Nuclear-retained long noncoding RNAs

描述(申请人提供)：我们最近发现了新的内含子衍生的长非编码RNAs(LncRNAs)，它们在两端都被snoRNA机制处理(我们将这些RNAs命名为sno-LncRNAs)。大多数snoRNA都是从内含子加工而来的。去分支后，核酸外切酶降解内含子，但不降解snoRNAs，因为这些内含子与阻止降解并导致snoRNP组装的特定因素相关联。Sno-IncRNAs是由含有两个SnoRNAs的内含子加工而来的。在核酸外切过程中，snoRNAs之间的序列不会降解，导致缺乏5‘帽结构或3’聚(A)尾巴的lncRNA积累。其中一些RNA可以在细胞中积累到非常高的水平，但以前被错过了，因为它们很长，它们来自内含子，并且它们没有Poly(A)尾巴。这些构成了一类全新的核lncRNA，这项提议是对它们进行更详细的研究。到目前为止的结果表明，只要内含子包含不是一个而是两个snoRNA基因，就可以产生sno-lncRNAs。到目前为止，在每一个被检查的细胞中都可以发现这样的RNA，而且似乎在组织中广泛表达。此外，编码其中一些最丰富的RNA的基因组区域(15q11-q13)在一种重要的人类遗传病Prader-Willi综合征中被特异性地缺失。我们将描述sno-lncRNA是如何形成的，与它们相关的蛋白质是什么，以及它们有多少。此外，我们将询问它们在细胞中的作用，并开始探讨它们在Prader-Willi综合征中可能扮演的角色。在第一个目标中，我们 将表征sno-lncRNAs的结构和表达。我们还将研究它们的亚核定位，并努力动态地跟踪它们的合成和定位，并贯穿整个细胞周期。最后，在这个目标中，我们将在几种不同的细胞类型中进行全基因组研究，并使用几种不同的方法来识别 更多的sno-lncRNA，其中一些将被更详细地研究。第二个目的是研究sno-lncRNAs的作用机制。特别感兴趣的是chr15 sno-lncRNAs与Fox1家族剪接调控因子的联系。令人信服的证据表明，这些lncRNA可能以高亲和力结合Fox2或Fox1。因此，它们可能作为分子“海绵”来滴定剪接因子，因为在初步实验中，我们发现它们改变了剪接模式。这一角度将被详细检查，并可能直接导致 对普雷德-威利病理学的新见解。我们还将询问一些sno-lncRNAs是否能像其他一些lncRNAs一样改变染色质结构。最后，由于这些新的RNA似乎相对稳定并严格保留在细胞核中，我们将开始利用我们对其表达的了解来生成一类新的载体，用于各种序列的核表达。

DESCRIPTION (provided by applicant): We have recently discovered novel intron-derived long noncoding RNAs (lncRNAs) that are processed on both ends by the snoRNA machinery (we named these sno-lncRNAs). Most snoRNAs are processed from introns. After debranching, exonucleases degrade the introns but not the snoRNAs because these associate with specific factors that prevent degradation and lead to snoRNP assembly. Sno-lncRNAs are processed from introns that contain two snoRNAs. During exonucleolytic trimming, the sequences between the snoRNAs are not degraded, leading to the accumulation of lncRNAs lacking 5' cap structures or 3' poly(A) tails. Some of these RNAs can accumulate to very high levels in cells but were missed before because they are long, they derive from introns, and they lack poly(A) tails. These constitute an entirely new class of nuclear lncRNAs and this proposal is to study them in greater detail. Results so far suggest that whenever introns contain not one but two snoRNA genes, sno-lncRNAs can be produced. Such RNAs can be found in every cell examined so far, and appear to be widely expressed in tissues. Further, the genomic region encoding some of the most abundant of these RNAs (15q11-q13) is specifically deleted in an important human genetic disease, Prader-Willi Syndrome. We will characterize how sno-lncRNAs are made, what proteins associate with them and how many of them there are. In addition, we will ask what they do in cells and begin to address their possible role in Prader-Willi Syndrome. In the first aim, we will characterize the structure and expression of sno-lncRNAs. We will also examine their sub-nuclear localization, and efforts will be made to follow their synthesis and localization dynamically and throughout the cell cycle. Finally in this aim, we will carry out genomewide studies in several different cell types and using several different approaches in order to identify more sno-lncRNAs, some of which will be studied in greater detail. The second aim is to investigate the mechanism of action of sno-lncRNAs. Of particular interest is the connection of the chr15 sno-lncRNAs to Fox1 family splicing regulators. Compelling evidence suggests that these lncRNAs may bind Fox2 and perhaps Fox1 with high affinity. Thus, they may act as molecular "sponges" to titrate splicing factors since in preliminary experiments we have found that they alter splicing patterns. This angle will be examined in detail and could lead directly to new insights into Prader-Willi pathology. We will also ask whether some sno-lncRNAs can alter chromatin organization like a number of other lncRNAs do. Finally, since these new RNAs appear to be relatively stable and strictly retained in the nucleus, we will begin to use our knowledge of their expression to generate a new class of vectors for nuclear expression of a variety of sequences.