DEGRADATION OF HAMMERHEAD RIBOZYMES BY HUMAN RIBONUCLEASES

Hammerhead ribozymes were used as substrates to examine endoribonucleo lytic activities in cell extracts and-cultured human cells. Primer-ext ension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA wer e cleaved at UA and CA dinucleotides by extracts. Preferred cleavage s ites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specifi c ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significant ly contaminated by extracellular nucleases. Placement of UA dinucleoti des adjacent to a ribozyme in mRNA led to excision of the ribozyme fro m long transcripts during incubation in extracts. UA dinucleotides als o made mRNA more labile than a control RNA when expressed from an endo genous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life whe n delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimid ine-specific ribonuclease family is involved in the intracellular degr adation of RNA, and they explain the paucity of UA dinucleotides in eu karyotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effectiv e hammerhead ribozymes.