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

Abstract

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

“…Previous studies have shown that b1 repeat transcription is not altered in a mutation of mop2, which encodes the second largest subunit of a Pol IV/Pol V-related complex (27). This suggests that Pol II might be the major polymerase contributing to b1 repeat transcription.…”

“…Studies in Tetrahymena suggest RDRs can also interact directly with Dicer proteins (41) and that this interaction is required for siRNA biogenesis. Our observations that mop1 is required to maintain the silent state (5) but that b1TR-siRNAs are not required to maintain silencing of B′ (27) suggest that mop1 may have a function in maintaining B′ silencing beyond generating b1TR-siRNAs.…”

“…Recent studies with mop2, which encodes the second largest subunit of Pol IV/PolV, demonstrated that the b1TR sequences generate siRNAs and that these are reduced in a mop2 mutant (27). To test if b1TR-siRNAs are also reduced in mop1, we examined deep sequencing data from small RNA libraries and performed Northern blots.…”

“…Northern blots revealed that both the active (B-I) and silenced (B′) alleles involved in paramutation (B-I and B′) and an allele (b) that does not participate in paramutation produce siRNAs, indicating that the b1TR-siRNAs are not sufficient for silencing in the tissues examined. Other experiments with a mop2 mutant, in which the b1TR-siRNAs are dramatically reduced but B′ silencing is not relieved, indicate that siRNAs are not required to maintain silencing (27). One possible explanation is that the b1TR-siRNAs are mediating the establishment of paramutation, which occurs very early in development, just after fertilization or in early embryogenesis (30).…”

“…Bidirectional transcription potentially generates dsRNA, the trigger molecule in a number of transcriptional and posttranscriptional gene regulation mechanisms that involve the processing of dsRNA into different classes of regulatory small RNAs (25,26). Recent experiments have shown that siRNAs are produced from the b1TR sequences in B′ (27).…”