Biochemical Mechanism of RNA Interference in Higher Organisms: A Dissertation

RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene silencing pathway found in eukaryotes, in which 21-nucleotide, small interfering RNAs (siRNAs) guide destruction of a corresponding target mRNA. RNAi is a natural mechanism for both genome surveillance and gene regulation. Moreover, siRNAs can be transfected into cultured mammalian cells, causing the sequence-specific ‘knock down’ of an mRNA. My work in the Zamore lab has centered around the Drosophila in vitro system and cultured mammalian cells to study the RNA interference (RNAi) pathway. small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC), which culminates in the cleavage of a complementary target mRNA. Previous work proved that certain structural features of siRNAs are essential for RNAi in flies, including the requirement for 5 ́ phosphates and 3 ́ hydroxyl groups. In cultured mammalian cells, the requirement for a 5 ́ phosphate also holds true, but we found no evidence to support the necessity for 3 ́ hydroxyls in either system. In addition, siRNAs can act as single strands entering the pathway downstream of double-stranded siRNAs, both of which are competent in directing the cleavage of its cognate mRNA at a single site. While these key features are a requirement for functional siRNAs, alone they do not determine the efficiency to which an siRNA can enter the RISC. In fact, both strands of an siRNA can enter RISC to a different degree as determined by the stabilities of the 5 ́ ends of the siRNA strand, a phenomenon termed ‘functional asymmetry’. This