Targeting mortalin using conventional and RNA-helicase-coupled hammerhead ribozymes.
Mortalin, also known as mot2/mthsp70/GRP75/PBP74, is a member of the heat-shock protein 70 family that is heat-uninducible. It is differentially distributed in cells that have normal and immortal phenotypes, has been localized to various subcellular sites, and has several binding partners and functions. Here, we describe the construction and use of mortalin-specific conventional and hybrid ribozymes to elucidate its crucial role in cell proliferation. Whereas conventional hammerhead ribozymes did not cause any repression of endogenous mortalin expression, RNA-helicase-linked hybrid ribozymes successfully suppressed the expression of mortalin, which resulted in the growth arrest of transformed human cells. We show that, first, RNA helicase-coupled hybrid ribozymes that have a linked unwinding activity can be used to target genes for which conventional hammerhead ribozymes are ineffective; second, the targeting of mortalin by RNA-helicase-coupled hybrid ribozymes causes growth suppression of transformed human cells and could be used as a treatment for cancer.
Wadhwa
Renu
R
Gene Function Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
Ando
Hiroshi
H
Kawasaki
Hiroaki
H
Taira
Kazunari
K
Kaul
Sunil C
SC
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002455:Cell Division; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D020365:RNA Helicases / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; C492001:HSPA9 protein, human; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; C080352:mortalin; D049452:Green Fluorescent Proteins; D012313:RNA; D008156:Luciferases; D020365:RNA Helicases