presentation-paper-review-p53-microRNA class presentation

1. Class Presentation by Tin Ho
Current Topics in Biosciences
1 Nov 2010
(cc) Tin Ho
http://creativecommons.org/licenses/by-nc-sa/2.5/

2. miRNA Overview
• Mature form is 18-25 nucleotides long
– single-stranded RNA, non-coding
– Post-transcriptional regulator
• Highly conserved for developmental genes
• Found in:
– Introns of protein-coding genes
– Long non-coding RNA transcripts
– Independent transcription units with core promoter
elements and poly-A signals
• Monocistronic and polycistronic
• miRNA DB has 700+ entries for human
– Biological relevance need to be determined
– 2-4% of genomes, largest class of regulators
hsa-mir-15a.
miRNAMap.mbc.nctu.edu.tw

3. Davis-Dusenbery B N , Hata A J Biochem 2010;148:381-392
© The Authors 2010. Published by Oxford University Press on behalf of the Japanese
Biochemical Society. All rights reserved
DGCR8
p68
p72
p53?
miRNA biogenesis pathway

4. miRNA Nomenclature
hsa – mir – 26 a – 2
Specie:
hsa = Homo sapiens
d = Drosophila
v = viral
r = precursor miRNA
R = mature miRNA
Slight variants
Identical miRNA
found in different
locations of genome.
A large number would mean
cluster (eg -214).
ID
Number = order of discovery.
Name = known function,
eg let7
Wikipedia: miRNA

5. miRNA’s normal pathway
Esquela-Kerscher et al. Nature Reviews Cancer 6, 259–269 (April 2006) | doi:10.1038/nrc1840

6. When tumor suppressor miRNA is deregulated…
Esquela-Kerscher et al. Nature Reviews Cancer 6, 259–269 (April 2006) | doi:10.1038/nrc1840

7. When oncogenic miRNA is deregulated…
Esquela-Kerscher et al. Nature Reviews Cancer 6, 259–269 (April 2006) | doi:10.1038/nrc1840

8. p53 Overview
• Transcription factor, central role
in genetic stability:
– Activate DNA repair
– Induce growth arrest
– Initiate apoptosis
• TP 53 gene is mutated in ~50%
of cancer
• p53 protein structure
– 393 amino acids
– 7 domains
• Approximage size comparison
of p53 and miRNA
Wikipedia: p53

9. p53’s Mechanism of Actions
Nature 460, 466-467 (23 July 2009)
Cancer: Three birds with one stone
Franck Toledo et al

10. Paper’s Framework & Hypothesis
• p53 is central to cell development
• p53 is often mutated in cancer, or its pathway altered
• miRNA is a key post-transcriptional factor
• miRNA is down regulated in many cancer
• Tumor tissues correlated to alteration of some miRNA-processing
factors, but no causal links had been established yet
• Authors proposes a direct connection between tumor suppressor
networks and miRNA biogenesis machinerary:
– p53 binds directly with miRNA-processing factors (Drosha Complex)
– p53 causes an up expression of miRNA when DNA is damaged
– Tumors is a causal factor in miRNA deregulation

11. HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
p53 increases expression of precursor and mature
miRNA post-transcriptionally when DNA is damaged
p53WT in HCT116 human colon cancer cell line p53-/- in HCT116 human colon cancer cell line
1a 1b

12. HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
p53, p68 and p72 are necessary to up-regulate miRNA
in response to DNA damage
siRNA knockdown for:
p53 = red line
p68 = blue line
p72 = gren line
Control = black line
1c

13. HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
Supplmental
Figure 1a
p53, p68 and p72 are necessary to up-regulate miRNA
in response to DNA damage

14. HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
p53 is associated with Drosha complex in DOX
IP with ectopic
p53 and Drosha in HCT116
IP with endogeneous
p53 and Drosha
2a

15. p53 binds with Drosha and pri-miRNA in DOX
HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
*DOX exposure to 8h in WI-38 cell; miR are primary miRNA
3a Nuclear run-on assay

16. HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
In-vitro pri-miRNA processing assay
Drosha complex immunoprecipitated from HCT116 cells
p53 facilitates Drosha processing in vitro

17. In vivo real time monitor shows Drosha is associated with p53
HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
In-vivo real-time monitoring of Drosha function via Florescent Proteins
Viral vector injects EGFP + short segment of pri-miR-143 or -145 into HCT115 cell
Increasing GFP intensity
Decreasing Drosha activity

18. Mutant p53 down-regulates miRNA processing
HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
Viral p53 insertion into p53-/- HCT116 cells
miR-16-1miR-143miR-206
4a

19. Mutant p53 hinders binding of Drosha to p68
HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
IncreasingGFPintensity
DecreasingDroshaactivity
4d
Fluorescence-activated cell sorting
analysis of p53-/- HCT116 cells
Immunoprecipitation assays

20. Mutant p53 decreases binding of miRNA
with p68 or Drosha
HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199
3b
4f

21. Summary
• p53 increases expression of miRNA post-transcriptionally when DNA
is damaged
• p53 needs p68, p72 to function
• p53 is associated with Drosha, and facilitates its processing of
primary miRNA to precursor miRNA
• Mutant p53 has opposing effect of wild type p53
•
•
•
p53 is a key player in the
miRNA biogenesis and tumor suppressor networks

22. Final Perspectives …
• miRNA and protein interactions are multifaceted
• Loss of mature miRNA causes reciprocal damage to DNA and
increases p53 activity
• How great a portion is pri-miRNA regulated by p53 needs further
research, other genes and pathways may have similar role:
– MYC protein directly regulates mir-17-92 pri-miRNA transcript
(unverified).
– mir-17-5p and miR-20a targets E2F1 transcription factor, which is a
controller of cell apoptosis
• ~200 miRNA gene can accurately classify cancer according to their
embryonic lineage (Lu et al)
• Anti-miRNA oligonucleotides (AMO) and antagomirs (AMOs
conjugate with cholesterol) may be effective controls to inhibit
miRNA.
Esquela-Kerscher et al. Nature Reviews Cancer 6, 259–269 (April 2006) | doi:10.1038/nrc1840

23. Thank you…

24. BACKUP SLIDES…
(cc) tin6150 (at) gmail.com

25. p53 as “Master Watchman”
Wikipedia: p53

26. Esquela-Kerscher et al. Nature Reviews Cancer 6, 259–269 (April 2006) | doi:10.1038/nrc1840

28. Esquela-Kerscher et al. Nature Reviews Cancer 6, 259–269 (April 2006) | doi:10.1038/nrc1840
MicroRNAs that are associated with human cancers

29. miRNA targets
• K-Ras mi-143
• CDK6: miR-16, miR-26a, 107, 145, 206

34. REDUNDANT SLIDES…

35. Paper Overview
• p53 interacts with Drosha complex, facilitate processing of pri-miRNA to pre-miRNA
• Up miRNA, down cell proliferation (ie, affected miRNA were tumor suppressors)
• DNA-binding domain is required for interaction with Drosha
– Probably binds to p68 RNA-helicase component of Drosha complex
• 3 types of missense mutation in DNA-binding domain of p53 that obliterate
transcriptonal activity
– Dec pri-miRNA processing by Drosha (this is new finding presented by this paper)
• P53 affects:
– Down Drosha’s activity to process pri-miRNA (when p53 is mutated)
• Correlates to human tumors that mutates p53
– Interferes Drosha assoc with p68 (p53 titrates it)
• P68 is a transcriptional co-regulator that is also involved in RNA splicing
• TP53 is transcribed into 9 mRNA
– Don’t know whether all of them are efficiently translated into proteins
• SNP on TP53 thicken the plot.
– MDM2, which encodes a major p53 inhibitor, affects age people get tumor
– SNP affects MDM2, which affects p53 concentration, in turn affects miRNA
• This dynamics likely have implications on when people develop cancer and their prognosis.

36. Intro …
paper reading…
• p53 enhances the post-transcriptional maturation of several miRNA
w/ growth-suppressive fn
– miR-16-1
– miR-143
– miR-145
• Shows interaction of p53 w/:
– Drosha processing complex (via:)
• DEAD-box RNA helicase p68 (DDX5)
• DEAD-box RNA …
– Transcriptionally inactive p53 mutants interfere above! (?? Mutated
p53 where domain binding no longer allows for transcription still binds to
Drosha and DDX5)

37. Paper Framework / Hypothesis
• Known roles of miRNA:
– Function as tumor suppressor and onncogenes
– Down regulated in many human cancer
– Genomic and epigenomic alterations can affect its expressions
– Deregulation of processing factors such as Dicer decreases miRNA
• Authors propose direct connection b/w tumor suppressor networks and miRNA
biogenesis:
– p53 binds directly with Drosha Complex / p68 RNA Helicase
– Tumors is a causal factor in miRNA deregulation
• Proof:
• p53 enhances post-transcriptional maturation of several miRNA with growth-
suppressive function in response to DNA damage (fig 1)
• Transcriptionally inactive p53 mutants interfere Dorsha complex and p68. (?? Mutated p53
where domain binding no longer allows for transcription still binds to Drosha and DDX5,
therefore it must use a different domain of p53 for this handiwork).
• Compare miRNA induced by DNA damage a/o p53 activation + p68/p72
– P72-dependent miRNA up reg in DNA damage cond (sup Fig 1)

38. Overview of Experiements
• Up DNA damage, up p72-dependent miRNA regulation
• Prior research (ref 18) showed relation of p53 and p68/p72
• Use HCT116 human colon cancer cell line, apply doxorubicin (DOX,
a potent p53 inducer), use qRT-PCR to see that miRNA were up
regulated Post-transcriptionally
– miR-34a (prev known, so diff pathway)
– miR-15a, 16-1, 23a, 26a, 103, 143, 145, 203, 206. (fig 1a)
• Investigate interaction b/w Drosha and p53

39. Overview of figures
1. Shows that p53 is correlated (and necessary) with increase of
several miRNA, as post-transcription event, when there are DNA
damage.
2. Shows that p53 is found to be bound to Drosha via use of Co-IP
that precipitates them out together,
3. Shows that p53 helps Drosha to up regulate pri-miRNA
4. Mutated p53 also hinters miRNA exp in a post-transcription
manner.

40. Thank you…