Regulation of transcript stability by splicing in non-coding gene regions

我们相信我们已经发现了一种可以调节基因表达的新机制。基因表达的调节使一个细胞与另一个细胞不同，并控制细胞对环境作出反应的许多方式。基因表达的差异调节，而不是基因功能的差异，也被认为是个体之间许多差异的基础。为了让细胞表达来自基因的蛋白质，它必须被转录成RNA，然后RNA被翻译成蛋白质。并不是一个基因内的所有序列都能进入最终的RNA：“蛋白质编码区”中散布着被称为“内含子”的区域，这些区域必须通过一个被称为剪接的过程去除，以重新构成蛋白质的序列。基因表达的调控通常是通过观察转录速率来研究的，但转录RNA的稳定性也很重要。转录本末端未被翻译的序列称为3′非翻译区（3′utr）。这些utr包含一系列调节因子的结合位点（例如被称为microrna的小调节RNA，或像CELF1这样的RNA结合蛋白），它们可以改变转录物的稳定性。另一个控制RNA稳定性的重要机制被称为无义介导的衰变（NMD）。这确保了含有基因失能突变的转录本被破坏。NMD还会导致错误拼接的转录本被破坏。因此，3' UTR中的剪接被认为会触发NMD。到目前为止，可能由3' UTR剪接产生的RNA序列一直被认为是人工产物，在很大程度上被忽视了。然而，我们已经发现了数千个这样的注销rna的大量表达，并且在许多情况下，删除的序列包含预测的microrna结合位点。这提出了两种假设：1)这种剪接确实触发了NMD，导致转录本的调控不稳定。2)这种剪接不会触发NMD，相反，去除UTR序列会调节mirna等RNA调节因子的敏感性。我们对这两种假设都有初步的证据。如果我们是正确的，那么这将代表一种新的、但基本的基因调控机制。在这里，我们建议重新分析来自不同组织、个体和实验室细胞系的数万个现有样本，以检查这种机制的普遍性，以及它与个体和细胞类型之间不同基因表达水平的关系。我们将在选定的候选rna和全基因组范围内测量在3' UTR上剪接的rna和没有剪接的rna之间转录稳定性的差异。我们将在具有正常NMD通路的细胞和禁用NMD通路的细胞中这样做，以确定它是否负责任何差异。我们还将通过操纵感兴趣的utr序列和RNA结合因子的水平来确定这种剪接是否调节对RNA结合因子的敏感性。最后，我们将通过测量剪接在促增殖刺激（如雌二醇）上的差异，来研究这种机制在主动调节中对促增殖刺激（如雌二醇）的反应中的作用。

We believe we have discovered a novel mechanism by which gene expression can be regulated. Regulation of gene expression makes one cell different from another and controls many of the ways in which cells respond to their environment. Differential regulation of gene expression, rather than differences in gene function, are also thought to underlie many of the differences between individuals. In order for a cell to express a protein from a gene it must be transcribed into RNA, which is then translated into a protein. Not all the sequence within a gene makes it into the final RNA: the "protein coding-regions" are interspersed with regions known as "introns" that must be removed by a process called splicing to reconstitute the sequence to make the protein. Regulation of gene expression is usually studied by looking at the rate of transcription, but the stability of the transcribed RNA is also important. Sequences at the end of the transcript that are not translated are called 3' Untranslated regions (3' UTRs). These UTRs contain binding sites for a range of regulatory factors (e.g. the small regulatory RNAs known as microRNAs, or RNA binding proteins such CELF1) that can alter transcript stability. Another important mechanism controlling RNA stability is called Nonsense Mediated Decay (NMD). This ensures that transcripts containing a gene-disabling mutation are destroyed. NMD also results in the destruction of transcripts that are incorrectly spliced. Splicing in the 3' UTR is thus thought to trigger NMD. Until now, RNA sequences that would have resulted from splicing in the 3' UTR have been regarded as artefacts and largely ignored. However, we have found substantial expression of thousands of such written-off RNAs, and in many cases the removed sequence contains predicted binding sites for microRNAs. This suggests two hypotheses: 1) This splicing does trigger NMD, causing a regulated destabilisation of the transcript. 2) This splicing does not trigger NMD and instead removal of UTR sequence regulates the sensitivity of RNA regulator factors such as miRNAs. We have preliminary evidence for examples of both these hypotheses. If we are correct then this will represent a novel, but fundamental mechanism of gene regulation. Here we propose to reanalyse tens of thousands of existing samples from different tissues, individuals and lab cell lines to examine the prevalence of this mechanism and how it relates to different gene expression levels between individuals and cell types. We will measure the difference in transcript stability between RNAs with splicing in the 3' UTR and those without, both in selected candidates and genome-wide. We will do this in cells with a normal NMD pathway and ones in which we have disabled the NMD pathway to determine if it is responsible for any differences. We will also ascertain whether this splicing regulates sensitivity to RNA binding factors by manipulation of the sequences of UTRs of interest and levels of the RNA binding factors. Finally we will examine the role of this mechanism in active regulation in response to pro-proliferative stimuli, such as the hormone estradiol, by measuring differences in splicing on this stimulus.