Characterization of an RNA Associated with Paramutation in Mice

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Methylation of the gene Rasgrf1, a paternally imprinted gene, is important for maintaining proper imprinting patterns and gene expression in mice. An important characteristic of Rasgrf1 is the differentially methylated domain (DMD). The paternal allele is methylated and expressed while the maternal allele is unmethylated and silent. Another important element of Rasgrf1 is a set of 41 nucleotides repeated 40 times that is located immediately downstream of the DMD. The repeats have shown to be necessary for proper regulation of methylation within the DMD. Mouse Igf2r, a maternally imprinted gene in mice, has a region known as Intron 2 or Region 2 (R2) that controls methylation and imprinting. Mutant mice called Lu mice were designed with R2 in place of the repeats seen at Rasgrf1. R2 was partially effective in taking the place of the repeats allowing the methylation and expression of the paternal Rasgrf1allele. For the most part, Lu mice had proper methylation of the paternal allele as seen in wild type mice, but the normally unmethylated, unexpressed maternal allele was activated in trans and aberrantly expressed. This phenomenon is similar to paramutation in corn that is mediated by RNA. In fact, an aberrant RNA was being produced in the mutated Rasgrf1 allele containing R2 and a recent study has data implicating that a piRNA pathway and DMD spanning RNAs may be controlling methylation. Whether or not the aberrant RNA is truly regulating methylation is unknown, but here in this study we sought to characterize the RNA to gain further clues. The technique of PCR using overlapping primers was used to determine the 3´ end of the aberrant RNA formed in Lu mice.

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