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Kong et al., PNAS 2008 Presenter: Chao Li

The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the target gene. Kong et al., PNAS 2008 Presenter: Chao Li. Mechanisms of miRNA suppression of gene expression. MiRNA-mediated mRNA decay - Deadenylation and subsequent decapping

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Kong et al., PNAS 2008 Presenter: Chao Li

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  1. The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the target gene Kong et al., PNAS 2008 Presenter: Chao Li

  2. Mechanisms of miRNA suppressionof gene expression • MiRNA-mediated mRNA decay - Deadenylation and subsequent decapping • Translational repression - Initiation - Post-initiation

  3. Major claims • Two type of miRNA-mediated translation repression Type I: Initiation Type II: Post-Initiation • Which type to use is determined by the promoter of the target gene

  4. What triggers the authors’ idea • Sucrose density gradient analysis is the major experimental technique • Disparate results exists. • Possible reasons: - sucrose gradient composition - different promoters

  5. Constructs -Luciferase allows easy non-invasion mesureing of gene expression Two commonly used 1. Firefly 2. Renilla -let-7 is a well studied repressive miRNA

  6. Repression efficiency Anti-Let-7 2’-O-methyl Oligonucleotide Blocking measuring Luciferase Activity Northern Blot

  7. The Polysomal Location of mRNAs That Are miRNA-Repressed Is Determined by the Promoter Sucrose density gradient Centrifugation Northern Blot to detect Luc in each gradient Re-probe the same membrane towards actin

  8. Does mRNA abundance matter? Remove enhance of SV40 promoter qPCR due to low level expression

  9. Possible explanations for the behavior of SV40 promoter • Complete inhibition of initiation, • A reduction in the rate of initiation, • Or a complete inhibition of translation elongation resulting in mRNAs harboring a single 80S ribosome. 

  10. Low concentration of cycloheximide was used to identify SV40 promoter related mechanism Similar redistribution of PABP mRNA, which is know to be repressed at the Initiation step, thus initiation rather Than elongation is the speed-liminting Step. miRNA-mediated repression reduces the frequeqncy of initiation, rather than completely blocking ths process

  11. Puromycin was used to identify TK promoter related mechanism Puromycin treatment resulted in a shift of the let-7 trargeted Renillla luciferase mRNA. Ribosomes can translocate on miRNA-repressed mRNA, thus miRNAs do not completely block elongation

  12. How about the endogenous 3’ UTR

  13. Target miRNA, site and corresponding constructs

  14. Repression efficiency and specificity

  15. Polysome analysis

  16. Unanswered Question • What’s the mechanism result in such differences? - Deferent modification to mRNA? - Recruit different factors which association with corresponding transcripts during later phases?

  17. Switching from Repression to Activation: MicroRNAs Can Up-Regulate Translation Vasudevan et al., Science 2007

  18. Background Introduction • TNFαis a key cytokine in inflammation and tumorigenesis. • AU-rich elements (ARE) are conserved sequences in mRNA 3’ UTRs that control gene expression posttranscriptionally.

  19. Background Introduction • The authors’ earlier work shows that the TNFαARE can be transformed by serum starvation into a translational activation signal. • Ago2 and FXR1 are required to such activation

  20. Screen out candidate miRNA miRBASE TNFaARE 5 Candidates Only miR369-3 tested positive

  21. miR369-3 is specifically required for TNFα ARE-mediated translation activation -less miR369-3 in Serum+ than - -si-pre369 siRNA works well against miR369-3/5 -knock down can be rescued with synthetic miR369-3 resistant to siRNA RPA assay

  22. Whether formation of base pairs between mir369-3 and the ARE is required -mutant ARE does not undergo translation up-regulation regardless of serum conditon -complementary mutation in miR369-3 can restore up-regulation -both seed sites mutation exhibited loss of up-regulation -both seed sites mutation can be restore at a dose dependent manner

  23. Affinity purified ARE mRNP revealed the involvement of miR369-3

  24. Any other microRNPs might be transformed into activation complexes upon cell cycle arrest? High-mobility group A2 (HMGA2) 3’ UTR contains seven sites for the endogenous Let-7 Artificial 3’ UTR(contain 4 CX) and Exogenouly added miRcxcr4 Knockdown of FXR1 or AGO2 Decrease the up-regulation

  25. Cell cycle synchronization helps distinguishing repression from basal translation Both ARE and CX+miRCXCR4 result in reppresion Both HMGA2+Let7 or mtHMGA2+mtLet7 result in reppresion Knockdown of miR369-3 release reppresion, while Synthetic miR369-3 can rescure AGO2 rather than FXR1 knockdown release reppresion

  26. Relationship between Ago-FXR1 and translation regulation Artificially ethering AGO2, FXR1 or AGO mutant (prp,paz10) to the 5B box reporter

  27. Conclusions • TNFα ARE recruits miR369-3 to mediate translation up-regulation • Base-pairing of miR369-3 is required to recruit the activating AGO2-FXR1 complex • MiRNAs oscillate between repression and activation in coordination with the cell cycle • FXR1 is found exclusively in the activation complex and activation by AGO2 tethering in serum-starved conditions requrired FXR1 expression

  28. Unanswered Questions • What’s the difference in activation/repression complex? Purification->Mass Spectrometry • What’s the mechanism behind activation?

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