miR-205 since that time. [score:5]
Additionally, let-7 is predicted to target several kinases and phosphatases in this pathway, and, importantly for the postulated involvement of let-7 in malignancy, the Fas ligand, TGF β receptor I, nerve growth factor and fibroblast growth factor 11. miR-9 has been described as a brain-specific miRNA [5], and recent evidence suggests that its expression is highest in fetal brain and oligodendrogliomas [54]. [score:5]
This is because this conservation pattern corresponds better to the inferred selection pattern of miR-544 than the inferred selection pattern of miR-9. One of the issues that has been extensively discussed in the miRNA literature is the question of the typical number of functional targets per miRNA, and the related question of what fraction of seed matches in 3' UTRs corresponds to functional target sites. [score:5]
In particular, whereas the target sites for the miRNAs on the right (miR-9 and miR-124a) tend to be shared between all mammals, and to some extent with chicken and opossum, the target sites for the miRNAs on the right (miR-544 and miR-205) are shared mostly among primates, but not with other mammals. [score:5]
These targets suggest that miR-9 may be involved in regulating the intercellular communication in the brain and the function of neural circuits. [score:4]
The top pathway associated with this miRNA is that of glutamate metabolism, in which miR-9 appears to target glutamate decarboxylase, glutamate dehydrogenase, glutamase, glutamate-cysteine ligase, glutamic-oxaloacetic transaminase 1, as well as glucosamine-phosphate N-acetyltransferase 1, 4-aminobutyrate aminotransferase, and phosphoribosyl pyrophosphate amidotransferase. [score:3]
The second most significant association for miR-9 is with with the focal adhesion pathway, in which many more genes appear to be targeted, among which collagen V α1, collagen IV α2, integrin 6, tenascin C, talin, trombospondin 2, and vinculin. [score:3]
For example, in the example above a site for miRNA miR-544 that is only conserved in primates would get considerably higher posterior probability than a site for miR-9 with the same conservation pattern. [score:1]
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5
[+] score: 26
Mir-9a is also expressed at high levels in epithelial cells adjacent to SOPs in proneural clusters, suppressing sens through miRNA/target interactions in the sens 3' UTR, and inhibiting neuronal fate in non-SOP cells [29]. [score:9]
Candidate loci ame-mir-9a, C3345, and C5152 were more strongly expressed in worker abdomens, while C1504 and ame-mir-71 were more strongly expressed in queen abdomens. [score:5]
For instance, we find that ame-mir-9a is among the most strongly caste-biased miRNAs, with much higher expression levels in adult worker thorax and abdomen than similar queen tissues, but higher levels of mir-9a occur in queen pupae (Figure 1). [score:3]
Of these SLS predictions, only ame-mir-9a and C5152 were tested for expression by RT-PCR, and both were validated. [score:3]
Interestingly, mir-9a controls sensory organ precursors (SOPs) in Drosophila, with loss of mir-9a function resulting in ectopic production of SOPs, while overexpression of mir-9a yields a severe diminution of SOPs. [score:3]
The SLS output contained five predictions with significant similarity to the HOM output (ame-mir-13a, ame-mir-276, ame-mir-305, ame-mir-92 and ame-mir-9a) and only two predictions with significant similarity to the top 25 MCE candidates, both of which were variants of C5152. [score:1]
This suggests possible roles for ame-mir-9a in influencing caste differences in honey bees. [score:1]
Ame-mir-9a. [score:1]
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6
[+] score: 26
This implies loss of an enhancer element(s) for directed expression in ventral mesoderm in the honeybee, as mesodermal expression is also found in vertebrates suggesting it is a more ancient pattern for mir-1. Mir-9a is a conserved microRNA in sequence but with differing functions in invertebrates and vertebrates. [score:6]
This implies loss of an enhancer element(s) for directed expression in ventral mesoderm in the honeybee, as mesodermal expression is also found in vertebrates suggesting it is a more ancient pattern for mir-1. Mir-9a is a conserved microRNA in sequence but with differing functions in invertebrates and vertebrates. [score:6]
In vertebrates, mir-9a has a quite different role in positively regulating neurogenesis, indicating that both its expression and function has changed significantly in the vertebrate group or this developmental role of mir-9a is particular to the insect group. [score:5]
Sequencing data indicated that Ame-mir-9a was expressed (Table 1) during honeybee early development. [score:4]
In vertebrates, however, while mir-9a is expressed in the developing brain it has a differing role, positively regulating neurogenesis [35, 36]. [score:4]
In situ hybridisation at stage 5, just prior to gastrulation, detected mir-9a throughout the head ectoderm, and then in broad ectodermal stripes across the middle of the embryo to the posterior terminus (Figure 2G). [score:1]
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7
[+] score: 20
Expression of miR-9 increases on a timescale equivalent to the down-regulation effects on KCNMA1 mRNA. [score:6]
miR-9 is predicted to target ALCOREX-containing KCNMA1 mRNAs based on the presence of and complementarity with an alternative 3'UTR (called 3'UTR-2.1) that is associated with ALCOREX-containing mRNAs (Pietrzykowski et al., 2008). [score:3]
These data identify miR-9 as a key regulator of BK channel ethanol tolerance although the in vivo consequences of this regulation remains to be tested, particularly in combination with beta subunit effects on sensitivity and tolerance. [score:3]
Very acutely, KCNMA1 expression in rats is decreased by miR-9 in response to ethanol treatment, but induction of KCNMA1 has been observed in mice in separate studies in specific brain regions 4 h after an injection of ethanol. [score:3]
The well-established roles for the microRNA miR-9 and the BK β subunits in altering BK function, and, specifically, in modifying the ethanol responsiveness of BK, make them excellent targets for study. [score:3]
Posttranscriptional regulation of BK channel splice variant stability by miR-9 underlies neuroadaptation to alcohol. [score:2]
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8
[+] score: 17
Interestingly, miR-9a was also highly expressed in winged S. avenae which has been reported to control the generation of sensory organs in Drosophila adult wing imaginal discs 46, but any potential effect any differences in sensory organ development or function within winged aphids remains unknown. [score:4]
Similarly, the expression of miR-7, Let-7, and miR-9a were 17.1-, 36.5-, 4.9- fold lower in wingless adults, respectively (Fig. 4D–F). [score:3]
The relative miRNA expression, including miR-277 (B), miR-1 (C), miR-7 (D), Let-7 (E), miR-9a (F), miR-315 (G), miR-8 (H), PC-5p-113190_15 (I), and PC-3p-2743_844 (J) at two wing morph was normalized to the wingless adult. [score:3]
Lane 1: 100 bp ladder marker; Lane 2: miR-315; Lane 4: miR-1; Lane 6: miR-9a; Lane 8: PC-5p-113190_15; Lane 10: PC-3p-2743_844; Lane 12: miR-7; Lane 14: miR-8; Lane 16: miR-277; Lane 18: Let-7. The other uneven lanes were negative controls for each target miRNA. [score:3]
Moreover, miR-8 influences cell survival and epithelial organization in Drosophila wings 49 and the miR-9a prevents apoptosis during Drosophila wing development. [score:2]
The RT-PCR amplified products for seven conserved miRNAs (Let-7, miR-1, miR-7, miR-277, miR-8, miR-9a and miR-315) and two novel miRNAs (PC-5p-113190_15 and PC-3p-2743_844) showed a single band in the expected size (60–100 bp) (Fig. 4A and S4). [score:1]
Overall, RT-qPCR results were consistent with RNA-seq analyses, except miR-9a. [score:1]
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9
[+] score: 11
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]
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]
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]
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]
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]
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]
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10
[+] score: 10
Other miRNAs from this paper: dme-mir-9c, dme-mir-9b
Together, these results suggest that increased miRNA‐9 levels alter the equilibrium of the miR9/CBX7 regulatory loop resulting in upregulation of p16 [INK4a] and senescence. [score:5]
Expression of miR‐9 repressed the luciferase activity of the reporter suggesting that miR9 did indeed target human CBX7 (Fig.   1B). [score:5]
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11
[+] score: 10
miR-9a is expressed in all life stages examined and its expression is reduced in adults, which is consistent with what was observed in D. melanogaster [21]. [score:5]
When D. melanogaster data are available for comparison as in the cases of let-7 and miR-9a, similar expression profiles were found between An. [score:3]
Northern analysis using total RNA from 17-day old females with antisense Locked Nucleic Acid (LNA) probes against 4 selected miRNAs (miR-9a, -14, -210, and let-7) all showed bands of the correct size, confirming cloning results (Figure 1, the last lane). [score:1]
Shown here are eight northern blots performed using Dig-labeled miRCURY LNA probes designed for hybridization to either miR-14, let-7, miR-9a, miR-210, or to one of the four novel miRNAs (miR-x1–x4). [score:1]
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12
[+] score: 9
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]
Six miRNAs (miR-279, miR-33, miR-79, miR-9, miR-S5 and miR-S12) were significantly down-regulated by more than twofold. [score:4]
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13
[+] score: 9
Other miRNAs from this paper: dme-mir-9c, dme-mir-9b
In a GBM mo del, miR-9 -mediated downregulation of PTCH1 resulted in the upregulation of Hh signaling and resistance to the standard-of-care drug, temozolomide that was reversed by treatment with the Smo inhibitor, vismodegib [141]. [score:9]
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14
[+] score: 8
In D. melanogaster, maternal mRNA clearing has been associated with the Mir-309 cluster, which includes Mir-9/4/79, Mir-5/6/2944, Mir-3/309 and Mir-279/286, which are mainly expressed between 9 and 12% of embryo development [21]. [score:3]
The first two waves of miRNA expression, represented by CoMod-A1 and CoMod-A2, involve Mir-279, which is associated with multiple biological processes [24], and let-7 and Mir-9, which are related with neural differentiation and function [25– 27]. [score:3]
Finally, Mir-9 and Mir-279, which are associated with maternal mRNA clearance in D. melanogaster [21], belong to CoMod-A2 and show high levels of expression in ED2. [score:2]
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15
[+] score: 7
The bifunctional microRNA miR-9/miR-9* regulates REST and CoREST and is downregulated in Huntington’s disease. [score:7]
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16
[+] score: 7
Members of the mir-9 family, particularly mir-9c, are particularly abundant in unfertilized eggs but lower expressed in early embryos (Figure 1). [score:3]
Last, the microRNA-9 family also targets unstable maternal transcripts. [score:3]
This indicates that mir-9 may be the first case of a maternal microRNA contributing to the degradation of maternal transcripts during MZT. [score:1]
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17
[+] score: 5
bmo-let-7b, bmo-let-7c, bmo-miR-9, bmo-miR-9*, bmo-miR-100-like, bmo-miR-263a, bmo-miR-31 and bmo-bantam were expressed in larva and pupa, but were not detected in moth; of these miRNAs, bmo-miR-9 and bmo-miR-9* are also complementary miRNAs. [score:3]
miRNAs having the most orthologs are mir-133 and mir-9, which are found in 25 and 23 animal species, including D. melanogaster and C. elegans. [score:1]
Weaver et al. (2007) demonstrated that some microRNAs function in the same or similar way in Drosophila and bee (e. g., mir-9a may control sensory organ precursors (SOPs) between Drosophila and bee) [52]. [score:1]
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18
[+] score: 5
In contrast, co-transfection with a miRNA not predicted to bind to the lar 3′UTR (miR-9a) had no effect on reporter expression. [score:3]
Again, as a negative control, co-transfection with a miRNA not predicted to bind to the wg 3′UTR (miR-9a) had no effect on luciferase reporter activity. [score:1]
In contrast, a miRNA with no predicted binding site (miR-9a) has no effect on Fluc activity. [score:1]
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19
[+] score: 4
Suh YS Bhat S Hong S-H Shin M Bahk S Cho KS Genome-wide microRNA screening reveals that the evolutionary conserved miR-9a regulates body growth by targeting sNPFR1/NPYRNat Commun. [score:4]
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20
[+] score: 4
These observations implicate that, orthologous miRs may regulate the orthologs of targeted genes in related species, such as, miR-9a controlling Sensory Organ Precursors (SOPs) in Drosophila and Bee [32]. [score:4]
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21
[+] score: 3
Other miRNAs from this paper: dme-mir-9c, dme-mir-9b
The shift to the production of ethanol-resistant BK channels has been documented in both the hypothalamo-neurohypophysial and medium spiny neurons of rats and is caused by the activation of a microRNA (mir9) that targets slo transcripts encoding ethanol-sensitive channels [31]. [score:3]
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22
[+] score: 3
The AMP expression phenotype for select miRNAs (miR-34, miR-92a, miR-9a and miR-989) is shown (Fig 1D and 1E). [score:3]
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23
[+] score: 2
A total of 109 mature miRNAs were detected (with >1 read per sample), with the most abundant miRNA, miR-9a-5p, roughly 12,000-fold more abundant than the levels of the least abundant miRNA reliably detected (miR-307a-3p). [score:1]
Our data show that the most abundant miRNA, miR-9a-5p, is about 12,000-fold more abundant than the levels of the least abundant miRNA reliably detected (miR-307a-3p). [score:1]
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24
[+] score: 2
However, the evolution of the mir-9 family is particularly complex and will be better understood as new genome sequences become available. [score:1]
We also observed that mir-9 and mir-279 microRNAs appear clustered in some insects (Apis mellifera and Tribolium castaneum according to miRBase), suggesting that an original cluster may have split in Drosophila. [score:1]
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25
[+] score: 2
Wang Y, Wang H, Li X, Li Y. Epithelial microRNA-9a regulates dendrite growth through Fmi-Gq signaling in Drosophila sensory neurons. [score:2]
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26
[+] score: 2
Interestingly, among miR-9 family members tested (miR-9bSP and miR-9cSP), only miR-9cSP was strongly uncovered by Df (Supplementary Fig. 1a), suggesting some degree of specialization for endogenous miRNA functions within the conserved family. [score:1]
Supporting this argument, several hits in the viability screen belonged to the K-box family (miR-2a, miR-2b and miR-2c, and miR-13a and miR-13b) and the miR-9 family (miR-9b and miR-9c). [score:1]
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27
[+] score: 2
miR-9a prevents apoptosis during wing development by repressing Drosophila LIM-only. [score:2]
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28
[+] score: 1
miR-9 is the most abundant human brain miRNA (Mattick and Makunin, 2005) and a recurring candidate from several AD profiling studies. [score:1]
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29
[+] score: 1
For instance, despite no difference in the total dme-miR-9a-5p amount in un-fractionated embryos, this specific miRNA becomes more polysome -associated in 7–8 h embryos (Log [2] fold = 3.3). [score:1]
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30
[+] score: 1
Among the old microRNAs, we have the mir-92, mir-184 and mir-9 families, which are conserved even in chordates. [score:1]
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31
[+] score: 1
Some of the miRNAs (miR-8, miR-9a, miR-10, miR-71, miR-252, miR-276, miR-281) are represented by both strands,-3p and-5p, as was also observed in the previous N6 library [14]. [score:1]
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32
[+] score: 1
For example, members of the microRNA family mir-1 are characteristic of muscle cells, while mir-9 sequences are expressed in the nervous system in both protostomes and deuterostomes (Christodoulou et al. 2010). [score:1]
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33
[+] score: 1
And ten pre-miRNAs also predicted mature parts on the star (*) arm (mir-305, mir-79, let-7, mir-2a-2, mir-8, mir-7, mir-9a, mir-316, mir-34, mir-12). [score:1]
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