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The bantam microRNA is associated with drosophila fragile X mental retardation protein and regulates the fate of germline stem cells.

Yang Y, Xu S, Xia L, Wang J, Wen S, Jin P, Chen D - PLoS Genet. (2009)

Bottom Line: We show that, like dFmr1, bantam is not only required for repressing primordial germ cell differentiation, it also functions as an extrinsic factor for germline stem cell maintenance.Furthermore, we find that bantam genetically interacts with dFmr1 to regulate the fate of germline stem cells.Collectively, our results support the notion that the FMRP-mediated translation pathway functions through specific miRNAs to control stem cell regulation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China.

ABSTRACT
Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP). We have previously demonstrated that dFmr1, the Drosophila ortholog of the fragile X mental retardation 1 gene, plays a role in the proper maintenance of germline stem cells in Drosophila ovary; however, the molecular mechanism behind this remains elusive. In this study, we used an immunoprecipitation assay to reveal that specific microRNAs (miRNAs), particularly the bantam miRNA (bantam), are physically associated with dFmrp in ovary. We show that, like dFmr1, bantam is not only required for repressing primordial germ cell differentiation, it also functions as an extrinsic factor for germline stem cell maintenance. Furthermore, we find that bantam genetically interacts with dFmr1 to regulate the fate of germline stem cells. Collectively, our results support the notion that the FMRP-mediated translation pathway functions through specific miRNAs to control stem cell regulation.

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Specific miRNAs associated with dFmr1 protein in Drosophila ovary.(A) Western blot shows that dFmr1 protein (dFmrp) was immunoprecipitated from wild-type Drosophila ovary. A dFmr1  mutant (dfmr13) was used as a negative control. (B) miRNA TaqMan assays of 72 known Drosophila miRNAs were performed in triplicate using both input and IP RNAs from both wild-type and dfmr13 mutants. The miRNAs that were enriched are shown in progressively brighter shades of red, and the miRNAs that were reduced in IP are shown in progressively brighter shades of green. The miRNAs shown in black were not changed. The fold of the change is indicated on both sides of the scale bar. The miRNAs that are specifically enriched in IP from wild-type ovary are shown. The data represent the average of two biological replicates (two independent immunoprecipitation experiments). (C) TaqMan assays of the bantam miRNA were performed in triplicate, and the enrichment of the bantam miRNA in dFmrp-IP (independent IP experiments from those presented in panel B) from wild-type ovary is shown. (D) Northern blot shows that the bantam miRNA is associated with dFmrp. Northern blots detecting the sense and anti-sense strands of the bantam miRNA in both input and IP RNAs from WT and dfmr13 mutants are shown.
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pgen-1000444-g001: Specific miRNAs associated with dFmr1 protein in Drosophila ovary.(A) Western blot shows that dFmr1 protein (dFmrp) was immunoprecipitated from wild-type Drosophila ovary. A dFmr1 mutant (dfmr13) was used as a negative control. (B) miRNA TaqMan assays of 72 known Drosophila miRNAs were performed in triplicate using both input and IP RNAs from both wild-type and dfmr13 mutants. The miRNAs that were enriched are shown in progressively brighter shades of red, and the miRNAs that were reduced in IP are shown in progressively brighter shades of green. The miRNAs shown in black were not changed. The fold of the change is indicated on both sides of the scale bar. The miRNAs that are specifically enriched in IP from wild-type ovary are shown. The data represent the average of two biological replicates (two independent immunoprecipitation experiments). (C) TaqMan assays of the bantam miRNA were performed in triplicate, and the enrichment of the bantam miRNA in dFmrp-IP (independent IP experiments from those presented in panel B) from wild-type ovary is shown. (D) Northern blot shows that the bantam miRNA is associated with dFmrp. Northern blots detecting the sense and anti-sense strands of the bantam miRNA in both input and IP RNAs from WT and dfmr13 mutants are shown.

Mentions: Given that dFmr1 plays an important role in the fate determination of germ cells, and that dFmrp physically associates with dAGO1, a key component in the miRNA pathway, we proposed that dFmr1 regulates the fate of GSCs through the miRNA pathway in Drosophila ovary [17]. To test this, we first determined whether dFmrp is associated with the endogenous miRNAs. We used the previously developed anti-dFmrp antibody to perform immunoprecipitation from the lysates of both wild-type (WT, w1118) and dfmr13 mutant ovaries (Figure 1A) [18]. RNAs from both the immunoprecipitated (IP) complex and input were isolated for miRNA TaqMan assays. We used TaqMan assays available from ABI that could detect a total of 72 known individual miRNAs. To identify the miRNAs specifically associated with dFmrp, we determined the level of each miRNA in both IP and input RNAs from WT and dfmr13 mutants. We identified the miRNAs that were consistently enriched by more than two-fold in WT-IP over both WT-Input and dfmr13-IP in two independent experiments, since such miRNAs are likely to be the ones specifically associated with dFmrp based on previous IP experiments (Figure 1B) [19]. Among the 72 miRNAs examined here, only selective miRNAs were enriched in WT-IP compared with both WT-Input and dfmr13-IP, suggesting that dFmrp is indeed associated with specific miRNAs, but not with endogenous miRNAs in general (Figure 1B). Since the bantam miRNA is among those miRNAs specifically associated with dFmrp in ovary, we further confirmed their specific association by quantitative RT-PCR and Northern blot (Figure 1C and 1D). The rest of this study focuses on the role of the bantam miRNA and its potential interaction with dFmr1 in regulating GSCs.


The bantam microRNA is associated with drosophila fragile X mental retardation protein and regulates the fate of germline stem cells.

Yang Y, Xu S, Xia L, Wang J, Wen S, Jin P, Chen D - PLoS Genet. (2009)

Specific miRNAs associated with dFmr1 protein in Drosophila ovary.(A) Western blot shows that dFmr1 protein (dFmrp) was immunoprecipitated from wild-type Drosophila ovary. A dFmr1  mutant (dfmr13) was used as a negative control. (B) miRNA TaqMan assays of 72 known Drosophila miRNAs were performed in triplicate using both input and IP RNAs from both wild-type and dfmr13 mutants. The miRNAs that were enriched are shown in progressively brighter shades of red, and the miRNAs that were reduced in IP are shown in progressively brighter shades of green. The miRNAs shown in black were not changed. The fold of the change is indicated on both sides of the scale bar. The miRNAs that are specifically enriched in IP from wild-type ovary are shown. The data represent the average of two biological replicates (two independent immunoprecipitation experiments). (C) TaqMan assays of the bantam miRNA were performed in triplicate, and the enrichment of the bantam miRNA in dFmrp-IP (independent IP experiments from those presented in panel B) from wild-type ovary is shown. (D) Northern blot shows that the bantam miRNA is associated with dFmrp. Northern blots detecting the sense and anti-sense strands of the bantam miRNA in both input and IP RNAs from WT and dfmr13 mutants are shown.
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Related In: Results  -  Collection

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pgen-1000444-g001: Specific miRNAs associated with dFmr1 protein in Drosophila ovary.(A) Western blot shows that dFmr1 protein (dFmrp) was immunoprecipitated from wild-type Drosophila ovary. A dFmr1 mutant (dfmr13) was used as a negative control. (B) miRNA TaqMan assays of 72 known Drosophila miRNAs were performed in triplicate using both input and IP RNAs from both wild-type and dfmr13 mutants. The miRNAs that were enriched are shown in progressively brighter shades of red, and the miRNAs that were reduced in IP are shown in progressively brighter shades of green. The miRNAs shown in black were not changed. The fold of the change is indicated on both sides of the scale bar. The miRNAs that are specifically enriched in IP from wild-type ovary are shown. The data represent the average of two biological replicates (two independent immunoprecipitation experiments). (C) TaqMan assays of the bantam miRNA were performed in triplicate, and the enrichment of the bantam miRNA in dFmrp-IP (independent IP experiments from those presented in panel B) from wild-type ovary is shown. (D) Northern blot shows that the bantam miRNA is associated with dFmrp. Northern blots detecting the sense and anti-sense strands of the bantam miRNA in both input and IP RNAs from WT and dfmr13 mutants are shown.
Mentions: Given that dFmr1 plays an important role in the fate determination of germ cells, and that dFmrp physically associates with dAGO1, a key component in the miRNA pathway, we proposed that dFmr1 regulates the fate of GSCs through the miRNA pathway in Drosophila ovary [17]. To test this, we first determined whether dFmrp is associated with the endogenous miRNAs. We used the previously developed anti-dFmrp antibody to perform immunoprecipitation from the lysates of both wild-type (WT, w1118) and dfmr13 mutant ovaries (Figure 1A) [18]. RNAs from both the immunoprecipitated (IP) complex and input were isolated for miRNA TaqMan assays. We used TaqMan assays available from ABI that could detect a total of 72 known individual miRNAs. To identify the miRNAs specifically associated with dFmrp, we determined the level of each miRNA in both IP and input RNAs from WT and dfmr13 mutants. We identified the miRNAs that were consistently enriched by more than two-fold in WT-IP over both WT-Input and dfmr13-IP in two independent experiments, since such miRNAs are likely to be the ones specifically associated with dFmrp based on previous IP experiments (Figure 1B) [19]. Among the 72 miRNAs examined here, only selective miRNAs were enriched in WT-IP compared with both WT-Input and dfmr13-IP, suggesting that dFmrp is indeed associated with specific miRNAs, but not with endogenous miRNAs in general (Figure 1B). Since the bantam miRNA is among those miRNAs specifically associated with dFmrp in ovary, we further confirmed their specific association by quantitative RT-PCR and Northern blot (Figure 1C and 1D). The rest of this study focuses on the role of the bantam miRNA and its potential interaction with dFmr1 in regulating GSCs.

Bottom Line: We show that, like dFmr1, bantam is not only required for repressing primordial germ cell differentiation, it also functions as an extrinsic factor for germline stem cell maintenance.Furthermore, we find that bantam genetically interacts with dFmr1 to regulate the fate of germline stem cells.Collectively, our results support the notion that the FMRP-mediated translation pathway functions through specific miRNAs to control stem cell regulation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China.

ABSTRACT
Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP). We have previously demonstrated that dFmr1, the Drosophila ortholog of the fragile X mental retardation 1 gene, plays a role in the proper maintenance of germline stem cells in Drosophila ovary; however, the molecular mechanism behind this remains elusive. In this study, we used an immunoprecipitation assay to reveal that specific microRNAs (miRNAs), particularly the bantam miRNA (bantam), are physically associated with dFmrp in ovary. We show that, like dFmr1, bantam is not only required for repressing primordial germ cell differentiation, it also functions as an extrinsic factor for germline stem cell maintenance. Furthermore, we find that bantam genetically interacts with dFmr1 to regulate the fate of germline stem cells. Collectively, our results support the notion that the FMRP-mediated translation pathway functions through specific miRNAs to control stem cell regulation.

Show MeSH
Related in: MedlinePlus