A novel RNAi library based on partially randomized consensus sequences of nuclear receptors: identifying the receptors involved in amyloid beta degradation.
Combinatorial gene inactivation using an RNAi library is a powerful approach to discovering novel functional genes. However, generation of a comprehensive RNAi library remains technically challenging. In this report, we describe a simple and novel approach to designing gene-family-specific RNAi libraries by targeting conserved motifs using degenerate oligonucleotides. We created an siRNA library in the pHUMU vector using partially randomized sequences targeting the consensus region in the ZnF_C4 signature motif of the nuclear hormone receptors and thus against the entire receptor superfamily. For proof of principle, we adapted a reporter assay to screen this library for receptors that might be involved in reducing amyloid beta peptide accumulation. We modified a previously described luciferase reporter assay to measure the amyloid beta precursor cleavages occurring only between beta- and gamma-secretase cleavage sites, thus excluding the major gamma-secretase activities that could generate neurotoxic Abeta peptides. Our screen using this assay identified siRNA vectors that specifically increase the Abeta40/42 cleavage and pointed to a potential receptor target, ROR-gamma. SiRNAs targeting other regions of ROR-gamma not only confirmed the observed reporter activity but also reduced the level of the toxic Abeta peptides. The results demonstrated a general principle for the creation and application of this RNAi library approach for functional gene discovery within a predefined protein family. The discovered negative effect of ROR-gamma on the degradation of the toxic Abeta peptides may also provide a potential drug target or targetable pathway for intervention of Alzheimer disease.
Yang
Jian-Ping
JP
Immusol, Inc., 10790 Roselle Street, San Diego, CA 92121, USA.
Fan
Wufang
W
Rogers
Cheryl
C
Chatterton
Jon E
JE
Bliesath
Joshua
J
Liu
Guohong
G
Ke
Ning
N
Wang
Cui-Ying
CY
Rhoades
Kristina
K
Wong-Staal
Flossie
F
Li
Qi-Xiang
QX
D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D002460:Cell Line; D015723:Gene Library; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism
D016229:Amyloid beta-Peptides; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear