Rebecca A Mosher

Argonaute (Ago) proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Ago proteins, but little is known about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes. Here we investigated the evolutionary dynamics of an Ago-binding platform in NRPE1, the largest subunit of RNA polymerase V. We compared NRPE1 sequences from > 50 species, including dense sampling of two plant lineages. This study demonstrates that the Ago-binding platform of NRPE1 retains Ago hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is the result of relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million yr. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism. The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.

PMID: 19942857;PMCID: PMC2797062;Abstract:

formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5′-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1Δ cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. © 2009 European Molecular Biology Organization.

PMID: 20129810;Abstract:

To understand how DNA sequence is translated to phenotype we must understand the epigenetic features that regulate gene expression. Recent research illuminates the complex interactions between DNA methylation, small RNAs, silencing of transposable elements, and genomic imprinting in the Arabidopsis (Arabidopsis thaliana) seed. These studies suggest that transposable elements reactivated in specific cells of the gametophyte and seed might enhance silencing of transposable elements in the germline and embryo. By sacrificing genomic integrity these cells might make an epigenetic rather than genetic contribution to the progeny. This research could have implications for interspecies hybridization, the evolution of genomic imprinting, and epigenetic communication from plant to progeny. © 2010 Elsevier Ltd. All rights reserved.

PMID: 20074090;Abstract:

Small RNAs recently emerged as ubiquitous regulators of gene expression. However, the most abundant class of small RNAs in flowering plants is poorly understood. Known as Pol IV-dependent (p4-)siRNAs, these small RNAs are associated with transcriptional gene silencing, transposable elements and heterochromatin formation. Recent research demonstrates that they are initially expressed in the maternal gametophyte and uniparentally expressed from maternal chromosomes in developing endosperm. This unique expression pattern links p4-siRNAs to double fertilization, parental genome interactions and imprinted gene expression. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

PMID: 18287047;PMCID: PMC2268599;Abstract:

DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context. © 2008 by The National Academy of Sciences of the USA.