TY - JOUR

T1 - Characterization of RNA interference in rat PC12 cells

T2 - requirement of GERp95

AU - Thonberg, Håkan

AU - Schéele, Camilla C

AU - Dahlgren, Cecilia

AU - Wahlestedt, Claes

PY - 2004/6/11

Y1 - 2004/6/11

N2 - Double-stranded RNA can initiate post transcriptional gene silencing in mammalian cell cultures via a mechanism known as RNA interference (RNAi). The sequence-specific degradation of homologous mRNA is triggered by 21-nucleotide RNA-duplexes termed short interfering RNA (siRNA). The homologous strand of the siRNA guides a multi-protein complex, RNA-induced silencing complex (RISC), to cleave target mRNA. Although the exact function and composition of RISC is still unclear, it has been shown to include several proteins of the Argonaute protein family. Here we report of a robust system to achieve RNAi in a cultured rat neuronal cell line, PC12. Targeting of neuropeptide Y mRNA by synthetic siRNA results in knock down of the mRNA levels with an IC50 of approximately 0.1 nM. The mRNA knockdown lasts for at least 96 h and is not dependent on protein synthesis. Further, PC12 cells were ablated of the rat Golgi-ER protein 95 kDa (GERp95), an Argonaute family protein, by siRNA methodology. After GERp95-ablation, sequential knockdown of NPY by siRNA was shown to be impaired. Thus, we report that the GERp95 protein is functionally required for RNAi targeting NPY in rat PC12 cells.

AB - Double-stranded RNA can initiate post transcriptional gene silencing in mammalian cell cultures via a mechanism known as RNA interference (RNAi). The sequence-specific degradation of homologous mRNA is triggered by 21-nucleotide RNA-duplexes termed short interfering RNA (siRNA). The homologous strand of the siRNA guides a multi-protein complex, RNA-induced silencing complex (RISC), to cleave target mRNA. Although the exact function and composition of RISC is still unclear, it has been shown to include several proteins of the Argonaute protein family. Here we report of a robust system to achieve RNAi in a cultured rat neuronal cell line, PC12. Targeting of neuropeptide Y mRNA by synthetic siRNA results in knock down of the mRNA levels with an IC50 of approximately 0.1 nM. The mRNA knockdown lasts for at least 96 h and is not dependent on protein synthesis. Further, PC12 cells were ablated of the rat Golgi-ER protein 95 kDa (GERp95), an Argonaute family protein, by siRNA methodology. After GERp95-ablation, sequential knockdown of NPY by siRNA was shown to be impaired. Thus, we report that the GERp95 protein is functionally required for RNAi targeting NPY in rat PC12 cells.

KW - Animals

KW - Argonaute Proteins

KW - Blotting, Northern

KW - Dose-Response Relationship, Drug

KW - Eukaryotic Initiation Factor-2

KW - Gene Silencing

KW - Kinetics

KW - Membrane Proteins

KW - Neuropeptide Y

KW - PC12 Cells

KW - RNA Interference

KW - RNA, Messenger

KW - Rats

KW - Receptors, Dopamine D2

KW - Recombinant Proteins

KW - Transfection

KW - Comparative Study

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.bbrc.2004.04.119

DO - 10.1016/j.bbrc.2004.04.119

M3 - Journal article

C2 - 15147961

VL - 318

SP - 927

EP - 934

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 4

ER -