75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

319 Biochemical Characterization Of Arabidopsis thaliana PPR proteins
Magalie UYTTEWAAL 1 , Nadege Arnal 1 , Jean Bigeard 1 , Anna Debicka 1 , Martine Quadrado 1 , Jean-Pierre Renou 2 ,
Hakim Mireau 1
1
INRA - Station de Genetique - Route de Saint-Cyr F78026 Versailles cedex France, 2 INRA - URGV - 2, rue
Gaston Cremieux 91057 Evry cedex
The expression of plastid and mitochondrial genomes is dependent on a large number of nucleus-encoded factors that were
shown to act predominantly at a posttranscriptional level. Genetic studies, carried in maize and Arabidopsis and positional
cloning of several cytoplasmic male sterility restorer genes (in rice, petunia and radish) revealed a predominant involvement of
PPR proteins in plant organellar RNA expression. They have been genetically linked to various processes like RNA stabilization,
processing, translation and editing. PPR proteins have been identified in various eukaryotes but this protein family has literally
exploded in higher plants, with over 450 members in Arabidopsis and rice. It was hypothesized that such expansion could
be correlated with the apparition a specific function in plant posttranscriptional processes like RNA editing. This large gene
family is characterised by the presence of tandem arrays of a 35-aminoacid motif. Because of its structural similarity to the
TPR (tetratricopeptide repeat) motif known to form interacting domains, the PPR motif is proposed to constitute highly specific
RNA binding domains that could recruit catalytic protein on a specific RNA sites. It makes no doubt that PPRs are key factors
of organellar gene expression but their molecular functions remain to be elucidated. To unravel the molecular roles of some
PPR proteins we decided to identify their interacting RNA and protein partners that may display known functions.
As a first step to select appropriate proteins, a biochemical screen looking for PPR proteins engaged in high molecular
protein complexes has been realised. About ten Arabidopsis PPR proteins were fused to short epitope tags (3HA, FLAG) and
to various versions of TAP tags allowing protein complex isolation. Analysis of stromal and mitochondrial extracts by size
exclusion chromatography allowed us to identify 5 PPR proteins involved in multi-protein complexes. Three PPRs have been
purified by tandem affinity purification and their putative partners needs to be identified by mass spectrometry. To identify
putative RNA associated to these proteins, we envisage to use a microarray-based strategy recently developed on the maize
CRP1 protein (method called RIP-Chip standing for RNA immunoprecipitation and chip hybridization). In our case, the
immunoprecipitation assays will be carried on the tagged selected PPRs and the coimmunoprecipitated RNA will be hybridized
on microarray slides covering the Arabidopsis mitochondrial and chloroplast genomes. Obtained results will be presented.
320 Genome-wide High Resolution Mapping and Functional Analysis of DNA Methylation in
Arabidopsis thaliana
Xiaoyu Zhang 1 , Junshi Yazaki 2 , Ambika Sundaresan 2 , Simon Chan 1 , Huaming Chen 2 , Lianna Johnson 1 , Paul Shinn 2 ,
Hiroshi Shiba 2 , Shawn Cokus 1 , Matteo Pellegrini 1 , Steve Jacobsen 1, 3 , Joseph Ecker 2
1
Dept. MCDB, UCLA, 2 The Salk Institute for Biological Studies, 3 The Howard Hughes Medical Institute,
UCLA
Cytosine DNA methylation is a conserved epigenetic silencing mechanism involved in many important biological
processes, including defense against transposons and other invading DNA, maintenance of chromosomal structure
and genome stability, establishment of parental imprinting, and regulation of gene expression. Previous studies have
largely focused on the establishment and maintenance of DNA methylation as well as its role in controlling individual
genes. However, a genome-wide analysis of DNA methylation or its function in regulating gene expression has not been
performed for any organism, thus greatly limiting our understanding of this important mechanism. Here we describe
the first such analysis using the model plant Arabidopsis. Methylated and unmethylated DNA were separated by two
biochemical methods and hybridized to whole-genome tiling microarrays, which allowed the genome-wide identification
of methylated regions with high resolution. Methylated DNA comprises ~20% of the Arabidopsis genome and is highly
enriched in heterochromatin. Methylation was also found in over 1/3 of all Arabidopsis genes and its distribution is
severely biased towards the 3’ end, whereas promoters are hypomethylated. Furthermore, methylated and unmethylated
genes have significantly different expression levels and tissue-specificity. Expression profiles were determined using
the same microarray platform for mutants severely impaired in DNA methylation. Drastic activation of transposons was
observed, as well as large-scale changes in gene expression, antisense transcription and intergenic non-coding RNA
accumulation.