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

总结 我的实验室的首要目标是确定一组非- 编码RNA（ncRNA）：核内富含U的小RNA（snRNA）。细胞snRNAs与Sm蛋白相关 形成核糖核蛋白颗粒（RNP），这些颗粒是负责 前体信使RNA（pre-mRNA）的剪接和3 ′-末端形成的加工步骤，以及它们的 扰动是几种疾病的基础。我的实验室和其他人最近的研究表明，这组 ncRNA在功能上比以前认为的更加多样化， 拼接和3端处理只是冰山一角。 最近，我们使用松鼠猴疱疹病毒（HVS）作为模型系统， 超越了前mRNA的加工这种灵长类动物g-疱疹病毒在潜伏感染细胞中表达7种snRNAs HSURs（Herpesvirus saimiri U-rich RNA）。我们关注了其中两种ncRNA，HSUR 1和2， 因为它们是不同HVS组中最高度保守的HSUR， 由相关的疱疹病毒ateles。HSUR 1和HSUR 2与宿主microRNA（miRNAs）和靶向miRNAs碱基配对 miRNA，miR-27，用于降解。我们实验室的工作表明，HSUR 2也与宿主mRNA碱基配对， 并将结合的miRNA与特定的mRNA连接起来使其不稳定。这构成了snRNA的第一个例子 前mRNA加工后的功能。我们还开发了iRICC，一种确定RNA结合伴侣的技术 的单个感兴趣的RNA和在体内介导RNA-RNA相互作用的序列。使用iRICC， HSUR 2结合位点主要位于靶mRNA的3 <$UTR区。我们也 表明HSUR 2不存在用于与大多数靶相互作用的“种子”或专门区域， 而是作为一种柔性接头，通过不同的碱基配对排列与不同的 mRNA。iRICC显示HSUR 1除了与靶基因的3个UTR结合外，还与编码序列大量结合 mRNA。这些发现表明HSUR 1可能通过不同的机制调节宿主基因的表达。 来自HSUR 2的员工最后，我们还开发了一种识别低丰度的方法， snRNAs，并发现哺乳动物细胞表达以前未知的snRNAs。 我们将结合遗传学、全基因组、分子生物学和生物化学方法， 进一步表征HSUR 1和HSUR 2功能的机械方面，并在功能上表征新的 细胞snRNAs。这项工作将促进我们对调节的机制的理解， 在病毒感染期间，这类基因在ncRNA上表达。它还将阐明新的机制， 在表达snRNA的广泛真核生物中的基因调控。

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.