Small RNA-mediated transgenerational genome surveillance

Small RNA-mediated transcriptional gene silencing is a fundamental cellular process in most eukaryotes including fungi, plants, flies, worms and mammals. One of the main tasks of small RNA-mediated transcriptional gene silencing is to neutralize the activity of transposons. However, the mechanisms by which cells distinguish between transposons and their own genomes and by which they selectively target the former with small RNAs are not fully understood. The ciliated protozoan Tetrahymena identifies transposon-derived sequences by a whole-genome transgenerational surveillance mechanism using small RNAs. Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. Many of the DNA segments that are eliminated are related to transposons, and DNA elimination is regulated by small RNAs (scnRNAs) approximately 28-30 nucleotides in length that are produced and function by an RNAi-related mechanism. The study proposed here will investigate how Tetrahymena epigenetically identifies transposon-derived sequences by small RNAs and will greatly increase our understanding of the process of small RNA-mediated self-nonself recognition in general eukaryotes. Our recent studies indicated that DNA elimination in Tetrahymena occurs through two epigenetic mechanisms: 1) biased scnRNA production from the eliminated sequences in the micronucleus and 2) selective degradation of scnRNAs that are complementary to the non-eliminated sequences in the parental macronucleus. We proposed that the former mechanism is transgenerationally regulated by the grandparental macronuclear genome and is epigenetically maintained as a chromatin state during vegetative growth and that the latter mechanism determines self and nonself sequences in the genome. This proposal aims to clarify these two key but poorly understood mechanisms in small RNA-mediated DNA elimination by artificially perturbing the epigenetic cycle and by genetically and biochemically characterizing novel factors involved in DNA elimination.

Transposable elements (TEs) are molecular parasites that able to move from one genome position to another. Cells in our body have a mechanism to silence these potentially harmful elements: locking TE into a closed form chromatin, called heterochromatin. In many eukaryotes, including humans, small RNAs, ~20-30 nucleotides in length, act as security guards to identify and silence TEs. The unicellular eukaryote Tetrahymena also induce heterochromatin formation on over 10,000 TE-related sequences but then they remove these sequences from the genome by the process called programmed DNA elimination. The aim of this project was to understand how small RNAs are produced, how they patrol the genome, and how they induce heterochromatin using the DNA elimination process of Tetrahymena as a model. We have clarified the detailed expression pattern of small RNAs and identified a transcriptional machinery that is necessary for the small RNA production. The DNA elimination process also includes degradation of small RNAs that are produced from non-eliminated sequences and we found that a protein degradation process is likely linked to this RNA turnover. Furthermore, we found that a novel class of small RNAs is produced in heterochromatin-dependent manner and is also involved in the DNA elimination process, indicating that a previously uncharacterized small RNA-heterochromatin positive feedback loop in DNA elimination. These new observations not only have opened a new avenue to investigate DNA elimination mechanism in Tetrahymena but also provided unique insights about how arms race between TE and host shapes genome evolution.