ORCID Profile
0000-0001-8204-7459
Current Organisations
Indiana University Bloomington
,
Howard Hughes Medical Institute - Indiana University
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Publisher: Elsevier BV
Date: 04-2014
Publisher: Oxford University Press (OUP)
Date: 05-2007
Abstract: 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.
Publisher: Wiley
Date: 2007
DOI: 10.1002/BMB.99
Abstract: Studies of protein function increasingly use multifaceted approaches that span disciplines including recombinant DNA technology, cell biology, and analytical biochemistry. These studies rely on sophisticated equipment and methodologies including confocal fluorescence microscopy, mass spectrometry, and X-ray crystallography that are beyond the scope of traditional laboratory courses. To equip the advanced undergraduate and beginning graduate students with an enabling base of knowledge and initial experience with advanced protein research methodologies, a laboratory course entitled Plant Cells and Proteins was developed in a partnership between Washington University and the Donald Danforth Plant Science Center in St. Louis. In this one semester course, 10-12 students obtain hands-on experience with plant tissue culture, gene transformation, subcellular localization of fluorescent recombinant proteins using confocal microscopy, purification of affinity-tagged recombinant proteins, isolation of total protein extracts, enzymatic assays, one- and two-dimensional gel electrophoresis, MALDI-TOF and ESI-Q-TOF mass spectrometry, protein crystallization, and X-ray diffraction. The course is taught as a series of modules, each led by an expert researcher. Students are evaluated based on a series of graded written reports and tests of their mastery of key concepts, interpretations, and the limitations of the experimental methods.
Publisher: Elsevier BV
Date: 07-2006
DOI: 10.1016/J.CELL.2006.05.032
Abstract: ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b the small nuclear RNA (snRNA) binding protein SmD3 and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.
Publisher: Cold Spring Harbor Laboratory
Date: 08-2012
Abstract: Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%–60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.
Location: United States of America
Location: United States of America
Location: United States of America
Location: United States of America
No related grants have been discovered for Craig Pikaard.