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

Initiation, Establishment, and Maintenance of Heritable MuDR Transposon Silencing in Maize Are Mediated by Distinct Factors

Paramutation and transposon silencing are two epigenetic phenomena that have intrigued and puzzled geneticists for decades. Each involves heritable changes in gene activity without changes in DNA sequence. Here we report the cloning of a gene whose activity is required for the maintenance of both silenced transposons and paramutated color genes in maize. We show that this gene, Mop1 (Mediator of paramutation1) codes for a putative RNA-dependent RNA polymerase, whose activity is required for the production of small RNAs that correspond to the MuDR transposon sequence. We also demonstrate that although Mop1 is required to maintain MuDR methylation and silencing, it is not required for the initiation of heritable silencing. In contrast, we present evidence that a reduction in the transcript level of a maize homolog of the nucleosome assembly protein 1 histone chaperone can reduce the heritability of MuDR silencing. Together, these data suggest that the establishment and maintenance of MuDR silencing have distinct requirements

Horizontal Transfer of a Plant Transposon

The majority of well-documented cases of horizontal transfer between higher eukaryotes involve the movement of transposable elements between animals. Surprisingly, although plant genomes often contain vast numbers of these mobile genetic elements, no evidence of horizontal transfer of a nuclear-encoded transposon between plant species has been detected to date. The most mutagenic known plant transposable element system is the Mutator system in maize. Mu-like elements (MULEs) are widespread among plants, and previous analysis has suggested that the distribution of various subgroups of MULEs is patchy, consistent with horizontal transfer. We have sequenced portions of MULE transposons from a number of species of the genus Setaria and compared them to each other and to publicly available databases. A subset of these elements is remarkably similar to a small family of MULEs in rice. A comparison of noncoding and synonymous sequences revealed that the observed similarity is not due to selection at the amino acid level. Given the amount of time separating Setaria and rice, the degree of similarity between these elements excludes the possibility of simple vertical transmission of this class of MULEs. This is the first well-documented example of horizontal transfer of any nuclear-encoded genes between higher plants

A Position Effect on the Heritability of Epigenetic Silencing

In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks

Loss of RNA–Dependent RNA Polymerase 2 (RDR2) Function Causes Widespread and Unexpected Changes in the Expression of Transposons, Genes, and 24-nt Small RNAs

Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA–dependent RNA polymerase 2 (RDR2) is a component of the RNA–directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA–seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2–sensitive and RDR2–resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant

Following Tetraploidy in Maize, a Short Deletion Mechanism Removed Genes Preferentially from One of the Two Homeologs

Following genome duplication and selfish DNA expansion, maize used a heretofore unknown mechanism to shed redundant genes and functionless DNA with bias toward one of the parental genomes

POPcorn: An Online Resource Providing Access to Distributed and Diverse Maize Project Data

The purpose of the online resource presented here, POPcorn (Project Portal for corn), is to enhance accessibility of maize genetic and genomic resources for plant biologists. Currently, many online locations are difficult to find, some are best searched independently, and individual project websites often degrade over time—sometimes disappearing entirely. The POPcorn site makes available (1) a centralized, web-accessible resource to search and browse descriptions of ongoing maize genomics projects, (2) a single, stand-alone tool that uses web Services and minimal data warehousing to search for sequence matches in online resources of diverse offsite projects, and (3) a set of tools that enables researchers to migrate their data to the long-term model organism database for maize genetic and genomic information: MaizeGDB. Examples demonstrating POPcorn's utility are provided herein