1. A validation of a two-step control model
MicroRNA structure plays a significant role in the specificity of microRNA-mediated RNA interference:
A validation of a two-step control model
Wu Yilong
National University of Singapore

2. Introduction

3. RNA interference (RNAi)
Used for regulation of genes to study diseases
Different types of interference:
Small interfering RNA (siRNA)
Piwi-interacting RNA (piRNA)
MicroRNA (miRNA)

4. Consideration MiRNA: Issues with current prediction models of binding
Nonspecific transcriptome matching
False positive prediction
A new model is required to better predict target sites in the genes

5. The new model proposed
AGO

6. Hypothesis & Objective

7. Hypothesis
MiRNA structure plays a significant role in the specific binding of miRNA::mRNA interaction
Objective
Experimentally verify this proposed model

8. Materials & Methods

9. Constructs generation Silencing activity test
Methodology
In-silico design
Selection of a proper target sequence
Design of mutant miRNAs
Constructs generation
Cloning of target sequence in Luciferase 3’ UTR
Cloning of miRNA precursors in vectors
Silencing activity test
Co-transfection of constructs into human cell lines
Performing firefly luciferase activity assay with renilla as internal standard

10. Results & Discussion

11. Relative DLR activity results
Bcl2 target site (5’)
loop 5’ 5’
***:p<0.001; **:p<0.01; *:p<0.05; .:p<0.10

12. Relative DLR activity results
p21 (3’ and 5’)& p27 target sites (3’ and 5’) 3’ 5’ 5’ 5’
3’&loop
p21
p27

13. Conclusion

14. Conclusion An acceptable validation of the proposed model
MiRNA structure plays a significant role in determining the specificity of miRNA-mediated RNA interference

15. Future Works

16. Future works Test more miRNA::mRNA interactions
Complete additional RNA structure prediction tools
Probe target and miRNA structure experimentally

17. Acknowledgements
This project was funded and supported by the Department of Microbiology, National University of Singapore. All the experiments were designed and performed under the guidance of Dr. Volker Patzel. It was also collaborated with Mr. Zhong Jun.

18. References (selected)
Grimson, A.; Farh, K.K.H.; Johnston, W.K.; Garrett-Engele, P.; Lim, L.P.; Bartel, D.P. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell , 27,
Long, D.; Lee, R.; Williams, P.; Chan, C.Y.; Ambros, V.; Ding, Y. Potent effect of target structure on microRNA function. Nat Struct Mol Biol. 2007, 14,
Kertesz, M.; Iovino, N.; Unnerstall, U.; Gaul, U.; Segal, E. The role of site accessibility in microRNA target recognition. Nat Genet. 2007, 39,
Patzel, V.; Sczakiel, G. Theoretical design of antisense RNA structures substantially improves annealing kinetics and efficacy in human cells. Nat Biotechnol. 1998, 16,
Patzel, V.; Rutz, S.; Dietrich, I.; Köberle, C.; Scheffold, A.; Kaufmann, S.H.E. Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency. Nat Biotechnol. 2005, 23,
Patzel, V.E.A. Guide Structure and miRNA Function. Under revision. National University of Singapore. 2009
Köberle, C.; Kaufmann, S.H.E.; Patzel, V. Selecting effective siRNAs based on guide RNA structure. Nat Protoc. 2006, 1,
Ding, Y., Chan, C. Y. & Lawrence, C. E. RNA secondary structure prediction by centroids in a Boltzmann weighted ensemble. RNA. 2005, 11,

19. Thank you!
Wu Yilong
National University of Singapore

20. Q&A
Wu Yilong
National University of Singapore

21. Supplementary
Select a proper domain on Bcl2 and insert it into firefly luciferase 3’ UTR
Bcl2
Bcl2+
Luciferase

22. Supplementary
Point mutation changes miRNA structures

23. Supplementary Criteria of miRNA design
Maintenance of complementarity with target
Shift or destroy nucleation site by changing structure
Minimum nucleotide changes
Legend: ) or (=paired nucleotides; =free nucleotides
|= W-C base pairs; := Wobble base pairs

24. Supplementary
miR16

25. Supplementary
miR106a

26. Supplementary
miR221

27. Supplementary
miR222

28. miRNA pathway Primary miRNA (pri-miRNA) generation
Processing of precursor
Uptake of mature miRNA
Seek for target to bind
Repression or degradation caused by seed match

29. The new model proposed Step 1: Target allows only a kind of nucleation
Step 2: Seed match control

30. The new model proposed