Biological functions and post-transcriptional regulation of microRNAs

microRNA（miRNAs）是小的非编码RNA，其被加载到Argonaute蛋白中以形成miRNA-Induced Silencing Complex（miRISC）的核心。miRNAs将miRISC引导至互补靶mRNA以沉默其表达。miRNA基因座中的突变破坏基因表达程序，因此可能导致包括癌症在内的各种疾病的发展。因此，了解miRNAs在正常发育中的功能和调控miRNAs的分子机制是至关重要的生物学问题。 了解miRNAs在胚胎发生中的生物学功能 虽然miRNAs在分化组织中的功能在C.在线虫和其他生物体中，仅了解少数动物miRNA的胚胎功能（综述于Kotagama等人，2023）。C.由于其明确的定型细胞谱系和强大的遗传工具，线虫是研究胚胎发育的极好模式生物。我们正在进行正向（诱变）和反向（RNAi）筛选microRNA家族突变表型的抑制因子，目前专注于高度保守的mir-51家族。 我们还利用CRISPR-Cas9介导的基因组编辑的力量来发现对发育至关重要的miRNA-靶标相互作用（Yang，et al. 2020）。了解胚胎表达的microRNA家族影响的生物网络将对如何控制基因表达以协调胚胎发生产生重要的见解。 定义miRNA和Argonaute周转的分子机制 miRNA生物合成和衰变速率的平衡控制着miRNA的丰度，从而控制着基因表达程序。先前的研究已经仔细阐明了miRNA生物合成的机制。然而，我们对miRNAs和miRISC是如何组成性地或以受调控的方式转变的知之甚少。这是我们对miRNA调控的理解中的一个主要空白，因此也是基因表达调控的一个主要空白。我们之前证明了咖啡因诱导的死亡（CID-1）是miRNA尿苷酸化所必需的，而F31C3.2（我们将其命名为GLD-2 Related-2）是miRNA腺苷酸化所必需的（Vieux，et al. 2021）。我们还发现，这些末端修饰在影响miRNA衰变速率方面并不起全局作用。最近，我们正在研究mir-35家族的调控衰变，这对胚胎发生至关重要，此后急剧下调。今年，我们发现该miRNA家族的衰变取决于其种子序列（核苷酸2-8），而不是miRNA序列的其他部分（Donnelly，et al. 2022）。这代表了一类新的miRNA衰变机制，可以在治疗上利用它来同时调节miRNA种子家族所有冗余成员的丰度。

MicroRNAs (miRNAs) are small noncoding RNAs that are loaded into Argonaute proteins to form the core of the miRNA-Induced Silencing Complex (miRISC). MiRNAs guide miRISC to complementary target mRNAs to silence their expression. Mutations in miRNA loci disrupt gene expression programs, and thus can contribute to the development of various diseases, including cancer. Consequently, understanding both the functions of miRNAs in normal development and the molecular mechanisms that regulate miRNAs are biological questions of critical importance. Understanding the biological functions of miRNAs during embryogenesis While the functions of miRNAs in differentiated tissues are well-studied in C. elegans and other organisms, the embryonic functions of only a few animal miRNAs are understood (reviewed in Kotagama, et al. 2023). C. elegans is an excellent model organism in which to study embryonic development due to its well-defined stereotypic cell lineage and powerful genetic tools. We are conducting forward (mutagenesis) and reverse (RNAi) screens for suppressors of microRNA family mutant phenotypes, currently focusing on the deeply conserved mir-51 family. We are also leveraging the power of CRISPR-Cas9-mediated genome editing to discover miRNA-target interactions that are essential to development (Yang, et al. 2020). Understanding the biological networks impacted by the embryonically-expressed microRNA families will yield important insights into how gene expression is controlled to coordinate embryogenesis. Defining the molecular mechanisms of miRNA and Argonaute turnover The balance of the rates of miRNA biogenesis and decay control miRNA abundance, and thus gene expression programs. Previous research has carefully elucidated mechanisms of miRNA biogenesis. However, we know very little about how miRNAs and miRISC are turned over either constitutively or in a regulated manner. This is a major gap in our understanding of miRNA regulation, and thus the regulation of gene expression. We previously demonstrated that Caffeine-Induced Death (CID-1) is necessary for uridylation of miRNAs, and F31C3.2 (which we named GLD-2 Related-2) is required for adenylation of miRNAs (Vieux, et al. 2021). We also found that these terminal modifications do not play a global role in influencing miRNA decay rates. More recently, we are investigating the regulated decay of the mir-35 family, which is essential for embryogenesis and sharply downregulated thereafter. This year, we showed that this miRNA family's decay is dependent upon its seed sequence (nucleotides 2-8), but not other parts of the miRNA sequence (Donnelly, et al. 2022). This represents a novel class of miRNA decay mechanism that may be harnessed therapeutically to modulate abundance of all redundant members of a miRNA seed family simultaneously.