Characterization of RNA interference in rat PC12 cells: requirement of GERp95.
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.
Thonberg
Håkan
H
Center for Genomics and Bioinformatics, Karolinska Institutet, Berzelius väg 35, S-171 77 Stockholm, Sweden. hakan.thonberg@cgb.ki.se
Schéele
Camilla C
CC
Dahlgren
Cecilia
C
Wahlestedt
Claes
C
D000818:Animals; D060565:Argonaute Proteins; D015152:Blotting, Northern; D004305:Dose-Response Relationship, Drug; D015852:Eukaryotic Initiation Factor-2; D020868:Gene Silencing / Q000187:drug effects; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D009478:Neuropeptide Y / Q000096:biosynthesis / Q000235:genetics; D016716:PC12 Cells; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C582332:Ago2 protein, rat; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D008565:Membrane Proteins; D009478:Neuropeptide Y; D012333:RNA, Messenger; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins