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. *ppt-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.

A. 斑马鱼与人类Adar蛋白的系统发育树。序列标注包含基因名称、染色体定位及登录号。为统一并简化命名规则，我们将各基因命名为Adar1-3，标注于每个进化枝的右侧；各Adar家族成员的相似性分值则标注于对应进化枝的上方。
B. 斑马鱼与人类Adar蛋白的序列保守性及基序分布情况。蛋白结构域包括：腺苷脱氨酶结构域（脱氨酶结构域，白色）、双链RNA结合基序（double-stranded RNA binding motif，缩写dsRBM，黑色）以及zDNA结合结构域（z_alpha，浅灰色）。
C-R. 原位杂交结果展示了不同发育阶段野生型（wild type，缩写WT）幼虫中4个adar基因的空间表达模式：受精后2天（days post fertilization，缩写dpf）组对应成像为C-D、F-G、I-J、L-M，受精后6 dpf组对应成像为E、H、K、N；成像视角分为侧视图（C、E、F、H、I、K、L、N、O、Q）与背视图（D、G、J、M、P、R）。信号主要定位于神经系统。O-R组为选取L与M图中黑色框区域的放大成像，可观察到2 dpf WT胚胎脊髓中adar2b（O-P）与adar3（Q-R）的表达情况。
S. 将分别融合了EGFP（enhanced green fluorescent protein，增强型绿色荧光蛋白）与MYC标签的斑马鱼Adar2a及Fmrp蛋白（分别命名为EGFP-Adar2a与MYC-Fmrp）瞬时转染HEK-293T细胞。采用免疫共沉淀实验检测Adar2a与Fmrp的相互作用，以肌动蛋白（Actin）作为阴性对照。细胞裂解液分别以抗肌动蛋白、抗MYC或抗EGFP抗体进行免疫沉淀，从复合物中纯化蛋白后通过SDS-PAGE（sodium dodecyl sulfate polyacrylamide gel electrophoresis，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳）进行分离。
T. 免疫沉淀前的转染产物经蛋白质印迹法（Western blot）检测蛋白含量，以针对MYC、EGFP及肌动蛋白的特异性抗体完成标记。
U. 通过计算序列同源性，预测了adar基因编码区（coding sequence，缩写CDS）中可被Fmrp识别的RNA识别元件（RNA recognition elements，缩写RREs）的数量。
V. RNA免疫沉淀（RNA immunoprecipitation，缩写RIP）实验结果显示，Fmrp可结合adar1。以抗肌动蛋白及抗MYC抗体开展RIP实验，提取对应RNA并通过PCR（polymerase chain reaction，聚合酶链反应）扩增adar1，同时以HEK293T细胞的总RNA作为对照。
W. 逆转录聚合酶链反应（reverse transcription polymerase chain reaction，缩写RT-PCR）实验结果显示，与WT幼虫（白色柱形）相比，6 dpf fmr1基因敲除（fmr1-/-）幼虫的4个adar基因mRNA表达水平均出现上调（灰色柱形）。数据以平均值±标准误（standard error of the mean，缩写SEM）表示，采用假设方差不齐的ppt检验，标记为*。
X. 以针对Adar2及作为内参的肌动蛋白的特异性抗体，通过Western blot检测Adar2的蛋白表达水平。结果显示，fmr1-/-脑组织中Adar2蛋白水平较对照升高约30%。