Induction of Gene Silencing by Hairpin RNA Expression inTetrahymena thermophilaReveals a Second Small RNAPathway

Abstract

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.

Dynamic distributions of long double-stranded RNA in Tetrahymena during nuclear development and genome rearrangements

“…We developed a method to test the sensitivity and specificity of J2 antibody in Tetrahymena using strains that contained a hairpin RNA transgene (encoding the green fluorescent protein, GFP) under the control of the cadmium-inducible promoter of the metallothionein gene. Ectopically expressed double-stranded RNA hairpins in Tetrahymena have been shown to trigger a second small RNA pathway for post-transcriptional gene silencing (Howard-Till and Yao, 2006). The immunofluorescent staining occurred clearly on the MACs of induced cells but not un-induced control cells under starvation condition (Fig.…”

“…Two sets of primers containing restriction enzyme cloning sites were used: PmeI-GFP-F1 and XmaI-GFP-R1 for the forward fragment amplification; ApaI-GFP-F2 and XhoI-GFP-R2 for the reverse fragment amplification (Table S1). The products were cloned into the pCRII-I3 vectors by the indicated restriction enzyme sites to form an inverted repeat, and this hairpin cassette was removed from the pCRII-I3 backbone by digestion with PmeI and ApaI and cloned into the pIBF rDNA vector (Howard-Till and Yao, 2006). The vector was introduced into mating cells of CU427 and CU428 by electroporation and the transformant strains were selected by their resistance to paromomycin, as conferred by the mutation on the pIBF rDNA vector.…”

Strand-asymmetric endogenous Tetrahymena small RNA production requires a previously uncharacterized uridylyltransferase protein partner

“…The need for Rdn1, Rdr1, and Dcr2 function in vegetative growth could reflect a requirement to prevent the accumulation of toxic intermediates of sRNA biogenesis. Aberrantly accumulating mRNA or mRNA-like transcripts could overwhelm the cellular machinery for RNA surveillance and degradation, or accumulating dsRNAs could constitutively activate viral defense (Howard-Till and Yao 2006).…”

The Tetrahymena meiotic chromosome bouquet is organized by centromeres and promotes interhomolog recombination

“…To create a cna1RNAi construct, fragment bp 4 to bp 388 of the CNA1 ORF was amplified from genomic DNA using PCR primers to add appropriate restriction sites for cloning back-to-back into the RNAi hairpin (hp) vector pD5H8-MTTSerH3-HP (Howard-Till and Yao, 2006). This vector contains rDNA, which is amplified in the Tetrahymena genome by autonomous replication, ensuring high copy numbers of inserted transgenic DNA, a neomycin resistance gene, and the Cd 2+ -inducible MTT1 metallothionein promoter (see Howard-Till and Yao, 2006).…”