Boodaghian Nouneh, Park Hyunsook, Cohen Susan E
Department of Biological Sciences, California State University, Los Angeles, Los Angeles, California.
Center for Circadian Biology, University of California, San Diego, San Diego, California.
J Biol Rhythms. 2024 Jun;39(3):308-317. doi: 10.1177/07487304241228333. Epub 2024 Feb 15.
Circadian rhythms are found widely throughout nature where cyanobacteria are the simplest organisms, in which the molecular details of the clock have been elucidated. Circadian rhythmicity in cyanobacteria is carried out via the KaiA, KaiB, and KaiC core oscillator proteins that keep ~24 h time. A series of input and output proteins-CikA, SasA, and RpaA-regulate the clock by sensing environmental changes and timing rhythmic activities, including global rhythms of gene expression. Our previous work identified a novel set of KaiC-interacting proteins, some of which are encoded by genes that are essential for viability. To understand the relationship of these essential genes to the clock, we applied CRISPR interference (CRISPRi) which utilizes a deactivated Cas9 protein and single-guide RNA (sgRNA) to reduce the expression of target genes but not fully abolish their expression to allow for survival. Eight candidate genes were targeted, and strains were analyzed by quantitative real-time PCR (qRT-PCR) for reduction of gene expression, and rhythms of gene expression were monitored to analyze circadian phenotypes. Strains with reduced expression of SynPCC7942_0001, , which encodes for the β-clamp of the replicative DNA polymerase, or SynPCC7942_1081, which likely encodes for a KtrA homolog involved in K transport, displayed longer circadian rhythms of gene expression than the wild type. As neither of these proteins have been previously implicated in the circadian clock, these data suggest that diverse cellular processes, DNA replication and K transport, can influence the circadian clock and represent new avenues to understand clock function.
昼夜节律广泛存在于自然界中,其中蓝细菌是最简单的生物体,其生物钟的分子细节已被阐明。蓝细菌中的昼夜节律是通过KaiA、KaiB和KaiC核心振荡器蛋白来实现的,这些蛋白能维持约24小时的时间。一系列输入和输出蛋白——CikA、SasA和RpaA——通过感知环境变化并为包括基因表达的全局节律在内的节律性活动计时来调节生物钟。我们之前的工作鉴定出了一组新的与KaiC相互作用的蛋白,其中一些由对生存至关重要的基因编码。为了了解这些必需基因与生物钟的关系,我们应用了CRISPR干扰(CRISPRi)技术,该技术利用失活的Cas9蛋白和单向导RNA(sgRNA)来降低靶基因的表达,但不完全消除其表达以确保存活。我们针对八个候选基因进行了操作,并通过定量实时PCR(qRT-PCR)分析菌株以检测基因表达的降低情况,并监测基因表达的节律以分析昼夜节律表型。编码复制性DNA聚合酶的β夹子的SynPCC7942_0001或可能编码参与钾运输的KtrA同源物的SynPCC7942_1081表达降低的菌株,其基因表达的昼夜节律比野生型更长。由于这些蛋白之前均未被认为与生物钟有关,这些数据表明不同的细胞过程,即DNA复制和钾运输,可影响昼夜节律,并代表了理解生物钟功能的新途径。