PDF(Pubmed)
Abstract：
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.
摘要：
短散布元件(SINE)在大多数多细胞生物的基因组中是常见的。它们通过RNA聚合酶III从包含框A和B的内部启动子转录。由于来自哺乳动物基因组的某些SINE的转录物可以被聚腺苷酸化，这样的转录物应包含AATAAA序列以及称为β-和τ-信号的那些。这项工作的目标之一是评估其他正弦部分是如何自主和独立的β和τ信号。从SINEB2和Ves中删除了β-和τ-信号之外的延伸区域，并使用衍生的构建体转染HeLa细胞，以评估其转录本的相对水平以及聚腺苷酸化效率。如果删除的区域影响框A和B，将带有外部启动子的U6RNA基因的5'侧翼区插入上游。B2中内部启动子的这种取代完全恢复了其转录。几乎所有测试的缺失/替换都没有降低转录本的聚腺苷酸化能力，表明β-和τ-信号的函数对相邻序列的依赖性较弱。对包含在β-和τ-信号之间插入的55bp外源序列的B2和Ves构建体的类似分析显示，与未插入的构建体相比，其转录物的聚腺苷酸化效率相等。获得的poly（A）-尾巴显着增加了寿命，从而增加了此类转录本的细胞水平。获得的数据突出了B2和VesSINE作为表达相对短序列的盒的潜力，可用于各种应用。
