Role of microRNAs in Regulating Gene Expression

microRNA 在调节基因表达中的作用

• 批准号: 8349351
• 负责人: Howard Young
• 金额: $ 44.95万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349351
• 关键词: Acylation; Affect; Binding; Binding Proteins; Binding Sites; Collaborations; Complex; Cytokine Gene; DNA Sequence Rearrangement; Development; Elements; Functional RNA; Gene Expression; Genes; Genetic Polymorphism; Goals; Hydroxyl Radical; Immune system; In Vitro; Indium; Interferon Type II; Laboratories; Mediating; Messenger RNA; MicroRNAs; Modeling; Mutate; Nature; Pathway interactions; Play; Post-Transcriptional Regulation; Primer Extension; Process; RNA; RNA-Induced Silencing Complex; Reporter; Reporting; Research; Role; Structure; Technology; Testing; Untranslated Regions; Vertebrates; cancer type; gene cloning; mRNA Stability; mRNA Transcript Degradation; research study; tumor

Segments of the IFN-g 3UTR are conserved across vertebrates and such conserved non-coding sequence (CNS) are hypothesized to have regulatory potential. Our analysis of the IFN-g gene sequence revealed that there is a promising target for miRNAs 29a and b binding downstream of the ARE element in the 3 UTR. This region is present in IFN-g genes cloned from many species, thus implicating this miRNA binding site as having a role in regulating IFN-g gene expression. Typically, both the ARE element and miRNA target sequences are known to be involved in post-transcriptional regulation of gene expression. While in the IFN-g 3 UTR, the ARE is located in the 5 end and the miRNA-29BS at the 3 end, there is no reported evidence supporting the model that miRNAs bind to the ARE region of IFN-g. To test this hypothesis, we generated several 3 IFN-g-UTR reporter constructs by mutating the ARE and miRNA-BS and our experiments revealed competing effects between the ARE and miRNA, affecting the stability of the mRNA. From these results, we hypothesize that the ARE mediated decay (AMD) and RNA induced silencing complex (RISC) pathways competitively interact and thereby regulate post-transcriptional control of IFN-g. We hypothesize that the RISC complex may inhibit the AMD process, by potentially interacting with ARE binding proteins. Our in vitro results led us to enlist the help of Dr. Bruce Shapiro (NCI), an expert in RNA structure. Dr. Shapiros laboratory analyzed the 3 UTR structure and evaluated whether binding of the miRNA altered 3 RNA structure. His laboratory provided computational evidence that binding of mir-29 in the region 1090-1109 leads to a rearrangement of the secondary structure of the mRNA. This structural change appears to be larger and different in nature compared to binding of miRNA in neighboring regions (see Figure 6). In particular, a long-distance interaction between the AU-rich region and the miRNA binding region seems to be modulated by the miRNA binding. The modulation appears to be in the reverse direction as initially expected: the long-distance interaction is predicted to be present upon binding of the miRNA and weakened or not present in the unbound form of the mRNA. To confirm that the predicted RNA structural changes upon binding of the miRNA actually occur, we initiated a collaboration with Dr. Stuart LeGrice, (NCI) utilizing SHAPE technology (selective 29-hydroxyl acylation analyzed by primer extension). This technology permits us to gain a better understanding of how miRNA binding may affect IFN-g RNA structure and determine if the predicted theoretical changes in RNA structure actually occur following interaction with the miRNAs. If this model is validated, we will then test whether the binding of the protein TTP, already reported to interact with the IFN-g ARE, is altered in the presence of the miRNA. We hypothesize that miRNA binding to the mRNA will interfere with the binding of TTP to the RNA, thus stabilizing the RNA. As part of this overall project, we are also searching for polymorphisms in the IFN-g gene that may effect gene expression by altering IFN-g mRNA stability.

IFN-g 3UTR 的片段在脊椎动物中是保守的，并且这种保守的非编码序列 (CNS) 被假设具有调节潜力。我们对 IFN-g 基因序列的分析表明，3 UTR 中 ARE 元件下游的 miRNA 29a 和 b 存在一个有希望结合的靶标。该区域存在于从许多物种克隆的 IFN-g 基因中，因此暗示该 miRNA 结合位点在调节 IFN-g 基因表达中发挥作用。通常，已知 ARE 元件和 miRNA 靶序列都参与基因表达的转录后调控。虽然在 IFN-g 3 UTR 中，ARE 位于 5 端，miRNA-29BS 位于 3 端，但没有报道证据支持 miRNA 与 IFN-g ARE 区域结合的模型。 为了检验这一假设，我们通过突变 ARE 和 miRNA-BS 生成了几种 3 IFN-g-UTR 报告基因构建体，我们的实验揭示了 ARE 和 miRNA 之间的竞争效应，影响了 mRNA 的稳定性。根据这些结果，我们假设 ARE 介导的衰变 (AMD) 和 RNA 诱导的沉默复合物 (RISC) 途径竞争性相互作用，从而调节 IFN-g 的转录后控制。我们假设 RISC 复合体可能通过与 ARE 结合蛋白相互作用来抑制 AMD 过程。我们的体外研究结果促使我们寻求 RNA 结构专家 Bruce Shapiro 博士 (NCI) 的帮助。 Shapiros 博士实验室分析了 3 UTR 结构并评估了 miRNA 的结合是否改变了 3 RNA 结构。他的实验室提供的计算证据表明 mir-29 在 1090-1109 区域的结合会导致 mRNA 二级结构的重排。与邻近区域的 miRNA 结合相比，这种结构变化似乎更大且性质不同（见图 6）。特别是，AU 丰富区域和 miRNA 结合区域之间的长距离相互作用似乎受到 miRNA 结合的调节。调节似乎与最初预期的相反方向：预计长距离相互作用在 miRNA 结合时出现，而在未结合的 mRNA 形式中减弱或不存在。为了确认 miRNA 结合后预测的 RNA 结构变化确实发生，我们利用 SHAPE 技术（通过引物延伸分析选择性 29-羟基酰化）与 Stuart LeGrice 博士 (NCI) 展开合作。这项技术使我们能够更好地了解 miRNA 结合如何影响 IFN-g RNA 结构，并确定预测的 RNA 结构理论变化是否在与 miRNA 相互作用后实际发生。如果该模型得到验证，我们将测试已报道与 IFN-g ARE 相互作用的蛋白质 TTP 的结合是否在 miRNA 存在时发生改变。我们假设 miRNA 与 mRNA 的结合会干扰 TTP 与 RNA 的结合，从而稳定 RNA。作为整个项目的一部分，我们还在寻找 IFN-g 基因中的多态性，这些多态性可能通过改变 IFN-g mRNA 稳定性来影响基因表达。