Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin

Abstract

RNA interference is an effective method to silence specific gene expression. Its application to mammalian cells, however, has been hampered by various shortcomings. Recently, it was reported that introduction of 22-bp double-stranded RNAs (dsRNAs) would specifically suppress expression of endogenous and heterogeneous genes in various mammalian cell lines. However, using this method, we failed to knock out proteins of interest effectively. Here we report the development of a stable and controllable method for generating dsRNA intracellularly. Tetracycline-responsive transactivator-containing cells were transfected with a vector capable of tetracycline-induced bidirectionally overexpressing sense and antisense RNA to form dsRNA in vivo. With this method, glutaredoxin, monitored by Western blot, was knocked out by overexpressing 290-base sense and antisense RNA in NIH 3T3 cells controlled by tetracycline or doxycycline. By using these glutaredoxin knocked-out cells, we have demonstrated that actin deglutathionylation plays a key role in growth factor-mediated actin polymerization, translocalization, and reorganization near the cell periphery.

Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones,

“…One-dimensional polyacrylamide gel electrophoresis and Western blotting of GRx1 were carried out as previously described using a rabbit polyclonal antibody to recombinant human GRx1 (23).…”

RNAi mediated MMP-1 silencing inhibits human chondrosarcoma invasion

“…Currently, RNAi has become a powerful and widely used tool for analysis of gene function in invertebrates and vertebrate animals [ 131. However, the effectiveness of protein attenuation by RNAi depends on the efficiency of cellular uptake of dsRNA, the half-life of the dsRNA inside cells, as well as the half-life of the protein to be silenced [31]. Typically the gene silencing produced by siRNA effects is shortlived, which severely limits its application to the study of metastasis.…”

Human Mitochondrial Glutaredoxin Reduces S-Glutathionylated Proteins with High Affinity Accepting Electrons from Either Glutathione or Thioredoxin Reductase

“…The 12-kDa cytosolic Grx1 has been extensively studied (18 -29), and three-dimensional structures have been determined for the reduced form and the glutathione-mixed disulfide intermediate (27,30,31). Grx1 is an electron donor for ribonucleotide reductase (5,6,24) and is involved in many different cellular processes like dehydroascorbate reduction (32), actin polymerization (33,34), protection against oxidative stress (35,36), apoptosis after acute cadmium exposure (37), and cellular differentiation (38). The second Grx (Grx2) is due to an alternative splicing mechanism targeted either to the nucleus or to mitochondria.…”