Nuclear-retained long noncoding RNAs

描述（由申请人提供）：我们最近发现了新的内含子衍生的长非编码RNA（lncRNA），其在两端被snoRNA机制加工（我们将这些sno-lncRNA命名为sno-lncRNA）。大多数snoRNA由内含子加工而成。脱支后，核酸外切酶降解内含子，但不降解snoRNA，因为这些与阻止降解并导致snoRNP组装的特定因子相关。Sno-lncRNA是从含有两个snoRNA的内含子加工而来的。在核酸外切酶剪切期间，snoRNA之间的序列不被降解，导致缺乏5'帽结构或3'聚（A）尾的lncRNA的积累。这些RNA中的一些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，并且它们缺乏poly（A）尾。这些构成了一类全新的核lncRNA，这项提议是更详细地研究它们。到目前为止的结果表明，每当内含子含有不是一个而是两个snoRNA基因，sno-lncRNA可以产生。这种RNA可以在迄今为止检查的每个细胞中找到，并且似乎在组织中广泛表达。此外，编码这些RNA中最丰富的一些RNA（15 q11-q13）的基因组区域在重要的人类遗传疾病Prader-Willi综合征中特异性缺失。我们将描述sno-lncRNA是如何产生的，与它们相关的蛋白质以及它们的数量。此外，我们将询问它们在细胞中的作用，并开始讨论它们在普拉德-威利综合症中可能发挥的作用。第一个目标，我们 将表征sno-lncRNA的结构和表达。我们还将研究它们的亚核定位，并将努力跟踪它们的合成和定位动态和整个细胞周期。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鉴定 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-lncRNA的作用机制。特别感兴趣的是chr 15 sno-lncRNA与Fox 1家族剪接调节子的连接。令人信服的证据表明，这些lncRNA可能结合Fox 2，也许Fox 1具有高亲和力。因此，它们可以作为分子“海绵”滴定剪接因子，因为在初步实验中，我们已经发现它们改变剪接模式。这个角度将被详细研究，并可能直接导致 普拉德-威利病理学的新见解我们还将询问一些sno-lncRNA是否可以像许多其他lncRNA那样改变染色质组织。最后，由于这些新的RNA看起来相对稳定并严格地保留在细胞核中，我们将开始利用我们对它们表达的了解来产生一类新的载体，用于核表达各种序列。

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.