Analysis and validation of CNBP target genes in Danio rerio

EZEQUIEL MARGARIT; PABLO ARMAS; NORA B CALCATERRA

Potrero de los Funes - San Luis

Congreso; 47th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2011

SAIB - Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

CNBP is a nucleic acid binding protein capable of chaperoning the formation of single-stranded DNA and RNA G-enriched sequences into guanine quadruplex structures (G4). CNBP is a conserved protein required for craniofacial development in zebrafish, mouse and chicken; however, its molecular targets remain unknown. By yeast inverse one hybrid assays with genomic Danio rerio and Mus musculus libraries we found 96 sequences as putative CNBP targets. We searched for a consensus motif between these sequences using several algorithms with the TMOD software and found a 14-nucleotide consensus string (NGGGGG(A/T)GGGGGGN). This sequence may fold as G4, thus reinforcing the notion that CNBP controls gene expression through G4 binding and/or folding promotion. On the other hand, a list of putative CNBP targets was retrieved from data of genes co-expressed with CNBP obtained from zebrafish, mouse and chicken microarrays (http://coxpresdb.jp/) and whole-mount in situ hybridization spatiotemporal patterns (http://zfin.org for zebrafish, http://www.emouseatlas.org/emage/ for mouse, and http://geisha.arizona.edu/ for chicken). The overall analysis resulted in a reduced list of nine putative CNBP targets. We have initiated the validation of these genes in vivo by studying their mRNA level and pattern by real time RT-PCR and whole-mount in situ hybridization in CNBP-depleted zebrafish embryos.