Factors Governing the Activity In Vivo of Ribozymes Transcribed by RNA Polymerase III

Abstract

In order to determine the parameters that govern the activity of a ribozyme in vivo, we made a systematic analysis of chimeric tRNAVal ribozymes by measuring their cleavage activities in vitro as well as the steady-state levels of transcripts, the half-lives of transcribed tRNAVal ribozymes, and their activities in both HeLa and H9 cells. These analyses were conducted by the use of transient expression systems in HeLa cells and stable transformants that express ribozymes. Localization of transcripts appeared to be determined by the higher-order structure of each transcribed tRNAVal ribozyme. Since colocalization of the ribozyme with its target RNA is important for strong activity of the ribozyme in vivo, the best system for tRNA-based expression seems to be one in which the structure of the transcript is different from that of the natural tRNA precursor so that processing of the tRNAValribozyme can be avoided. At the same time, the structure of the transcript must be similar enough to allow recognition, probably by an export receptor, so that the transcript can be exported to the cytoplasm to ensure colocalization with its target. In the case of several tRNAVal ribozymes that we constructed, inspection of computer-predicted secondary structures enabled us to control the export of transcripts. We found that only a ribozyme that was transcribed at a high level and that had a sufficiently long half-life, within cells, had significant activity when used to withstand a challenge by human immunodeficiency virus type 1.

Targeting mortalin using conventional and RNA‐helicase‐coupled hammerhead ribozymes

“…It forms two domains: the scaffold (domain 2) on which the ribozyme is built, and the active centre (the catalytic domain; known as domain 1; Cech & Uhlenbeck, 1994; Hammann & Lilley, 2002). During the past two decades, the mechanism of action of hammerhead ribozymes, describing the requirement for divalent metal ions, definition of the catalytic domains, and the sequence specificity, which is usually referred to as the target site, has been shown (Kawasaki et al ., 1996; Koseki et al ., 1999; Takagi et al ., 2001; Kato et al ., 2001; Akashi et al ., 2002; Hammann & Lilley, 2002; Pyle, 2002). The activity of ribozymes in vivo is dependent on the level of expression at which they are effective, their specificity, and their intracellular stability, target colocalization and the accessibility of target sites (Yarus, 1999).…”

Inhibition of type I procollagen production by tRNAVal CTE‐HSP47 ribozyme

“…The former modification, the usage of the tRNA Val promoter, was to increase the efficiency of transport of Rz from nucleus to cytoplasm where Rz actually function to cleave the target mRNA 24. The latter modification was to enhance Rz activity by resolving RNA structure.…”

“…However, there is a crucial drawback for ordinary ribozymes such as hammerhead‐type or hairpin‐type in that they may remain in the nucleus and fail to be transported to the cytoplasm where they exert their function 23. To circumvent this problem, Koseki et al recently constructed an expression vector of ribozyme driven by the tRNA Val promoter, which allowed export of transcribed ribozymes from the nucleus to the cytoplasm and confirmed that the expression of ribozyme was indeed observed in the cytoplasm 24, although the ribozyme activity, measured as mRNA cleavage activity, was unexpectedly low 23–26. The most plausible reason for this low activity was that the secondary or tertiary structures of the target mRNA block ribozyme binding to the target sequences.…”

Ribozymes and DNAzymes – molecular structure, mechanism action and application in gene therapy

“…Z tego względu regulowane w komórkach docelowych promotory typu Pol II mają znaczącą przewagę w terapii genowej. Ekspresja rybozymów z promotorów Pol II przebiega w cytoplazmie, w której jest obecny docelowy mRNA [99]. Na przykład promotor rodopsyny (typu Pol II) został wykorzystany w celu osiągnięcia wysokiego poziomu transkrypcji rybozymów, których ekspresja jest ograniczona do komórek pręcikowych fotoreceptorów siatkówki [26].…”