Antisense oligonucleotides reach mRNA targets via the RNA matrix: downregulation of the 5-HT1A receptor.
Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on the ease of design, efficiency of (intra)cellular delivery, ODN stability, and target specificity. Equally essential is a detailed understanding of the mechanism of antisense action. To address these issues, we employed phosphorothioate ODNs directed against specific regions of the mRNA of the serotonin 5HT1A receptor, governed by sequence and structure. We demonstrate that rather than various intracellular factors, the gene sequence per se primarily determines the antisense effect, since 5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells are all efficiently downregulated following ODN delivery via a cationic lipid delivery system. The data also reveal that the delivery system as such is a relevant parameter in ODN delivery. Antisense ODNs bound extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than a steric blockage of the mRNA impedes protein expression. The specificity of the antisense approach, as described in this study, is reflected by the effective functional downregulation of the 5-HT1A receptor.
Shi
Fuxin
F
Department of Membrane Cell Biology, Faculty of Medical Sciences, University of Groningen, Antonius Deusinglaan 1,9713 AV Groningen, The Netherlands.
Visser
Willy H
WH
de Jong
Natasja M J
NM
Liem
Robert S B
RS
Ronken
Eric
E
Hoekstra
Dick
D
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D008854:Microscopy, Electron; D015530:Nuclear Matrix / Q000378:metabolism*; D020320:Oligoribonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D044282:Receptor, Serotonin, 5-HT1A / Q000096:biosynthesis* / Q000235:genetics; D058829:Serotonin 5-HT1 Receptor Antagonists; D014162:Transfection / Q000379:methods
D020320:Oligoribonucleotides, Antisense; D012333:RNA, Messenger; D058829:Serotonin 5-HT1 Receptor Antagonists; D044282:Receptor, Serotonin, 5-HT1A