Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences

• Published in: Nature biotechnology 2000, Vol.18 (1), p.58-61
• Main Authors: Ma, Michael, Benimetskaya, Lyuba, Lebedeva, Irina, Dignam, Jeanne, Takle, Garry, Stein, C.A.
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions - genetics; Agriculture; bcl-X Protein; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Bladder; Blotting, Western; Cancer; Down-Regulation; Endoribonucleases - metabolism; Enzyme Activation; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Humans; Industrial applications and implications. Economical aspects; Isoenzymes - genetics; Isoenzymes - metabolism; Life Sciences; Miscellaneous; Nucleic Acid Conformation; oligonucleotides; Oligoribonucleotides - chemistry; Oligoribonucleotides - genetics; Phosphatidylethanolamines - metabolism; Protein expression; Protein Kinase C - genetics; Protein Kinase C - metabolism; Protein Kinase C-alpha; Proteins; Proto-Oncogene Proteins c-bcl-2 - analysis; Proto-Oncogene Proteins c-bcl-2 - genetics; ribonuclease H; Ribonuclease H - metabolism; Ribonuclease P; RNA Processing, Post-Transcriptional - genetics; RNA, Antisense - chemistry; RNA, Antisense - genetics; RNA, Antisense - physiology; RNA, Catalytic - metabolism; RNA, Guide - chemistry; RNA, Guide - genetics; RNA, Guide - physiology; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Transfer - chemistry; RNA, Transfer - genetics; RNA, Transfer - metabolism; Substrate Specificity; Transfection - methods; Transfer RNA; tRNA; Tumor Cells, Cultured; Urinary Bladder Neoplasms - enzymology; Urinary Bladder Neoplasms - genetics; Urinary Bladder Neoplasms - pathology
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/71924

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-α mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-α 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.