Academic journal article Genetics

Unstable RNAi Effects through Epigenetic Silencing of an Inverted Repeat Transgene in Chlamydomonas Reinhardtii

Academic journal article Genetics

Unstable RNAi Effects through Epigenetic Silencing of an Inverted Repeat Transgene in Chlamydomonas Reinhardtii

Article excerpt

ABSTRACT

RNA interferences in the unicellular green alga, Chlamydomonas reinhardtii, can be silenced. We have used the silencing of a transgene (aadA) that confers resistance to spectinomycin to investigate the mechanisms responsible for silencing by an artificial inverted repeat (IR) of the aadA gene. The IR construct provided strong silencing, but the RNAi efficiency varied among subclones of a single RNAi-transformed strain with successive cell divisions. Northern blot analyses revealed an inverse correlation between the copy number of the hairpin RNA and the spectinomycin resistance of the subclones. There is an inverse correlation between the efficiency of RNAi and the frequency of methylated CpG (*CpG) in the silenced region. No significant methylated cytosine was observed in the target aadA gene, which suggests the absence of RNA-directed DNA methylation in trans. Several experiments suggest the existence of an intrinsic IR sequence-dependent but a transcription-independent DNA methylation system in C. reinhardtii. The correlation between the *CpG levels and the IR transcript implies the existence of IR DNA-dependent DNA methylation. Treatment of RNAi-induced cells with a histone deacetylase inhibitor, Trichostatin A, rapidly increased the amount of the hairpin RNA and suggests that transcription of the silencer construct was repressed by *CpG-related silencing mechanisms.

RNA interference (RNAi) provides a means of silencing genes that protects eukaryotic cells against transposable elements and viruses that produce double-stranded RNAs. In addition, the introduction of transgenes can also elicit RNAi. The major biochemical reactions of RNA interference are shared among various eukaryotes (Cerutti and Casas-Mollano 2006). These reactions include the Piwi and the Dicer family of proteins. However, additional pathways are not identical among eukaryotes. In some organisms that include Caenorhabditis elegans, Schizosaccharomyces pombe, and Arabidopsis thaliana, there is a pathway to convert the target mRNA into dsRNA by siRNA-primed RNA dependent RNA polymerase (RdRP) (Cerutti 2003; Cerutti and Casas-Mollano 2006). In these organisms, the newly generated dsRNA is also recognized by Dicer to produce a large amount of secondary siRNA, which leads to efficient degradation of the target mRNA (Dalmay et al. 2000). Variation exists in the duration of effective RNAi among organisms. In C. elegans, RNAi can even be passed through the germ line to subsequent generations (Grishok et al. 2000). However, in the unicellular green alga, Chlamydomonas reinhardtii, it is often reported that silencing efficiency decreases over variable numbers of cell divisions without rearrangement of the silencer DNA construct (e.g., Koblenz et al. 2003; Schroda 2006).For example, 106-bp long dsRNA worked successfully to knock down centrin (a protein located in multiple places in the flagellar apparatus) and cells became aflagellate (Koblenz et al. 2003). However, the effect was transient and most transformants restored two flagella within 200 days after transformation when they were grown continuously in liquid medium. The AGG2 and AGG3 genes were silenced within 20-30 days in liquid medium (Iomini et al. 2006).

In some eukaryotes, DNA sequences that are complementary to the siRNA are methylated. The siRNA dependent de novo methylation is called RNA-directed DNA methylation (RdDM). Generally, cytosine methylation is detected in symmetric CpG sequences, because only this type of methylation is efficiently preserved by maintenance-type methyltransferases and inherited by daughter cells. In land plants, cytosine methylation at CpHpG sites (where H equals A, T, or C) is faithfully inherited by daughter cells in addition to the symmetric CpG sites (Tariq and Paszkowski 2004; Chan et al. 2005). In A. thaliana, >30% of total C's in the genome are methylated (Adams 1990), while in the case of C. reinhardtii (Hattman et al. 1978) and its closely related multicellular relative, Volvox carteri (Babinger et al. …

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