A journal club style presentation on a publication about the effect of microRNAs and pseudogenes on tumor gene regulation. (Note: The animations in the slides do not work on SlideShare, please download the PowerPoint file to view.)

1. A Coding-Independent Function of
Gene and Pseudogene mRNAs
Regulates Tumour Biology
by Poliseno, Salmena, and Pandolfi
Krishna Doppalapudi, Lucas Man,
Samantha Margulies, Patty Yau

2. 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 PTEN
genes that have lost their ability to be
translated into protein
▫ Once considered nonfunctional PTENP1
• PTEN and PTENP1 mRNA transcripts
show homology
▫ Dark grey area is the highly conserved
region
▫ Colored lines show similar miRNA
binding sites

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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
siRNA function in RNA
interferencemature miRNA for
requires to degrade mRNA,
function
introducing siRNA can have the
F. Silencing both PTEN and
effect of “knocking-down” or
PTENP1 showed strongest
“silencing” genes
increase in cell proliferation
▫ Suggests additive roles for
PTEN and PTENP1 in
growth suppression

14. 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

15. 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

16. 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

17. Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3
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

18. Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3
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 = 0.0092
2. r = 0.6056, P = 0.0129

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

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

21. 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

22. 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/pse
udogene.asp
• Nature, Vol 465, 24 June 2010, A coding-
independent function of gene and pseudogene
mRNAs regulates tumour biology

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