specificity of argonaute protein association with mirna
DESCRIPTION
Specificity of argonaute protein association with miRNA. Molly Blatz Dr. Jim Carrington Dr. Sunny Gilbert. Importance of small RNA. Gene regulation Growth and development Therapeutics Agriculture. miRNA Functions. miRNA precursor. Dicer. miRNA. RISC proteins. - PowerPoint PPT PresentationTRANSCRIPT
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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