Deciphering the 24 nt siRNA Biogenesis and Chromatin Modification Network in Plants
EMSL Project ID
39905
Abstract
Small RNAs, including short interfering RNAs (siRNAs) and micro RNAs (miRNAs) play essential roles in development, nuclear organization of the genome, transposon taming, and defense against viral and bacterial attack. Plants have an extensive repertoire of proteins involved in small RNA biogenesis and signaling. These include two plant-specific DNA-dependent RNA polymerases, abbreviated as Pol IV and Pol V, six RNA-dependent RNA polymerases (RDRs) that generate the double-stranded precursors of small RNAs, four dicer endonucleases (DCLs) that cut the double-stranded RNA precursors into small RNAs, and ten Argonaute family proteins (AGOs) that use the small RNAs as guides to seek out complementary sequences. My laboratory is focused on the network of proteins involved in RNA-directed DNA methylation and silencing of transposons, DNA viruses and endogenous repeats. Pol IV is a key activity of this network, generating transcripts that are made double stranded by RDR2, diced by DCL3 into 24 nt siRNAs and loaded into AGO4. Independent of siRNA biogenesis, Pol V generates noncoding transcripts to which siRNA-AGO4 complexes bind. In subsequent steps that are not understood, the de novo DNA methyltransferase, DRM2 and histone modifying activities are recruited by AGO4-siRNA complexes to target loci, generating highly condensed heterochromatin that is refractive to transcription by conventional polymerases, such as Pol II or Pol III.
We are using a combined biochemical, genetic, and genomic approach to decipher the siRNA-directed DNA methylation network. In collaboration with Ljiljana Pasa-Tolic's talented group at EMSL, we used LC-MS/MS to determine the complete subunit compositions of affinity-purified Pol IV and Pol V, revealing them to be specialized forms of Pol II, the enzyme responsible for mRNA synthesis [1]. The mass spec analyses also indicated that Pol IV and RDR2 interact, which we have confirmed in vivo using reciprocal co-immunoprecipitation assays (manuscript in revision). We hypothesize that mechanistic coupling of DNA-dependent RNA polymerase IV and RNA-dependent RNA polymerase 2 allows double-stranded RNA to be efficiently generated from a DNA template.
In continued collaboration with Dr. Pasa-Tolic and her team, we now propose a new and more ambitious goal, which is to tackle the analysis of the entire network as we know it: RDR2, DCL3, AGO4, DRD1, DMS3, CLSY1, KTF1 and the unknown proteins that associate with them. These studies will dovetail with work in my laboratory involving purification of complexes containing these proteins using column chromatography (by FPLC), yeast two-hybrid screens for interacting proteins, high-throughput sequencing analyses of protein-DNA interactions involving the likely DNA binding proteins of the network (Pol IV, Pol V, CLSY1, DRD1, DMS3, DRM2), cell biological assays of protein localization and co-localization, and functional analyses of newly identified network proteins. Through this multi-pronged approach, our goal is to elucidate the complexity of the 24 nt siRNA biogenesis and chromatin modification network and gain insight into the cross-talk and coordination of protein complexes comprising the network. EMSL's involvement is crucial, as we lack the expertise or instrumentation for these analyses otherwise. Overall, the project fits with EMSL's call for proposals in the realm of Biological Interactions and Dynamics, specifically for the analysis and reconstruction of intercellular and intracellular networks.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2010-10-01
End Date
2013-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Haag JR, and CS Pikaard. 2011. "Multisubunit RNA Polymerases IV and V: Purveyors of Non-Coding RNA for Plant Gene Silencing ." Nature Reviews. Molecular Cell Biology 12:483-492. doi:10.1038/nrm3152
Haag JR, TS Ream, M Marasco, CD Nicora, AD Norbeck, L Pasa-Tolic, and CS Pikaard. 2012. "In vitro transcription activities of Pol IV, Pol V and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing." Molecular Cell 48(5):811-8. doi:10.1016/j.molcel.2012.09.027
He XJ, YF Hsu, O Pontes, J Zhu, J Lu, RA Bressan, CS Pikaard, C Wang, and JK Zhu. 2009. "NRPD4, a Protein Related to the RPB4 Subunit of RNA Polymerase II, is a Component of RNA Polymerases IV and V and is Required for RNA-directed DNA methylation ." Genes & Development 23:318-330. doi:10.1101/gad.1765209
Jeremy R. Haag, Thomas S. Ream, Michelle Marasco, Carrie D. Nicora, Angela D. Norbeck, Ljiljana Pasa-Tolic and Craig S. Pikaard. In vitro transcription activities of Pol IV, Pol V and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing. (submitted)
Pikaard, Craig S., Jeremy R. Haag, Olga M.F. Pontes, Todd Blevins and Ross Cocklin (2013). A transcription fork model for Pol IV and Pol V-dependent RNA-directed DNA methylation. Cold Spring Harb Symp Quant Biol (in press).
Tan EH, T Blevins, TS Ream, and CS Pikaard. 2012. "Functional Consequences of Subunit Diversity in RNA Polymerases II and V." Cell Reports 1(3):208-214. doi:10.1016/j.celrep.2012.01.004
Thomas Ream, Jeremy Haag and Craig S. Pikaard (2013). Plant multisubunit RNA polymerases IV and V. In: Nucleic Acid Polymerases, edited by Katsuhiko Murakami and Michael Trakselis. Springer (in press).
Tucker, Sarah L., Joshua Reece, Thomas S. Ream, and Craig S. Pikaard (2010). Evolutionary history of plant multisubunit RNA Polymerases IV and V: subunit origins via genome-wide and segmental gene duplications, retrotransposition and lineage-specific sub-functionalization. Cold Spring Harb Symp Quant Biol. 75:285-97.
Wierzbicki AT, R Cocklin, A Mayampurath, R Lister, MJ Rowley, BD Gregory, JR Ecker, H Tang, and CS Pikaard. 2012. "Spatial and Functional Relationships Among Pol V-Associated loci, Pol IV-Dependent siRNAs, and Cytosine Methylation in the Arabidopsis Epigenome." Genes & Development 26(16):1825-1836. doi:10.1101/gad.197772.112