Interaction between tumour necrosis factor alpha ribozyme and cellular proteins. Involvement in ribozyme stability and activity.
Ribozymes are RNA molecules that cleave other RNA molecules. Thus, ribozymes offer a new way of inhibiting expression of specific genes whose nucleotide sequences are known. Intracellular stability of ribozymes is an important factor for their efficacy. We previously showed that hammerhead ribozyme directed against mRNA of tumour necrosis factor alpha (TNF alpha) slowly acquires resistance to degradation in cultured human cells. In order to explain this resistance, we now report on endogenous cellular protein(s) that bind to TNF alpha-ribozyme (TNF alpha-Rz) in solution to form stable complexes during native gel electrophoresis. Suppression of the effects of ribonucleases in the cytoplasmic extracts allowed approximately 80% of the input ribozyme RNA to be recovered in the form of complexes, indicating that complex formation protected the ribozyme from degradation. Treatment of the ribozyme-protein complexes with proteinase K prior to electrophoresis led to the recovery of full-length ribozyme. Interestingly, ribozyme-protein complexes retained cleavage activity, suggesting that the binding is in reversible equilibrium. Analysis of protein cytoplasmic extracts for binding to sub-fragments of TNF alpha-Rz demonstrated that protein binds to a conformational epitope formed by an interaction between the 5' end of TNF alpha-Rz and its catalytic domain. Competition of the ribozyme-protein binding with a ribozyme construct containing DNA instead of RNA at the 5' end, indicated that the ribose phosphate backbone of the 5' end is required for strong binding. The protein responsible for the formation of the complex with low electrophoretic mobility was found to be specific for the TNF alpha-Rz, since ribozyme for HIV-1 integrase gene (Int-Rz), or for human interleukin-2 (IL2-Rz) did not compete significantly with the TNF alpha-Rz binding. Covalent linkage of the IL2-Rz to the 3' end of TNF alpha-Rz, or to the proposed RNA protein binding site conferred protein binding and enhanced the stability and activity of the chimeric molecules. The RNA epitope identified in this study, through its endogenous protein binding, may serve as an oligonucleotide cassette for enhancing the in vivo stability and activity of other RNA molecules in general. This RNA epitope will also be useful in the study of RNA-protein interactions.
Sioud
M
M
Institute of Immunology and Rheumatology, Rikshospitalet, Oslo, Norway.
D001483:Base Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D007376:Interleukin-2; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D014409:Tumor Necrosis Factor-alpha