RESUMEN
The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that, for different guide-RNA sequences, slicing rates of perfectly complementary bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.
Asunto(s)
Proteínas Argonautas , Conformación de Ácido Nucleico , ARN Guía de Sistemas CRISPR-Cas , Complejo Silenciador Inducido por ARN , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/química , Humanos , Complejo Silenciador Inducido por ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/química , Cinética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Interferencia de ARN , Secuencia de Bases , Células HEK293RESUMEN
The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that for different guide-RNA sequences, slicing rates of perfectly complementary, bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.