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Specificity of argonaute protein association with miRNA
Molly BlatzDr. Jim CarringtonDr. Sunny Gilbert
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Importance of small RNA
• Gene regulation• Growth and development• Therapeutics• Agriculture
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miRNA Functions
Dicer
RISC proteins
Target RNA degradation
Translation arrest
miRNA
miRNA precursor
(Zamore, Bartel, Sharp, Carrington labs)
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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.
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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
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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.
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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.
miR390: AAGCUCAGGAGGGAUAGCGCC Binds AGO7
miR390-11U: AAGCUCAGGAUGGAUAGCGCC Binds AGO1 ???
miR172: AGAAUCUUGAUGAUGCUGCAU Binds AGO1
miR172-11G: AGAAUCUUGAGGAUGCUGCAU Binds AGO1 ???
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How• Construct mutant miRNAs.• Infiltrate Nicotania benthamiana with constructs
containing miRNAs, tagged proteins, and appropriate controls.
• Co-immunoprecipitation• Small RNA gels
HA-AGO Tissue
Immunoprecipitateusing HA antibody
IP Fraction
Small RNA gels
Lyse Cells Input Fraction
Small RNA gels
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Position 11U Results
miR172-11G associates with AGO1
11U is not a specificity determinant.
Vectorin HA
HA-AGO1 in HA
HA-AGO7 in HA
Vector
miR172
miR172-11G
Vector
miR390
miR390-11U
Vectorin HA
HA-AGO1 in HA
HA-AGO7 in HA
miR390-11U associates with AGO7 and not AGO1
HA-AGO Tissue
Immunoprecipitateusing HA antibody
IP Fraction
Small RNA gels
Lyse Cells Input Fraction
Small RNA gels
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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.
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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
miR390-5’G: GAGCUCAGGAGGGAUAGCGCC
miR390: AAGCUCAGGAGGGAUAGCGCC
miR390-19C: AAGCUCAGGAGGGAUAGCCCC
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Alteration of miR390
• Prediction: Both miR390-5’G and miR390-19C should bind AGO7 and accumulate as well as endogenous miR390.
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miR390 Alteration Results
miR390-5’G creates a 19 nucleotide small RNA
miR390-19C accumulates more
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miR390-5’G associates with AGO7 even though it is a 19 nucleotide small RNA.
miR390 Alteration Results
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Future Plans
• Create miR390 with single point mutations at every position.
• Test nonconserved miRNAs.
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Acknowledgement
• Howard Hughes Medical Institute• Dr. Jim Carrington• Dr. Sunny Gilbert• The Carrington Lab