Exploring the specificity and mechanisms of siRNA-mediated gene silencing in mammalian cells

Abstract

Complementary short interfering RNAs (siRNAs) are routinely used to knockdown gene expression. siRNAs bind to their target sequence and guide transcript cleavage and subsequent degradation. This type of silencing is associated with equivalent levels of RNA and protein knockdown. siRNA-mediated knockdown was originally thought to be highly specific. However, the downregulation of non-target mRNAs has been observed following transfection of siRNAs in human cells. Many of these RNA changes are due to siRNA binding to partially complementary sequences within nontargeted transcripts and therefore are termed "off-target" effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing a variety of base pairing interactions in the 9th, 10th, and 11th positions of two siRNA binding sites located in a reporter gene. This region was chosen because siRNA-mediated transcript cleavage occurs between the 10th and 11th positions of the mRNA:siRNA duplex. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 resulted in a two-fold or more mRNA decrease, with varying degrees of protein knockdown. mRNA and protein analysis revealed combinations for which the resulting mRNA and protein levels did not correlate. Although siRNA-mediated transcript cleavage is catalyzed by the endonuclease Argonaute 2 (Ago2), knockdown of Ago2 expression did not affect mRNA knockdown for imperfectly complementary combinations. These results indicate that off-target mRNA reductions are likely attributable to Ago2-independent degradation processes. Using the same reporter system we have also uncovered instances in which complementary siRNAs resulted in high protein/RNA knockdown ratios with dramatic protein silencing.

(cont.) For these particular combinations, the disparity between RNA and protein knockdown is dependent on Ago2 function. This may suggest that the sequences within atypical complementary mRNA:siRNA combinations result in unproductive cleavage by Ago2, leading to persistent binding and enhanced silencing through translational repression. Our findings demonstrate that differences within complementary target sequences can lead to differences in the type of silencing mechanisms that result. The results presented in this dissertation also provide a better understanding of how off-target effects mediate mRNA and protein knockdown of unrelated transcripts, with meaningful implications for those using siRNAs as a tool.