2000 Ambros 2004 Bartel 2004 Bagga et al. MicroRNAs (miRNAs) are a large family of 21-22-nt noncoding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation ( Olsen and Ambros 1999 Reinhart et al.
Cleavage on the target RNA sequence in RNAi is known to occur between +10 and +11 positions of the guide siRNA (the 5′-end of the guide siRNA being assigned +1) ( Elbashir et al. 2001 Hutvagner and Zamore 2002a Martinez et al. Subsequently, the siRNA duplex goes through an ATP-dependent unwinding process and one strand over the other is often preferentially loaded onto the RNA-induced silencing complex (RISC) that endonucleolytically cleaves target RNA at sites completely complementary to the siRNA ( Hammond et al. Long dsRNA is first converted to 21-23-nucleotide (nt) small interfering RNA (siRNA) by Dicer ( Bernstein et al. RNA interference (RNAi) is a sequence-specific RNA cleavage process triggered by double-stranded RNA (dsRNA) (for review, see Sontheimer 2005 Tomari and Zmore 2005). These findings give rise to a new model in which AGO2 is directly involved in RISC formation as “Slicer” of the passenger strand of the siRNA duplex. We show that upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Slicer activity is reconstituted with recombinant full-length AGO1. Here we show that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. In Drosophila, AGO2 was shown to possess the Slicer activity. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage (“Slicer”) activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. Argonaute proteins play important yet distinct roles in RNA silencing.
0 kommentar(er)
