RNA 解旋酶 RHAU（DHX36）抑制转录因子 PITX1 的表达。

The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1.

机构信息

Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada, University of Manitoba, Winnipeg, Manitoba, Canada, Department of Biochemistry and Molecular Biology, University of British Columbia, V6T 1Z4 Vancouver, British Columbia, Canada and Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2.

出版信息

Nucleic Acids Res. 2014 Mar;42(5):3346-61. doi: 10.1093/nar/gkt1340. Epub 2013 Dec 24.

DOI:10.1093/nar/gkt1340

PMID:24369427

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3950718/

Abstract

RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3'-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non-quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3'-untranslated region.

摘要

RNA 解旋酶与富含 AU 元件（RHAU）（DHX36）是一种 DEAH（天冬氨酸、谷氨酸、丙氨酸、组氨酸）盒 RNA 解旋酶，可结合和解旋 DNA 和 RNA 中的 G4-四链体。为了检测 RHAU 的新 RNA 靶标，我们进行了 RNA 共免疫沉淀筛选，发现 PITX1 信使 RNA（mRNA）特异性和高度富集。PITX1 是一种同源盒转录因子，在发育和癌症中都有作用。初级序列分析在 PITX1 mRNA 的 3'非翻译区中鉴定出三个可能的四链体。这些序列中的每一个，当被分离时，都会形成稳定的四链体结构，与 RHAU 相互作用。我们提供的证据表明，这些四链体存在于内源性 mRNA 中；然而，我们发现 RHAU 通过非四链体形成区域与 mRNA 连接。通过小干扰 RNA 敲低 RHAU 会导致 PITX1 蛋白水平显著增加，而 mRNA 只有微小变化，这表明 RHAU 在翻译调节中发挥作用。miRNA 机制的组成部分的参与得到了类似的支持，并且在 Dicer 和联合 RHAU/Dicer 敲低时，PITX1 蛋白表达呈非加性增加。我们还证明了 Argonaute-2（一种关键的 RNA 诱导沉默复合物成分）的参与对于介导 RHAU 依赖性 PITX1 蛋白水平变化是必需的。这些结果表明 RHAU 在含有四链体的 3'非翻译区的 miRNA 介导的翻译调节中发挥了新的作用。