Nucleic Acids Research Advance Access originally published online on September 15, 2009
Nucleic Acids Research 2009 37(20):6950-6959; doi:10.1093/nar/gkp764
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Nucleic Acids Research, 2009, Vol. 37, No. 20 6950-6959
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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The 5'-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex
Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Husargatan 3, S-751 23 Uppsala, Sweden
*To whom correspondence should be addressed. Tel: +46 18 471 4164; Fax: +46 18 50 98 76; Email: goran.akusjarvi{at}imbim.uu.se
Received December 16, 2008. Revised August 25, 2009. Accepted August 31, 2009.
Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.
Present addresses:Ning Xu, Department of Medicine, Unit of Dermatology and Venereology, Karolinska Institutet, Stockholm, Sweden.
Sofia Gkountela, Department of Molecular, Cell and Developmental Biology, University of Los Angeles, CA, USA.