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

我们NSERC资助的研究计划的长期目标是促进我们对“HECT结构域和RCC 1样结构域”（HERC）蛋白质家族的进化和生物学功能的理解。在过去的5年里，我们进行了小HERC家族的进化，结构和功能分析。我们表明，这个家族具有> 4.76亿年前的古老海洋起源，并且在整个脊椎动物进化过程中得到很好的保护，指出了HERC在生物学中的潜在基础作用（Paparisto et al.，2017年，提交）。为了支持这一观点，我们的团队和其他人已经表明，HERC蛋白影响各种细胞功能，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，我们的小组发现HERC蛋白可以差异性地抑制RanGTP依赖性核输出途径（Woods et al.，2014）。在接下来的5年里，我们计划表征这种HERC介导的RanGTP依赖性核输出途径抑制的起源，进化和生物学功能。近年来，microRNAs（miRNAs）的基因调控活性受到了广泛关注。MicroRNA和处理它们的基因在物种间高度保守，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miRNAs主要通过RanGTP依赖性输出途径从细胞核输出到细胞质，我们测试并发现HERC蛋白可以有效地阻断miRNAs的核输出（未发表）。** 我们的工作假设是HERC蛋白通过一种新的、进化上保守的机制调节基因表达，该机制涉及miRNA核输出的调节。为了解决这一假设，我们建议确定的关键蛋白参与RanGTP依赖的核出口的功能受HERC表达，映射的关键蛋白质-蛋白质的相互作用和残基所需的这种活动，并表征鸟嘌呤核苷酸交换因子的活性重组HERC蛋白使用建立在体外试验。我们还将通过使用RNA测序确定它们对miRNA和mRNA表达谱的影响来研究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上不同的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，并表征一种潜在的新的转录后基因调控机制，该机制靶向miRNA的核输出。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演变，以及它们的演变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.