Germ Line Transcripts Are Processed by a Dicer-Like Protein That Is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila

Abstract

Abundant ∼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 (ΔDCL1) 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 ΔDCL1 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 ΔDCL1 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, ΔDCL1 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.

ACKNOWLEDGMENTS

This work was supported by National Science Foundation research grant MCB-0131421 and by National Institutes of Health research grant GM069593 to D.L.C.

We thank Washington University Biology 3492 students (Eric Archer, Robert Cooper, Laura Ernst, Michael Evenson, Annabel Fu, Xiaoou Pan, and Andrew Schmerling) for technical assistance with the project. We also thank Martin Gorovsky (University of Rochester) for providing plasmid pMNBL and K. Mochizuki (University of Rochester) for providing the ΔTWI1 strains, sharing unpublished data, and comments on our manuscript. Preliminary Tetrahymena genome sequence data were obtained from The Institute for Genomic Research website (http://www.tigr.org).