Laboratory of Eukaryotic Genome Evolution, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov St., Moscow, 119991, Russian Federation.
Genome. 2018 May;61(5):367-370. doi: 10.1139/gen-2017-0223. Epub 2018 Feb 2.
Short nuclear 4.5SI RNA can be found in three related rodent families. Its function remains unknown. The genes of 4.5SI RNA contain an internal promoter of RNA polymerase III composed of the boxes A and B. Here, the effect of the sequence immediately upstream of the mouse 4.5SI RNA gene on its transcription was studied. The gene with deletions and substitutions in the 5'-flanking sequence was used to transfect HeLa cells and its transcriptional activity was evaluated from the cellular level of 4.5SI RNA. Single-nucleotide substitutions in the region adjacent to the transcription start site (positions -2 to -8) decreased the expression activity of the gene down to 40%-60% of the control. The substitution of the conserved pentanucleotide AGAAT (positions -14 to -18) could either decrease (43%-56%) or increase (134%) the gene expression. A TATA-like box (TACATGA) was found at positions -24 to -30 of the 4.5SI RNA gene. Its replacement with a polylinker fragment of the vector did not decrease the transcription level, while its replacement with a GC-rich sequence almost completely (down to 2%-5%) suppressed the transcription of the 4.5SI RNA gene. The effect of plasmid sequences bordering the gene on its transcription by RNA polymerase III is discussed.
短核 4.5SI RNA 可在三个相关的啮齿动物家族中发现。其功能仍不清楚。4.5SI RNA 的基因包含 RNA 聚合酶 III 的内部启动子，由框 A 和 B 组成。在这里，研究了小鼠 4.5SI RNA 基因 5'侧翼序列中紧邻序列对其转录的影响。使用缺失和取代 5'侧翼序列的基因转染 HeLa 细胞，并从细胞水平评估 4.5SI RNA 的转录活性。紧邻转录起始位点（位置-2 到-8）的单核苷酸取代将基因的表达活性降低至对照的 40%-60%。保守五核苷酸 AGAAT（位置-14 到-18）的取代可以降低（43%-56%）或增加（134%）基因表达。在 4.5SI RNA 基因的位置-24 到-30 处发现了一个 TATA 样框（TACATGA）。用载体的多接头片段取代它不会降低转录水平，而用富含 GC 的序列取代它几乎完全（降低到 2%-5%）抑制了 4.5SI RNA 基因的转录。讨论了基因边界的质粒序列对 RNA 聚合酶 III 转录的影响。
