Role of Bicaudal-C in Left-Right Axis Formation and Kidney Morphogenesis

The KH domain RNA binding protein Bicaudal-C is known to play a role in anterior-posterior (AP) patterning in Drosophila by acting on oskar mRNA. In mammals, its expression is induced at late gastrulation in the mesodermal layer of the ventral node. Renal cysts arise in mice and frogs lacking Bicaudal-C. Polycystic diseases and left-right (LR) axis malformations are frequently linked to cilia defects. However, a role for BicC in cilia function has not been demonstrated. Here, I report that targeted inactivation of BicC confirms a role in inducing polycystic kidney disease (PKD). In addition, this targeted mutation was found to randomize left-right (LR) asymmetry by disrupting the planar alignment of motile cilia required for cilia-driven fluid flow. Furthermore, depending on its SAM domain, BicC can uncouple Dvl2 signaling from the canonical Wnt pathway, which has been implicated in antagonizing planar cell polarity (PCP). The SAM domain is shown to concentrate BicC in cytoplasmic structures harboring RNA-processing bodies (P-bodies) and Dvl2. P-bodies are implicated degrading target mRNAs that are silenced by microRNAs. These results suggest a model whereby BicC links the orientation of cilia to PCP, possibly by regulating RNA silencing in P-bodies. In the kidney, BicC is localized in proximal tubules. The cystic phenotype is accompanied by increased levels of cAMP and of Adcy6 protein. BicC is proposed to inhibit target mRNA at the translation-initiation stage by regulating the assembly of miRNP/P-bodies.

Regulation of the mRNA silencing activity of Bicaudal-C

Florian Urs Bernet

Cilia dysfunction is a common factor underlying left-right axis malformation and the pathogenesis of virtually all known renal cystic diseases. Mutations in the RNA-binding protein Bicaudal-C (Bicc1) provoke cystic kidneys reminiscent of polycystic kidney disease (PKD), and ectopic Wnt/b-catenin signaling during left-right axis development. Renal cysts in these mice have been linked to defective miRNA-mediated silencing of specific mRNAs, including adenylate cyclase 6 (AC6) and protein kinase A inhibitor (PKIa), but little is known about how Bicc1 activities are regulated. Prompted by the overlapping phenotypes of mice lacking Bicc1 or Inversin (Invs), the protein defective in patients with nephronophthisis type II, we investigated a potential epistatic relationship. We found that Invs acts as an inhibitor of Bicc1 induced silencing and that Invs is required for Bicc1 localization to the Invs compartmentent in the proximal cilium. Molecular analysis demonstrated that Invs interacts with the Bicc1 KH domains required for mRNA binding. The Bicc1/Invs complex prevented the recruitment of target mRNA and their incorporation into miRNA-induced silencing complexes (miRISC). To test whether Bicc1 activity is regulated in vivo, we monitored Bicc1 targets in inv/inv mutant mouse kidneys lacking Invs. While AC6, the RNA helicase Ddx5 (a novel Bicc1 target) and PKA signaling were dramatically downregulated in inv/inv mutant kidneys, these effects were suppressed in Bicc1-/-; inv/inv double mutants, demonstrating that loss of Invs leads to ectopic activation of Bicc1. In autosomal dominant polycystic kidney disease (ADPKD), fluid-filled cysts develop in both kidneys due to defective calcium-permeable mechanosensory complexes of polycystin-1 and -2 encoded by PKD1 and PKD2. Studies in zebrafish suggest that Pkd2/Ca2+ signaling stimulates calmodulin kinase 2 (CaMK2). To further elucidate a role for Ca2+ in Bicc1 regulation, we mutated specific residues that are predicted to be phosphorylated by CaMK2. We found that CaMK2 phosphorylated Bicc1 on T724 and S805 and enabled miRISC assembly, at least in part by stimulating Bicc1 binding to nascent miRNAs. In addition, elevated Ca2+ concentrations prevented in vitro binding of Bicc1 to Invs. Based on these data we suggest a dual role for Ca2+ in regulating Bicc1. Elevated intracellular Ca2+ levels may induce the dissociation of a Bicc1/Invs complex and activate CaMK2 to enable Bicc1 mediated silencing of target mRNAs.

EPFL2015

Functional analysis of Bicc1 and novel interacting proteins in renal tubule cell metabolism and polycystic kidneys

Lucía Carolina Leal Esteban

Genetic disorders known as ciliopathies develop polycystic kidneys, including autosomal dominant and autosomal recessive polycystic kidney diseases (ADPKD and ARPKD). Several signaling pathways including the cAMP/PKA pathway are implicated in driving cystic growth. However, the mechanisms underlying cysts formation is still elusive. Renal cysts also arise in patients and in mouse models with mutations in the RNA-binding protein Bicaudal-C (Bicc1). Bicc1 binds specific mRNAs such as adenylate cyclase VI (AC6) mRNA for translational repression. Conducting a structure-function analysis, we demonstrated that Bicc1 self-polymerizes through its sterile alpha motif (SAM domain) to form cytoplasmic clusters, which specifically localize and silence bound mRNAs. Altered glucose and lipid metabolism also sustain cystic growth in polycystic kidneys, but the cause of these perturbations is unclear. Here, we found that Bicc1 is essential for normal expression of the gluconeogenic enzymes FBP1 and PEPCK specifically in kidneys, and to maintain euglycemia. In a proteomic interactome screen using a knock-in cell line, we found that TAP-tagged Bicc1 binds the C-Terminal to Lis-Homology domain (CTLH) complex, the mammalian orthologue of the glucose-induced degradation (Gid) complex that triggers degradation of FBP1 in S. cerevisiae. Bicc1 stimulates FBP1 expression in vitro in cultured murine inner medullary collecting duct (mIMCD3) cells, albeit independently of its interaction with the CTLH complex. Instead, Bicc1 and CTLH complex seem to mutually downregulate each other. Bicc1 interactome is also enriched with metabolic related proteins. Here we found that Bicc1 coimmunoprecipitates with rate-limiting metabolic enzymes and contributes to maintain their protein levels, and that deletion of Bicc1 significantly alters the renal metabolome while enhances autophagy. Altogether, these results implicate Bicc1 as an important metabolic regulator in the etiology of polycystic kidneys.

EPFL2017

Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow

Daniel Constam, Charlotte Maisonneuve, Thomas Alois Weber

Polycystic diseases and left-right (LR) axis malformations are frequently linked to cilia defects. Renal cysts also arise in mice and frogs lacking Bicaudal C (BicC), a conserved RNA-binding protein containing K-homology (KH) domains and a sterile alpha motif (SAM). However, a role for BicC in cilia function has not been demonstrated. Here, we report that targeted inactivation of BicC randomizes left-right (LR) asymmetry by disrupting the planar alignment of motile cilia required for cilia-driven fluid flow. Furthermore, depending on its SAM domain, BicC can uncouple Dvl2 signaling from the canonical Wnt pathway, which has been implicated in antagonizing planar cell polarity (PCP). The SAM domain concentrates BicC in cytoplasmic structures harboring RNA-processing bodies (P-bodies) and Dvl2. These results suggest a model whereby BicC links the orientation of cilia with PCP, possibly by regulating RNA silencing in P-bodies.