|
Fig. 4 Celf1 binds to dmrt2a mRNA in vitro and in vivo. (A) Sequences of RNA oligonucleotide probes used in vitro binding assay. Underlines mark UGU repeats and capital letters indicate substituted nucleotides. (B) Binding of Celf1 with specific RNA was tested by in vitro binding assay. Lane 1: dmrt2a_wildtype; lane2: dmrt2a_3p mut; lane 3: dmrt2a_7p mut; lane 4: empty; lane 5: s0 (negative control); lane 6: EDEN (positive control). Interaction between Celf1 and either dmrt2a_wildtype (lane 1) or EDEN (lane 6) was detected. (C) In vivo interaction between Celf1 with dmrt2a (upper left panel), myod (upper right panel), rbpja (middle left panel), rbpja (middle right panel), β-actin (lower left panel) or cyclinA1 (lower right panel) mRNAs. Column 1: RIP control sample using normal serum from uninjected embryos; column 2: RIP sample using Celf1 antiserum (Celf1 AS) from uninjected embryos; column 3: RIP sample using Celf1 AS from embryos injected with dmrt2a-TP-MO. Celf1 interacted with dmrt2a, myod, rbpja and rbpjb mRNAs. dmrt2a-TP-MO specifically blocked the interaction of Celf1 with dmrt2a mRNA.