Analysis of Argonaute 4-Associated Long Non-Coding RNA in Arabidopsis thaliana Sheds Novel Insights into Gene Regulation through RNA-Directed DNA Methylation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material and Growth Conditions
2.2. RNA Extraction and cDNA Synthesis
2.3. Expression Analysis by Semi-Quantitative RT-PCR and RT-qPCR
2.4. Sequence-Specific RT-PCR
2.5. Nuclear RNA-Immunoprecipitation
2.6. Chromatin-Immunoprecipitation Assay
2.7. 5’ End Labeling and SDS-PAGE
2.8. Template-Switch cDNA Library Preparation and Expression Analysis of AGO4- and AGO1-Associated RNA by PCR
2.9. Deep Sequencing
2.10. Bioinformatics
2.11. Western Blot Analysis
2.12. Bisulphite Sequencing
2.13. 5’ Rapid Amplification of cDNA Ends
2.14. Infection of Arabidopsis Plants with Fusarium Oxysporum
2.15. Data Access
3. Results
3.1. Transgenic FLAG:AGO4 Functions Like Endogenous AGO4
3.2. Anti-FLAG:AGO4 RNA Immunoprecipitation Gives Highly Enriched AGO4-Specific RNA Species
3.3. Establishment of an AGO4-Associated Non-Coding RNA Landscape by RNA-IP Seq
3.4. FLAG:AGO4 RNA-IP Seq Data Show Enrichment in RNA Reads from Noncoding Regions of Genes
3.5. AGO4-Associated lncRNAs Overlap with AGO4 and POL V ChIP-Seq Data, TEs and AGO4-IP sRNAs
3.6. AGO4-Associated lncRNAs Show Partial Dependence on POL V for Transcription
3.7. nrpe1 Plants Show CHH Hypomethylation in Genomic Loci that Generate AGO4-Associated and POL V Dependent lncRNAs
3.8. The FLAG:AGO4 RNA-IP Seq Data Reveal Novel RdDM Target Genes
3.9. RdDM Is Involved in the Activation of Stress-Responsive Genes
3.10. RdDM/AGO4-Activated Stress-Response Genes Are Induced upon Fusarium Oxysporum Infection
3.11. Repression or Activation of Gene Expression by RdDM May Depend on the Location of the Target Site in the Gene
3.12. AGO4-Associated lncRNAs Derived from Gene Body Are Associated with H3K4me3 Marks
4. Discussion
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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FLAG:AGO4 RNA-IP | Negative RNA-IP (+15 Cycles) | Nuclear RNA | |
---|---|---|---|
# reads after adaptor trim | 18,878,339 | 10,799,912 | 17,537,595 |
# reads map to Chr 1-5 | 15,905,349 (84%) | 1,071,294 (13%) | 6,307,354 (36%) |
# reads map to Chr C & M | 50,238 (0.3%) | 8,087,305 (75%) | 3,467,242 (20%) |
% total mapped reads | 84.3% | 88% | 56% |
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Au, P.C.K.; Dennis, E.S.; Wang, M.-B. Analysis of Argonaute 4-Associated Long Non-Coding RNA in Arabidopsis thaliana Sheds Novel Insights into Gene Regulation through RNA-Directed DNA Methylation. Genes 2017, 8, 198. https://doi.org/10.3390/genes8080198
Au PCK, Dennis ES, Wang M-B. Analysis of Argonaute 4-Associated Long Non-Coding RNA in Arabidopsis thaliana Sheds Novel Insights into Gene Regulation through RNA-Directed DNA Methylation. Genes. 2017; 8(8):198. https://doi.org/10.3390/genes8080198
Chicago/Turabian StyleAu, Phil Chi Khang, Elizabeth S. Dennis, and Ming-Bo Wang. 2017. "Analysis of Argonaute 4-Associated Long Non-Coding RNA in Arabidopsis thaliana Sheds Novel Insights into Gene Regulation through RNA-Directed DNA Methylation" Genes 8, no. 8: 198. https://doi.org/10.3390/genes8080198