MicroRNA对无义突变mRNA的监控和降解机制及其功能研究

Premature termination codon (PTC) can arise in messenger RNA (mRNA) as a consequence of mutations, errors in transcription, faulty splicing, or programmed rearrangements. Synthesis of C-terminally truncated proteins could be detrimental to the cell and related to many human diseases, such as in β-thalassemia, von Willebrand disease and factor X deficiency. In animals, nonsense-mediated decay (NMD) act as evolutionarily conserved cellular survilliance system to eliminate mRNAs containing PTC and thus helps limit the synthesis of abnormal proteins. In current model, NMD distinguishes normal termination codons from PTC on the basis of their location relative to exon-exon junction. However, PTCs located in the last exon of mRNA still can elicit NMD in both human tissue and culture cells by unknown mechanisms. MicroRNAs (miRNAs), which are only ~22 nucleotides in length, can regulate the expression of more than half of the genes in eukaryotic organism. In animals, miRNA downregulate gene expression by a combination of two distinct mechanisms: translational repression and accelerated decay caused by rapid poly(A) tail removal. In contrast to siRNA (RNAi), which can target almost any part of an mRNA and be fully functional, almost all identified target sites for endogenous miRNAs are located in 3' untranslated regions (3' UTRs) of mRNA. This has been established by extensive bioinformatic sequence analyses and experimental approaches. Here we propose an alternaitive NMD mechanism mediated by miRNA. In the presence of a downstream PTC, the miRNA target sites originally located in the ORF become part of the 3'UTR, which allow miRNA-programmed RISC to remain attached to the target mRNA to effectively silence translation and cause mRNA degradation in cis. We will use APC gene，in which the PTC mutants are often located in the last exon, as an example to study the mechanism and function of miRNA in NMD and its relations to colorectal cancer.

DNA复制中的突变以及mRNA剪接加工过程中发生错误等都可能产生含有提前终止密码子（PTC）的异常mRNA，如果不能被及时识别并降解，翻译产生羧端截短的蛋白质产物将会干扰正常蛋白质的功能，导致包括癌症在内的多种疾病。目前研究较多的是由外显子连接复合体（EJC）介导的无义突变mRNA的降解（NMD，Nonsense-mediated decay），但尚有大量生物学和病理现象难以用该模型进行解释。本申请提出miRNA在细胞内一种新功能的假设：miRNA可能是细胞内mRNA质量监控和降解系统的一部分，通过识别PTC上游原本位于mRNA编码区中的miRNA靶位点，从而参与对细胞内无义突变mRNA的翻译抑制和降解，保护细胞免受羧端截短蛋白质产物的损害。对此，我们将以APC基因为主要研究对象，结合临床病理样本分析，揭示细胞内miRNA识别和降解无义突变mRNA的分子机制，以及与相关肿瘤发生间的关联。

DNA复制中的突变以及mRNA在转录和剪接加工过程中发生错误，都会产生带有提前终止密码子（无义突变）的异常mRNA，翻译产生的羧端截短蛋白质产物将干扰正常蛋白质的功能，导致包括癌症在内的多种疾病。生物体内具有精密高效的监控机制来识别和清除这些有害的mRNA，目前研究较多的是由外显子连接复合体EJC介导的对含有提前终止密码子mRNA的降解（nonsense-mediated mRNA decay，NMD）。miRNA是哺乳动物细胞中调控基因表达的一类小分子非编码小RNA，主要识别位于mRNA 3’UTR区的靶位点，调控超过三分之一基因的表达,参与了包括细胞分化、增殖、凋亡、代谢等几乎所有生物过程，直接影响生物体的生长发育和执行功能，是细胞内基因表达调控网络的重要组成部分，并且大量研究表明miRNA的表达异常与多种人类疾病密切相关。我们研究证明mRNA中的蛋白质编码区（ORF）中存在大量潜在的miRNA靶位点，但由于核糖体的翻译作用而不能有效发挥抑制功能。mRNA出现提前终止密码子导致原本在ORF中不能被miRNA调节的潜在的靶位点成为了具有活性的靶位点，在相应miRNA存在的情况下，这些无义突变的mRNA将被降解或翻译将被抑制。我们发现与结直肠癌相关的抑癌基因APC的无义突变体的表达能够被miNMD有效抑制，而经典的NMD机制则无法识别它们。对HCT-116细胞系进行外显子组和转录组测序分析并结合实验验证发现，细胞内相当一部分天然存在的无义突变mRNA是miNMD的底物。由此我们提出一种新的由miRNA介导的mRNA质量监控系统——miRNA除了负责调节细胞内大量基因的正常表达以外，还作为mRNA质量监控和降解系统的一部分，与EJC介导的NMD协同作用，保护细胞免受无义突变产生的有害截短蛋白质产物的损害。此项研究工作发表在Elife（2014）。Elife同期刊登述评“Stop that nonsense!”介绍了该研究工作。