RESUMO
Canonical ß-catenin-dependent Wnt signal transduction is important for several biological phenomena, such as cell fate determination, cell proliferation, stem cell maintenance and anterior-posterior axis formation. The hallmark of canonical Wnt signaling is the translocation of ß-catenin into the nucleus where it activates gene transcription. However, the mechanisms regulating ß-catenin nuclear localization are poorly understood. We show that Simplet/Fam53B (Smp) is required for Wnt signaling by positively regulating ß-catenin nuclear localization. In the zebrafish embryo, the loss of smp blocks the activity of two ß-catenin-dependent reporters and the expression of Wnt target genes, and prevents nuclear accumulation of ß-catenin. Conversely, overexpression of smp increases ß-catenin nuclear localization and transcriptional activity in vitro and in vivo. Expression of mutant Smp proteins lacking either the nuclear localization signal or the ß-catenin interaction domain reveal that the translocation of Smp into the nucleus is essential for ß-catenin nuclear localization and Wnt signaling in vivo. We also provide evidence that mammalian Smp is involved in regulating ß-catenin nuclear localization: the protein colocalizes with ß-catenin-dependent gene expression in mouse intestinal crypts; siRNA knockdown of Smp reduces ß-catenin nuclear localization and transcriptional activity; human SMP mediates ß-catenin transcriptional activity in a dose-dependent manner; and the human SMP protein interacts with human ß-catenin primarily in the nucleus. Thus, our findings identify the evolutionary conserved SMP protein as a regulator of ß-catenin-dependent Wnt signal transduction.
Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Western Blotting , Humanos , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Luciferases , Camundongos , Camundongos Transgênicos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Wnt/genéticaRESUMO
Translational regulation is heavily employed during developmental processes to control the timely accumulation of proteins independently of gene transcription. In particular, mRNA poly(A) tail metabolism in the cytoplasm is a key determinant for balancing an mRNA's translational output and its decay rate. Noncanonical poly(A) polymerases (PAPs), such as germline development defective-2 (GLD-2), can mediate poly(A) tail extension. Little is known about the regulation and functional complexity of cytoplasmic PAPs. Here we report the discovery of Caenorhabditis elegans GLD-4, a cytoplasmic PAP present in P granules that is orthologous to Trf4/5p from budding yeast. GLD-4 enzymatic activity is enhanced by its interaction with GLS-1, a protein associated with the RNA-binding protein GLD-3. GLD-4 is predominantly expressed in germ cells, and its activity is essential for early meiotic progression of male and female gametes in the absence of GLD-2. For commitment into female meiosis, both PAPs converge on at least one common target mRNA-i.e., gld-1 mRNA-and, as a consequence, counteract the repressive action of two PUF proteins and the putative deadenylase CCR-4. Together our findings suggest that two different cytoplasmic PAPs stabilize and translationally activate several meiotic mRNAs to provide a strong fail-safe mechanism for early meiotic progression.