Production and Processing of siRNA Precursor Transcripts from the Highly Repetitive Maize Genome

Mutations affecting the maintenance of heritable epigenetic states in maize identify multiple RNA–directed DNA methylation (RdDM) factors including RMR1, a novel member of a plant-specific clade of Snf2-related proteins. Here we show that RMR1 is necessary for the accumulation of a majority of 24 nt small RNAs, including those derived from Long-Terminal Repeat (LTR) retrotransposons, the most common repetitive feature in the maize genome. A genetic analysis of DNA transposon repression indicates that RMR1 acts upstream of the RNA–dependent RNA polymerase, RDR2 (MOP1). Surprisingly, we show that non-polyadenylated transcripts from a sampling of LTR retrotransposons are lost in both rmr1 and rdr2 mutants. In contrast, plants deficient for RNA Polymerase IV (Pol IV) function show an increase in polyadenylated LTR RNA transcripts. These findings support a model in which Pol IV functions independently of the small RNA accumulation facilitated by RMR1 and RDR2 and support that a loss of Pol IV leads to RNA Polymerase II–based transcription. Additionally, the lack of changes in general genome homeostasis in rmr1 mutants, despite the global loss of 24 nt small RNAs, challenges the perceived roles of siRNAs in maintaining functional heterochromatin in the genomes of outcrossing grass species

A Novel Snf2 Protein Maintains trans-Generational Regulatory States Established by Paramutation in Maize

Paramutations represent heritable epigenetic alterations that cause departures from Mendelian inheritance. While the mechanism responsible is largely unknown, recent results in both mouse and maize suggest paramutations are correlated with RNA molecules capable of affecting changes in gene expression patterns. In maize, multiple required to maintain repression (rmr) loci stabilize these paramutant states. Here we show rmr1 encodes a novel Snf2 protein that affects both small RNA accumulation and cytosine methylation of a proximal transposon fragment at the Pl1-Rhoades allele. However, these cytosine methylation differences do not define the various epigenetic states associated with paramutations. Pedigree analyses also show RMR1 does not mediate the allelic interactions that typically establish paramutations. Strikingly, our mutant analyses show that Pl1-Rhoades RNA transcript levels are altered independently of transcription rates, implicating a post-transcriptional level of RMR1 action. These results suggest the RNA component of maize paramutation maintains small heterochromatic-like domains that can affect, via the activity of a Snf2 protein, the stability of nascent transcripts from adjacent genes by way of a cotranscriptional repression process. These findings highlight a mechanism by which alleles of endogenous loci can acquire novel expression patterns that are meiotically transmissible

Diversity of Pol IV Function Is Defined by Mutations at the Maize rmr7 Locus

Mutations affecting the heritable maintenance of epigenetic states in maize identify multiple small RNA biogenesis factors including NRPD1, the largest subunit of the presumed maize Pol IV holoenzyme. Here we show that mutations defining the required to maintain repression7 locus identify a second RNA polymerase subunit related to Arabidopsis NRPD2a, the sole second largest subunit shared between Arabidopsis Pol IV and Pol V. A phylogenetic analysis shows that, in contrast to representative eudicots, grasses have retained duplicate loci capable of producing functional NRPD2-like proteins, which is indicative of increased RNA polymerase diversity in grasses relative to eudicots. Together with comparisons of rmr7 mutant plant phenotypes and their effects on the maintenance of epigenetic states with parallel analyses of NRPD1 defects, our results imply that maize utilizes multiple functional NRPD2-like proteins. Despite the observation that RMR7/NRPD2, like NRPD1, is required for the accumulation of most siRNAs, our data indicate that different Pol IV isoforms play distinct roles in the maintenance of meiotically-heritable epigenetic information in the grasses

Multiple Trans-Sensing Interactions Affect Meiotically Heritable Epigenetic States at the Maize pl1 Locus

Interactions between specific maize purple plant1 (pl1) alleles result in heritable changes of gene regulation that are manifested as differences in anthocyanin pigmentation. Transcriptionally repressed states of Pl1-Rhoades alleles (termed Pl′) are remarkably stable and invariably facilitate heritable changes of highly expressed states (termed Pl-Rh) in Pl′/Pl-Rh plants. However, Pl′ can revert to Pl-Rh when hemizygous, when heterozygous with pl1 alleles other than Pl1-Rhoades, or in the absence of trans-acting factors required to maintain repressed states. Cis-linked features of Pl1-Rhoades responsible for these trans-sensing behaviors remain unknown. Here, genetic tests of a pl1 allelic series identify two potentially separate cis-linked features: one facilitating repression of Pl-Rh and another stabilizing Pl′ in trans. Neither function is affected in ethyl-methanesulfonate-induced Pl1-Rhoades derivatives that produce truncated PL1 peptides, indicating that PL1 is unlikely to mediate trans interactions. Both functions, however, are impaired in a spontaneous Pl1-Rhoades derivative that fails to produce detectable pl1 RNA. Pl′-like states can also repress expression of a pl1-W22 allele, but this repression is not meiotically heritable. As the Pl′ state is not associated with unique small RNA species representing the pl1-coding region, the available data suggest that interactions between elements required for transcription underlie Pl1-Rhoades epigenetic behaviors

Locus-specific paramutation in Zea mays is maintained by a PICKLE-like chromodomain helicase DNA-binding 3 protein controlling development and male gametophyte function.

Paramutations represent directed and meiotically-heritable changes in gene regulation leading to apparent violations of Mendelian inheritance. Although the mechanism and evolutionary importance of paramutation behaviors remain largely unknown, genetic screens in maize (Zea mays) identify five components affecting 24 nucleotide RNA biogenesis as required to maintain repression of a paramutant purple plant1 (pl1) allele. Currently, the RNA polymerase IV largest subunit represents the only component also specifying proper development. Here we identify a chromodomain helicase DNA-binding 3 (CHD3) protein orthologous to Arabidopsis (Arabidopsis thaliana) PICKLE as another component maintaining both pl1 paramutation and normal somatic development but without affecting overall small RNA biogenesis. In addition, genetic tests show this protein contributes to proper male gametophyte function. The similar mutant phenotypes documented in Arabidopsis and maize implicate some evolutionarily-conserved gene regulation while developmental defects associated with the two paramutation mutants are largely distinct. Our results show that a CHD3 protein responsible for normal plant ontogeny and sperm transmission also helps maintain meiotically-heritable epigenetic regulatory variation for specific alleles. This finding implicates an intersection of RNA polymerase IV function and nucleosome positioning in the paramutation process

Rmr6 Maintains Meiotic Inheritance of Paramutant States in Zea mays

Paramutation generates heritable changes affecting regulation of specific alleles found at several Zea mays (maize) loci that encode transcriptional regulators of anthocyanin biosynthetic genes. Although the direction and extent of paramutation is influenced by poorly understood allelic interactions occurring in diploid sporophytes, two required to maintain repression loci (rmr1 and rmr2), as well as mediator of paramutation1 (mop1), affect this process at the purple plant1 (pl1) locus. Here we show that the rmr6 locus is required for faithful transmission of weakly expressed paramutant states previously established at both pl1 and red1 (r1) loci. Transcriptional repression occurring at both pl1 and booster1 (b1) loci as a result of paramutation also requires Rmr6 action. Reversions to highly expressed, nonparamutant states at both r1 and pl1 occur in plants homozygous for rmr6 mutations. Pedigree analysis of reverted pl1 alleles reveals variable latent susceptibilities to spontaneous paramutation in future generations, suggesting a quantitative nature of Rmr6-based alterations. Genetic tests demonstrate that Rmr6 encodes a common component required for establishing paramutations at diverse maize loci. Our analyses at pl1 and r1 suggest that this establishment requires Rmr6-dependent somatic maintenance of meiotically heritable epigenetic marks