Efficient and specific knockdown of small non-coding RNAs in mammalian cells and in mice.
Hundreds of small nuclear non-coding RNAs, including small nucleolar RNAs (snoRNAs), have been identified in different organisms, with important implications in regulating gene expression and in human diseases. However, functionalizing these nuclear RNAs in mammalian cells remains challenging, due to methodological difficulties in depleting these RNAs, especially snoRNAs. Here we report a convenient and efficient approach to deplete snoRNA, small Cajal body RNA (scaRNA) and small nuclear RNA in human and mouse cells by conventional transfection of chemically modified antisense oligonucleotides (ASOs) that promote RNaseH-mediated cleavage of target RNAs. The levels of all seven tested snoRNA/scaRNAs and four snRNAs were reduced by 80-95%, accompanied by impaired endogenous functions of the target RNAs. ASO-targeting is highly specific, without affecting expression of the host genes where snoRNAs are embedded in the introns, nor affecting the levels of snoRNA isoforms with high sequence similarities. At least five snoRNAs could be depleted simultaneously. Importantly, snoRNAs could be dramatically depleted in mice by systematic administration of the ASOs. Together, our findings provide a convenient and efficient approach to characterize nuclear non-coding RNAs in mammalian cells, and to develop antisense drugs against disease-causing non-coding RNAs.
Department of Core Antisense Research, ISIS Pharmaceuticals, Inc. 1896 Rutherford Rd. Carlsbad, CA 92008, USA. lliang@isisph.com
Xue-hai
XH
Liang
Timothy A
TA
Vickers
Shuling
S
Guo
Stanley T
ST
Crooke
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000032:analysis / Q000737:chemistry; D012335:RNA, Ribosomal / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D016376:Oligonucleotides, Antisense; D012335:RNA, Ribosomal; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar