Laboratory of Eukaryotic Genome Evolution, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov St., Moscow 119991, Russia.
Int J Mol Sci. 2023 Sep 27;24(19):14600. doi: 10.3390/ijms241914600.
Short Interspersed Elements (SINEs) are common in the genomes of most multicellular organisms. They are transcribed by RNA polymerase III from an internal promoter comprising boxes A and B. As transcripts of certain SINEs from mammalian genomes can be polyadenylated, such transcripts should contain the AATAAA sequence as well as those called β- and τ-signals. One of the goals of this work was to evaluate how autonomous and independent other SINE parts are β- and τ-signals. Extended regions outside of β- and τ-signals were deleted from SINEs B2 and Ves and the derived constructs were used to transfect HeLa cells in order to evaluate the relative levels of their transcripts as well as their polyadenylation efficiency. If the deleted regions affected boxes A and B, the 5'-flanking region of the U6 RNA gene with the external promoter was inserted upstream. Such substitution of the internal promoter in B2 completely restored its transcription. Almost all tested deletions/substitutions did not reduce the polyadenylation capacity of the transcripts, indicating a weak dependence of the function of β- and τ-signals on the neighboring sequences. A similar analysis of B2 and Ves constructs containing a 55-bp foreign sequence inserted between β- and τ-signals showed an equal polyadenylation efficiency of their transcripts compared to those of constructs without the insertion. The acquired poly(A)-tails significantly increased the lifetime and thus the cellular level of such transcripts. The data obtained highlight the potential of B2 and Ves SINEs as cassettes for the expression of relatively short sequences for various applications.
短散在元件 (SINEs) 在大多数多细胞生物的基因组中很常见。它们由 RNA 聚合酶 III 从包含 A 盒和 B 盒的内部启动子转录。由于哺乳动物基因组中的某些 SINEs 的转录物可以被多聚腺苷酸化,因此这些转录物应该包含 AATAAA 序列以及称为β-和 τ-信号的序列。这项工作的目的之一是评估其他 SINE 部分β-和 τ-信号的自主性和独立性。从 SINEs B2 和 Ves 中删除了β-和 τ-信号之外的扩展区域,并使用衍生的构建体转染 HeLa 细胞,以评估它们的转录物的相对水平及其多聚腺苷酸化效率。如果删除的区域影响了 A 盒和 B 盒,则用外部启动子插入 U6 RNA 基因的 5'侧翼区。这种在 B2 中替代内部启动子完全恢复了其转录。几乎所有测试的缺失/替换都没有降低转录物的多聚腺苷酸化能力,表明β-和 τ-信号的功能对邻近序列的依赖性较弱。对含有插入在β-和 τ-信号之间的 55 个碱基对的外源序列的 B2 和 Ves 构建体的类似分析表明,与没有插入的构建体相比,其转录物的多聚腺苷酸化效率相等。获得的多(A)尾显著增加了这些转录物的寿命,从而增加了它们在细胞内的水平。获得的数据突出了 B2 和 Ves SINEs 作为用于各种应用的相对短序列表达的盒的潜力。