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MicroRNA in Developing and Germinating Arabidopsis thaliana Seeds

MicroRNA in Developing and Germinating Arabidopsis thaliana Seeds. Jing Sun Mentor: Dr. Hiro Nonogaki. Importance of Seeds. Environment Protection of the embryo Dispersal of offspring Agriculture and food. http://osulibrary.oregonstate.edu/video/agri55.html.

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MicroRNA in Developing and Germinating Arabidopsis thaliana Seeds

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  1. MicroRNA in Developing and Germinating Arabidopsis thaliana Seeds Jing Sun Mentor: Dr. Hiro Nonogaki

  2. Importance of Seeds • Environment • Protection of the embryo • Dispersal of offspring • Agriculture and food http://osulibrary.oregonstate.edu/video/agri55.html www.psln.com/pete/images.htm www.invest.moldova.md/_1economy.html www.julianessamphotography.com/professional/pages/vegetables.htm

  3. Project Goal To examine the involvement of microRNA in gene expression in Arabidopsis thaliana seeds and siliques www.bpmp.cnrs.fr/

  4. What are MicroRNAs? • Discovered in 1993 by Dr. Victor Ambros and colleagues • Originally for viral studies • Involved in posttranscriptional gene regulation Photo by Amanda Weatherman http://dms.dartmouth.edu/news/2001/25oct2001.shtml

  5. MicroRNA Double Stranded Hairpin Loop Pri-microRNA Pre-microRNA How are MiRNAs Produced? Single Stranded RNA

  6. DNA RNA Protein Transcription Translation MiRNA Function in Gene Regulation mRNA cleavage

  7. DNA RNA Protein Transcription Translation MiRNA Function in Gene Regulation mRNA cleavage

  8. Biological Function of MiRNA • Developmental control • Environmental response • Homeostasis www.britishcouncil.org/coratia-science.htm http://www.mun.ca/biochem/course/3107/Topics/Flower_development.html

  9. Methodology Overview Screening Extraction, Purification, Separation Detection & Identification GUS Expression Functional Analysis

  10. RNA Extraction Phenol-SDS Extraction Method Isopropanol Fractionation High Molecular Weight RNA (18S RNA, 28S RNA, rRNA, mRNA) Low Molecular Weight RNA (5S RNA, tRNA, siRNA, microRNA)

  11. ppt1 ppt3 ppt1 ppt3 0% 20 0% 20 - - 50% 50% (kb) (kb) 10 10 2.0 2.0 28S 28S rRNA rRNA 18S 18S rRNA rRNA 1.0 1.0 0.25 0.25 Small Small RNAs RNAs RNA Purification & Separation

  12. AP AP AP D Digoxigenin label D * * * * G U C G G U U C C U A C U G A A C G G C U C A G C C A A G G A U G A C U U G C C G A RNA Probe Detection Alkaline Phosphatase Anti – DIG Ab Extra bases Antisense probe Target miRNA

  13. Microarrays Examines the whole genome But not yet commercially available for microRNA www.bpp.msu.edu/ Seed/SeedArray.htm

  14. Membrane Probe solution (170 µ l) miR172 miR161 miR162 miR168 miR170 miR171 Screening of Expressed miRNAs

  15. MicroRNAs Expressed Developing siliques Micro RNA

  16. Germination Germination 100 100 - - - - 50 50 Relative Expression (%) Relative Expression (%) 156 156 157 157 158 158 159 159 161 161 168 168 170 170 171 171 160 160 162 162 164 166 167 169 172 173 173 163 165 166 167 169 172 163 164 165 319 319 394 394 395 395 396 396 398 398 399 399 390 390 391 391 393 393 397 397 microRNA MicroRNAs Expressed

  17. Germination Germination 100 100 - - Relative Expression (%) Relative Expression (%) - - 50 50 156 156 157 157 158 158 159 159 160 160 161 161 162 162 168 168 170 170 171 171 173 173 163 163 164 164 165 165 166 166 167 167 169 169 172 172 319 319 390 390 391 391 393 393 394 394 395 395 396 396 397 397 398 398 399 399 Developing Silique 1.2 1 0.8 Relative Intensity 0.6 0.4 0.2 0 170 171 172 173 310 390 391 393 394 395 396 397 398 399 MicroRNA Target MicroRNAs miR393: TIR/F-BOX miR396: GRF miR156: SPL 13 miR157: SPL 5 miR160: ARF

  18. 6 hr 12 hr 18 hr 24 hr When Target Gene Expressed • Northern Blotting 6 hr 12 hr 18 hr 24 hr • Reverse Transcription Polymerase Chain Reaction (RT-PCR)

  19. Target Gene Promoter Reporter Gene GUS Selection Marker Antibiotic resistance Kanamycin Where Gene Expressed

  20. Localization GUS Expression – vascular bundles GUS Expression – stem, flower, silique

  21. Functional Analysis • Mutate microRNA binding site • Observe phenotype (Palatnik et al., 2003; Nature 425: 257-263)

  22. MicroRNA Resistant Target Wild Type Control Wild Type

  23. MicroRNA Resistant Target Wild Type Wild Type

  24. MicroRNA Resistant Target

  25. Germination Germination - - - - 50 50 Summary • Screened for microRNAs in developing and germinating seeds 100 • Target gene expression • GUS expression target genes • Characterizing mutations target genes

  26. Acknowledgements Dr. Hiro Nonogaki Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Chris Mathews Ernest and Pauline Jaworski Summer Research Grant Dr. Jim Carrington Tanja Homrichhausen Po-Pu (Eric) Liu Jessica Hewitt Nobuya Koizuka Ruth Martin

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