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Molecular and Cellular Biology, December 2006, p. 8731-8742, Vol. 26, No. 23
0270-7306/06/$08.00+0     doi:10.1128/MCB.01430-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Induction of Gene Silencing by Hairpin RNA Expression in Tetrahymena thermophila Reveals a Second Small RNA Pathway{triangledown}

Rachel A. Howard-Till1,2 and Meng-Chao Yao1,3*

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109,1 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195,2 Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan3

Received 2 August 2006/ Returned for modification 24 August 2006/ Accepted 18 September 2006

Unlike in other eukaryotes, in which it causes gene silencing, RNA interference (RNAi) has been linked to programmed DNA deletion in the ciliate Tetrahymena thermophila. Here we have developed an efficient method to inducibly express double-stranded RNA hairpins and demonstrated that they cause gene silencing through targeted mRNA degradation in all phases of the life cycle, including growth, starvation, and mating. This technique offers a new tool for gene silencing in this model organism. Induction of RNA hairpins causes dramatic upregulation of Dicer and Argonaute family genes, revealing a system capable of rapidly responding to double-stranded RNA. These hairpins are processed into 23- to 24-nucleotide (nt) small RNAs, which are distinctly different from the 28- to 30-nt small RNAs known to be associated with DNA deletion. Thus, two different small RNA pathways appear to be responsible for gene silencing and DNA deletion. Surprisingly, expression of the RNA hairpin also causes targeted DNA deletion during conjugation, although at low efficiencies, which suggests a possible crossover of these two molecular paths.

* Corresponding author. Mailing address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., P.O. Box 19024, Seattle, WA 98109. Phone: (206) 667-5005. Fax: (206) 667-6526. E-mail: mcyao{at}fhcrc.org.

{triangledown} Published ahead of print on 25 September 2006.

Molecular and Cellular Biology, December 2006, p. 8731-8742, Vol. 26, No. 23
0270-7306/06/$08.00+0     doi:10.1128/MCB.01430-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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