Keystone Symposia 2010

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These are the slides for my presentation at the Keystone microRNA meeting 2010.

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  • First mention that both BMP and TGF-b do this, and that this is the first instance of post-transcriptional regulation of microRNAs due to extracellular signal Also inducate that thus, TGF-B and BMP can regulate gene expression through two ways, one is through transcription and the second is through post-transcriptional regulation of microRNA expression , say that TGF-B signaling promotes the association of the Smads with pri-miRNAs which promotes their processing by drosha, onced processed the pre miRNA is transported out of the nucleus where it undergoes dicer processing to generate the mature miRNA which can alter gene expression post transcriptionally
  • The fold induction of miRNA expression was then clustered. Because miR-21 was similarly induced by both BMP4 and TGF-b we chose the cluster of miRNAs induced by both growth factors this group included miR-21. We hypothesized that smad-regulated miRNAs may share a defining feature which allows them to be regulated. In order to begin to think about the mechanism, I need to tell you a bit more details about the general structure of pri-miRNAs. We compared the sequence and predicted structures of the cluster one miRNAs using motif detection programs. Interestingly this analysis identified a motif which was centrally located in the mature miRNA, and composed of the sequence CAGAC. Given that this is the same sequence which smad optimally binds in DNA, we thought that this sequence could be important for the selection of miRNAs for post-transcriptional processing. Thus we used a sequence motif finding program to identify over-represented sequences present in the cluster 1 miRNAs. Interestingly, the consensus motif CAGAC was identified in the mature miRNA.
  • Remember to say fold induction by BMPEmphasize that m3 maintains the secondary structure of the hairpin.
  • When correctly positioned, the CAGAC sequence can provide Smad mediated processing.
  • Keystone Symposia 2010

    1. 1. Smad Proteins Bind a Conserved RNA Sequence to Promote microRNA Maturation by Drosha <br />Brandi N. Davis, PhD<br />Tufts Medical Center <br />Mentor: Akiko Hata, PhD<br />
    2. 2. TGF-b/BMP induce expression of miR-21 to promote Smooth Muscle cell differentiation<br />
    3. 3. miR-21 is post-transcriptionally induced by BMP/TGF-b<br />Davis et al. Nature 2008<br />
    4. 4. TGF-b/BMP stimulation promotes the processing of miR-21/199a by Drosha<br /><ul><li> The Smads are required
    5. 5. Smads associate with p68, Drosha & DGCR8 on the pri-miRNA
    6. 6. MicroRNA expression can be rapidly modulated in response to cellular stimuli
    7. 7. p53
    8. 8. Estrogen Receptor</li></ul>Davis et al. Nature 2008<br />
    9. 9. A conserved sequence is present in miRNAs induced by BMP/TGF-β<br />
    10. 10. The CAGAC sequence is required for Smad-mediated miRNA processing<br />Pri-miR-21<br />WT<br />loop<br />5’ <br />m1<br />m2<br />m3<br />
    11. 11. Insertion of the CAGAC sequence can confer Smad-mediated miRNA processing<br />C. Elegans miR-84 WT<br />C. Elegans miR-84 5’<br />C. Elegans miR-84 3’<br />C. Elegans miR-84 mid<br />miR-21 WT<br />WT<br />5’<br />3’<br />WT<br />mid<br />C. Elegans miR-84<br />miR-21<br />
    12. 12. Endogenous, CAGAC-containing miRNAs are post-transcriptionally regulated by BMP/TGF-b<br />
    13. 13. Smads directly & sequence-specifically associate with CAGAC containing miRNAs<br />Synthesized miRNA<br />Purified <br />recombinant protein<br />Precipitate protein & <br />associated nucleic acids<br />Detect by qRT-PCR<br />p68<br />Smad:<br />MH1<br />MH2<br />
    14. 14. Smads directly associate with specific sequence elements to promote miRNA processing <br />BMP/TGF-b<br />p68<br />Drosha<br />DGCR8<br />Pri-miRNA<br />Pre-miRNA<br />
    15. 15. Acknowledgements<br />Akiko Hata, PhD<br />Hara Kang, PhD<br />Aaron Hilyard<br />Peter Nguyen<br />Giorgio Lagna, PhD<br />MC Chan, PhD<br />Connie Wu<br />Dahai Wang, PhD<br />JemPrakash<br />Nicole Neuman, PhD<br /><ul><li>JohnKryikas, PhD
    16. 16. JohnCastellot, PhD
    17. 17. LarryFeig, PhD
    18. 18. Craig Hunter, PhD
    19. 19. Judy Lieberman, MD, PhD
    20. 20. NavinKapur, MD</li>

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