Alissa M Anderson | PubMed Health

PubMed2006

The germ line limited M element of Tetrahymena is targeted for elimination from the somatic genome by a homology-dependent mechanism.

A RNA interference (RNAi) like mechanism is involved in elimination of thousands of DNA segments from the developing somatic macronucleus of Tetrahymena, yet how specific internal eliminated sequences (IESs) are recognized remains to be fully elucidated. To define requirements for DNA rearrangement, we performed mutagenesis of the M element, a well-studied IES. While sequences within the macronucleus-retained DNA are known to determine the excision boundaries, we show that sequences internal to these boundaries are required to promote this IES's rearrangement. However, this element does not contain any specific sequence required in cis as removal of its entire left or right side was insufficient to abolish all rearrangement. Instead, rearrangement efficiency correlated with the overall size of the M element sequence within a given construct, with a lower limit of nearly 300 bp. Also, the observed minimal region necessary to epigenetically block excision supports this size limit. Truncated M element constructs that exhibited impaired rearrangement still showed full transcriptional activity, which suggests that their defect was due to inefficient recognition. This study indicates that IESs are targeted for elimination upon their recognition by homologous small RNAs and further supports the idea that DNA elimination is a RNAi-related mechanism involved in genome surveillance.

Animals↗

PubMed2005

Germ line transcripts are processed by a Dicer-like protein that is essential for developmentally programmed genome rearrangements of Tetrahymena thermophila.

Abundant approximately 28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (DeltaDCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These DeltaDCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation DeltaDCL1 cells die without eliminating the germ line-limited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicer-related gene is required for programmed DNA rearrangements. Surprisingly, DeltaDCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.

Animals↗