Specificity of argonaute protein
association with miRNA
Molly Blatz
Dr. Jim Carrington
Dr. Sunny Gilbert
Importance of small RNA
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Gene regulation
Growth and development
Therapeutics
Agriculture
miRNA Functions
miRNA precursor
Dicer
miRNA
RISC proteins
(Zamore, Bartel,
Sharp, Carrington
labs)
Translation
arrest
Target RNA
degradation
Background
• Small RNA silencing is a mode of gene
regulation.
• Argonaute proteins are the catalytic
components of RISC.
• miRNAs guide AGOs to their targets.
• The nucleotide at the 5’ end is one source of
miRNA specificity.
5’ nucleotide specificity of AGO’s
Kim, N. (2008) Cell Montgomery, et. al. (2008) Cell; Mi et. Al. (2008) Cell; Takeda, et. Al. (2008) Plant Cell Physiology
What We Know
• 5’ nucleotide specificity rule has exceptions.
• There are miRNA’s that associate with AGO1
and lack 5’U.
• AGO7 has no known 5’end nucleotide
specificity.
– Instead it only binds miR390.
• GOAL: Understand the rules of miRNA
association with AGO’s.
miRNA Sequence
• miRNA’s that deviate from AGO1, 5’U rule
contain U at eleventh position.
• Hypothesis: A uracil at position 11 is
important for association with AGO1.
miR172: AGAAUCUUGAUGAUGCUGCAU
miR172-11G: AGAAUCUUGAGGAUGCUGCAU
miR390:
AAGCUCAGGAGGGAUAGCGCC
miR390-11U: AAGCUCAGGAUGGAUAGCGCC
Binds AGO1
Binds AGO1 ???
Binds AGO7
Binds AGO1 ???
How
• Construct mutant miRNAs.
• Infiltrate Nicotania benthamiana with constructs
containing miRNAs, tagged proteins, and
appropriate controls.
HA-AGO Tissue
• Co-immunoprecipitation
Input
Lyse Cells
Fraction
• Small RNA gels
Immunoprecipitate
using HA antibody
IP Fraction
Small RNA gels
Small RNA gels
Position 11U Results
Vector
in HA
HA-AGO1 HA-AGO7
in HA
in HA
HA-AGO Tissue
Vector
miR172-11G associates
Lyse Cells
with AGO1
miR172
Input
Fraction
Immunoprecipitate
miR172-11G
Vector
in HA
miR390-11U associates
with AGO7 and not AGO1
using HA antibody
HA-AGO1
in HA
IP Fraction
HA-AGO7
in HA
Small RNA gels
Vector
Small RNA gels
miR390
miR390-11U
11U is not a specificity determinant.
Determine how non-5’U miRNAs
are specified
• Too much background miR390 and miR172
• Make new system using a miRNA that is
distinguishable from endogenous miRNA but
is still processed efficiently.
Alteration of miR390
• Eliminate competition with other AGOs.
• Purine and pyrimidine conservation.
• Changed position 19 from a G to C
– Choose miRNA over miRNA*
• Changed position 1 from an A to a G
– 5’ end of miR390 doesn’t play a role in association.
miR390:
AAGCUCAGGAGGGAUAGCGCC
miR390-19C: AAGCUCAGGAGGGAUAGCCCC
miR390:
AAGCUCAGGAGGGAUAGCGCC
miR390-5’G:
GAGCUCAGGAGGGAUAGCGCC
Alteration of miR390
• Prediction: Both miR390-5’G and miR390-19C
should bind AGO7 and accumulate as well as
endogenous miR390.
miR390 Alteration Results
miR390-5’G creates a 19 nucleotide small RNA
miR390-19C accumulates more
miR390 Alteration Results
miR390-5’G associates with AGO7 even
though it is a 19 nucleotide small RNA.
Future Plans
• Create miR390 with single point mutations at
every position.
• Test nonconserved miRNAs.
Acknowledgement
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Howard Hughes Medical Institute
Dr. Jim Carrington
Dr. Sunny Gilbert
The Carrington Lab