| Small RNAs May Cause Genomic Rearrangements in Tetrahymena | |||
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The protozoa Tetrahymena are ciliated, unicellular organisms that divide by binary fission. However, they can also conjugate and exchange their genetic material prior to cell division. Interestingly, Tetrahymena have two nuclei--a micronucleus and a macronucleus. These two nuclei serve different functions. The polyploid macronucleus acts as the transcription center of the cell during vegetative growth. The diploid and transcriptionally inert micronucleus, on the other hand, acts as the germline nucleus during conjugation. In conjugation, two Tetrahymena of different mating types form a temporary junction under starving conditions. The diploid micronucleus of each of these two cells then divides by meiosis--three of the meiotic products are destroyed, while the remaining one undergoes a mitotic division to produce two identical zygotic nuclei in each mating cell. One of the two zygotic nuclei then moves from one mating cell to the other and fuses with the meiotic product of that cell to produce a diploid nucleus. Subsequently, the diploid nucleus divides by mitosis--one mitotic product gives rise to a new diploid micronucleus while the other gives rise to a new polyploid macronucleus. The old polyploid macronucleus, in the meantime, is destroyed by apoptosis (1, 2). In order for the diploid micronucleus to generate the new polyploid macronucleus, two interesting and peculiar recombination events take place. First, approximately 6000 internal eliminated sequences (IES) ranging from 0.5 to >20 kb get removed from the 5 pairs of micronucleus chromosomes. Second, these chromosomes are broken down into 200-300 minichromosomes (by deletions of small < 50 nt breakage eliminated sequences or BES), onto which telomeres are added. Up until now, the mechanism of these recombination events had remained a mystery. Now in a paper published in the 20 September 2002 issue of Cell, Martin Gorovsky and colleagues provide evidence, which suggests that small RNA molecules may be involved in the above recombination events (3). In particular the authors show that a gene called TWI1 is responsible for 1) the elimination of IES sequences and chromosome breakage and, 2) the accumulation of small RNA molecules (~28 nt) in conjugating Tetrahymena thermophilia. The authors propose a model to explain how these small RNA molecules-- termed scan RNAs (scnRNAs)--could lead to IES and BES elimination. The Approach Gorovsky and colleagues first cloned a gene, termed TWI1, from the early mating cells of Tetrahymena thermophilia. Interestingly, TWI1 turned out to be a member of the PPD gene family--a family of genes with conserved Piwi and PAZ domains, some of which are involved in post-transcriptional gene silencing (PTGS) and RNA interference (RNAi) (e.g. AGO1 in plants, qde-2 in fungi, and rde-1 in worms). The authors then investigated the role of TWI1 in conjugation. They first looked at the temporal expression pattern of TWI1. Interestingly, TWI1 mRNA was detected in conjugating cells, but not in starved or growing cells--these results suggested that TWI1 has a role in conjugation. In order to further investigate this connection, the authors knocked out the gene (by using a paromomycin cassette) from Tetrahymena macronuclei. (NOTE: The chromosomes of the polyploid macronucleus segregate by a process called amitosis, where approximately half of the alleles of a locus are distributed randomly to each daughter cell. By this so-called "phenotypic assortment", eventually some Tetrahymena cells would end up exclusively with the TWI1 knockout allele--these cells can be selected by subjecting cells to step-wise and increasing concentrations of paromomycin.) They then determined that these TWI1 knockout cells could undergo conjugation; however, the resulting progeny were not viable. Subsequent PCR analysis suggested that cell death resulted from defects in IES and BES elimination. The fact that a number of PPD family members are involved in PTGS and RNAi, processes that are mediated via ~21 nt siRNAs, prompted the authors to investigate whether TWI1 mediated these DNA rearrangements through small RNA molecules. To this end, the authors looked for the presence of small RNA molecules in conjugating Tetrahymena cells. Remarkably they found the existence of a population of ~28 nt RNAs in conjugating WT, but not in TWI1 mutant, Tetrahymena. The Scan RNA Model The above results suggest that ~28 nt small RNA molecules might be involved in IES and BES elimination, events that take place during the conversion of the old diploid micronucleus to the new polyploid macronucleus. The authors proposed a model, called "the scan RNA model", to account for this involvement (see figure below). Previously it had been shown that both strands of the IES are transcribed from Tetrahymena micronuclei early in conjugation. The authors suggested that these transcripts (and possibly similar transcripts from the BES) would first hybridize and form dsRNAs. These dsRNAs would be then be broken down into ~28 nt "scan RNAs" by a DICER-like enzyme. The scan RNAs would subsequently be transferred from the old micronucleus to the old macronucleus (where IES elimination and chromosome breakage have already taken place)--this transfer could possibly be mediated via TWI1. There, any scan RNA with homology to the old macronuclear DNA (i.e. those outside of the deleted, recombination DNA regions) would be destroyed--an RNase H-like enzyme, which destroys RNA in an RNA-DNA hybrid, could possibly mediate this event. The remaining scan RNAs (i.e. those targeting the recombination regions) would then go from the old macronucleus to the new macronucleus--again, possibly in association with TWI1. There, they would bind to homologous DNA regions. This binding would then specifically target the DNA recombination regions for elimination, either directly (e.g. by attracting recombination enzymes) or indirectly (e.g. by promoting chromatin remodeling).
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