siRNA binding proteins of microglial cells: PKR is an unanticipated ligand

✍ Scribed by Zhiren Zhang; Toni Weinschenk; Ketai Guo; Hermann J. Schluesener

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 299 KB
• Volume: 97
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.20716

✦ Synopsis

Abstract

Small interfering RNA (siRNA), double‐stranded RNA (dsRNA) 21–23 nucleotides (nt) long with two nt 3′ overhangs, has been shown to mediate powerful sequence‐specific gene silence in mammalian cells through RNA interference (RNAi). Due to its high efficiency and high specificity siRNA has been used as a powerful post genomic tool and a potent therapeutic candidate. However, there is still a lot to learn about the mobility of siRNA inside cells and the cellular factors that might interfere with the specificity and activity of siRNA. Microglia are the brain's effector cells of the innate immune system and suitable targets in the development of novel therapeutic strategies. Here, we show the cellular uptake and intracellular distribution of siRNA in murine microglial N9 cells. siRNA was internalized by microglial N9 cells without transfection reagent and mainly localized to the endosomes However, no significant gene silencing effects were observed. Its cellular uptake and cellular distribution pattern were similar with that of a same length single stranded DNA (ssDNA). Further, cellular binding proteins of siRNA were purified and identified by mass spectrometry. Negative control siRNA and siRNA targeted to β‐actin were used in this part of experiment. Most of the siRNA binding proteins for negative control siRNA and siRNA targeted to β‐actin were dsRNA‐binding proteins, such as dsRNA‐dependent protein kinase R (PKR). Furthermore, both control siRNA and siRNA targeted to β‐actin activated PKR in N9 cells, which suggest that siRNA might cause off‐target effects through activation of PKR. J. Cell. Biochem. 97: 1217–1229, 2006. © 2005 Wiley‐Liss, Inc.