Lyudmila Sidorenko

An RNA-dependent RNA polymerase is required for paramutation in maize

Paramutation is an allele-dependent transfer of epigenetic information, which results in the heritable silencing of one allele by another. Paramutation at the b1 locus in maize is mediated by unique tandem repeats that communicate in trans to establish and maintain meiotically heritable transcriptional silencing. The mop1 (mediator of paramutation1) gene is required for paramutation, and mop1 mutations reactivate silenced Mutator elements. Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic developmental phenotypes. Here we report the map-based cloning of mop1, an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP in plants that is associated with the production of short interfering RNA (siRNA) targeting chromatin. Nuclear run-on assays reveal that the tandem repeats required for b1 paramutation are transcribed from both strands, but siRNAs were not detected. We propose that the mop1 RDRP is required to maintain a threshold level of repeat RNA, which functions in trans to establish and maintain the heritable chromatin states associated with paramutation.

Distinct size distribution of endogeneous siRNAs in maize: Evidence from deep sequencing in the mop1-1 mutant.

Small RNAs from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of RDR2. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop1-1 loss-of-function mutant by using Illuminas sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic ≈22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.

Transgene-induced RNA interference as a tool for plant functional genomics

RNA interference (RNAi) is a powerful tool for functional genomics in a number of species. The logistics and procedures for doing high-throughput RNAi to investigate the functions of large numbers of genes in Arabidopsis thaliana and in Zea mays are described. Publicly available plasmid vectors that facilitate the stable chromosomal integration of inverted repeat transgenes that trigger RNAi have been used to generate more than 50 independent transgenic lines each in Arabidopsis and maize. Analysis of mRNA abundance of the targeted genes in independent lines transformed with distinct constructs indicates that the success of RNAi-induced silencing is gene dependent. mRNA levels were not detectably reduced for some genes, but were dramatically reduced for a number of genes targeted. A common pattern was that multiple independent lines transgenic for the same construct showed the same extent of silencing. This chapter describes the procedures used to generate and test transgenic lines mediating RNAi in Arabidopsis and maize.

A Dominant Mutation in mediator of paramutation2, One of Three Second-Largest Subunits of a Plant-Specific RNA Polymerase, Disrupts Multiple siRNA Silencing Processes

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes.

RNA-mediated trans-communication can establish paramutation at the b1 locus in maize

Paramutation is the epigenetic transfer of information between alleles that leads to the heritable change of expression of one allele. Paramutation at the b1 locus in maize requires seven noncoding tandem repeat (b1TR) sequences located ∼100 kb upstream of the transcription start site of b1, and mutations in several genes required for paramutation implicate an RNA-mediated mechanism. The mediator of paramutation (mop1) gene, which encodes a protein closely related to RNA-dependent RNA polymerases, is absolutely required for paramutation. Herein, we investigate the potential function of mop1 and the siRNAs that are produced from the b1TR sequences. Production of siRNAs from the b1TR sequences depends on a functional mop1 gene, but transcription of the repeats is not dependent on mop1. Further nuclear transcription assays suggest that the b1TR sequences are likely transcribed predominantly by RNA polymerase II. To address whether production of b1TR-siRNAs correlated with paramutation, we examined siRNA production in alleles that cannot undergo paramutation. Alleles that cannot participate in paramutation also produce b1TR-siRNAs, suggesting that b1TR-siRNAs are not sufficient for paramutation in the tissues analyzed. However, when b1TR-siRNAs are produced from a transgene expressing a hairpin RNA, b1 paramutation can be recapitulated. We hypothesize that either the b1TR-siRNAs or the dsRNA template mediates the trans-communication between the alleles that establishes paramutation. In addition, we uncovered a role for mop1 in the biogenesis of a subset of microRNAs (miRNAs) and show that it functions at the level of production of the primary miRNA transcripts.

RNA-Dependent RNA Polymerase Is Required for Enhancer-Mediated Transcriptional Silencing Associated With Paramutation at the Maize p1 Gene

Paramutation is the ability of an endogenous gene or a transgene to heritably silence another closely related allele or gene. At the maize p1 (pericarp color1) gene, paramutation is associated with decreases in transcript levels and reduced pigmentation of the endogenous allele that normally specifies red seed coat (pericarp) and cob pigmentation. Herein we demonstrate that this silencing occurs at the transcriptional level and that a specific enhancer fragment from p1 is sufficient to induce all aspects of paramutation. Further, we demonstrate that a mutation in the mop1 gene (mediator of paramutation1), which encodes a RNA-dependent RNA polymerase, is absolutely required for establishing the silencing associated with p1 paramutation. In contrast to its effects on other paramutation loci, the mop1 mutation does not immediately reactivate a previously silenced allele; several generations in the presence of the mop1 mutation are required. In addition, the mop1 mutation was also able to release tissue-specific silencing of another p1 allele that does not participate in paramutation, but does contain a tandem repeated structure and is likely regulated through epigenetic mechanisms. These results demonstrate that RNA-mediated gene-silencing mechanisms play key roles in p1 paramutation and the spectrum of roles for MOP1 is broadened to include tissue-specific expression patterns.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

Long-distance cis and trans interactions mediate paramutation.

This chapter focuses on the paramutation at two maize loci, b1 and p1. The chapter describes the recent experiments that have identified cis-acting regions required for paramutation at these two loci and compares the similarities and differences in the results. A model for the mechanism of paramutation is presented that incorporates these recent findings and discusses the possible functions for paramutation. Paramutation-like interactions can occur between two homologous transgenes, or a transgene and a homologous endogenous gene. At b1 , paramutation is fully penetrant and the resulting B ’ alleles are extremely stable, independent of how the alleles are maintained (homozygous or heterozygous with B-I or neutral alleles). Similar to the b1 paramutation, the paramutable P1-rr (red pericarp and red cob) allele can change spontaneously to a lower expression state, P1-pr (patterned pericarp and red cob). Models that attempt to explain paramutation must account for various features. Certain enhancer sequences are associated with the sequences required for paramutation in two different genes. The high-expressing alleles can be silenced spontaneously.

Diverse gene-silencing mechanisms with distinct requirements for RNA polymerase subunits in Zea mays.

In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes—mop1-1, Mop2-1, and mop3-1—suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation.

Maize Unstable factor for orange1 Is Required for Maintaining Silencing Associated with Paramutation at the pericarp color1 and booster1 Loci

To understand the molecular mechanisms underlying paramutation, we examined the role of Unstable factor for orange1 (Ufo1) in maintaining paramutation at the maize pericarp color1 (p1) and booster1 (b1) loci. Genetic tests revealed that the Ufo1-1 mutation disrupted silencing associated with paramutation at both p1 and b1. The level of up regulation achieved at b1 was lower than that at p1, suggesting differences in the role Ufo1-1 plays at these loci. We characterized the interaction of Ufo1-1 with two silenced p1 epialleles, P1-rr′ and P1-prTP, that were derived from a common P1-rr ancestor. Both alleles are phenotypically indistinguishable, but differ in their paramutagenic activity; P1-rr′ is paramutagenic to P1-rr, while P1-prTP is non-paramutagenic. Analysis of cytosine methylation revealed striking differences within an enhancer fragment that is required for paramutation; P1-rr′ exhibited increased methylation at symmetric (CG and CHG) and asymmetric (CHH) sites, while P1-prTP was methylated only at symmetric sites. Both silenced alleles had higher levels of dimethylation of lysine 9 on histone 3 (H3K9me2), an epigenetic mark of silent chromatin, in the enhancer region. Both epialleles were reactivated in the Ufo1-1 background; however, reactivation of P1-rr′ was associated with dramatic loss of symmetric and asymmetric cytosine methylation in the enhancer, while methylation of up-regulated P1-prTP was not affected. Interestingly, Ufo1-1–mediated reactivation of both alleles was accompanied with loss of H3K9me2 mark from the enhancer region. Therefore, while earlier studies have shown correlation between H3K9me2 and DNA methylation, our study shows that these two epigenetic marks are uncoupled in the Ufo1-1–reactivated p1 alleles. Furthermore, while CHH methylation at the enhancer region appears to be the major distinguishing mark between paramutagenic and non-paramutagenic p1 alleles, H3K9me2 mark appears to be important for maintaining epigenetic silencing.