Specificity of argonaute protein association with miRNA

Molly BlatzDr. Jim CarringtonDr. Sunny Gilbert

Importance of small RNA

• Gene regulation• Growth and development• Therapeutics• Agriculture

miRNA Functions

Dicer

RISC proteins

Target RNA degradation

Translation arrest

miRNA

miRNA precursor

(Zamore, Bartel, Sharp, Carrington labs)

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.

miR390: AAGCUCAGGAGGGAUAGCGCC Binds AGO7

miR390-11U: AAGCUCAGGAUGGAUAGCGCC Binds AGO1 ???

miR172: AGAAUCUUGAUGAUGCUGCAU Binds AGO1

miR172-11G: AGAAUCUUGAGGAUGCUGCAU Binds AGO1 ???

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

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

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

miR390-5’G: GAGCUCAGGAGGGAUAGCGCC

miR390: AAGCUCAGGAGGGAUAGCGCC

miR390-19C: AAGCUCAGGAGGGAUAGCCCC

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

miR390-5’G associates with AGO7 even though it is a 19 nucleotide small RNA.

miR390 Alteration Results

Future Plans

• Create miR390 with single point mutations at every position.

• Test nonconserved miRNAs.

Acknowledgement

• Howard Hughes Medical Institute• Dr. Jim Carrington• Dr. Sunny Gilbert• The Carrington Lab