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 www.invest.moldova.md/_1economy.html www.psln.com/pete/images.htm www.julianessamphotography.com/professional/pages/vegetables.htm http://osulibrary.oregonstate.edu/video/agri55.html

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

6. mRNA cleavage MiRNA Function in Gene Regulation DNARNAProtein TranslationTranscription

7. mRNA cleavage MiRNA Function in Gene Regulation DNARNAProtein TranslationTranscription

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 Low Molecular Weight RNA (5S RNA, tRNA, siRNA, microRNA) High Molecular Weight RNA (18S RNA, 28S RNA, rRNA, mRNA)

11. RNA Purification & Separation ppt1 ppt3 10 0.25 2.0 1.0 (kb) 28SrRNA 18SrRNA SmallRNAs 0% 20-50% ppt1 ppt3 10 0.25 2.0 1.0 (kb) 28SrRNA 18SrRNA SmallRNAs 0% 20-50%

12. RNA Probe Detection TargetmiRNA Antisenseprobe Digoxigeninlabel C A G C C A A G G A U G A C U U G C C G A GUC G GU C UA CUG A A C G G CU D D *** * Extra bases Anti – DIG Ab AlkalinePhosphatase AP

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

14. Membrane Probe solution (170µl) miR162 miR161 miR172 miR171miR170 miR168 Screening of Expressed miRNAs

15. MicroRNAs Expressed Developing siliques Micro RNA

16. 156157158159 160 161 162 163 164 165 166167 168 169 170171 172 173 319 390391393 394395396 397 398399 Relative Expression (%) 100 50 - - Germination 156157158159 160 161 162 163164165 166167 168 169 170171 172 173 319 390391393 394395396 397 398399 Relative Expression (%) 100 50 - - Germination microRNA MicroRNAs Expressed

17. Target MicroRNAs Developing Silique 0 0.2 0.4 0.6 0.8 1 1.2 170171172173310390391393394395396397398399 MicroRNA Relative Intensity miR156: SPL 13 miR157: SPL 5 miR160: ARF miR393: TIR/F-BOX miR396: GRF

18. When Target Gene Expressed Reverse Transcription Polymerase Chain Reaction (RT-PCR) 6 hr 12 hr 18 hr 24 hr Northern Blotting 6 hr12 hr18 hr24 hr

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

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

23. MicroRNA Resistant Target Wild Type

24. MicroRNA Resistant Target

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

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