Analysis and validation of CNBP target genes in Danio rerio

MARGARIT, E.; CALCATERRA, N. B.; ARMAS, P.

Potrero de los Funes

Congreso; XLVII Annual Meeting of the Argentine Society for Biochemestry and Molecular Biology Research.; 2011

Society for Biochemestry and Molecular Biology Research.

CNBP is a nucleic acid binding protein capable of chaperoning theformation of single-stranded DNA and RNA G-enriched sequencesinto guanine quadruplex structures (G4). CNBP is a conservedprotein required for craniofacial development in zebrafish, mouseand chicken; however, its molecular targets remain unknown. Byyeast inverse one hybrid assays with genomic Danio rerio and Musmusculus libraries we found 96 sequences as putative CNBPtargets. We searched for a consensus motif between thesesequences using several algorithms with the TMOD softwareand found a 14-nucleotide consensus string(NGGGGG(A/T)GGGGGGN). This sequence may fold as G4,thus reinforcing the notion that CNBP controls gene expressionthrough G4 binding and/or folding promotion. On the other hand, alist of putative CNBP targets was retrieved from data of genes coexpressedwith CNBP obtained from zebrafish, mouse and chickenmicroarrays (http://coxpresdb.jp/) and whole-mount in situhybridization spatiotemporal patterns (http://zfin.org for zebrafish,http://www.emouseatlas.org/emage/ for mouse, andhttp://geisha.arizona.edu/ for chicken). The overall analysisresulted in a reduced list of nine putative CNBP targets. We haveinitiated the validation of these genes in vivo by studying theirmRNA level and pattern by real time RT-PCR and whole-mount insitu hybridization in CNBP-depleted zebrafish embryos.