Sarah Warburton and Dr. Barry Willardson, Chemistry and Biochemistry
Our lab has recently described an interaction of Phosducin-like protein (PhLP) with the cytoplasmic chaperonin complex (CCT), a sixteen polypeptide complex required for the native folding of actin and tubulin and many other cellular proteins. We found that the binding of PhLP blocked CCT-dependent folding of firefly luciferase in vitro and that over expression of PhLP in Chinese hamster ovary (CHO) cells inhibited actin folding by 80%. Thus, it appears that PhLP is the first known regulator of CCT-dependent protein folding.
A logical follow-up is to perform the converse experiment by decreasing PhLP expression and measuring the effects on CCT activity. Methods to silence genes in cells in culture have had mixed success. A novel technique to silence gene expression using double-stranded interfering RNA (siRNA) has been very successful in silencing genes in C. elegans and Drosophila.1, 2 This method was used to silence PhLP expression as an initial step towards determining the effect of PhLP on CCT activity.
Specific sequences within the coding region of luciferase were reported to have somewhat better siRNA capability than others, thus different 21 base pair sequences will be tested for maximal siRNA activity.3 Single-stranded RNA corresponding to a 21 base pair region of the PhLP encoding region with a two base pair 3’ thymidine overhang were synthesized by Bob Schackmann from the University of Utah. Single stranded RNA corresponding to a 21 base pair region of the green fluorescent protein (GFP) encoding region was also synthesized to be used as a control for the experiment. The sequence of the synthesized primers is shown below:
PhLP sense: 5’ CAG UGG GAA GAU GAU UCU GAA TT 3’
PhLP antisense: 5’ UUC AGA GUC AUC UUC CCA CUG TT 3’
GFP sense: 5’ AGC AGC ACG ACU UCU UCA AGU TT 3’
GFP antisense: 5’ ACU UGA AGA AGU CGU GCU GCU TT 3’
The primers were deprotected, purified and lyophilized before they were sent to us from the University of Utah. Upon receiving the primers, each was brought to a concentration of 10 g/l using nuclease-free water. Annealing of complementary strands to form double-stranded RNA was performed by placing equal amount of the complimentary strands in annealing buffer, heating to 95C and allowing the mixture to slowly cool to room temperature. To confirm the annealing of complimentary strands the annealed RNA, along with a single stranded control, was run on a 3% agarose gel.
siRNA, at a concentration of 200 nM, was transiently transfected into CHO cells using Oligofectamine. Cells were harvested at 12, 24, 36 and 48 hours to test for decreased expression of PhLP. Control transfections using GFP siRNA, which have no siRNA activity in CHO cells, were performed in parallel for comparison purposes.
The cell lysate from each sample was run out on a 10% SDS polyacrylamide gel and transferred to nitrocellulose paper. Immunoblot analysis using a PhLP specific antibody was used to test for decreased PhLP expression at time intervals up to 48 hours. Immunoblot analysis of CHO cells transfected with siRNA showed that there was no decrease in PhLP expression. The experiment was repeated numerous times yielding the same result.
We decided to repeat the transfection in human embryonic kidney (HEK 293) cells which have also been previously shown to support siRNA. However, results showed that no decrease in PhLP expression was observed.
Because different sequences within the coding region of previous silenced genes have better siRNA capabilities than others, a new sequence to the PhLP encoding region will be synthesized to measure its siRNA capability. Once a sequence is found which effectively decreases PhLP expression levels this sequence will be used to measure the effect of decreasing PhLP levels on the rate of CCT-dependent actin folding.
- Elbashir, S.M.; Harboth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001, 411(6836), 494-498.
- Fire, A.; Xu, S.; Montgomery, M.K.; Kostas, S.A.; Driver, S.E.; Mello, C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391(6696), 806-811.
- Elbashir, S.M.; Lendeckel, W.; Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001, 15(2), 188-200.