Data_Sheet_3_Dynamic RNA Regulation in the Brain Underlies Physiological Plasticity in a Hibernating Mammal.CSV

Hibernation is a physiological and behavioral phenotype that minimizes energy expenditure. Hibernators cycle between profound depression and rapid hyperactivation of multiple physiological processes, challenging our concept of mammalian homeostasis. How the hibernator orchestrates and survives these extremes while maintaining cell to organismal viability is unknown. Here, we enhance the genome integrity and annotation of a model hibernator, the 13-lined ground squirrel. Our new assembly brings this genome to near chromosome-level contiguity and adds thousands of previously unannotated genes. These new genomic resources were used to identify 6,505 hibernation-related, differentially-expressed and processed transcripts using RNA-seq data from three brain regions in animals whose physiological status was precisely defined using body temperature telemetry. A software tool, squirrelBox, was developed to foster further data analyses and visualization. SquirrelBox includes a comprehensive toolset for rapid visualization of gene level and cluster group dynamics, sequence scanning of k-mer and domains, and interactive exploration of gene lists. Using these new tools and data, we deconvolute seasonal from temperature-dependent effects on the brain transcriptome during hibernation for the first time, highlighting the importance of carefully timed samples for studies of differential gene expression in hibernation. The identified genes include a regulatory network of RNA binding proteins that are dynamic in hibernation along with the composition of the RNA pool. In addition to passive effects of temperature, we provide evidence for regulated transcription and RNA turnover during hibernation. Significant alternative splicing, largely temperature dependent, also occurs during hibernation. These findings form a crucial first step and provide a roadmap for future work toward defining novel mechanisms of tissue protection and metabolic depression that may 1 day be applied toward improving human health.

冬眠是一种旨在最小化能量消耗的生理和行为表型。冬眠动物在深度抑郁和多个生理过程的快速超活化之间循环，这对我们关于哺乳动物稳态的概念提出了挑战。冬眠动物如何调控行动并在这极端条件下维持细胞至有机体的生存能力尚不为人知。在本研究中，我们对模式冬眠动物，13线地松鼠的基因组完整性和注释进行了增强。我们新的组装将这一基因组提升至接近染色体水平连续性，并增加了数千个之前未注释的基因。这些新的基因组资源被用于通过RNA测序数据从动物三个大脑区域识别出6,505与冬眠相关的、差异表达的及处理过的转录本，这些动物的生理状态是通过体温遥测精确定义的。为了促进进一步的数据分析和可视化，我们开发了名为squirrelBox的软件工具。squirrelBox包含了一套全面的工具，用于快速可视化基因水平和聚类群体的动态变化，k-mer和结构域的序列扫描，以及基因列表的交互式探索。利用这些新工具和数据，我们首次解析了季节性效应与温度依赖性效应在冬眠期间对大脑转录组的影响，突出了精心时间点样本对于研究冬眠中差异基因表达的重要性。所识别的基因包括在冬眠期间动态变化的RNA结合蛋白的调控网络，以及RNA池的组成。除了温度的被动影响外，我们还提供了冬眠期间调控转录和RNA周转的证据。冬眠期间还发生了显著的交替剪接，这主要依赖于温度。这些发现构成了一个关键的第一步，为未来工作提供了路线图，旨在定义新的组织保护机制和代谢抑制机制，这些机制可能有一天会被应用于改善人类健康。