Kim Minlee, Kogan Nicole, Slack Frank J
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.
Institute for RNA Medicine, Department of Pathology, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA, USA.
Oncotarget. 2016 Mar 15;7(11):11770-84. doi: 10.18632/oncotarget.7599.
Multiple RNA-binding proteins and non-coding RNAs, such as microRNAs (miRNAs), are involved in post-transcriptional gene regulation through recognition motifs in the 3' untranslated region (UTR) of their target genes. The KRAS gene encodes a key signaling protein, and its messenger RNA (mRNA) contains an exceptionally long 3' UTR; this suggests that it may be subject to a highly complex set of regulatory processes. However, 3' UTR-dependent regulation of KRAS expression has not been explored in detail. Using extensive deletion and mutational analyses combined with luciferase reporter assays, we have identified inhibitory and stabilizing cis-acting regions within the KRAS 3' UTR that may interact with miRNAs and RNA-binding proteins, such as HuR. Particularly, we have identified an AU-rich 49-nt fragment in the KRAS 3' UTR that is required for KRAS 3' UTR reporter repression. This element contains a miR-185 complementary element, and we show that overexpression of miR-185 represses endogenous KRAS mRNA and protein in vitro. In addition, we have identified another 49-nt fragment that is required to promote KRAS 3' UTR reporter expression. These findings indicate that multiple cis-regulatory motifs in the 3' UTR of KRAS finely modulate its expression, and sequence alterations within a binding motif may disrupt the precise functions of trans-regulatory factors, potentially leading to aberrant KRAS expression.
多种RNA结合蛋白和非编码RNA，如微小RNA（miRNA），通过其靶基因3'非翻译区（UTR）中的识别基序参与转录后基因调控。KRAS基因编码一种关键的信号蛋白，其信使RNA（mRNA）含有异常长的3'UTR；这表明它可能受到一套高度复杂的调控过程的影响。然而，尚未详细探讨KRAS表达的3'UTR依赖性调控。通过广泛的缺失和突变分析结合荧光素酶报告基因检测，我们在KRAS 3'UTR中鉴定出了可能与miRNA和RNA结合蛋白（如HuR）相互作用的抑制性和顺式作用稳定区域。特别地，我们在KRAS 3'UTR中鉴定出一个富含AU的49个核苷酸的片段，它是KRAS 3'UTR报告基因抑制所必需的。该元件包含一个miR-185互补元件，并且我们表明miR-185的过表达在体外抑制内源性KRAS mRNA和蛋白。此外，我们鉴定出另一个49个核苷酸的片段，它是促进KRAS 3'UTR报告基因表达所必需的。这些发现表明，KRAS 3'UTR中的多个顺式调控基序精细地调节其表达，并且结合基序内的序列改变可能破坏反式调控因子的精确功能，从而可能导致KRAS表达异常。
