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. 2006;2006(4):64347.
doi: 10.1155/JBB/2006/64347.

Dicer-derived microRNAs are utilized by the fragile X mental retardation protein for assembly on target RNAs

Isabelle Plante  1 , Laetitia Davidovic, Dominique L Ouellet, Lise-Andrée Gobeil, Sandra Tremblay, Edouard W Khandjian, Patrick Provost
Affiliations

Dicer-derived microRNAs are utilized by the fragile X mental retardation protein for assembly on target RNAs

Isabelle Plante et al. J Biomed Biotechnol. 2006.

Abstract

In mammalian cells, fragile X mental retardation protein (FMRP) has been reported to be part of a microRNA (miRNA)-containing effector ribonucleoprotien (RNP) complex believed to mediate translational control of specific mRNAs. Here, using recombinant proteins, we demonstrate that human FMRP can act as a miRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of miRNAs on specific target RNA sequences. The miRNA assembler property of FMRP was abrogated upon deletion of its single-stranded (ss) RNA binding K-homology domains. The requirement of FMRP for efficient RNA interference (RNAi) in vivo was unveiled by reporter gene silencing assays using various small RNA inducers, which also supports its involvement in an ss small interfering RNA (siRNA)-containing RNP (siRNP) effector complex in mammalian cells. Our results define a possible role for FMRP in RNA silencing and may provide further insight into the molecular defects in patients with the fragile X syndrome.

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Figures

Figure 1
Figure 1
FMRP can act as an acceptor of miRNA derived from Dicer cleavage of pre-miRNA. (a) Recombinant FMRP interacts with Dicer RNA cleavage products. 32P-labeled pre-miR-31 was incubated in the absence or presence of recombinant Dicer, without (left) or with (center) MgCl2. The samples were analyzed by nondenaturing (left) or denaturing (center) PAGE and autoradiography. RNA derived from Dicer cleavage was gel-purified and incubated with increasing amounts of recombinant FMRP (0.35–1.4 μg), without or with BSA (20 μg), prior to EMSA analysis (right). M indicates a 10-nt RNA size marker. (b) FMRP preferentially interacts with miRNAs. 32P-labeled green fluorescent protein (GFP) siRNA (left), hsa-miR-196a-1 miRNA:miRNA* duplex (center), or miRNA (right) was incubated in the absence or presence of increasing amounts of recombinant FMRP. The samples were analyzed by EMSA and autoradiography, and analyzed quantitatively by PhosphorImaging. Bound RNA was expressed as a bound to unbound ratio.
Figure 1
Figure 1
FMRP can act as an acceptor of miRNA derived from Dicer cleavage of pre-miRNA. (a) Recombinant FMRP interacts with Dicer RNA cleavage products. 32P-labeled pre-miR-31 was incubated in the absence or presence of recombinant Dicer, without (left) or with (center) MgCl2. The samples were analyzed by nondenaturing (left) or denaturing (center) PAGE and autoradiography. RNA derived from Dicer cleavage was gel-purified and incubated with increasing amounts of recombinant FMRP (0.35–1.4 μg), without or with BSA (20 μg), prior to EMSA analysis (right). M indicates a 10-nt RNA size marker. (b) FMRP preferentially interacts with miRNAs. 32P-labeled green fluorescent protein (GFP) siRNA (left), hsa-miR-196a-1 miRNA:miRNA* duplex (center), or miRNA (right) was incubated in the absence or presence of increasing amounts of recombinant FMRP. The samples were analyzed by EMSA and autoradiography, and analyzed quantitatively by PhosphorImaging. Bound RNA was expressed as a bound to unbound ratio.
Figure 2
Figure 2
FMRP preferentially facilitates natural miRNA:target RNA complex formation. FMRP mediates miRNA (a) and siRNA (b) annealing to specific RNA targets. (a) 32P-labeled cel-let-7 (left) or hsa-miR-196a-1 (right) was incubated with a specific lin-41 or HOXB8 RNA target, respectively, in the absence or presence of increasing amounts of FMRP, or BSA at 4 : 1 (protein:RNA) molar ratio. (b) The RNA strand annealing assays using a single 32P-labeled strand of Dicer (left) or CLP (right) siRNA duplex incubated with a specific or nonspecific RNA target were performed as in (a). Control hybridization was conducted at 37°C for 10 mina or 65°C for 30 minb. RNA complexes were analyzed by nondenaturing 10% PAGE and autoradiography. B, BSA; NS, nonspecific target.
Figure 2
Figure 2
FMRP preferentially facilitates natural miRNA:target RNA complex formation. FMRP mediates miRNA (a) and siRNA (b) annealing to specific RNA targets. (a) 32P-labeled cel-let-7 (left) or hsa-miR-196a-1 (right) was incubated with a specific lin-41 or HOXB8 RNA target, respectively, in the absence or presence of increasing amounts of FMRP, or BSA at 4 : 1 (protein:RNA) molar ratio. (b) The RNA strand annealing assays using a single 32P-labeled strand of Dicer (left) or CLP (right) siRNA duplex incubated with a specific or nonspecific RNA target were performed as in (a). Control hybridization was conducted at 37°C for 10 mina or 65°C for 30 minb. RNA complexes were analyzed by nondenaturing 10% PAGE and autoradiography. B, BSA; NS, nonspecific target.
Figure 3
Figure 3
FMRP mediates miRNA annealing to specific RNA targets through its KH domains. RNA strand annealing properties of the FMRP I304N (a) and ΔKHT (b) mutants. (a) 32P-labeled hsa-miR-196a-1 miRNA was incubated with a specific HOXB8 RNA target in the absence or presence of FMRP or FMRP I304N or with BSA at 4 : 1 (protein:RNA) molar ratio. (b) The RNA strand annealing assays using the FMRP ΔKHT mutant were performed as in (a). Control hybridization was conducted at 37°C for 10 mina or 65°C for 30 minb. RNA complexes were analyzed by nondenaturing 10% PAGE and autoradiography. B, BSA; NS, nonspecific lin-41 RNA target.
Figure 3
Figure 3
FMRP mediates miRNA annealing to specific RNA targets through its KH domains. RNA strand annealing properties of the FMRP I304N (a) and ΔKHT (b) mutants. (a) 32P-labeled hsa-miR-196a-1 miRNA was incubated with a specific HOXB8 RNA target in the absence or presence of FMRP or FMRP I304N or with BSA at 4 : 1 (protein:RNA) molar ratio. (b) The RNA strand annealing assays using the FMRP ΔKHT mutant were performed as in (a). Control hybridization was conducted at 37°C for 10 mina or 65°C for 30 minb. RNA complexes were analyzed by nondenaturing 10% PAGE and autoradiography. B, BSA; NS, nonspecific lin-41 RNA target.
Figure 4
Figure 4
FMRP is required for efficient RNA silencing in mammalian cells. (a) Expression of FMRP in wild-type and Fmr1 KO (TSV-40) cells was verified by immunoblot analysis, in parallel with actin. (b) Fmr1 KO (TSV-40) and wild-type (Naïves) cells were cotransfected with psiSTRIKE encoding Rluc shRNA and psiCHECK reporter construct (n = 6). (c) Fmr1 KO cell lines STEK TSV-40 (n = 2), STEK 3T3A (n = 2), STEK 3T6A (n = 1), and STEK TpBSVE (n = 2), and wild-type Naïves (n = 6) and NIH/3T3 (n = 5) cell lines were cotransfected as in (b). Results of Rluc activity were normalized with Fluc activity and expressed as a percentage of Rluc activity obtained with an shRNA directed against a sequence deleted in the Rluc reporter mRNA. Results were expressed as mean ± SEM and analyzed by analysis of variance followed by unpaired Student's t test. ***P < .001.
Figure 4
Figure 4
FMRP is required for efficient RNA silencing in mammalian cells. (a) Expression of FMRP in wild-type and Fmr1 KO (TSV-40) cells was verified by immunoblot analysis, in parallel with actin. (b) Fmr1 KO (TSV-40) and wild-type (Naïves) cells were cotransfected with psiSTRIKE encoding Rluc shRNA and psiCHECK reporter construct (n = 6). (c) Fmr1 KO cell lines STEK TSV-40 (n = 2), STEK 3T3A (n = 2), STEK 3T6A (n = 1), and STEK TpBSVE (n = 2), and wild-type Naïves (n = 6) and NIH/3T3 (n = 5) cell lines were cotransfected as in (b). Results of Rluc activity were normalized with Fluc activity and expressed as a percentage of Rluc activity obtained with an shRNA directed against a sequence deleted in the Rluc reporter mRNA. Results were expressed as mean ± SEM and analyzed by analysis of variance followed by unpaired Student's t test. ***P < .001.
Figure 4
Figure 4
FMRP is required for efficient RNA silencing in mammalian cells. (a) Expression of FMRP in wild-type and Fmr1 KO (TSV-40) cells was verified by immunoblot analysis, in parallel with actin. (b) Fmr1 KO (TSV-40) and wild-type (Naïves) cells were cotransfected with psiSTRIKE encoding Rluc shRNA and psiCHECK reporter construct (n = 6). (c) Fmr1 KO cell lines STEK TSV-40 (n = 2), STEK 3T3A (n = 2), STEK 3T6A (n = 1), and STEK TpBSVE (n = 2), and wild-type Naïves (n = 6) and NIH/3T3 (n = 5) cell lines were cotransfected as in (b). Results of Rluc activity were normalized with Fluc activity and expressed as a percentage of Rluc activity obtained with an shRNA directed against a sequence deleted in the Rluc reporter mRNA. Results were expressed as mean ± SEM and analyzed by analysis of variance followed by unpaired Student's t test. ***P < .001.
Figure 5
Figure 5
FMRP is required for ss siRNA-induced RNA silencing. Fmr1 KO (TSV-40) and wild-type (Naïves) cells were cotransfected with ss antisense Rluc siRNA (asRluc) or Rluc siRNA duplex (siRluc) and psiCHECK reporter construct (n = 6). Results were expressed as mean ± SEM and analyzed as described in the legend of Figure 4. *P < .05; **P < .01; ***P < .001.
Figure 6
Figure 6
The paralog FXR1P shares the RNA strand annealing properties of FMRP. (a)-(b) RNA strand annealing assays with FXR1P were performed and analyzed as described in the legend of Figure 2. (c) Expression of FXR1P in wild-type and Fxr1 KO cells was verified by immunoblot analysis, in parallel with actin. (D) Fxr1 KO and wild-type (Naïves) cells were cotransfected with psiSTRIKE encoding Rluc shRNA and psiCHECK reporter construct (n = 6). Results were expressed as mean ± SEM and analyzed as described in the legend of Figure 4. *P < .05; **P < .01.
Figure 6
Figure 6
The paralog FXR1P shares the RNA strand annealing properties of FMRP. (a)-(b) RNA strand annealing assays with FXR1P were performed and analyzed as described in the legend of Figure 2. (c) Expression of FXR1P in wild-type and Fxr1 KO cells was verified by immunoblot analysis, in parallel with actin. (D) Fxr1 KO and wild-type (Naïves) cells were cotransfected with psiSTRIKE encoding Rluc shRNA and psiCHECK reporter construct (n = 6). Results were expressed as mean ± SEM and analyzed as described in the legend of Figure 4. *P < .05; **P < .01.
Figure 6
Figure 6
The paralog FXR1P shares the RNA strand annealing properties of FMRP. (a)-(b) RNA strand annealing assays with FXR1P were performed and analyzed as described in the legend of Figure 2. (c) Expression of FXR1P in wild-type and Fxr1 KO cells was verified by immunoblot analysis, in parallel with actin. (D) Fxr1 KO and wild-type (Naïves) cells were cotransfected with psiSTRIKE encoding Rluc shRNA and psiCHECK reporter construct (n = 6). Results were expressed as mean ± SEM and analyzed as described in the legend of Figure 4. *P < .05; **P < .01.
Figure 6
Figure 6
The paralog FXR1P shares the RNA strand annealing properties of FMRP. (a)-(b) RNA strand annealing assays with FXR1P were performed and analyzed as described in the legend of Figure 2. (c) Expression of FXR1P in wild-type and Fxr1 KO cells was verified by immunoblot analysis, in parallel with actin. (D) Fxr1 KO and wild-type (Naïves) cells were cotransfected with psiSTRIKE encoding Rluc shRNA and psiCHECK reporter construct (n = 6). Results were expressed as mean ± SEM and analyzed as described in the legend of Figure 4. *P < .05; **P < .01.
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