Molecular Genetic Analysis of Non-Catalytic Pol IV and V Subunits

Among eukaryotes, plants have the distinction of encoding multisubunit RNA polymerases used exclusively for RNA directed DNA Methylation: RdDM) in addition to Pol I, II, and III. In Arabidopsis thaliana, Pol IV is required for the biogenesis of 24nt siRNAs whereas Pol V transcription is needed for cytosine methylation of the DNA sequences corresponding to these siRNAs. The ancestry of Pol IV and V can be traced back to Pol II, and Pol II, IV and V still utilize multiple non-catalytic subunits encoded by the same genes. Genetic analysis of non-catalytic subunits that are highly similar reveals that these subunits are not necessarily redundant. For instance, NRPB9b but not its 97% similar paralog, NRPB9a is required for RdDM. Likewise, Pol IV and Pol V-specific 7th largest subunits are very similar yet have different involvements in RdDM. In some of the non-catalytic subunit mutants of Pol IV, 24nt siRNA accumulation is not dramatically reduced, yet RNA silencing is disrupted. This contrasts with Pol IV catalytic subunit mutants in which siRNA biogenesis and RdDM are coordinately disrupted. Taken together, these results suggest that Pol IV might possess functions in RdDM that are in addition to, and separable from siRNA biogenesis. Differences in Pol V subunit composition based on the use of alternative non-catalytic subunit variants might also have functional consequences for RdDM. The evidence suggests that alternative non-catalytic subunits in Pol IV and V are likely to influence interactions with other proteins needed for RdDM

Point Mutations in Centromeric Histone Induce Post-zygotic Incompatibility and Uniparental Inheritance.

The centromeric histone 3 variant (CENH3, aka CENP-A) is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type

Functional consequences of subunit diversity in RNA polymerases II and V

SummaryMultisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V

Catastrophic chromosomal restructuring during genome elimination in plants.

Genome instability is associated with mitotic errors and cancer. This phenomenon can lead to deleterious rearrangements, but also genetic novelty, and many questions regarding its genesis, fate and evolutionary role remain unanswered. Here, we describe extreme chromosomal restructuring during genome elimination, a process resulting from hybridization of Arabidopsis plants expressing different centromere histones H3. Shattered chromosomes are formed from the genome of the haploid inducer, consistent with genomic catastrophes affecting a single, laggard chromosome compartmentalized within a micronucleus. Analysis of breakpoint junctions implicates breaks followed by repair through non-homologous end joining (NHEJ) or stalled fork repair. Furthermore, mutation of required NHEJ factor DNA Ligase 4 results in enhanced haploid recovery. Lastly, heritability and stability of a rearranged chromosome suggest a potential for enduring genomic novelty. These findings provide a tractable, natural system towards investigating the causes and mechanisms of complex genomic rearrangements similar to those associated with several human disorders

Naturallyoccurring differences in cenh3 affect chromosome segregation in zygotic mitosis of hybrids

The point of attachment of spindle microtubules to metaphase chromosomes is known as the centromere. Plant and animal centromeres are epigenetically specified by a centromere-specific variant of Histone H3, CENH3 (a.k.a. CENP-A). Unlike canonical histones that are invariant, CENH3 proteins are accumulating substitutions at an accelerated rate. This diversification of CENH3 is a conundrum since its role as the key determinant of centromere identity remains a constant across species. Here, we ask whether naturally occurring divergence in CENH3 has functional consequences. We performed functional complementation assays on cenh3-1, a null mutation in Arabidopsis thaliana, using untagged CENH3s from increasingly distant relatives. Contrary to previous results using GFP-tagged CENH3, we find that the essential functions of CENH3 are conserved across a broad evolutionary landscape. CENH3 from a species as distant as the monocot Zea mays can functionally replace A. thaliana CENH3. Plants expressing variant CENH3s that are fertile when selfed show dramatic segregation errors when crossed to a wild-type individual. The progeny of this cross include hybrid diploids, aneuploids with novel genetic rearrangements and haploids that inherit only the genome of the wild-type parent. Importantly, it is always chromosomes from the plant expressing the divergent CENH3 that missegregate. Using chimeras, we show that it is divergence in the fast-evolving N-terminal tail of CENH3 that is causing segregation errors and genome elimination. Furthermore, we analyzed N-terminal tail sequences from plant CENH3s and discovered a modular pattern of sequence conservation. From this we hypothesize that while the essential functions of CENH3 are largely conserved, the N-terminal tail is evolving to adapt to lineage-specific centromeric constraints. Our results demonstrate that this lineage-specific evolution of CENH3 causes inviability and sterility of progeny in crosses, at the same time producing karyotypic variation. Thus, CENH3 evolution can contribute to postzygotic reproductive barriers

An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs.

BACKGROUND: Small RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus. RESULTS: Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases. CONCLUSIONS: Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2

Non-Motor and Motor Features in LRRK2 Transgenic Mice

10.1371/journal.pone.0070249PLoS ONE87-POLN

Improvement in the Reproducibility and Accuracy of DNA Microarray Quantification by Optimizing Hybridization Conditions

BACKGROUND: DNA microarrays, which have been increasingly used to monitor mRNA transcripts at a global level, can provide detailed insight into cellular processes involved in response to drugs and toxins. This is leading to new understandings of signaling networks that operate in the cell, and the molecular basis of diseases. Custom printed oligonucleotide arrays have proven to be an effective way to facilitate the applications of DNA microarray technology. A successful microarray experiment, however, involves many steps: well-designed oligonucleotide probes, printing, RNA extraction and labeling, hybridization, and imaging. Optimization is essential to generate reliable microarray data. RESULTS: Hybridization and washing steps are crucial for a successful microarray experiment. By following the hybridization and washing conditions recommended by an oligonucleotide provider, it was found that the expression ratios were compressed greater than expected and data analysis revealed a high degree of non-specific binding. A series of experiments was conducted using rat mixed tissue RNA reference material (MTRRM) and other RNA samples to optimize the hybridization and washing conditions. The optimized hybridization and washing conditions greatly reduced the non-specific binding and improved the accuracy of spot intensity measurements. CONCLUSION: The results from the optimized hybridization and washing conditions greatly improved the reproducibility and accuracy of expression ratios. These experiments also suggested the importance of probe designs using better bioinformatics approaches and the need for common reference RNA samples for platform performance evaluation in order to fulfill the potential of DNA microarray technology