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

UBE3A-ATS长非编码RNA是人类染色体15q11q13上的Prader-Willi/Angelman综合征印记基因簇(保守在小鼠7C上)中UBE3A印记表达的表观遗传调控的候选宏RNA。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着重要作用。它们抑制顺式基因的表达，要么是通过RNA转录物本身，比如X失活中的Xist，要么是通过与蛋白质编码基因，如Airn，Kcnq1ot1或Nespas的转录重叠。Ube3a-ATS的表达局限于大脑中神经元的父系染色体，导致Ube3a在父系染色体上的沉默，以及Ube3a在母体染色体上的印记表达。UBE3A-ATS是与Ube3a反义转录的，并与整个Ube3a基因和启动子重叠，因此目前尚不清楚Ube3a-ATS是通过其转录抑制Ube3a在父本等位基因上的表达，还是通过转录重叠来抑制Ube3a的表达。本提案中描述的实验旨在确定Ube3a-ATS和Ube3a之间转录重叠的必要性和程度，以供Ube3a-ATS沉默。UBE3A-ATS将在三个位置过早终止，从而产生(1)与Ube3a没有转录重叠或(2)覆盖Ube3a的3端的部分重叠或(3)除Ube3a启动子外与Ube3a完全转录重叠的等位基因。Ube3a-ATS的表达和Ube3a的印记将通过等位基因特异性表达分析在分化为神经元的小鼠胚胎干细胞中确定，该分析使用的是在使用或通过基因操作引入的细胞中存在的单核苷酸多态性。

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.