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
www.psln.com/pete/images.htm
www.julianessamphotography.com/professional/pages/vegetables.htm
www.invest.moldova.md/_1economy.html
Project Goal
To examine the involvement of microRNA
in gene expression in Arabidopsis thaliana
seeds and siliques
www.bpmp.cnrs.fr/
What are MicroRNAs?
• Discovered in 1993 by
Dr. Victor Ambros and colleagues
• Originally for viral studies
Photo by Amanda Weatherman
http://dms.dartmouth.edu/news/2001/25oct2001.shtml
• Involved in posttranscriptional gene regulation
How are MiRNAs Produced?
Single Stranded RNA
Double Stranded
Hairpin Loop
Pri-microRNA
MicroRNA
Pre-microRNA
MiRNA Function in Gene Regulation
DNA
RNA
Transcription
mRNA cleavage
Protein
Translation
MiRNA Function in Gene Regulation
DNA
RNA
Transcription
mRNA cleavage
Protein
Translation
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
Methodology Overview
Screening
Extraction, Purification, Separation
Detection & Identification
GUS Expression
Functional Analysis
RNA Extraction
Phenol-SDS Extraction Method
Isopropanol Fractionation
High Molecular
Weight RNA
Low Molecular
Weight RNA
(18S RNA, 28S RNA,
rRNA, mRNA)
(5S RNA, tRNA,
siRNA, microRNA)
RNA Purification & Separation
ppt1 ppt3
(kb)
0%
20 -50%
10
2.0
1.0
0.25
28S rRNA
18S rRNA
Small RNAs
RNA Probe Detection
AP
AP
Alkaline Phosphatase
Anti – DIG Ab
Extra bases
D
D
Digoxigenin label
*
*
G U C G G U U C C U AC U GAAC G G C
U
*
*
Antisense probe
CAG C CAAG GAU GAC U U G C C GA
Target miRNA
Microarrays
Examines the
whole genome
But not yet
commercially available
for microRNA
www.bpp.msu.edu/ Seed/SeedArray.htm
Screening of Expressed miRNAs
Membrane
Probe solution (170
µl)
MicroRNAs Expressed
Developing siliques
Developing Silique
1.2
1
1
Relative Intensity
1.2
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
MicroRNA
MicroRNA
Micro RNA
399
398
397
396
395
394
393
391
390
310
173
172
171
170
169
168
167
166
165
164
163
162
161
160
159
158
157
0
156
Relative Intensity
Developing Silique
50 -
Relative Expression (%)
MicroRNAs Expressed
100 -
Germination
399
398
397
396
395
394
393
391
390
319
173
172
171
170
169
168
167
166
165
164
163
162
161
160
159
158
157
156
microRNA
Target MicroRNAs
Developing Silique
Relative Intensity
1.2
miR393: TIR/F-BOX
miR396: GRF
1
0.8
0.6
0.4
0.2
0
170 171 172 173 310 390 391 393 394 395 396 397 398 399
Relative Expression (%)
MicroRNA
Germination
100 -
miR156: SPL 13
miR157: SPL 5
50 -
miR160: ARF
399
398
397
396
395
394
393
391
390
319
173
172
171
170
169
168
167
166
165
164
163
162
161
160
159
158
157
156
When Target Gene Expressed
• Northern Blotting
6 hr
12 hr 18 hr
24 hr
• Reverse Transcription Polymerase Chain Reaction
(RT-PCR)
6
hr
12
hr
18
hr
24
hr
Where Gene Expressed
Target Gene Promoter
Reporter Gene GUS
Selection Marker
Antibiotic resistance
Kanamycin
Localization
GUS Expression – vascular bundles
GUS Expression – stem, flower, silique
Functional Analysis
• Mutate microRNA binding site
• Observe phenotype
(Palatnik et al., 2003; Nature 425: 257-263)
MicroRNA Resistant Target
Wild Type
Control
Wild Type
MicroRNA Resistant Target
Wild Type
Wild Type
MicroRNA Resistant Target
Summary
• Screened for microRNAs in developing and germinating seeds
Germination
100 -
• Target gene expression
50-
• GUS expression target genes
• Characterizing mutations target genes
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