Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements

PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

PTCH1基因（PTCH1）编码一种具有12次跨膜结构的受体，在Hedgehog-Gli信号通路中发挥负调控功能。PTCH1生殖系突变可引发Gorlin综合征（Gorlin syndrome），该疾病以发育异常与肿瘤易感性为主要特征。常染色体显性遗传模式以及PTCH1单倍剂量不足的相关证据表明，机体存在针对patched同源物1（PTC1）蛋白水平的精细调控机制，以精准调控该信号通路。鉴于5'非翻译区（5' untranslated region, 5'UTR）在蛋白质表达中的关键作用，本研究旨在深入探究PTCH1转录本1b的5'UTR内的顺式调控元件。此前的研究中，(CGG)n多态性是被重点关注的潜在调控元件，但相关研究结果存在不一致性，且重复序列数目与疾病风险之间未发现明确关联。本研究通过在人类细胞系中使用荧光素酶报告基因构建体，证实CGG重复序列数目在信使RNA（mRNA）水平与蛋白质水平上，均对基因表达无显著影响。研究团队观察到5'UTR的长度存在个体变异，且在信号通路激活后，相关转录本的丰度发生显著改变。我们发现，仅存在于较长5'UTR中的上游起始密码子（uAUGs）可负向调控PTC1长亚型（PTC1-L）的表达量。通过多种方法鉴定并定位至CGG重复序列区域的内部核糖体进入位点（internal ribosome entry site, IRES），可抵消uAUGs的调控效应，使PTC1-L在缺氧等应激条件下仍能正常合成。通过多聚核糖体谱分析与蛋白质印迹（Western blot）实验，我们观察到缺氧培养的HEK 293T细胞中，PTCH1b mRNA的相对翻译效率显著升高。本研究所有结果均表明，PTCH1b的5'UTR顺式调控元件特征在Hedgehog-Gli信号通路的调控中，发挥着此前未被阐明的、极为复杂的作用。