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

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Alemán, Lourdes Maria

Advisor dc:contributor.advisor

• Phillip A. Sharp.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/45146

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/45146