A Coding-Independent Function of Gene and Pseudogene mRNAs Regulates Tumour Biologyby Poliseno, Salmena, and Pandolfi

Krishna Doppalapudi, Lucas Man, Samantha Margulies, Patty Yau

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

Introduction• PTEN is a tumor suppressor gene

▫ Its protein product is a phosphatase which regulates the cell cycle by preventing cells from dividing too rapidly

▫ Mutations in this gene are found in many cancers

• PTENP1 is the pseudogene of PTEN▫ Pseudogenes are relatives of known

genes that have lost their ability to be translated into protein

▫ Once considered nonfunctional• PTEN and PTENP1 mRNA transcripts

show homology ▫ Dark grey area is the highly conserved

region ▫ Colored lines show similar miRNA

binding sites

PTEN

PTENP1

Intro

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

Introduction•microRNAs: class of small non-coding

RNAs•miRNAs bind to sequences in the 3’ UTR

of target mRNAs, resulting in gene silencing

Intro

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

Competition for miRNA binding

•miRNAs can bind to either PTEN or PTENP1 pseudogene

miRNAs

PTEN

PTENP1

Intro

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

Main Points of the Paper

1. PTENP1 is also targeted by PTEN-targeting miRNAs

2. The 3’ UTR of PTENP1 has tumor suppressive activity

3. PTENP1 levels influence downregulation of PTEN in cancer cells

4. PTEN/PTENP1 model should work for other genes

Main Pts

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

1. PTENP1 is targeted by PTEN-targeting miRNAs

A. PTEN is protected from miRNA binding by PTENP1

B. Homology▫ PTENP1 is one kb

shorter▫ Only 18 mismatches

throughout the entire coding sequence

C. Similar miRNA binding sites

▫ Perfectly conserved seed matches for miRNA 20, 21, 214, 19, 26

Results: Fig. 1

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

1. PTENP1 is targeted by PTEN-targeting miRNAs

D. In prostate cancer cells, miRNAs 19b and 20a suppressed PTEN and PTENP1 transcript abundance

▫ siLuc is used as a control▫ Blue and red bars show a

decrease in mRNA E. miRNA inhibitors (Imix)

increased PTENP1 and PTEN transcript abundance

▫ IC used as a control Without inhibitors

▫ Inhibitors cause derepression- miRNAs will not be able to silence the mRNA transcripts

Results: Fig. 1

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

A. PTENP1 3’ UTR expression derepressed PTEN transcription and translation

▫ Method: PTENP1 3’ UTR inserted into DU145 cells using retroviral vectors

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

B. Elevated PTEN expression is related to reduced phospho-AKT activity

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

B. Elevated PTEN expression is related to reduced phospho-AKT activity

C. Effect of derepressed PTEN is growth inhibition

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

B. Elevated PTEN expression is related to reduced phospho-AKT activity

C. Effect of derepressed PTEN is growth inhibition

D. PTENP1 3’ UTR is a more potent growth suppressor than PTEN

▫ Could function as a decoy for miRNAs that bind to other targets with tumour suppressive activities

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

E. Disruption of DICER blunts derepression of PTEN by PTENP1 3’ UTR

▫ Suggests PTENP1 3’ UTR requires mature miRNA for function

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

E. Disruption of DICER blunts derepression of PTEN by PTENP1 3’ UTR

▫ Suggests PTENP1 3’ UTR requires mature miRNA for function

F. Silencing both PTEN and PTENP1 showed strongest increase in cell proliferation

▫ Suggests additive roles for PTEN and PTENP1 in growth suppression

siRNA function in RNA interference to degrade mRNA, introducing siRNA can have the effect of “knocking-down” or “silencing” genes

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

E. Disruption of DICER blunts derepression of PTEN by PTENP1 3’ UTR

▫ Suggests PTENP1 3’ UTR requires mature miRNA for function

F. Silencing both PTEN and PTENP1 showed strongest increase in cell proliferation

▫ Suggests additive roles for PTEN and PTENP1 in growth suppression

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

G. siRNA knockdown decreased PTEN and PTENP1 mRNA

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

2. The 3’ UTR of PTENP1 has tumor suppressive activity

G. siRNA knockdown decreased PTEN and PTENP1 mRNA

H. siRNA knockdown also decreased PTEN protein abundance

Results: Fig. 2

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

3. PTENP1 levels influence downregulation of PTEN in cancer cells

A., B. PTEN and PTENP1 expression are co-regulated

Method: Quantitative RT PCR, measured by green fluorescence (Taqman)

Direct correlation between PTEN and PTENP1 expression in both normal tissue and tumour samples (r = 0.8087 and 0.7538; P<0.0001)

C. In colon cancer samples, independent copy number losses occur specifically in PTENP1 gene

Copy number losses in PTENP1 occur due to selective pressure in cancer

Results: Fig. 3

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

3. PTENP1 levels influence downregulation of PTEN in cancer cells

D. PTEN expression is downregulated in cancer cells

E. PTENP1 transcript levels regulate PTEN expression

▫ Direct correlation between log ratio of PTENP1 copy number vs. log10 PTEN expression intensity

1. r = 0.6105, P = 0.00922. r = 0.6056, P = 0.0129

Results: Fig. 3

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

4. Model should work for other genes

A. PTEN 3’ UTR derepresses PTENP1 mRNA and shows growth suppression

B. KRAS and its pseudogene show similar regulatory mechanisms.

▫ It is known that K1P 3’ UTR overexpression leads to increased levels of KRAS, and consequently cell proliferation and accelerated cell growth

Results: Fig. 4

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

4. Model should work for other genes

C. RNAs X and Y in this figure can be thought of as a pseudogene and its cognate protein coding gene.▫ In Case 1, the

downregulation of RNA X leads to increased binding sites for the miRNA on RNA Y, causing RNA Y to be repressed.

▫ In Case 2, both RNA X and RNA Y are in equilibrium

▫ In Case 3, RNA X is overexpressed and act as an miRNA sponge, this derepresses RNA Y

Results: Fig. 4

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

Conclusion

•Scientists have discovered a new dimension by which cellular and tumor biology can be regulated

•Pseudogenes were once considered non-functional, but are now known to affect mRNA transcript abundance through a miRNA binding mechanism

Conclusion

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

References

•http://www.jyi.org/features/ft.php?id=392•http://

www.answersingenesis.org/tj/v17/i2/pseudogene.asp

•Nature, Vol 465, 24 June 2010, A coding-independent function of gene and pseudogene mRNAs regulates tumour biology

Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

Questions