Department of Life and Environmental Science, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
BMC Biotechnol. 2011 Jul 25;11:79. doi: 10.1186/1472-6750-11-79.
The group I intron, a ribozyme that catalyzes its own splicing reactions in the absence of proteins in vitro, is a potential target for rational engineering and attracted our interest due to its potential utility in gene repair using trans-splicing. However, the ribozyme activity of a group I intron appears to be facilitated by RNA chaperones in vivo; therefore, the efficiency of self-splicing could be dependent on the structure around the insert site or the length of the sequence to be inserted. To better understand how ribozyme activity could be modulated in cultured mammalian cells, a group I intron was inserted into a short hairpin RNA (shRNA), and silencing of a reporter gene by the shRNA was estimated to reflect self-splicing activity in vivo. In addition, we appended a theophylline-binding aptamer to the ribozyme to investigate any potential effects caused by a trans-effector.
shRNA-expression vectors in which the loop region of the shRNA was interrupted by an intron were constructed to target firefly luciferase mRNA. There was no remarkable toxicity of the shRNA-expression vectors in Cos cells, and the decrease in luciferase activity was measured as an index of the ribozyme splicing activity. In contrast, the expression of the shRNA through intron splicing was completely abolished in 293T cells, although the silencing induced by the shRNA-expressing vector alone was no different from that in the Cos cells. The splicing efficiency of the aptamer-appended intron also had implications for the potential of trans-factors to differentially promote self-splicing among cultured mammalian cells.
Silencing by shRNAs interrupted by a group I intron could be used to monitor self-splicing activity in cultured mammalian cells, and the efficiency of self-splicing appears to be affected by cell-type specific factors, demonstrating the potential effectiveness of a trans-effector.
在缺乏蛋白质的体外环境中催化自身剪接反应的 I 组内含子是一种潜在的理性工程目标，由于其在使用反式剪接进行基因修复中的潜在用途而引起了我们的兴趣。然而，I 组内含子的核酶活性似乎在体内受到 RNA 伴侣的促进；因此，自我剪接的效率可能取决于插入位点周围的结构或要插入的序列的长度。为了更好地了解核酶活性如何在培养的哺乳动物细胞中被调节，将 I 组内含子插入短发夹 RNA（shRNA）中，并通过 shRNA 对报告基因的沉默来估计体内自我剪接活性。此外，我们将茶碱结合适体附加到核酶上，以研究任何可能由反式效应物引起的潜在影响。
构建了 shRNA 表达载体，其中 shRNA 的环区被内含子打断，以靶向萤火虫荧光素酶 mRNA。在 Cos 细胞中，shRNA 表达载体没有明显的毒性，并且将荧光素酶活性的降低作为核酶剪接活性的指标进行测量。相比之下，尽管单独表达 shRNA 的表达载体引起的沉默与 Cos 细胞没有不同，但在 293T 细胞中，内含子剪接表达的 shRNA 完全被废除。附加适体的内含子的剪接效率也暗示了反式因子在不同培养的哺乳动物细胞中促进自我剪接的潜力。
由 I 组内含子打断的 shRNA 沉默可用于监测培养的哺乳动物细胞中的自我剪接活性，并且自我剪接的效率似乎受到细胞类型特异性因素的影响，证明了反式效应物的潜在有效性。
