Fig. 4

Fmrp-Adar interaction in zebrafish.

A. Phylogenetic tree of zebrafish and human Adar proteins. Sequences are labeled with gene names, chromosomal locations, and accession numbers. To standardize and simplify the nomenclature, we named the genes Adar1-3, as indicated on the right side of each clade. Similarity values of each Adar member appear on top of each clade. B. Sequence conservation and motif distribution of Adar proteins in zebrafish and humans. Protein domains: adenosine deaminase domain (deaminase, white), double-stranded RNA binding motif (dsRBM, black) and zDNA binding domain (z_alpha, light grey). C-R. In situ hybridization showing lateral (C, E, F, H, I, K, L, N, O, Q) and dorsal (D, G, J, M, P, R) views of the spatial expression pattern of all four adar genes in 2 dpf (C-D, F-G, I-J, L-M) and 6 dpf (E, H, K, N) WT larvae. Expression is detected primarily in the nervous system. O-R. Selected regions (black frames in L and M) show adar2b (O-P) and adar3 (Q-R) expression in the spinal cord of 2 dpf WT embryo. S. HEK-293T cells were transiently transfected with the zebrafish proteins Adar2a and Fmrp fused to EGFP and MYC, respectively (EGFP-Adar2a and MYC-Fmrp). Co-immunoprecipitation was used to detect Adar2a and Fmrp interaction. Actin was used as a negative control. The cell lysate was immunoprecipitated with anti-actin, anti-MYC, or anti-EGFP. Proteins were purified from the complexes and separated by SDS-PAGE. T. Western blot shows the protein content following the transfection prior to the immunoprecipitation. The proteins were detected with specific antibodies against MYC, EGFP, and actin. U. Computational sequence homology predicted the number of RNA recognition elements (RREs) in the CDS of adar genes that are recognized by Fmrp. V. RNA immunoprecipitation (RIP) assays show that Fmrp binds adar1. PCR amplification of adar1 on RNA extracted from a RIP experiment conducted with anti-Actin and anti-MYC antibodies, and on total RNA extracted from HEK293T cells. W. RT-PCR assays showed that the mRNA expression levels of all four adar genes increased in 6 dpf fmr1-/- larvae (grey bars) when compared with WT larvae (white bars). Values are represented as means ± SEM. *p<0.05, **p<0.005, two-way t-test assuming unequal variances. X. Adar2 protein expression was analyzed by Western blot with specific antibodies against Adar2 and actin as a loading control. Elevated Adar2 protein levels of approximately 30% are present in fmr1-/- brains.