translational inhibition in arabidopsis thaliana
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Translational Inhibition in Arabidopsis thaliana. Russell Burke Dr. James Carrington Botany and Plant Pathology. Significance. microRNAs are 21 to 24 nucleotide RNAs that serve in post-transcriptional regulation in nearly all eukaryotic organisms - PowerPoint PPT PresentationTRANSCRIPT
Translational Inhibition in Arabidopsis thaliana
Russell BurkeDr. James CarringtonBotany and Plant Pathology
Significance• microRNAs are 21 to 24 nucleotide RNAs that serve
in post-transcriptional regulation in nearly all eukaryotic organisms
• They have large roles in growth, development, stress response and antiviral defense in plants
• Having a greater knowledge of miRNAs will allow us to better understand growth and development of agriculturally useful plants
• microRNA regulation in plants is also very similar to animals and may lead to a better understanding of cancer and other human diseases
RNA Silencing Pathways – Basic Mechanisms and Functions
dsRNA or RNA foldback
RNA Dependent RNAPolymerase
Dicer-like
Small RNADuplex
Argonaute
Target mRNA
RNA-induced silencing complex
RDR3 RDR2
RDR6
DCL1DCL2 DCL3
DCL4
AGO1AGO2
AGO3AGO4 AGO5
AGO6
AGO7AGO8AGO9 AGO10
RDR1
RDR5 RDR4
Major Small RNA Biogenesis and Effector
Families
RNA Silencing Pathways – Basic Mechanisms and Functions
dsRNA or RNA foldback
RNA Dependent RNAPolymerase
Dicer-like
Small RNADuplex
Argonaute
Target mRNA
RNA-induced silencing complex
DRBDCL3
RDR2
24 nt siRNA pathway
DRB4DCL4
RDR6
tasiRNA (21 nt siRNA) pathway
HYL1DCL1SE
Small RNA Classes Function Through Sets of ARGONAUTEs
miRNA pathway
Targets – messenger RNA, a few noncoding RNA
Targets – messenger RNA, virus RNA, transposons
Targets – transposons, repeated sequences
DRBDCL3
RDR2
24 nt siRNA pathway
DRB4DCL4
RDR6HYL1DCL1SE
Small RNA Classes Function Through Sets of ARGONAUTEs
miRNA pathway
An
ARF8 mRNA
AGO1miR167
Posttranscriptional silencing
Targets – messenger RNA, a few noncoding RNA
Targets – messenger RNA, virus RNA, transposons
Targets – transposons, repeated sequences
tasiRNA (21 nt siRNA) pathway
DRBDCL3
RDR2
AGO4 AGO6
DRB4DCL4
RDR6
AGO2 AGO5
HYL1DCL1SE
AGO1 AGO10
Small RNA Classes Function Through Sets of ARGONAUTEs
AGO7 AGO3 AGO8 AGO9
24 nt siRNA pathwaymiRNA pathway
An
ARF8 mRNA
AGO1miR167
Transcriptional silencing
Posttranscriptional silencing
Targets – messenger RNA, a few noncoding RNA
Targets – messenger RNA, virus RNA, transposons
Targets – transposons, repeated sequences
tasiRNA (21 nt siRNA) pathway
DRBDCL3
RDR2
AGO4 AGO6
DRB4DCL4
RDR6
AGO2 AGO5
HYL1DCL1SE
AGO1 AGO10
Small RNA Classes Function Through Sets of ARGONAUTEs
AGO7 AGO3 AGO8 AGO9
24 nt siRNA pathwaymiRNA pathway
An
ARF8 mRNA
AGO1miR167
Posttranscriptional silencing
Targets – messenger RNA, a few noncoding RNA
Targets – messenger RNA, virus RNA, transposons
Targets – transposons, repeated sequences
tasiRNA (21 nt siRNA) pathway
Col-0 ago1-25
Broderson et al. (2008) Science
Translational inhibition found in Arabidopsis mutants
Problems with translational inhibition experiment• Protein levels are not quantified• Experiments are done in mutant Arabidopsis plants
• Difficult to know all the consequences of anago1 mutant• Only 3 specific cases of potential inhibition are shown
Broderson et al. (2008) Science
Translational inhibition found in Arabidopsis mutants
Exogenous Infiltration of Nicotiana benthamiana
Protein Assay
mRNA Analysis
Transiently express GFP in N. benthamiana using a 35S promoter• Allows us to express RNA and proteins in a consistent manner• Avoids consequences of AGO1 mutation and other endogenous background• We can use the same microRNA to target various GFP constructs
GFP
Exogenous Infiltration of Nicotiana benthamiana
• Designed two different GFP mRNAs, that are both targets of an artificial microRNA• Perfect complementation between the microRNA and mRNA target leads to
cleavage of the mRNA• By changing the serine codon in the target site, the mRNA is not cleaved by the
microRNA
GFP GFPser2
GUS
GFP GFPser2
amiR GFP amiR GFP
GFP mRNA
GFP
Exogenous Infiltration of Nicotiana benthamiana
GFP
GFPser2
GFP-am
iR
GFPser2-am
iR0
2
4
6
8
10
12
Protein Levels
GFP
GFPser2
GFP-am
iR
GFPser2-am
iR0
2
4
6
8
10
12
GFP mRNA levels
Exogenous Infiltration of Nicotiana benthamiana
GFP GFPser2
GUS
GFP GFPser2
amiR GFP amiR GFP
GFP mRNA
GFP
5’GATATTGGCGCGGCTCAATCA 3’ |||||||||||||||||||||3’CTATAACCGCGCCGAGTTAGT 5’
5’TTGCTTACTCTCTTCTGTCA 3’ ||||||||||||||||||3’CACGAGTGAGAGAAGACAGT 5’
miR171
SCL6-IV Target
35S
Sensor Constructs
targetsmGFP
miR156
SPL3 Target
5’AAGGGGTTTCCTGAGATCACA 3’ ||||||| ||||||| ||||3’TTCCCCACTGGACTCTTGTGT 5’miR398
CSD1 Target
5’CTACTGCCGCTACTGCTACCA 3’ |||||||||||||||||||3’AATGGTGGCGATGACGATGGT 5’miR834
CIP4 Target
TUMV 3’ utr
5’GATATTGGCGATTCTCAATCA 3’ |||||||||| ||||||||3’CTATAACCGCGCCGAGTTAGT 5’
5’TTGCTTACTAGATTCTGTCA 3’ ||||||| ||||||||3’CACGAGTGAGAGAAGACAGT 5’
miR171
SCL6-IV mTarget
miR156
SPL3 mTarget
5’AAGGGGTTTCAGTAGATCACA 3’ ||||||| | ||| ||||3’TTCCCCACTGGACTCTTGTGT 5’miR398
CSD1 mTarget
5’CTACTGCCGCGCATGCTACCA 3’ |||| ||||| |||||||3’AATGGTGGCGATGACGATGGT 5’miR834
CIP4 mTarget
Next steps in GFP translational repression
Constructs can be introduced into Arabidopsis where translation of GFP can be analyzed endogenously
Acknowledgements
• Howard Hughes Medical Institute• Cripps Scholarship Fund, College of Science• Mentors:
– Dr. James Carrington– Josh Cuperus
• Kevin Ahern