Argonaute proteins associate with small RNAs that guide mRNA degradation, translational repression, or a combination of both. The human Argonaute family has eight members, four of which (Ago1 through Ago4) are closely related and coexpressed in many cell types. To understand the biological function of the different Ago proteins, we set out to determine if Ago1 through Ago4 are associated with miRNAs as well as RISC activity in human cell lines. Our results suggest that miRNAs are incorporated indiscriminately of their sequence into Ago1 through Ago4 containing microRNPs (miRNPs). Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. Exogenously introduced siRNAs also associate with Ago2 for guiding target RNA cleavage. The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay.

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.

contributed equally to this work Duplexes of 21±23 nucleotide (nt) RNAs are the sequence-speci®c mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate ef®cient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3¢ overhangs were the most ef®cient triggers of sequence-speci®c mRNA degradation. Substitution of one or both siRNA strands by 2¢-deoxy or 2¢-O-methyl oligonucleotides abolished RNAi, although multiple 2¢-deoxynucleotide substitutions at the 3¢ end of siRNAs were tolerated. The target recognition process is highly sequence speci®c, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is de®ned by the 5¢ end of the guide siRNA rather than its 3¢ end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

No abstract

No abstract