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Human Argonaute-2 (Ago-2)
For All Your RNA Slicing Needs
Evan Bruss, T.J. Davis, Jack Hermsen, Justin Johnson, Robert Laughlin, Maurice Lucré, Chad Marable, Brett Poniewaz, Virginia Tuncel, Gina Wade, Michael Weeden
Teacher: David Sampe
Mentor: Mark McNally, Ph.D., Medical College of Wisconsin, Microbiology and Molecular Genetics
The Story of
Human Argonaute-2
Human cells have the remarkable capability to regulate protein
production by degrading target mRNA by two pathways: RNA
interference (RNAi) and micro RNA (miRNA). Central to these pathways
is the protein Argonaute-2 (Ago-2). In the RNAi pathway, small RNAs
derived from viruses are used by Ago-2 to slice virus mRNA, protecting
the cells from infection. In the miRNA pathway, Ago-2 utilizes naturally
occurring miRNA to slice cellular mRNAs to control protein production.
Ago-2 works by binding small (~22 nucleotide) regulatory RNAs (siRNA
and miRNA) that target mRNA by base pairing. Ago-2 binds to the
phosphate backbone of regulatory RNA within the RNA Interference
Silencing Complex (RISC), which is then guided to the mRNA target by
the regulatory RNA. The RNase domain of Ago-2 (containing His807,
Asp669, Asp597, and Glu637 in its active site) then “slices” the target to
initiate degradation. Researchers have found that they can make their
own synthetic siRNA sequences to insert within cells that target a
specific RNA of their choosing. This may reduce the level of diseasecausing proteins (for example, in breast cancer). Determining the
structure of Ago-2 allowed researchers to understand how this enzyme
functions in the siRNA/miRNA pathways to break down target RNA. The
Brown Deer High School SMART (Students Modeling A Research Topic)
Team has designed a model of Ago-2 using 3D printing technology to
investigate its structure-function relationship.
How Ago-2 Works
siRNA Pathway
miRNA Pathway
1a. In the small interfering RNA
pathway (siRNA), a virus
introduces its genetic material
as double stranded RNA
(dsRNA) into a cell.
1b. In the micro RNA pathway
(miRNA), the DNA is
transcribed into primary miRNA. Pri-miRNA is single
stranded, but assumes a
secondary structure in the form
of a hairpin shape.
2a. A protein called Dicer dices 2b. Dicer converts pre-miRNA
to miRNA.
the dsRNA into 21-22
nucleotide segments.
3. The RNA unwinds, as only the antisense strand is used.
Because Ago-2 recognizes and binds to the 21 nucleotide RNA
on the backbone, it can grab any RNA of that size. The
miRNA/siRNA targets the RNA-Induced Silencing Complex (RISC)
to the mRNA where Ago-2 slices the mRNA towards the center
of the RNA hybrid.
4a. The sliced portions of the 4b. Occasionally in the miRNA
target RNA are quickly broken pathway, perfect base pairing is
down into nucleotides by
not achieved. In these special
RNases in the cell.
cases, an unknown mechanism
stops translation of the target
RNA, effectively inactivating it.
The SMART Team Program is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant
Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Stabilization of the
Domains of Ago-2
1a. Virus Implants dsRNA
1b. DNA Transcription
of Pri-miRNA
Virus
Linear pri-miRNA
Pri-miRNA
Dicer
Nucleus
Pre-miRNA
Drosha
2a. Formation of 2b. Formation of
miRNA by Dicer
siRNA by Dicer
Ago-2
3. Slicing of Target
RNA in RISC
Does Ago-2 structure change upon miRNA binding? To determine if
there was a change in Ago-2 structure, researchers used an approach
called “protease sensitivity.” Differences in protease (thermolysin)
accessibility determine structural changes that researchers can see using
gel electrophoresis. In the experiment, thermolysin, was used to break
down the protein into its domains. The results of the gel electrophoresis
showed that several domains of Ago-2 were separated when miRNA was
not present. When Ago-2 is bound to miRNA, the Ago-2 structure is
stabilized and does not cleave into separate domains. Based on the
experiment, there is a major structure change in Ago-2 when miRNA is
bound.
Ago-2 Domain Functions
The PIWI domain acts as an RNase, slicing the target RNA into pieces.
This domain contains the active site (His807, Asp669, Asp597, and
Glu637) and comprises the area between amino acids 573 and 859.
The PAZ domain holds the 3’ end of the template RNA. This domain is
located between amino acids 226 and 347.
The MID domain holds the 5’ end of the template RNA. This domain is
located between amino acids 450 and 573.
The N domain plays an important role in unwinding the RNA duplex
during formation of RISC. This domain is located between amino acids
1 and 175.
The L2 domain is a “linker” region between the PAZ and MID domains.
It stretches from amino acid 347 to 450.
RISC
The target RNA binds to the PAZ and MID domains. When it is
present, it helps to hold the domains of Ago-2 together.
Conclusions
4a. RNase Degradation
4b. Translation Repressed
Imperfect Base Pairing
Ribosome
Blockage
 Ago-2 has domain structures that include: PIWI, MID, PAZ, and the
N-terminus.
 The MID and PAZ domains are oriented such that only ~21 nucleotide
RNAs can bind.
 Ago-2 binds to the phosphate backbone of miRNA and siRNA so that
the nucleotides can bind with complementarity to the target mRNA.
 The target mRNA gets “sliced” at the PIWI domain because the active
site within it acts like an RNase.
References
Elkayam, E., Kuhn, C., Tocilj, A., Haase, A. D., Greene, E. M., Hannon, G. J., Joshua-Tor, L. (2012). The Structure of Human Argonaute-2 in Complex with miR-20a. Cell 150: 100-110.
Ender, C., Meister, G. (2010). Argonaute Proteins at a Glance. Journal of Cell Science. 123: 1819-1823.
Kuhn, C., Joshua-Tor, L. (2013). Eukaryotic Argonautes come into focus. Trends in Biochemical Sciences. 38(5): 263-271.
Kwak, P.B., Tomari, Y. (2012). The N Domain of Argonaute drives duplex unwinding during RISC assembly. Nature Structural and Molecular Biology. 19(2): 145-151.
Aldoaldoz. (2010, April 10). Euclid Icosahedron 3. Retrieved February 24, 2015, from https://commons.wikimedia.org/wiki/File:Euclid_Icosahedron_3.svg