Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Data generated from this project are disseminated through various websites. The Arabidopsis Small RNA Project (ASRP) database (http://asrp.cgrb.oregonstate.edu/db/) is operated in the public domains for anyone seeking genomic-wide small RNA data. All high-throughput sequencing data generated during the duration of this project are available from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). Computational analysis programs created to process and analyzed high-throughput sequencing data are available to the public. The Cache-ASsisted Hash Search - XOR Logic (CASHX) programs were developed to efficiently map and quantify short sequencing reads. The programs run as an Illumina data processing pipeline. All papers from data produced on this grant are available on the Carrington lab website (http://jcclab.science.oregonstate.edu/). Three graduate students have been involved with various aspects of this grant. Taiowa Montgomery completed his PhD and is currently a postdoctoral researchers. Noah Fahlgren and Joshua Cuperus are currently graduate students in the Molecular and Cellular Biology Program. The project is involved with training of undergraduate students by enabling a front-line participation in research. The long tradition in the Carrington lab of exposing undergraduate students to research in the wet laboratory has expanded to include exposing undergraduates to computational research and development. Undergraduate students are an integral part the Arabidopsis Small RNA Project (ASRP) database and associated website. Two students, Tyler Backman, and Zachary Miller, have been involved with computational biology and programming. Many undergraduate have been involved with wetbench work resulting publications. Tyler Backman was the first author contributor to the ASRP database paper (NAR (2008) 36, D982-5). PARTICIPANTS: Carrington, James - P.I. Howell, Miya - Postdoctoral Researcher, cis- and trans- regulatory factors in the ta-siRNA pathway. Gilbert, Sunny - Postdoctoral Researcher, cis- and trans- regulatory factors in the ta-siRNA pathway. Montgomery, Taiowa - Graduate Student, cis- and trans- regulatory factors in the ta-siRNA pathway. Fahlgren, Noah - Graduate Student, computational analysis of ta-siRNAs. Cuperus, Joshua - Graduate Student, genetic analysis of ta-siRNA pathways. Sullivan, Christopher - Computational Scientist, , computational program development, development and maintenance of computer hardware. Backman, Tyler - Undergraduate computer programmer. Hansen, Jesse - Undergraduate researcher, cis- and trans- regulatory factors in the ta-siRNA pathway. Alexander, Amanda - Undergraduate researcher, cis- and trans- regulatory factors in the ta-siRNA pathway. Miller, Zachary - Undergraduate computer programmer, Timmermans, Marja, Cold Spring Harbor Laboratory, Collaborator. TARGET AUDIENCES: Research and development personnel at university and industrial settings with an interest in plant biotechnology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project seeks to understand the mechanisms of trans-acting siRNA biogenesis and function using biochemical and genetic approaches. Trans-acting siRNA (tasiRNA), which function as posttranscriptional regulators in plants, form through a refined RNA interference mechanism. miRNA-guided cleavage triggers entry of the tasiRNA precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 (RDR6) pathway, and sets the register for phased, 21-nt ta-siRNA formation by DICER-LIKE4 (DCL4). Aim 1, focusing on development of synthetic tasiRNAs to study biogenesis and effector mechanisms of both TAS1 and TAS3 tasiRNAs, was fully achieved during year 1. Aim 2 focuses on factors that facilitate biogenesis of tasiRNAs. We specifically focused on the role of AGO7, which is uniquely required by TAS3 (not TAS1). We show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5' adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5' nucleotide discrimination function for some, although not all, ARGONAUTEs. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation-cleavage of primary transcripts. miR173 was unique from other miRNAs in its ability to initiate TAS1c-based syn-tasiRNA formation. A single miR173 target site was sufficient to route non-TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with AGO1 during TAS1 and TAS2 tasiRNA formation, and provide data indicating that the miR173-AGO1 complex possesses unique functionality that many other miRNA-AGO1 complexes lack. We have made significant progress towards finishing aim 2. Aim 3 focuses on genetic screens for mutants that lose TAS1 and TAS3 specific functions. TAS1c and TAS3a syn-tasiRNA transgenic Arabidopsis seeds were mutagenized using 0.2% ethyl methanosulfate (EMS) solution. Plants mutant for one or more component of ta-siRNA necessary for syn-tasiRNA biogenesis or activity were selected for based on loss of photobleaching. Mutants have been categorized into six classes based on their small RNA profile, dominance and phenotype: Class 1, normal small RNA profile; Class 2, loss of all ta-siRNA; Class 3, specific loss of syn-tasiRNA; Class 4, loss of all small RNA; Class 5, loss of initiator microRNAs; and Class 6, specific loss of TAS3 ta-siRNAs. Many mutants have been recovered, including 52B2, which did not map to any known protein coding genes. High-throughput Illumina sequencing was used to identify the causative mutation in the 52B2 mutant, revealing a point mutation in the foldback sequence of miR390a. The sequencing-based strategy saved 8-10 months over traditional map-based cloning strategies. We continue to screen novel mutants using high-throughput sequencing.
Publications
- Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL, Chapman EJ, Fahlgren N, Allen E, Carrington JC. (2008) Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA Formation. Cell 133, 128-141.
- Backman TW, Sullivan CM, Cumbie JS, Miller ZA, Chapman EJ, Fahlgren N, Givan SA, Carrington JC, Kasschau KD. (2008) Update of ASRP: the Arabidopsis Small RNA Project database. Nucleic Acids Res 36, D982-5.
- Montgomery, T.A., Yoo, S.J., Fahlgren, N., Gilbert, S.D., Howell, M.D., Sullivan, C., Alexander, A., Nguyen, G., Allen, E., Ahn, J.H., and Carrington, J.C. 2008. miR173-AGO1 complexes initiate phased siRNA formation in plants. Proc. Natl. Acad. Sci. USA 105, in press.
- Montgomery TA, Carrington JC. (2008) Splicing and dicing with a SERRATEd edge. Proc Natl Acad Sci U S A 105, 8489-90.
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Progress 09/01/06 to 08/31/07
Outputs
This project seeks to understand the mechanisms of trans-acting siRNA biogenesis and function using biochemical and genetic approaches. Trans-acting siRNA (tasiRNA), which function as posttranscriptional regulators in plants, form through a refined RNA interference mechanism. miRNA-guided cleavage triggers entry of the tasiRNA precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 (RDR6) pathway, and sets the register for phased, 21-nt ta-siRNA formation by DICER-LIKE4 (DCL4). Aim 1 focuses on development of synthetic tasiRNAs to study biogenesis and effector mechanisms of both TAS1 and TAS3 tasiRNAs, which represent the two major types of tasiRNAs in plants. siRNAs directed against Phytoene desaturase mRNA were successfully produced from modified TAS1 and TAS3 constructs, yielding a photobleached phenotype that was dependent on RDR6, DCL4, and in the case of syn-TAS3, AGO7. Using the syn-tasiRNA system, TAS1 and TAS3 were shown to require miR173 and miR390,
respectively for initiation cleavage. This aim was fully achieved. Aim 2 focuses on factors that facilitate biogenesis of TAS3 tasiRNAs. We specifically focused on the role of AGO7, which is uniquely required by TAS3 (not TAS1). miR390-ARGONAUTE7 (AGO7) complexes were shown to function in distinct modes at two target sites in TAS3a transcripts to form ta-siRNAs that regulate mRNAs encoding several AUXIN RESPONSE FACTORs (ARFs). Interaction assays revealed that AGO7 possesses high specificity for miR390. The miR390-AGO7 complex functions in a slicer mode for processing near the 3' end of TAS3a transcripts, and in a non-slicing mode near the 5' end. The requirement for AGO7 slicer function, but not the non-slicer function, could be bypassed through introduction of alternate miRNA target sites. We proposed that miR390-AGO7 functions to process TAS3a transcripts, and to recruit RDR6 through a stable association with the non-cleaved target site. These were very surprising results, as the
basis for selectivity of small RNAs for specific AGO proteins is entirely unclear. We have made significant progress towards finishing aim 2. Aim 3 focuses on genetic screens for mutants that lose TAS1 and TAS3 specific functions. TAS1c and TAS3a syn-tasiRNA transgenic Arabidopsis seeds were mutagenized using 0.2% ethyl methanosulfate (EMS) solution. Plants mutant for one or more component of ta-siRNA necessary for syn-tasiRNA biogenesis or activity were selected for based on loss of photobleaching. Mutant screening involves multiple steps: small RNA profiling, restorative cross to wild-type (Col-0), and phenotype analyses, as well as selective complementation crosses. Mutants have been categorized into six classes based on their small RNA profile, dominance and phenotype: Class 1, normal small RNA profile; Class 2, loss of all ta-siRNA; Class 3, specific loss of syn-tasiRNA; Class 4, loss of all small RNA; Class 5, loss of initiator microRNAs; and Class 6, specific loss of TAS3
ta-siRNAs. We are at an early stage in characterizing these mutants, but it is very clear that we will obtain over 100 mutants in each screen.
Impacts
This work will have significant impact to understanding the factors will lead to better, more useful methods to apply silencing-based methods in useful agricultural settings. Plants contain a series of gene regulation mechanism based on RNA, which 21-24-nucleotide RNAs serve as guides to negatively regulate target genes, viruses and other elements. This project focuses on mechanistic studies to understand a recently discovered form of RNA silencing, based on ta-siRNAs.
Publications
- Fahlgren N, Montgomery TA, Howell MD, Allen E, Dvorak SK, Alexander AL, Carrington JC. (2006) Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr Biol 16, 939-44.
- Howell MD, Fahlgren N, Chapman EJ, Cumbie JS, Sullivan CM, Givan SA, Kasschau KD, Carrington JC. (2007) Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNA- and tasiRNA-directed targeting. Plant Cell 19, 926-942.
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