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Nucleic Acids Res. 2014 Apr;42(8):5256-69. doi: 10.1093/nar/gku153. Epub 2014 Feb 20.

Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells.

Author information

1
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, Department of Biotechnology, Faculty of Engineering, Maebashi Institute of Technology, 460-1 Kamisadori-cho, Maebashi-shi, Gunma 371-0816, Japan and Department of Molecular Medicine and Anatomy, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.

Abstract

Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5' proximal nucleotides, which include seed sequence (positions 2-8 from the 5' end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the trans-activation response RNA-binding protein (TRBP). The terminal RNA (positions 19-21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15-18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.

PMID:
24561616
PMCID:
PMC4005638
DOI:
10.1093/nar/gku153
[Indexed for MEDLINE]
Free PMC Article
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