The mechanism of Ube3a imprinting: Relevance of sense - antisense transcriptional overlap between Ube3a and Ube3a-ATS

UBE 3A-ATS长链非编码RNA是一种候选的宏RNA，用于表观遗传学调控人染色体15 q11 q13上Prader-Willi / Angelman综合征印迹基因簇（在小鼠染色体7 C上保守）中UBE 3A的印迹表达。这种长的非编码RNA作为基因表达的表观遗传调节因子发挥主要作用。它们通过RNA转录本身（如X失活中的Xist）或通过与蛋白质编码基因（如Airn、Kcnq 1 ot 1或Nespas）的转录重叠来顺式沉默基因表达。Ube 3a-ATS的表达局限于脑中神经元的父染色体，导致父染色体上Ube 3a的沉默和脑中来自母染色体的Ube 3a的印记表达。Ube 3a-ATS是Ube 3a的反义转录，与整个Ube 3a基因和启动子重叠，因此尚不清楚Ube 3a-ATS是否通过其转录本或转录重叠沉默父本等位基因上的Ube 3a表达。本实验旨在确定Ube 3a-ATS和Ube 3a之间转录重叠的必要性和程度，以便通过Ube 3a-ATS进行沉默。Ube 3a-ATS将在三个位置处过早终止，从而产生具有（1）与Ube 3a无转录重叠或（2）覆盖Ube 3a的3端的部分重叠或（3）除Ube 3a启动子外与Ube 3a完全转录重叠的等位基因。Ube 3a-ATS的表达和Ube 3a的印迹将通过使用单核苷酸多态性在分化成神经元的鼠胚胎干细胞中进行等位基因特异性表达分析来确定，所述单核苷酸多态性存在于所使用的细胞中或通过遗传操作引入。

The UBE3A-ATS long non-coding RNA is a candidate macroRNA for epigenetic regulation of the imprinted expression of UBE3A in the Prader-Willi / Angelman syndrome imprinted gene cluster on human chromosome 15q11q13 (conserved on mouse chromosome 7C). Such long non-coding RNAs play a major role as epigenetic regulators of gene expression. They silence gene expression in cis, either by the RNA transcript itself, like Xist in X inactivation, or by transcriptional overlap with protein-coding genes, like Airn, Kcnq1ot1 or Nespas. Expression of Ube3a-ATS is restricted to the paternal chromosome of neurons in the brain, leading to silencing of Ube3a on the paternal chromosome and imprinted expression of Ube3a from the maternal chromosome in the brain. Ube3a-ATS is transcribed antisense to Ube3a and overlaps the entire Ube3a gene and promoter, so it is not clear whether Ube3a-ATS silences Ube3a expression on the paternal allele by its transcript or by transcriptional overlap. The experiments described in this proposal aim to determine the necessity and extent of transcriptional overlap between Ube3a-ATS and Ube3a for silencing by Ube3a-ATS. Ube3a-ATS will be terminated prematurely at three positions, creating alleles with (1) no transcriptional overlap with Ube3a or (2) partial overlap covering the 3 end of Ube3a or (3) complete transcriptional overlap with the Ube3a apart from the Ube3a promoter. Expression of Ube3a-ATS and imprinting of Ube3a will be determined by allele-specific expression analysis in murine embryonic stem cells differentiated into neurons using single nucleotide polymorphisms that are either present in the cells used or introduced by genetic manipulation.