Neuroepigenetics Laboratory, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy.
Central RNA Laboratory, Istituto Italiano di Tecnologia, Genova, Italy.
Methods Mol Biol. 2022;2434:63-87. doi: 10.1007/978-1-0716-2010-6_4.
SINEUP is a new class of long non-coding RNAs (lncRNAs) which contain an inverted Short Interspersed Nuclear Element (SINE) B2 element (invSINEB2) necessary to specifically upregulate target gene translation. Originally identified in the mouse AS-Uchl1 (antisense Ubiquitin carboxyl-terminal esterase L1) locus, natural SINEUP molecules are oriented head to head to their sense protein coding, target gene (Uchl1, in this example). Peculiarly, SINEUP is able to augment, in a specific and controlled way, the expression of the target protein, with no alteration of target mRNA levels. SINEUP is characterized by a modular structure with the Binding Domain (BD) providing specificity to the target transcript and an effector domain (ED)-containing the invSINEB2 element-able to promote the loading to the heavy polysomes of the target mRNA. Since the understanding of its modular structure in the endogenous AS-Uchl1 ncRNA, synthetic SINEUP molecules have been developed by creating a specific BD for the gene of interest and placing it upstream the invSINEB2 ED. Synthetic SINEUP is thus a novel molecular tool that potentially may be used for any industrial or biomedical application to enhance protein production, also as possible therapeutic strategy in haploinsufficiency-driven disorders.Here, we describe a detailed protocol to (1) design a specific BD directed to a gene of interest and (2) assemble and clone it with the ED to obtain a functional SINEUP molecule. Then, we provide guidelines to efficiently deliver SINEUP into mammalian cells and evaluate its ability to effectively upregulate target protein translation.
SINEUP 是一类新的长链非编码 RNA（lncRNA），它包含一个必需的反向短散在核元件（SINE）B2 元件（invSINEB2），用于特异性地上调靶基因翻译。最初在小鼠 AS-Uchl1（反义泛素羧基末端酯酶 L1）基因座中被鉴定出来，天然的 SINEUP 分子与它们的有意义的蛋白质编码靶基因（在本例中为 Uchl1）头对头排列。特别的是，SINEUP 能够以特定和受控的方式增强靶蛋白的表达，而不会改变靶 mRNA 的水平。SINEUP 的特点是具有模块化结构，结合域（BD）为靶转录本提供特异性，效应域（ED）包含 invSINEB2 元件，能够促进靶 mRNA 加载到重多核糖体上。自其在内源性 AS-Uchl1 ncRNA 中的模块化结构被理解以来，通过为感兴趣的基因创建一个特定的 BD，并将其放置在 invSINEB2 ED 的上游，已经开发出了合成的 SINEUP 分子。因此，合成的 SINEUP 是一种新型的分子工具，可能用于任何工业或生物医学应用，以增强蛋白质的产生，也可能作为单倍不足驱动疾病的治疗策略。在这里，我们描述了一个详细的方案，用于（1）设计一个针对感兴趣基因的特定 BD，（2）将其与 ED 组装和克隆，以获得功能性的 SINEUP 分子。然后，我们提供了指导方针，以有效地将 SINEUP 递送到哺乳动物细胞，并评估其有效上调靶蛋白翻译的能力。
