title: Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences.
abstract: Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.
authors.affiliation: Innovir Laboratories, VimRx Pharmaceuticals, Wilmington, DE 19808, USA.
authors.forename: M
authors.initials: M
authors.lastname: Ma
authors.forename: L
authors.initials: L
authors.lastname: Benimetskaya
authors.forename: I
authors.initials: I
authors.lastname: Lebedeva
authors.forename: J
authors.initials: J
authors.lastname: Dignam
authors.forename: G
authors.initials: G
authors.lastname: Takle
authors.forename: C A
authors.initials: CA
authors.lastname: Stein
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D015153:Blotting, Western; D015536:Down-Regulation; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D043262:Ribonuclease P; D013379:Substrate Specificity; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D051020:bcl-X Protein; D058727:RNA, Small Untranslated; D000099221:Protein Kinase C zeta
chemical_list: D020413:3' Untranslated Regions; D004722:Endoribonucleases; D007527:Isoenzymes; D009843:Oligoribonucleotides; D010714:Phosphatidylethanolamines; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D019253:Proto-Oncogene Proteins c-bcl-2; D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012343:RNA, Transfer; D016914:Ribonuclease H; D043262:Ribonuclease P; D051020:bcl-X Protein; D058727:RNA, Small Untranslated; D000099221:Protein Kinase C zeta; C494811:BCL2L1 protein, human; C020888:1,2-dielaidoylphosphatidylethanolamine; C496628:PRKCA protein, human; C468368:RPP14 protein, human