Analysis and validation of CNBP target genes during zebrafish development.

MARGARIT, E.; ARMAS, P.; ALLENDE, M. L.; CALCATERRA, N.B.

Montevideo

Congreso; 6th International Meeting of the Latin American Society of Developmental Biology.; 2012

Latin American Society of Developmental Biology.

CNBP is a nucleic acid binding protein capable of chaperoning the formation of guanine-enriched single-stranded DNA and RNA 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 DNA of Danio rerio and Mus musculus libraries, we selected 76 and 193 clones, respectively, which were later sequenced resulting in 96 (11 and 85) unique clones. These clones were in silico mapped to the zebrafish and mouse genomes to define their location in or nearby annotated genes and putative promoter regions, resulting in a total of 203 genes that may be CNBP targets. When analyzed using the Quadparser software (www.quadruplex.org), 80 % of the clones contained putative G4 structures that in comparison to the genomes of zebrafish and mouse had a 2-fold higher G4 frequency, meaning that CNBP prefers sequences with G4 formation potential. Also, we searched for a consensus motif among 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. Gene Ontology indices for a subset of 148 genes of the 203 identified were searched and registered for the processes related to CNBP function. Moreover, 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 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. To confirm the action of CNBP on these putative targets during development, zebrafish embryos were micro-injected with Morpholinos against CNBP and were analyzed both by real time qRT-PCR and whole-mount in situ hybridization. Three genes (wnt5b, smarca5, tbx2b) showed changes in their mRNA levels (one increased, two decreased) pointing them as novel CNBP gene targets.