Identifying regulatory uORFs as a targetable axis for hereditary disease

摘要（来自父母补助金） 该提案的广泛目标是通过计算识别，验证，然后靶向治疗 蛋白质编码基因的5'非翻译区（UTR）内的干预调控元件， 称为上游开放阅读框（uORF）。这样做的目的是调节蛋白质 从选定的基因输出，提供了一种新的，具有广泛潜力的翻译方法。 uORF是mRNA 5′端非翻译区的一段序列，可以启动和终止翻译 蛋白质编码（CDS）起始密码子的上游。罕见的uORF已经被证明可以产生 潜在的功能性微肽，而另一些则通过调节翻译来影响蛋白质表达 下游蛋白质编码序列的速率。我们最近的工作表明，uORF表现出 强负选择，以保留它们的起始和终止密码子，但不保留编码的 序列，支持这样的观点，即uORF通过进化得以保留，因为它们的 对蛋白质基因产物的顺式调节作用。利用基因数据库，我们发现， 影响与疾病表型相关的uORF起始/终止密码子（PheWAS分析）， 验证了它们对下游基因的蛋白质（而不是RNA）水平的预期作用。 在约50%的人类蛋白质编码基因中已经发现了潜在的uORF，其中许多基因 具有多于一个uORF。虽然阻断一些uORF会减少CDS蛋白的输出，但我们的研究表明， 未公开的数据显示靶向uORF可用于增加CDS翻译。作为 对于蛋白水平升高的潜在病例，我们将重点放在遗传性肺动脉 高血压（PAH），一种目前无法治愈的致命疾病，最常见的是由BMPR 2引起的 单倍不足通过阻断BMPR 2 uORF，我们能够增加BMPR 2蛋白水平， 高达220%。基于我们已发表和未发表的结果，我们提出了一套综合的 计算和实验方法，以系统地检测，优先考虑，验证和 用于治疗干预的靶向uORF，重点关注与单倍不足相关的基因 和ASO作为干预措施。

Abstract (from parent grant) The broad goal of this proposal it to computationally identify, validate, then target for therapeutic intervention regulatory elements within the 5' untranslated regions (UTR) of protein coding genes, known as upstream open reading frames (uORFs). In so doing, we aim to modulate the protein output from selected genes, offering a novel, translational approach with broad potential. uORFs are segments of 5′UTR mRNA sequences that can initiate and terminate translation upstream of protein-coding (CDS) start codons. Rare uORFs have been shown to produce potentially functional micro-peptides, while others affect protein expression by tuning translation rates of downstream protein-coding sequences. Our recent work showed that uORFs exhibit strong negative selection for preserving their start and stop codons, but not the encoded sequence, favoring the notion that uORFs have been retained through evolution because of their cis regulatory effect on protein gene products. Using genetic databases, we showed that variants affecting uORF start/stop codons associate with disease phenotypes (PheWAS analysis) and validated their expected effect on the protein (but not RNA) levels of the downstream genes. Potential uORFs have been identified in ~50% of all human protein-coding genes, many of which harbor more than one uORF. While blocking some uORFs decreases CDS protein output, our unpublished data show that targeting uORFs can be used to increase CDS translation. As a potential case for increasing protein levels, we focused on heritable pulmonary arterial hypertension (PAH), a fatal condition with no current cure, most commonly driven by BMPR2 haploinsufficiency. By blocking a BMPR2 uORF, we were able to increase BMPR2 protein levels by up to 220%. Based on our published and unpublished results we propose an integrative set of computational and experimental approaches to systematically detect, prioritize, validate and target uORFs for therapeutic intervention, focusing on genes associated with haploinsufficiency and ASOs as an intervention.