Endogenously expressed multimeric self-cleaving hammerhead ribozymes ablate mutant collagen in cellulo.
Hammerhead ribozymes are small catalytic RNA molecules that can be targeted to any RNA molecule containing a putative cleavage site. We developed a vector (pCOLZ) that uses the COL1A1 promoter to drive expression of a self-cleaving multimeric ribozyme (M8Rz547) and its monomeric counterpart (Rz547). The ribozymes were stably coexpressed in MC3T3-E1 osteoblasts expressing a truncated COL1A1 target transcript. The multimeric ribozyme exhibited self-cleavage to derivative fragments, including monomers. Increased expression of ribozymes was found in cells expressing the multimeric ribozyme. A modest reduction of truncated target transcript and protein was seen in cells expressing the ribozyme monomer, while nearly complete ablation of target transcript and protein occurred in cells expressing the ribozyme multimer. A reversion to a more normal collagen phenotype, measured as an increase in fibril diameter and restored fibrillar architecture, and a decreased rate of collagen turnover were seen in cells expressing the ribozyme multimer.
Peace
Belinda E
BE
Cincinnati Children's Hospital Research Foundation, ML 7016, Cincinnati, OH 45229-3039, USA. belinda.peace@cchmc.org
Florer
Jane B
JB
Witte
David
D
Smicun
Yoel
Y
Toudjarska
Iva
I
Wu
George
G
Kilpatrick
Michael W
MW
Tsipouras
Petros
P
Wenstrup
Richard J
RJ
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D000090602:Collagen Type I, alpha 1 Chain; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009154:Mutation; D010006:Osteoblasts / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000502:physiology*
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D016337:RNA, Catalytic; C427204:hammerhead ribozyme