Division of Neurobiology, Medical Research Council Laboratory of Molecular Biology, CB2 0QH Cambridge, United Kingdom;
Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, CB2 0QH Cambridge, United Kingdom.
Proc Natl Acad Sci U S A. 2018 Dec 26;115(52):E12388-E12397. doi: 10.1073/pnas.1811438115. Epub 2018 Nov 28.
The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional-translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.
视交叉上核(SCN)是哺乳动物的主要生物钟,协调生理和行为的日常节律。昼夜节律计时围绕着自我维持的转录-翻译负反馈环(TTFL),其中 CLOCK 和 BMAL1 驱动负调节因子 Period 和 Cryptochrome(Cry)的表达。Cry1 和 Cry2 的全局缺失会使 TTFL 失活,导致下游行为的节律性丧失。我们利用这种高度可行的生物学方法进一步开发遗传密码扩展(GCE)作为翻译开关,以实现对 SCN 中生物相关蛋白 Cry1 的可逆控制。这采用了一种正交的氨酰-tRNA 合成酶/tRNA 对,通过腺相关病毒(AAV)载体递送到 SCN,允许将非典型氨基酸(ncAA)掺入携带异位琥珀终止密码子的 AAV 编码的 Cry1 蛋白中。因此,翻译通读和 Cry1 的表达取决于通过培养基或饮用水供应的 ncAA,并且通过突触素依赖性氨酰 tRNA 合成酶的表达限制在神经元中。在 Cry 缺失的 SCN 切片中,神经元中 ncAA 激活 Cry1 翻译会立即并剂量依赖性地启动 TTFL 昼夜节律,在 ncAA 去除后迅速消散。此外,在 Cry 缺失的小鼠中,通过 SCN 神经元的遗传激活 TTFL 会迅速且可逆地引发昼夜行为,其节律幅度由转导的 SCN 神经元数量决定。因此,Cry1 并不指定昼夜节律电路和能力的发展,但对于其不稳定和快速可逆的激活是必不可少的。这证明了使用基于 GCE 的翻译切换对哺乳动物行为进行可逆控制,这是一种具有广泛神经生物学意义的方法。