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

背景资料：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大部分被称为“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，其中小散布核元件（西内斯）是这些元件中最大的一组。有趣的是，大量的这些SINE元件被转录成非编码RNA（SINE RNA）。然而，这些SINE RNA的功能在很大程度上仍然未知。特定类别的SINE RNA，如B2和Alu RNA，最近已显示通过结合RNA聚合酶II抑制特定靶基因的转录。在以前的研究中，我们发现，在施加环境刺激后，这些RNA被广泛加工和不稳定，这导致它们无法结合和抑制RNA聚合酶II，因此，它增加了一个额外的靶基因表达调控层。然而，SINE RNA的加工机制和调节这一过程的因素仍不清楚。目的：我建议的研究计划旨在解剖的SINE RNA的RNA加工的机制。它将集中在1）确定触发这种加工和调节RNA加工速率的因素，2）确定和研究SINE RNA加工的RNA产物在调节细胞应激反应中的功能，包括线性和环状RNA片段，以及3）研究SINE RNA序列的变化如何影响它们的加工，以更好地了解该过程的顺序要求。研究方法：为了实现这些目标，我和我的学员将采用和定制一系列RNA基因组学和生物信息学方法，例如用于RNA测序的文库构建方法和适用于重复、短和环状RNA研究的生物信息学管道。这些方法包括我们先前开发的用于估计SINE RNA加工的短RNA测序以及分别用于鉴定线性和环状RNA的RNA测序和circRNA测序。该研究计划还将采用RNA生物化学和细胞培养技术，在体外和细胞中测试SINE RNA与控制SINE RNA加工的许多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”的非编码基因组部分的功能。它将允许更好地理解SINE RNA在响应细胞应激和调节对细胞功能至关重要的基因表达中的作用。

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.