1. Suzuki, Hiroshi I., Akihiro Katsura, Takahiko Yasuda, Toshihide Ueno, Hiroyuki Mano, Koichi
Sugimoto, and Kohei Miyazono et al. Nature 2015
2.
3. Introduction
• Asymmetry has crucial effects on the function of small RNAs and the specificity of RNA interference (RNAi).
• In the biogenesis of mammalian endogenous miRNAs, after cleavage of primary miRNA transcripts (pri-miRNAs) by
Drosha and Dicer, one miRNA strand in a given 21- to 25-nt RNA duplex is incorporated as the guide strand into
Argonaute (Ago) proteins during formation of the RNA-induced silencing complex (RISC), whereas the other strand (the
passenger strand, also referred to as miRNA*) is discarded
• The selection of the guide strand is an asymmetric, nonrandom process
• One important known parameter for asymmetric selection is the thermodynamic asymmetry of the RNA duplex. According
to the thermodynamic stability (TS) rule, the strand with the less stably base-paired 5′ terminus is preferentially selected
• Dcr-2–R2D2 heterodimer has been proposed to sense the thermodynamic asymmetry of siRNA duplexes during fly Ago2–
RISC assembly. R2D2 binds to the thermodynamically more stable end, thereby positioning Dcr-2 at the less stable end
• It has recently been suggested that guide-strand selection of endogenous and exogenous siRNAs occurs independently of
R2D2. In addition, mammalian Dicer is dispensable for activities of exogenous siRNAs and asymmetry sensing
4. Dicer
dsRNA fragments
cleaved by dicer
RNA loaded into
argonaute
RNA Induced Silencing Complex (RISC)
Passenger strand degraded
(RISC)
RISC can now target and
cleave mRNA
guide strand less
stable 5’ end
5. General nature of microRNA asymmetry in mammals
• Functional profiling of the miR-181 and miR-30 families, which include asymmetry-fixed miRNAs (miR-181a, miR-181b and
miR-181d; and miR-30a, miR-30b, miR-30c, miR-30d and miR-30e) and arm-switching miRNAs (miR-181c and miR-384)-
overall sequence similarity
• Also analyzed other arm-switching miRNAs (miR-142 and miR-154)
• 5p arms consistently show potent activities, whereas 3p arms show varying activities that depend on ΔΔG = (ΔG5p
-ΔG3p
)
• The latter trend correlated well with the empirical TS (thermodynamic stability) rule, in which the strand with the
less stably base-paired 5′-terminus is preferentially selected
• Closer sequence inspection showed that the majority of these 5p arms started with 5′-U or 5′-A, whereas 3p arms
frequently began with 5′-C
2 H-bonds
3 H-bonds
(Uracil)
6. • From these findings, the following models were constructed describing the general nature of mammalian miRNA
asymmetry
• First- Two miRNA-duplex termini contain two separate types of asymmetry information: 5′-nucleotide (5′-nt) identity
and thermodynamic stability
• Second- Asymmetry is determined by two independent rules in superposition: (i) the 5′-nt selection rule, in which a
strand beginning with 5′-U or 5′-A is discretely preferred and (ii) the TS rule, in which the strand with the more
unstable 5′ end is favored with continual variation.
7. To test this hypothesis systematically, we prepared a series of miRNA precursors
We selected diverse miRNA species with the following characteristics
-typical miRNA-duplex structures with 2-nt overhangs
-low activities in HEK 293T cells and
-relative thermodynamic stabilities with ordered intervals of 1–3 kcal/mol to cover the entire dynamic range of
thermodynamic asymmetry of endogenous miRNA duplexes.
• Mutated the 5′ nucleotides of 5p or 3p arms within these pri-miRNAs to 5′-U or 5′-A, or 5′-G or 5′-C. The resultant wild-type
(WT) and mutant pri-miRNAs covered four lattice-like patterns of 5′-nt identity and a range of relative thermodynamic
stabilities
• In extensive luciferase reporter assays with these pri-miRNAs, the observed asymmetry clearly coincided with our
hypothesis, thus unveiling unique features of miRNA asymmetry in vivo
8. Conservation of asymmetry pattern in four Ago proteins
• In mammals, four Ago proteins (Ago1–Ago4) form the RISC with miRNAs
• Although all Ago proteins have been shown to execute miRNA-mediated translational
repression, Ago2 is notable because it alone has 'slicer' activity, in which perfectly matched
targets are cleaved
• Asymmetry patterns for Ago1–Ago4 were essentially similar to the pattern observed previously..
These results exclude the possibility that each Ago differentially contributes to these rules and
suggest a minor role for Ago2 slicer activity in asymmetric selection.
9. Luciferase reporter assays showing
asymmetry patterns of Ago1–Ago4 RISC.
Samples are from mouse Ago-null E7 ES
cells (with Ago1–Ago4 knocked out)
expressing an HA-tagged single human Ago
(Ago1 (a), Ago2 (b), Ago3 (c) and Ago4 (d))
and transfected with pri-miRNA expression
vector and corresponding sensor vector.
Relative arm ratios were determined by
comparison of inhibitory activities of 5p and
3p arms
slicer activity Ago2
10. The PAZ domain of Ago2 is dispensable for asymmetry
sensing
• Recent reports have suggested that Drosophila R2D2 and mammalian Dicer can be bypassed in asymmetric selection,
thereby complicating mechanisms for sensing thermodynamic asymmetry.
• We hypothesized the presence of a ‘G drive’ (referring to Gibbs free energy) and an ‘N drive’ (referring to 5′ nucleobase),
two other proteins or domains responsible for the two rules.
• Ago2 holds 5′ and 3′ ends of mature miRNAs by its MID and PAZ domains, respectively
11. • As a mediator of the 5′-nt rule, the MID domain of Ago2 has been
proposed to select 5′-U or 5′-A through its nucleotide-selection loop
• TRBP or PACT has been proposed to transfer thermodynamically stable
ends to the PAZ domain
• ( TRBP is a double strand RNA binding protein that is
required for the recruitment of Ago2 to the small
interfering RNA (siRNA) bound by Dicer)
• if the PAZ domain receives or favors stable duplex ends, the
coexistence of the 5′-nt and TS rules could be explained by randomly
determined RNA recognition by either the MID or the PAZ domain
12. • To test this, RNA immunoprecipitation (RIP) coupled with quantitative reverse-transcription PCR (qRT-PCR) analysis
(RIP- qRT-PCR) of Ago2-bound RNAs whose 5p and 3p arms are equivalently selected by the 5′-nt and TS rules.
• However, deletion of the PAZ domain failed to disrupt the TS-rule implementation and asymmetry pattern
• Conversely, deletion of the MID domain strongly reduced RNA-binding capacity for both miRNA species
• Mutation of residues close to 3’ binding site (R277, R280 and K335) -located 20 Å away from the 3′-end–binding pocket
for the guide strand- did not affect asymmetric sensing
•
• These results were inconsistent with the previous model in which the PAZ domain receives stable ends from TRBP and
PACT, and they collectively suggest that the PAZ domain is dispensable for asymmetry sensing.
13. Twin-drive model: recognition of 5′-nt and 5′-phosphates
• The twin drive model occurs in the MID domain of the Argonaute protein
• Subsequently, they hypothesized that the phosphate-binding pocket (tract) and nucleotide-selection loop of the MID
domain function as the G drive and N drive, respectively, and simultaneously select strands with thermodynamically
unstable ends
• This twin drive model was supported by doing RIP-qRT-PCR
14. Twin Drive Model
• Mutations of Y529, K533, K566, Q548,R792 and C793 and the 524-GKT-526 motif attenuated the Twin Drive Model
• This occurred both in miRNA and siRNA
• RIP-qRT-PCR shows the expression in siRNA with regards to mutations
15. Global Modeling of small-RNA asymmetry
• Consistently with the results from in vitro binding assays, the rank order of the most to the least favorable
nucleobase for the 5′-nt rule was U, A, G, C,
• Quantitative analysis of luciferase reporter assays also supported this trend
• No discernable difference was found between the 4 Ago proteins, even though Ago 2 has a slicer ability
16. Global modeling of small-RNA asymmetry
• 5p/3p ratios of a diverse group of miRNAs generally reflected superposition of the 5′-nt and TS rules
• G Drive: K566, Q548, R792, C793 (Gibbs Free)
• N Drive: 524-GKT-526, 523-PGKTP-527 (5”-nt)
• Mutations in the phosphate-binding pocket (K566, Q548, R792, C793) and the nucleotide-selection loop (524-GKT-526
motif for N Drive) attenuated the 5p/3p ratio for siRNA.
Fig. 7
Repeat 3
times
Resuspend
and wash
Etc.
RT
PCR
18. Source
Suzuki, Hiroshi I., Akihiro Katsura, Takahiko Yasuda, Toshihide Ueno, Hiroyuki Mano, Koichi Sugimoto, and Kohei Miyazono.
"Small-RNA Asymmetry Is Directly Driven by Mammalian Argonautes." Nature (2015): n. pag. 22 June 2015. Web. 23 June
2015.
