Lisa M. Smith

An SNF2 Protein Associated with Nuclear RNA Silencing and the Spread of a Silencing Signal between Cells in Arabidopsis

The silencing phenotype in Arabidopsis thaliana lines with an inverted repeat transgene under the control of a phloem-specific promoter was manifested in regions around veins due to a mobile signal of silencing. Genetic analysis implicates RNA-DEPENDENT RNA POLYMERASE2 (RDR2) and an RNA polymerase IVa subunit gene (NRPD1a) in the signaling mechanism. We also identified an SNF2 domain–containing protein (CLASSY1) that acts together with RDR2 and NRPD1a in the spread of transgene silencing and in the production of endogenous 24-nucleotide short interfering RNAs (siRNAs). Cytochemical analysis indicates that CLASSY1 may act in the nucleus with NRPD1a and RDR2 in the upstream part of RNA silencing pathways that generate a double-stranded RNA substrate for Dicer-like (DCL) nucleases. DCL3 and ARGONAUTE4 act in a downstream part of the pathway, leading to endogenous 24-nucleotide siRNA production, but are not required for intercellular signaling. From genetic analysis, we conclude that another downstream part of the pathway associated with intercellular signaling requires DCL4 and at least one other protein required for 21-nucleotide trans-acting siRNAs. We interpret the effect of polymerase IVa and trans-acting siRNA pathway mutations in terms of a modular property of RNA silencing pathways.

Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata

Transposable elements (TEs) are often the primary determinant of genome size differences among eukaryotes. In plants, the proliferation of TEs is countered through epigenetic silencing mechanisms that prevent mobility. Recent studies using the model plant Arabidopsis thaliana have revealed that methylated TE insertions are often associated with reduced expression of nearby genes, and these insertions may be subject to purifying selection due to this effect. Less is known about the genome-wide patterns of epigenetic silencing of TEs in other plant species. Here, we compare the 24-nt siRNA complement from A. thaliana and a closely related congener with a two- to threefold higher TE copy number, Arabidopsis lyrata. We show that TEs—particularly siRNA-targeted TEs—are associated with reduced gene expression within both species and also with gene expression differences between orthologs. In addition, A. lyrata TEs are targeted by a lower fraction of uniquely matching siRNAs, which are associated with more effective silencing of TE expression. Our results suggest that the efficacy of RNA-directed DNA methylation silencing is lower in A. lyrata, a finding that may shed light on the causes of differential TE proliferation among species.

MicroRNA Gene Evolution in Arabidopsis lyrata and Arabidopsis thaliana

A whole-genome analysis of MIRNA from Arabidopsis thaliana and close relative Arabidopsis lyrata suggests that evolutionarily young MIRNA are diverging in sequence and function more rapidly than are more deeply conserved MIRNA. These and other results shed light on the birth, divergence, and death of MIRNA genes in plants. MicroRNAs (miRNAs) are short regulatory RNAs processed from partially self-complementary foldbacks within longer MIRNA primary transcripts. Several MIRNA families are conserved deeply through land plants, but many are present only in closely related species or are species specific. The finding of numerous evolutionarily young MIRNA, many with low expression and few if any targets, supports a rapid birth-death model for MIRNA evolution. A systematic analysis of MIRNA genes and families in the close relatives, Arabidopsis thaliana and Arabidopsis lyrata, was conducted using both whole-genome comparisons and high-throughput sequencing of small RNAs. Orthologs of 143 A. thaliana MIRNA genes were identified in A. lyrata, with nine having significant sequence or processing changes that likely alter function. In addition, at least 13% of MIRNA genes in each species are unique, despite their relatively recent speciation (∼10 million years ago). Alignment of MIRNA foldbacks to the Arabidopsis genomes revealed evidence for recent origins of 32 families by inverted or direct duplication of mostly protein-coding gene sequences, but less than half of these yield miRNA that are predicted to target transcripts from the originating gene family. miRNA nucleotide divergence between A. lyrata and A. thaliana orthologs was higher for young MIRNA genes, consistent with reduced purifying selection compared with deeply conserved MIRNA genes. Additionally, target sites of younger miRNA were lost more frequently than for deeply conserved families. In summary, our systematic analyses emphasize the dynamic nature of the MIRNA complement of plant genomes.

The Capsella rubella genome and the genomic consequences of rapid mating system evolution

The shift from outcrossing to selfing is common in flowering plants, but the genomic consequences and the speed at which they emerge remain poorly understood. An excellent model for understanding the evolution of self fertilization is provided by Capsella rubella, which became self compatible <200,000 years ago. We report a C. rubella reference genome sequence and compare RNA expression and polymorphism patterns between C. rubella and its outcrossing progenitor Capsella grandiflora. We found a clear shift in the expression of genes associated with flowering phenotypes, similar to that seen in Arabidopsis, in which self fertilization evolved about 1 million years ago. Comparisons of the two Capsella species showed evidence of rapid genome-wide relaxation of purifying selection in C. rubella without a concomitant change in transposable element abundance. Overall we document that the transition to selfing may be typified by parallel shifts in gene expression, along with a measurable reduction of purifying selection.

Reference-guided assembly of four diverse Arabidopsis thaliana genomes

We present whole-genome assemblies of four divergent Arabidopsis thaliana strains that complement the 125-Mb reference genome sequence released a decade ago. Using a newly developed reference-guided approach, we assembled large contigs from 9 to 42 Gb of Illumina short-read data from the Landsberg erecta (Ler-1), C24, Bur-0, and Kro-0 strains, which have been sequenced as part of the 1,001 Genomes Project for this species. Using alignments against the reference sequence, we first reduced the complexity of the de novo assembly and later integrated reads without similarity to the reference sequence. As an example, half of the noncentromeric C24 genome was covered by scaffolds that are longer than 260 kb, with a maximum of 2.2 Mb. Moreover, over 96% of the reference genome was covered by the reference-guided assembly, compared with only 87% with a complete de novo assembly. Comparisons with 2 Mb of dideoxy sequence reveal that the per-base error rate of the reference-guided assemblies was below 1 in 10,000. Our assemblies provide a detailed, genomewide picture of large-scale differences between A. thaliana individuals, most of which are difficult to access with alignment-consensus methods only. We demonstrate their practical relevance in studying the expression differences of polymorphic genes and show how the analysis of sRNA sequencing data can lead to erroneous conclusions if aligned against the reference genome alone. Genome assemblies, raw reads, and further information are accessible through http://1001genomes.org/projects/assemblies.html.

Mutational analysis of a heterogeneous nuclear ribonucleoprotein A2 response element for RNA trafficking.

Cytoplasmic transport and localization of mRNA has been reported for a range of oocytes and somatic cells. The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) is a 21-nucleotide segment of the myelin basic protein mRNA that is necessary and sufficient for cytoplasmic transport of this message in oligodendrocytes. The predominant A2RE-binding protein in rat brain has previously been identified as hnRNP A2. Here we report that an 11-nucleotide subsegment of the A2RE (A2RE11) was as effective as the full-length A2RE in binding hnRNP A2 and mediating transport of heterologous RNA in oligodendrocytes. Point mutations of the A2RE11 that eliminated binding to hnRNP A2 also markedly reduced the ability of these oligoribonucleotides to support RNA transport. Oligodendrocytes treated with antisense oligonucleotides directed against the translation start site of hnRNP A2 had reduced levels of this protein and disrupted transport of microinjected myelin basic protein RNA. Several A2RE-like sequences from localized neuronal RNAs also bound hnRNP A2 and promoted RNA transport in oligodendrocytes. These data demonstrate the specificity of A2RE recognition by hnRNP A2, provide direct evidence for the involvement of hnRNP A2 in cytoplasmic RNA transport, and suggest that this protein may interact with a wide variety of localized messages that possess A2RE-like sequences.

JMJ14, a JmjC domain protein, is required for RNA silencing and cell-to-cell movement of an RNA silencing signal in Arabidopsis

JMJ14 is a histone H3 Lys4 (H3K4) trimethyl demethylase that affects mobile RNA silencing in an Arabidopsis transgene system. It also influences CHH DNA methylation, abundance of endogenous transposon transcripts, and flowering time. JMJ14 acts at a point in RNA silencing pathways that is downstream from RNA-dependent RNA polymerase 2 (RDR2) and Argonaute 4 (AGO4). Our results illustrate a link between RNA silencing and demethylation of histone H3 trimethylysine. We propose that JMJ14 acts downstream from the Argonaute effector complex to demethylate histone H3K4 at the target of RNA silencing.

Transposon variants and their effects on gene expression in Arabidopsis.

Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA–mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs, and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24-nt-long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA–targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants.

Putative Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis

RNA silencing in plants and some animals has a non–cell-autonomous effect due to an RNA signal that moves between cells or organs. To identify unique factors involved in this process, we analyzed a group of Arabidopsis mutants with defective spread of RNA silencing from a transgene expressed specifically in the phloem. These mutants accumulated reduced amounts of small interfering (si)RNA from the transgene locus and from endogenous loci TAS1, TAS2, and an inverted repeat locus IR71. The defect in TAS1 and TAS2 siRNA biogenesis is in the processing of a long siRNA precursor. We mapped the mutations to a gene encoding the Arabidopsis homolog of a protein, TEX1, which is involved in intracellular transport of RNA in animals. TEX1 is a component of the THO/TREX complex, and we show that the Arabidopsis TEX1 interacts with other predicted components of a THO/TREX complex. Correspondingly, we found at least two other components of the Arabidopsis THO core complex that are involved in RNA silencing. To reconcile the effect of these mutations on transgene and endogenous gene siRNA, we propose a mechanism in which THO/TREX processes or transports a long RNA molecule so that it can be a template for secondary siRNA production.

Layout impact on the performance of a locally strained PMOSFET

We present a study on the layout dependence of a SiGe S/D PMOSFET technology. While 65% increase in drive current is obtained for 45nm gate length transistors with large active areas, measurements and simulations show that this improvement may be seriously degraded when transistor dimensions, such as the source-drain length (L/sub s/d/) and the device width are further scaled. TDDB and NBTI measurements show that the oxide reliability is not degraded for this technology.