INTERACTION BETWEEN TUMOR-NECROSIS-FACTOR-ALPHA RIBOZYME AND CELLULARPROTEINS - INVOLVEMENT IN RIBOZYME STABILITY AND ACTIVITY

Ribozymes are RNA molecules that cleave other RNA molecules. Thus, rib ozymes offer a new way of inhibiting expression of specific genes whos e 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 al pha (TNF alpha) slowly acquires resistance to degradation in cultured human cells. In order to explain this resistance, we now report on end ogenous cellular protein(s) that bind to TNF alpha-ribozyme (TNF alpha -Rz) in solution to form stable complexes during native gel electropho resis. Suppression of the effects of ribonucleases in the cytoplasmic extracts allowed approximately 80% of the input ribozyme RNA to be rec overed in the form of complexes, indicating that complex formation pro tected the ribozyme from degradation. Treatment of the ribozyme-protei n complexes with proteinase K prior to electrophoresis led to the reco very of full-length ribozyme. Interestingly, ribozyme-protein complexe s retained cleavage activity, suggesting that the binding is in revers ible 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-pr otein 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 fo rmation of the complex with low electrophoretic mobility was found to be specific for the TNF alpha-Rz, since ribozyme for HIV-1 integrase g ene (Int-Rz) or for human interleukin-2 (IL2-Rz) did not compete signi ficantly 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 activit y of the chimeric molecules. The RNA epitope identified in this study through its endogenous protein binding, may serve as an oligonucleotid e cassette for enhancing the in vivo stability and activity of other R NA molecules in general. This RNA epitope will also be useful in the s tudy of RNA-protein interactions.