Poster abstracts

Poster number 39 submitted by Vishalakshi Krishnan

In vitro assembly of human RISC

Vishalakshi Krishnan (University of Michigan), Renata Afi Rawlings (University of Michigan)

Abstract:
RNA interference (RNAi) is a mechanism that enables sequence specific regulation of gene expression at the RNA level. Exogenous long double-stranded RNAs function as initiating substrates for the RNAi pathway. They are cleaved into short interfering RNA (siRNA) duplexes by an endogenous RNase III-type enzyme called Dicer. The siRNA duplexes get loaded into the effector RNA Induced Silencing Complex (RISC) by Dicer and are unwound in a strand specific manner during RISC assembly. This single-stranded siRNA guides RISC to effect sequence specific degradation of target mRNA ¹.

RISC assembly has been extensively studied in cell extracts of embryos of the fruit fly Drosophila ³, but little is known about the interaction of siRNAs, Dicer, and RISC proteins in mammalian cells. To identify the component proteins of human RISC and other siRNA containing complexes, we performed native (non-denaturing polyacrylamide) gel-shift assays after incubating cytosolic HeLa cell extract with radiolabeled siRNA duplexes for varying time periods under buffered conditions. Three distinct siRNA-protein complexes, R1, R2, and R3, with distinct gel mobility were observed at all incubation times, but with varying relative abundances. On studying the kinetics of these complexes, the following rates were obtained: rate of formation of R1 and R3 =7.83 ± 4.50 e-4 sec ^-1 and 4.33 ± 0.83 e-4 sec ^-1 respectively, and the rate of disappearance of R2 =2.23 ± 1.00 e-3 sec ^-1. These complexes were subjected to mass spectrometry analysis. In addition, Western Blotting was performed on these complexes for certain proteins known to be involved in the RNAi pathway. Dicer was found to be present in R1, and the content of Dicer in the complex decreased with increasing incubation time. This is consistent with the hypothesis that dicer gets displaced from the mature RISC complex ^{1, 2}. The other components of RISC are yet to be identified.

References:
1. Witold Filipowicz (2005) RNAi: The Nuts and Bolts of the RISC Machine, Cell, Vol. 122, 17–20.
2. ,Hutvágner G, Zamore PD (2002) RNAi: nature abhors a double-strand, Curr Opin Genet Dev. 12(2):225-32.
3. John W. Pham, Janice L. Pellino, Young Sik Lee , Richard W. Carthew, and Erik J. Sontheimer (2004), A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila, Cell, Vol. 117, 83–94
`

Keywords: RNAi, siRNA, RISC