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I guess I'm blogging too much when I see a hot paper mentioned at the Corner before I've heard about it from my colleagues. Lolle et al. describe a high reversion rate at the Hothead (HTH) locus in Arabidopsis thaliana, the E. coli of the plant world. For those who are not geneticists, there are different versions of the HTH gene, called alleles. All caps indicates the "wild-type" allele (wt). Lower case indicates a mutant allele; in this study they use several different alleles: hth-4, hth-8, and hth-10. Arabidopsis is diploid, which means that each plant has two HTH alleles, and they can be found as HTH/HTH (homozygous wt), HTH/hth (heterozygous), or hth/hth (homozygous mutant). Arabidopsis is a self-fertilizing plant. Lolle et al., self-fertilized hth/hth. The progeny will get one HTH allele from the pollen and one from the ovule, equivalent to how we get one copy of most genes from mom and the other from dad. Because there were no HTH alleles in this self-fertilization, normally we'd expect hth/hth plants to give only hth/hth offspring. But that's not what happened! About 10% of the offspring were HTH/hth. The HTH allele came from somewhere. Lolle et al., then addressed several posibilities for the source of the HTH allele, ranging from contamination to hidden copies of pieces of the HTH gene elsewhere in the genome. These were not found. They also asked if this switching was something special about the HTH locus, or if other parts of the genome were also affected. To do this, they crossed their hth plants with a different isolate (ecotype) of Arabidopsis, which has lots of single nucleotide polymorphisms (differences), or SNPs. The F1 plants are the progeny of this cross, and are all HTH/hth, and are also heterozygous for every SNP. Selfing these give F2 plants, which will be 25% hth/hth, 50% HTH/hth, and 25% HTH/HTH. They chose hth/hth F2 plants, each of which is a mix of 1:2:1 genotypes for each SNP. Analyzing the SNPs, they could find hth/hth plants that were also homozygous for each SNP...they took these, and selfed them to give F3 plants. If the reversion from space phenomenon was confined to the HTH locus, all of these other SNPs should breed true and stay homozygous in the F3 plants. Alternatively, if the reversion phenomenon is genome-wide, then they should see the SNP loci changing to the missing allele some of the time. That's what happened...SNP alleles that were not in the genomic DNA reappeared in the progeny. This only happened if the plants were hth/hth. Reed Cartwright at De Rerum Natura suggests that the hth conversion data could be accounted for by selection for HTH, particularly at the level of pollen. However, this would not explain the conversions of the SNPs, which are found to go in both allelic directions. Lolle et al., argue that there has to be lingering nucleic acid sequence out there somewhere, and propose that it could be RNA. This is mostly by the classic Sherlock Holmes reasoning - if DNA is eliminated as a suspect, then RNA becomes the best candidate, no matter how odd that is superficially. In fact, it may not be as odd as it sounds: Although the model proposed above might represent an extraordinary view of inheritance, each of the processes required by the model has been demonstrated previously. Research performed over the past few years in post-transcriptional gene silencing or RNA interference has shown that silencing induced by double-stranded RNA can persist for several generations in Caenorhabditis elegans10 and can spread through the organism in plants, fungi and animals11, 12. This strongly implies that some form of nucleotide sequence information must be stably replicated and transmitted outside the DNA genome, which is consistent with a requirement for an RNA-directed RNA polymerase in post-transcriptional gene silencing13-15. Replication of the sequence cache could allow it to persist for several generations; this is consistent with the fact that hth mutations retain their ability to revert even after multiple generations in the homozygous state (S.J.L. and R.E.P., unpublished observations). Experiments to detect changes in the reversion frequency after several generations of homozygous propagation are in progress. Both double-stranded RNA and microRNA have been implicated in the sequence-specific modification of the nuclear genome by DNA methylation16-18 and it has been possible to perform site-directed mutagenesis by the introduction of chimaeric RNA-DNA oligonucleotides in both plant and animal cells19-21. We propose that a mechanism combining these individual steps could explain the exceptional inheritance we see in hth mutants as well as the unusual results observed by earlier workers.The paper suggests lots of interesting followups, which I'm sure are going to be in the next grant from the Pruitt lab, if they aren't already being done. An obvious experiment is to look for the missing sequences in the RNA of the plants by RT-PCR. This is probably technically doable, but harder than it sounds, depending on how large the cache RNAs are, if they exist. Also, there is the question of why the effect is only seen in hth/hth plants. Assuming for the moment that there is a population of cache RNAs, the authors suggest that lack of HTH function provides a general stress that increases gene conversion, copying information from the cache RNA to the genomic DNA. This predicts that other stresses might cause similar gene conversions in HTH/HTH plants. Alternatively, one of the normal functions of HTH may be in preventing the buildup of cache RNAs. Replicating dsRNAs are thought to occur in the mechanism for RNAi . Normal turnover of untranslated pre-mRNAs and mRNAs may keep them from being attacked by the RNAi system, which is thought to be an antiviral defense. Perhaps hth/hth is defective in RNA turnover, leading to a general buildup of siRNAs...but with so many competing RNAs, perhaps the RISC complex doesn't turn off everything in the genome. In this model, the phenomenon would be a consequence of dysfunction of a system evolved for a very different purpose - degrading RNA viruses. Perhaps the most interesting possibility (of the few that I can think of anyway), is that RNAi and stress-induced conversion from cache RNAs are both manifestations of some other system that evolved for a different purpose - DNA repair. One of the basic problems in repair is distinguishing which strand in a DNA mismatch is the one that was changed. Perhaps checking which sequence is in the transcribed RNA was one solution.
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