Dissecting the mechanisms underlying RNA processing and function of non-coding RNAs derived by SINE elements.

背景：超过97%的人类和小鼠基因组不编码蛋白质(非编码基因组)，直到最近，这种非编码基因组的很大一部分被称为垃圾DNA，被认为没有功能重要性。超过50%的非编码基因组由重复元件组成，其中小的散布核元件(Sine)构成了这些元件中最大的一组。有趣的是，这些正弦元素中的大量被转录成非编码RNA(Sine RNAs)。然而，这些正弦RNA的功能在很大程度上仍不清楚。最近研究表明，特定类型的正弦RNA，如B2和Alu RNA，通过与RNA聚合酶II结合而抑制特定靶基因的转录。在先前的研究中，我们揭示了在环境刺激作用后，这些RNA被广泛加工和不稳定，导致它们无法结合和抑制RNA聚合酶II，从而增加了对靶基因表达的额外一层调节。然而，Sine RNA的加工机制和调节这一过程的因素仍然不清楚。*目的：我提出的研究计划旨在剖析Sine RNAs的RNA加工机制。研究方法：为了实现这些目标，我和我的学员们将采用并定制一系列RNA基因组学和生物信息学方法，例如RNA测序的文库构建方法和适应重复、短和环状RNA研究的生物信息学管道。这些方法包括我们以前开发的用于估计正弦RNA加工的短RNA测序，以及分别用于识别线性和环状RNA的RNA测序和CircRNA测序。拟议的研究计划还将利用RNA生物化学和细胞培养技术，在体外和细胞内测试Sine RNA和一些控制Sine RNA加工的蛋白质之间的相互作用。*影响：该研究计划将揭示以前被认为是“垃圾DNA”的非编码基因组的一部分的功能。这将有助于更好地理解到目前为止被低估的Sine RNAs在应对细胞压力和调节基因表达方面的作用，这些基因表达对细胞功能至关重要。

Background: More than 97% of human and mouse genomes do not encode for proteins (non-coding genome) and until recently, a large part of this non-coding genome was referred as "junk DNA" that was believed to have no functional importance. More than 50% of the non-coding genome consists of repetitive elements, with small interspersed nuclear elements (SINEs) making the largest group of these elements. Interestingly, a large number of these SINE elements are transcribed into non-coding RNAs (SINE RNAs). However, the function of these SINE RNAs remains largely unknown. Specific classes of SINE RNAs, such as the B2 and Alu RNAs, have been recently shown to inhibit transcription of specific target genes by binding RNA Polymerase II. In a previous study, we revealed that after application of environmental stimuli, these RNAs are extensively processed and destabilized, which results in their inability to bind and inhibit RNA Polymerase II and thus, it adds an additional layer of regulation of expression of target genes. However, the mechanism of processing of SINE RNAs and the factors regulating this process remain unclear.******Objectives: My proposed research program aims to dissect the mechanisms underlying RNA processing of SINE RNAs. It will focus on 1) identifying the factors that trigger this processing and regulate RNA processing rate, 2) identifying and studying the function of the RNA products of the SINE RNA processing in regulation of cellular stress response, including both linear and circular RNA fragments, and 3) investigating how changes in the sequence of SINE RNAs affect their processing, to understand better the sequence requirements for this process.******Research Approach: To achieve these aims, my trainees and I will employ and customize a series of RNA genomics and bioinformatics approaches, such as library construction approaches for RNA sequencing and bioinformatics pipelines adapted to the study of repetitive, short and circular RNAs. These approaches include our previously developed short-RNA sequencing for estimation of the SINE RNA processing as well as RNA-sequencing and circRNA-sequencing for identification of linear and circular RNAs, respectively. The proposed research program will also employ RNA biochemistry and cell culture techniques to test in vitro and in cells the interaction between SINE RNAs and a number of proteins that control SINE RNA processing.******Impact: This research program will shed light to the function of a part of the non-coding genome previously believed to be "junk DNA". It will allow for a better understanding of the under-estimated until now role of SINE RNAs in response to cellular stress and regulation of gene expression that are critical for cell function.