Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter.
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.
Thompson
J D
JD
Ribozyme Pharmaceuticals Inc., Boulder, CO 80301, USA.
Ayers
D F
DF
Malmstrom
T A
TA
McKenzie
T L
TL
Ganousis
L
L
Chowrira
B M
BM
Couture
L
L
Stinchcomb
D T
DT
D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics*; D015347:RNA Probes; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics
D015347:RNA Probes; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D012320:RNA Polymerase III