Final Report Summary - TETRAHYMENA (RNA directed DNA elimination in Tetrahymena) Transposable elements are molecular parasites that are able to move from one genome position to another. Cells in our body are equipped with a mechanism to silence these potentially harmful elements: locking transposable element into a closed type of chromatin known as heterochromatin. Chemically, both transposable elements and other parts of the genome are merely stretches of DNA. How are cells able to distinguish junk from precious DNA? Evidence suggests that tiny RNAs, ~20-30 nucleotides in length, act as security guards to identify transposable elements in many different eukaryotes. In this project, we use the protozoan Tetrahymena as a model to study how these short RNAs lock transposable elements into heterochromatin.During their sexual reproduction, Tetrahymena eliminates, ~10,000 Internal Eliminated Sequences (IESs). Many IESs are related to transposable elements. Heterochromatin components, including histone H3 methylated on lysine 9 (H3K9me) or on lysine 27 (H3K27me), and the chromodomain protein Pdd1p are specifically associated with eliminated IES sequences and are essential for DNA elimination. We previously demonstrated that small (~29nt) scan (scn) RNAs are also required for the elimination of DNA. scnRNAs are produced by the Dicer protein Dcl1p and associate with the Argonaute protein Twi1p. Dcl1p and Twi1p are required for accumulation and/or targeting of H3K9me/H3K27me/Pdd1p and for DNA elimination. Thus, heterochromatin is formed downstream of the RNAi-related mechanism in the DNA elimination pathway.In this project, we have revealed that:1) 2’-O-methylation stabilizes scnRNA and ensures DNA elimination. This was one of the first demonstration that 3’-terminal 2’-O-methylation on a selected class of small RNAs regulates the function of a specific RNAi pathway.2) There is a quality control mechanism of Twi1p-scnRNA complex that only functionally matured complex is imported to the nucleus. Although emerging evidence suggests that many RNAi-related processes act in the nucleus, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. This was the first demonstration of the state-dependent the nuclear import of an Argonaute protein.3) The pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and by the selective degradation of scnRNAs in the parental macronucleus. Our study indicated that DNA elimination in Tetrahymena is possibly epigenetically and trans-generationally regulated not only by the parental somatic genome sequence, but also by the somatic genomes of grandparents.4) A domesticated PiggyBac transposese catalyzes DNA elimination. Although several domesticated transposon proteins are known to be involved in host genome regulations in other eukaryotes, their evolutional histories are not clear. Ciliated protozoa may preserve traceable “fossil” records that help illuminate how a transposon has been domesticated to regulate eukaryotic genome.
