April 1, 2010;
The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity.
The RNA-binding protein Bicaudal C
is an important regulator of embryonic development in C. elegans, Drosophila and Xenopus. In mouse, bicaudal C
) mutants are characterized by the formation of fluid-filled cysts in the kidney
and by expansion of epithelial ducts
. This phenotype is reminiscent of human forms of polycystic kidney
). Here, we now provide data that Bicc1
functions by modulating the expression of polycystin 2
), a member of the transient receptor potential (TRP) superfamily. Molecular analyses demonstrate that Bicc1
acts as a post-transcriptional regulator upstream of Pkd2
. It regulates the stability of Pkd2
mRNA and its translation efficiency. Bicc1
antagonized the repressive activity of the miR-17 microRNA family on the 3''UTR of Pkd2
mRNA. This was substantiated in Xenopus, in which the pronephric defects of bicc1
knockdowns were rescued by reducing miR-17 activity. At the cellular level, Bicc1
protein is localized to cytoplasmic foci that are positive for the P-body markers GW182
. Based on these data, we propose that the kidney
phenotype in Bicc1
(-/-) mutant mice is caused by dysregulation of a microRNA-based translational control mechanism.
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References [+] :
Fig. 3. Bicc1 regulates Pkd2. (A-C) qPCR analysis for mouse Pkd1, Pkd2 and Pkhd1 mRNA levels in Bicc1+/+ (dark gray) and Bicc1−/− (light gray) littermates. The averages and s.d. from six kidney pairs at E15.5 and four pairs at E18.5 are shown (*, P<0.05, Student's t-test). (D) Western blot analysis comparing Pkd2 protein levels in E15.5 kidneys from two Bicc1 mouse litters (#55 and #65) of the indicated genotypes using the Pkd2-specific antibody from Santa Cruz. Actin served as a loading control. (E) Quantification of multiple Pkd2 western blot analyses comparing several mouse litters at E15.5 and normalized to actin. Average values and s.d. are indicated (*, P<0.05, Student's t-test). (F) Whole-mount in situ hybridization for Pkd2 mRNA on uninjected and xBicC-MO1+2-injected Xenopus embryos at stage 39.
Fig. 4. Bicc1 is epistatic to Pkd2. (A-A′) Analysis of the expression of nbc-1 in the Xenopus late distal tubule at stage 39 by whole-mount in situ hybridization of uninjected control embryos, and embryos radially injected with xBicC-MO1+2 in the presence or absence of a single injection of 2 ng pkd2 mRNA. (B) Quantification of the expression of nbc-1 in the late distal tubule from the experiments shown in A-A′. Black, bilateral expression; white, no expression; gray, unilateral expression rescued by co-injected mRNA. The number of embryos analyzed is indicated. (C-D) Reciprocal experiments to those in A-B using Xenopus embryos injected with either Pkd2-MO alone or together with pkd2-myc or bicc1 mRNA. Co-injection with pkd2-myc rescued nbc-1 expression, whereas co-injection with bicc1 did not. (E) Flow diagram outlining the proposed mechanism of Bicc1 activity.
Fig. 7. Cross-talk between Bicc1 and the miR-17 miRNA family. (A) Alignment of the Xenopus miR-17 family members. Mature forms are highlighted in yellow. The sequence targeted by the miR-17 antisense MO (miR-17-MO) is indicated by the black line. The nucleotides shared between miR-17-MO and the individual members are indicated in red. (B-B′) Analysis of the expression of nbc-1 by whole-mount in situ hybridization of uninjected control embryos, embryos injected with xBicC-MO1+2 alone or with miR-17-MO. Arrowheads indicate the expression of nbc-1 in the Xenopus late distal tubule. Note that this expression domain is rescued upon co-injection of the two antisense MOs. (C) Quantification of the experiments shown in B-B′. Black, bilateral expression; white, reduced or no expression; gray, unilateral, rescued expression in the late distal tubule. (D,D′) Models for the post-transcriptional regulation of Pkd2 mRNA by the miR-17 family in the absence or presence of Bicc1.
Fig. 3. Pkd2 expression at stg. 39
Fig. S5. pkd2 mRNA and protein expression in Xenopus. (A) Whole-mount in situ hybridization detecting expression of pkd2 mRNA in the Xenopus pronephros. Inset is a magnified image of the pronephric tubules and the nephrostomes (arrowheads). (B-C′′) Immunofluorescence using antibodies against Pkd2 (AB9088, Millipore; red) and acetylated α-tubulin (green). Note that Pkd2 is expressed in the cilia of the duct (B-B′′) and those of the multiciliated cells in the nephrostomes (C-C′′) present in the Xenopus pronephros. (D-E′′) Immunofluorescence analysis of the cilia in the pronephric tubules using antibodies against Pkd2 (red) and acetylated α-tubulin (green) comparing uninjected Xenopus embryos with embryos injected with Pkd2-MO (400 fmol) at stage 40. Nuclei were counterstained with DAPI.
Fig. S6. Loss of Pkd2 results in a PKD-like phenotype in Xenopus embryos. (A-B′) Xenopus embryos microinjected with antisense MOs against pkd2 (Pkd2-MO) developed a PKD-like phenotype. Morphological analysis showed severe edema formation (A,A′), while histological analysis using Hematoxylin and Eosin staining detected dilated pronephric tubules (B,B′). en, endoderm; no, notochord; nt, neural tube; pn, pronephros; so, somites, (C) Schematic of the Xenopus pronephros indicating the segments expressing the marker genes nbc-1 and lim1 (lhx1). (D-E′′) Whole-mount in situ hybridization of Xenopus embryos injected with xBicC-MO1+2, Pkd2-MO or uninjected controls at stage 39 with nbc-1 (D-D′′) and Lim1 (E-E′′). Arrowheads indicate the expression of these two genes in the late distal tubule that is lost in the antisense MO-injected embryos.
Fig. S7. Expression of pronephric marker genes in pkd2 morphants. (A-E′) Whole-mount in situ hybridization of uninjected control and Pkd2-MO-injected Xenopus embryos at stage 39 with Nephrin (A,A′), xSGLT-1K (B,B′), NKCC2 (C,C′), NCC (D,D′) and β1-Na/K ATPase (E,E′) mRNA. Note that the expression of NCC, a marker for late distal tubule and pronephric duct, was reduced in Pkd2-MO-injected embryos. (F) Schematic of the Xenopus pronephros indicating the markers analyzed.
, Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. The International Polycystic Kidney Disease Consortium. 1995, Pubmed