Origins and evolution of a novel mechanism of post-transcriptional gene regulation by the HERC family

我们nserc资助的研究项目的长期目标是推进我们对“HECT结构域和rcc1样结构域包含”（HERC）蛋白家族的进化和生物学功能的理解。在过去的5年里，我们对HERC小家族进行了进化、结构和功能分析。我们发现，这个家族在4.76亿年前有一个古老的海洋起源，并且在脊椎动物的进化过程中得到了很好的保护，这表明herc在生物学中具有潜在的基础作用（Paparisto et al., 2017，提交）。为了支持这一观点，我们的团队和其他人已经证明HERC蛋白影响多种细胞功能，包括免疫反应、精子发生、神经发育、DNA损伤修复和细胞生长。最近，我们的研究小组发现HERC蛋白可以不同地抑制rangtp依赖的核输出途径（Woods et al., 2014）。在接下来的5年里，我们将研究herc介导的rangtp依赖性核输出通路抑制的起源、进化和生物学功能。******近年来，微rna （micro RNAs, miRNAs）的基因调控活性备受关注。MicroRNA和处理它们的基因在物种中高度保守，被认为是基因调控的重要和进化上古老的组成部分，影响宿主转录组的60%。由于mirna主要通过rangtp依赖的输出途径从细胞核输出到细胞质，我们测试并发现HERC蛋白可以有效阻断mirna的核输出（未发表）。******我们的工作假设是HERC蛋白通过一种涉及miRNA核输出调节的新颖且进化保守的机制调节基因表达。为了解决这一假设，我们提出确定参与rangtp依赖性核输出的关键蛋白，其功能受HERC表达的影响，绘制关键蛋白相互作用和该活性所需的残基，并利用已建立的体外实验表征重组HERC蛋白的鸟嘌呤核苷酸交换因子活性。我们还将通过RNA测序来确定HERC蛋白对miRNA和mRNA表达谱的影响，从而研究HERC蛋白的生物学作用。最后，我们将使用类似的方法来确定进化多样化的HERC蛋白是否抑制rangtp依赖的核输出，以及该机制是否保守。******影响：我们的研究将深入了解HERC蛋白的生物学功能，并描述靶向mirna核输出的转录后基因调控的潜在新机制。重要的是，我们的研究将有助于阐明复杂的基因调控网络如何进化，以及它们的进化如何导致表型变化和物种形成。

The long-term goal of our NSERC-funded research program is to advance our understanding of the evolution and biological functions of the “HECT domain and RCC1-like domain-containing” (HERC) family of proteins. Over the last 5 years, we performed evolutionary, structural and functional analyses of the small HERC family. We showed that this family has an ancient marine origin of >476 million years ago, and is well-conserved throughout vertebrate evolution, pointing to a potentially fundamental role for HERCs in biology (Paparisto et al., 2017, submitted). In support of this notion, our group and others have shown that HERC proteins impact a variety of cellular functions including the immune response, spermatogenesis, neurodevelopment, DNA damage repair and cell growth. Recently, our group discovered that HERC proteins can differentially inhibit the RanGTP-dependent nuclear export pathway (Woods et al., 2014). Over the next 5 years, we propose to characterize the origin, evolution and biological function of this HERC-mediated inhibition of the RanGTP-dependent nuclear export pathway.******In recent years, much attention has been focused on the gene regulatory activity of micro RNAs (miRNAs). MicroRNA and the genes that process them are highly conserved across species and are thought to be a vital and evolutionarily ancient component of gene regulation, influencing >60% of a host's transcriptome. Since miRNAs are mainly exported to the cytoplasm from the nucleus by the RanGTP-dependent export pathway, we tested and discovered that HERC proteins can potently block the nuclear export of miRNAs (unpublished). ******Our working hypothesis is that HERC proteins modulate gene expression by a novel and evolutionarily conserved mechanism involving the regulation of miRNA nuclear export. To address this hypothesis, we propose to identify the key proteins involved in RanGTP-dependent nuclear export whose functions are affected by HERC expression, map the key protein-protein interactions and residues required for this activity, and characterize the guanine nucleotide exchange factor activity of recombinant HERC proteins using established in vitro assays. We will also investigate the biological role of HERC proteins by determining their impact on miRNA and mRNA expression profiles using RNA sequencing. Lastly, we will use a similar approach to determine if evolutionarily diverse HERC proteins inhibit RanGTP-dependent nuclear export and whether the mechanism is conserved.******Impact: Our research will provide insight into the biological function(s) of the HERC proteins and characterize a potentially novel mechanism of post-transcriptional gene regulation that targets nuclear export of miRNAs. Importantly, our research will help elucidate how complex gene regulatory networks evolve and how their evolution results in phenotypic change and speciation.