Functional Antagonism Between CELF and Mbnl Proteins in Cytoplasm and Nucleus [hearts]

The CUG-BP and ETR-3-like factor 1 (Celf1) RNA binding protein plays an important role in heart and muscle development, and is over-expressed in the disease myotonic dystrophy. Celf1 has known roles in regulation of RNA splicing, RNA stability, and protein translation. To identify transcriptome-wide targets of the Celf1 protein in heart, we performed RNA-Seq of polyA+ RNA from mice inducibly expressing Celf1 in the muscle. Mice were engineered to express the reverse tetracycline trans-activator (rtTA2S-M2) from the rate myosin light chain 1/3 promoter/enhancer, and an N-terminal Flag-tagged version of the LYLQ isoform of human Celf1 from a tet-inducible promoter. Mice were fed doxycycline to induce Celf1 expression in muscle, and gastrocnemius muscles were harvested from 3 mice each at 12 hour, 24 hour, 72 hour, and 7 day time points. To account for potential doxycycline-dependent effects, control mice were fed doxycycline for 72 hours but these mice did not contain the tet-inducible Celf1 cassette. In total, 15 gastrocnemius samples were analyzed by RNA-Seq.

CUG-BP与ETR-3样因子1（CUG-BP and ETR-3-like factor 1，简称Celf1）RNA结合蛋白在心脏与肌肉发育过程中发挥关键作用，且在肌强直性营养不良（myotonic dystrophy）患者体内呈现过表达状态。已知Celf1可调控RNA剪接、RNA稳定性及蛋白质翻译进程。为鉴定心脏中Celf1蛋白的全转录组靶点，我们对肌肉中可诱导表达Celf1的小鼠的聚腺苷酸化RNA（polyA+ RNA）开展了RNA测序（RNA-Seq）实验。我们构建了两类基因工程小鼠：一类通过大鼠肌球蛋白轻链1/3启动子/增强子（rat myosin light chain 1/3 promoter/enhancer）表达反向四环素反式激活因子（reverse tetracycline trans-activator, rtTA2S-M2）；另一类通过四环素诱导型启动子（tet-inducible promoter）表达人源Celf1的LYLQ亚型的N端Flag标签融合蛋白。给小鼠喂食多西环素（doxycycline）以诱导肌肉中Celf1的表达，分别在12小时、24小时、72小时及7天四个时间点各采集3只小鼠的腓肠肌（gastrocnemius muscles）组织。为排除多西环素本身可能带来的非特异性影响，我们设置了对照组小鼠：该组小鼠同样喂食多西环素72小时，但不携带四环素诱导型Celf1表达盒。本次研究共获得15份腓肠肌样本，均通过RNA-Seq完成分析。