Novel functions for Sm-class RNAs in the regulation of gene expression

摘要 我的实验室的首要目标是定义一组非 编码RNAs(NcRNAs)：富含U的小核RNAs(SnRNAs)。与Sm蛋白相关的细胞内单链RNA 形成核糖核蛋白颗粒(RNPs)，RNPs是负责 前体信使RNA(Pre-mRNA)的剪接和3‘端形成的加工步骤及其 扰动是几种疾病的根源。我的实验室和其他人最近的研究表明，这群人 NcRNAs在功能上比之前认为的更加多样化，而且在 剪接和3美分末端加工只是冰山一角。 使用疱疹病毒塞米里病毒(HHS)作为模型系统，我们最近展示了SnRNAs执行功能 超出了前信使核糖核酸的加工。这种灵长类g疱疹病毒在潜伏感染的细胞中表达七个小分子RNA。 称为HSURs(疱疹病毒富含U-RNAs)。我们重点研究了其中的两个ncRNA，HSUR1和HSUR2， 因为它们是不同HV组中最保守的HSURs，并且仅有两个表达 由相关的疱疹病毒阿特莱斯。带有宿主microRNAs(MiRNAs)的HSUR1和HSUR2碱基对(MiRNAs)和靶标microRNAs MiRNA，miR-27，用于降解。我们实验室的工作表明，HSUR2也与宿主mRNAs发生碱基配对 并且链接将miRNAs与特定的mRNAs结合起来，以破坏它们的稳定。这构成了SnRNA的第一个示例 作用于前信使核糖核酸。我们还开发了iRICC，这是一种确定RNA结合伙伴的技术 单个感兴趣的RNA和在体内介导RNA-RNA相互作用的序列。使用iRICC，我们展示了 HSUR2结合位点主要位于靶mRNAs的3°非翻译区。我们也 表明HSUR2不存在用于与大多数靶点相互作用的“种子”或专门化区域， 而是作为一个灵活的适配器，通过不同的碱基配对排列与不同的 MRNAs。IRICC发现，HSUR1除了与3？UTRs的靶基因外，还与编码序列大量结合 MRNAs。这些发现提示HSUR1可能通过不同的机制调节宿主基因的表达 与HSUR2雇用的人不同。最后，我们还开发了一种识别低丰度的方法。 并发现哺乳动物细胞表达以前未描述的SnRNAs。 我们将使用遗传学、全基因组、分子生物学和生化方法的组合来 进一步刻画HSUR1和HSUR2功能的机械方面，并从功能上刻画小说 细胞内的单链RNA。这项工作将促进我们对调节的基础机制的理解 病毒感染期间ncRNAs上这类基因的表达。它还将阐明新的机制 在广泛的真核生物中表达短链RNA的基因调控。

Summary The overarching goal of my laboratory is to define the functions and mechanisms of a group of non- coding RNAs (ncRNAs): the small nuclear, U-rich RNAs (snRNAs). Cellular snRNAs associate with Sm proteins to form ribonucleoprotein particles (RNPs) that are essential components of cellular machineries responsible for the precursor messenger RNA (pre-mRNA) processing steps of splicing and 3¢-end formation, and their perturbation underlies several diseases. Recent research from my lab and others suggests that this group of ncRNAs is more functionally diverse than previously thought and that the canonical functions of snRNAs in splicing and 3¢-end processing constitute only the tip of the iceberg. Using Herpesvirus saimiri (HVS) as a model system, we recently showed that snRNAs perform functions beyond pre-mRNA processing. This primate g-herpesvirus expresses seven snRNAs in latently infected cells called HSURs (Herpesvirus saimiri U-rich RNAs). We have focused on two of these ncRNAs, HSURs 1 and 2, because they are the most highly conserved HSURs among different HVS groups and the only two expressed by the related Herpesvirus ateles. HSUR1 and HSUR2 basepair with host microRNAs (miRNAs) and target one miRNA, miR-27, for degradation. Work from our laboratory showed that HSUR2 also basepairs with host mRNAs and links bound miRNAs with specific mRNAs to destabilize them. This constitutes the first example of an snRNA function after pre-mRNA processing. We also developed iRICC, a technique to determine RNA binding partners of a single RNA of interest and the sequences mediating RNA-RNA interactions in vivo. Using iRICC we showed that HSUR2 binding sites reside mostly in the 3¢ untranslated regions (3¢UTRs) of target mRNAs. We also showed that HSUR2 does not present a “seed” or specialized region that is used to interact with most targets, but rather acts as a flexible adaptor that interacts through different base-pairing arrangements with different mRNAs. iRICC revealed that HSUR1 profusely binds to coding sequences in addition to 3¢UTRs of target mRNAs. These findings suggest that HSUR1 might regulate host gene expression through mechanisms different from those employed by HSUR2. Finally, we have also developed an approach for identifying low-abundance snRNAs and discovered that mammalian cells express previously uncharacterized snRNAs. We will use a combination of genetic, genome-wide, molecular biology and biochemical approaches to further characterize mechanical aspects of HSUR1 and HSUR2 function and to functionally characterize novel cellular snRNAs. This work will advance our understanding of the mechanisms underlying the regulation of gene expression by this class on ncRNAs during viral infection. It will also illuminate novel mechanisms of gene regulation in the broad range of eukaryotes that express snRNAs.