Class Presentation by Tin HoCurrent Topics in Biosciences

1 Nov 2010(cc) Tin Ho

http://creativecommons.org/licenses/by-nc-sa/2.5/

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

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

DGCR8p68p72p53?

miRNA biogenesis pathway

miRNA Nomenclature

hsa – mir – 26 a – 2

Specie:hsa = Homo sapiensd = Drosophilav = viral

r = precursor miRNAR = mature miRNA

Slight variants

Identical miRNAfound in differentlocations of genome.A large number would meancluster (eg -214).

IDNumber = order of discovery.Name = known function,eg let7

Wikipedia: miRNA

miRNA’s normal pathway

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

When tumor suppressor miRNA is deregulated…

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

When oncogenic miRNA is deregulated…

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

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

p53’s Mechanism of Actions

Nature 460, 466-467 (23 July 2009)Cancer: Three birds with one stone

Franck Toledo et al

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

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

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 linep68 = blue linep72 = gren lineControl = black line

1c

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 miRNAin response to DNA damage

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

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

HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199

In-vitro pri-miRNA processing assayDrosha complex immunoprecipitated from HCT116 cells

p53 facilitates Drosha processing in vitro

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 ProteinsViral vector injects EGFP + short segment of pri-miR-143 or -145 into HCT115 cell

Increasing GFP intensityDecreasing Drosha activity

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-

1

miR

-143

miR

-206

4a

Mutant p53 hinders binding of Drosha to p68

HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199

Incr

easi

ng G

FP in

tens

ityD

ecre

asin

g D

rosh

a ac

tivity

4d

Fluorescence-activated cell sorting analysis of p53-/- HCT116 cells

Immunoprecipitation assays

Mutant p53 decreases binding of miRNAwith p68 or Drosha

HI Suzuki et al. Nature 460, 529-533 (2009) doi:10.1038/nature08199

3b

4f

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

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

?Thank you…

BACKUP SLIDES…

(cc) tin6150 (at) gmail.com

p53 as “Master Watchman”

Wikipedia: p53

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

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

MicroRNAs that are associated with human cancers

miRNA targets

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

REDUNDANT SLIDES…

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.

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)

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)

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

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-miRNA4. Mutated p53 also hinters miRNA exp in a post-transcription

manner.

?Thank you…