Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.
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.
Martinez
Javier
J
Department of Cellular Biochemistry, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany.
Patkaniowska
Agnieszka
A
Urlaub
Henning
H
Lührmann
Reinhard
R
Tuschl
Thomas
T
D001483:Base Sequence; D020868:Gene Silencing; D018390:Gene Targeting; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism*; D016372:RNA, Antisense / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000032:analysis / Q000378:metabolism*
D016372:RNA, Antisense; D034741:RNA, Small Interfering; D012313:RNA