Pseudogene Journal Club Presentation

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A journal club style presentation on a publication about the effect of microRNAs and pseudogenes on tumor gene regulation.
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  • PTENP1 3’ UTR inserted into DU145 cells using retroviral vectorsPTENP1 3’ UTR expression derepressed PTEN transcription and translation
  • Pseudogene Journal Club Presentation

    1. 1. A Coding-Independent Function ofGene and Pseudogene mRNAsRegulates Tumour Biologyby Poliseno, Salmena, and Pandolfi Krishna Doppalapudi, Lucas Man, Samantha Margulies, Patty Yau
    2. 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. 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. 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. 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. 6. Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion 1. PTENP1 is targeted by PTEN- targeting miRNAsA. PTEN is protected from miRNA binding by PTENP1B. Homology ▫ PTENP1 is one kb shorter ▫ Only 18 mismatches throughout the entire coding sequenceC. Similar miRNA binding sites ▫ Perfectly conserved seed matches for miRNA 20, 21, 214, 19, 26
    7. 7. Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion 1. PTENP1 is targeted by PTEN- targeting miRNAsD. 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 mRNAE. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 23. Intro Main Pts Results: Fig. 1 Fig. 2 Fig. 3 Fig. 4 Conclusion

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