Thompson J D, Ayers D F, Malmstrom T A, McKenzie T L, Ganousis L, Chowrira B M, Couture L, Stinchcomb D T
Ribozyme Pharmaceuticals Inc., Boulder, CO 80301, USA.
Nucleic Acids Res. 1995 Jun 25;23(12):2259-68. doi: 10.1093/nar/23.12.2259.
RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.
RNA聚合酶III（pol III）转录本在所有细胞中都很丰富。因此，pol III启动子可能是在许多不同细胞类型中表达高水平外源RNA（如反义RNA、诱饵RNA和核酶）的理想选择。我们通过修饰转录本的3'末端使其与5'末端杂交，将源自人meti tRNA的pol III启动子表达的重组RNA积累提高了100倍以上。这种末端双链体包括初级野生型meti tRNA转录本中存在的8个核苷酸的前导序列，该序列在加工成成熟tRNA的过程中通常会被去除。在用编码含有这种修饰的pol III转录单元的逆转录病毒载体稳定转导的细胞中分析了抗HIV核酶的表达。在所有测试的细胞系中都观察到重组pol III核酶转录本的高积累。由于转录本积累增强，在从稳定转导的人T细胞系中提取的总RNA中很容易检测到核酶切割活性。一个称为“TRZ”的pol III转录单元针对核酶切割活性进行了进一步优化。本文报道的改进的pol III转录单元可能有助于表达多种功能性和治疗性RNA。
