Analysis of human small nucleolar RNAs (snoRNA) and the development of snoRNA modulator of gene expression vectors.
Human small nucleolar RNAs (snoRNAs) that copurify with nucleoli isolated from HeLa cells have been characterized. Novel fibrillarin-associated snoRNAs were detected that allowed the creation of a new vector system for the targeted knockdown of one or more genes in mammalian cells. The snoMEN (snoRNA modulator of gene expressioN) vector technology is based on snoRNA HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets. Gene-specific knockdowns are demonstrated for endogenous cellular proteins and for G/YFP-fusion proteins. Multiplex snoMEN vectors coexpress multiple snoRNAs in one transcript, targeted either to different genes or to different sites in the same gene. Protein replacement snoMEN vectors can express a single transcript combining cDNA for a tagged protein with introns containing cognate snoRNAs targeted to knockdown the endogenous cellular protein. We foresee applications for snoMEN vectors in basic gene expression research, target validation, and gene therapy.
Ono
Motoharu
M
Wellcome Trust Centre for Gene Regulation and Expression, and Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.
Yamada
Kayo
K
Avolio
Fabio
F
Scott
Michelle S
MS
van Koningsbruggen
Silvana
S
Barton
Geoffrey J
GJ
Lamond
Angus I
AI
D002466:Cell Nucleolus / Q000235:genetics; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D009154:Mutation; D034622:RNA Interference; D020537:RNA, Small Nucleolar / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002868:Chromosomal Proteins, Non-Histone; D020537:RNA, Small Nucleolar; C526471:SMN1 protein, human; D055533:Survival of Motor Neuron 1 Protein; C047458:fibrillarin; D049452:Green Fluorescent Proteins