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MicroRNA Target Prediction Using Muscle Atrophy Genes As Models

Caltech Wold Lab. Mentors: Dr. Barbara Wold Diane Trout Brandon King Gilberto Hernandez, M.D. Qing Yuan. MicroRNA Target Prediction Using Muscle Atrophy Genes As Models. MicroRNA and MicroRNA Target Prediction Programs. What are microRNAs?

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MicroRNA Target Prediction Using Muscle Atrophy Genes As Models

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  1. Caltech Wold Lab Mentors: Dr. Barbara Wold Diane Trout Brandon King Gilberto Hernandez, M.D. Qing Yuan MicroRNA Target Prediction Using Muscle Atrophy Genes As Models

  2. MicroRNA and MicroRNA Target Prediction Programs What are microRNAs? What biological function or functions do they perform? With which biomolecules do they interact? How do microRNA target detection programs predict mRNA/target interaction? What information do microRNA target detection programs provide?

  3. MicroRNAs: Gene Regulation at the Post-transcriptional Level MicroRNAs are small (17 to 25 nt.) RNA molecules which regulate gene expression by degrading mRNAs of certain genes or interfering with translational machinery of mRNAs. mRNA Degradation mRNA Suppression RISC - RNA induced silencing complex UTR - untranslated region of an mRNA Images from Bartel. (2004) Cell, Vol 116: 281-297

  4. microRNA2 microRNA2 mRNA 3’ UTR Known target previously unknown target mRNA1 3’ UTR microRNA microRNA1 microRNA1 microRNA1 microRNA1 microRNA1 microRNA1 mRNA2 3’ UTR mRNA3 3’ UTR MicroRNA Target Prediction Programs rely on MicroRNA Targeting Promiscuity One microRNA can bind to the 3’ UTR of an mRNA. 2. Multiple microRNAs can bind to the 3’ UTR of one mRNA. 3. A single microRNA can have many distinct mRNA targets.

  5. Region of High Complementarity 3’ 5’ TGACGTA mRNA mRNA 3’UTR miR-A miR-A miR-A miR-A AUUGCAU microRNA miR-B miR-B miR-B miR-B 5’ 3’ miR-C miR-C miR-C MicroInspector: MicroRNA Target Prediction Using Databases of Known MicroRNAs 1 2 Predicted Structure of mRNA:microRNA Complex mRNA microRNA U Output

  6. NASA Biological Interest: Muscle Atrophy Causes: Prolonged disuse Microgravity Disease Result: Upregulation of muscle protein degradation genes, such as MuRF-1 and MAFbx (ubiquitin ligases) --> Loss of muscle mass

  7. SRF HDAC4 prolif. diff. Evidence of MicroRNA Involvement in Transcriptional Regulation of Muscle Differentiation A Model MEF2 - muscle-related transcription factor HDAC4 - inhibitor of muscle differentiation SRF - myoblast proliferation MyoD - myogenic differentiation MicroRNAs regulate genes which make muscle. Could microRNAs regulate genes which destroy muscle? miR-1/133 clusters miR-133 miR-1 MEF2 MyoD Myoblast Myotube See Chen et al. 2006 for more information

  8. MuRF-1 MAFbx SRF HDAC4 prolif. diff. Potential MicroRNA Involvement in Muscle Degradation miR-1/133 clusters miR-133 miR-1 microRNA-regulated? MyoD MEF2 Could microRNA target detection programs be used to identify the microRNAs regulating MuRF-1 and MAFbx? Myoblast Myotube Chen et al. 2006

  9. migo: Identifying Genes with Multiple Common microRNA Binding Sites List of Genes: Gene1 Gene2 Gene3 miRBase … miRNA 1 miRNA 3 miRNA 5 miRNA 2 … • Created by Diane Trout from the Caltech Wold Lab • Identification of microRNA binding sites by known microRNAs for multiple genes • Visualization of binding site profile for genes using TreeView Microinspector microRNA target detection XClust 2-D hierarchical clustering TreeView 2-D hierarchical clustering Microinspector - Tabler Lab XClust - Eisen Lab TreeView - Eisen Lab

  10. migo Visualization Problem • Linkage analysis: how subtrees are combined single, average and complete • XClust bug identical entries not grouped together immediately problem avoided by using complete linkage analysis instead of average linkage analysis • Alternative: PyCluster offers different types of linkage analysis; user can avoid the bug associated with average linkage analysis

  11. list of microRNAs which target one or multiple mRNA transcript submitted Gene Info (retrieved by migo from NCBI) migo Screenshot: Results Viewed Using TreeView migo mRNA:microRNA binding profile

  12. migo Screenshot: Results Viewed Using TreeView

  13. MuRF1 MAFbx microLink - A First Addition To Migo • It allows the user to use genes for positive or negative control to select or exclude microRNA candidates. • It allows the user to visually inspect results and select strong microRNA candidates with ease. miRs

  14. migo How is migo different from MicroInspector? a list of microRNAs and their putative binding sites a mRNA sequence or a Gene ID Disadvantage: High Number of False Positives a list of microRNAs shared between every two genes a list of Gene IDs Helps the user select microRNA candidates

  15. microLink Screenshot Figure 1: a microLink analysis for a list of genes (GUI) • Center is the target gene which the user wants to examine. • On the peripheral are the other genes also submitted. • Thickness of each line connecting every two genes reflect the number of microRNAs they have in common.

  16. microLink Screenshot (Cont.) Figure 3: a microLink analysis in text format (right) Figure 4: binding site positions and mRNA:microRNA interaction free energy (below)

  17. Future Work • Complete the graphical user interface • Improve the visualization scheme for migo • Implement migo’s own microRNA target detection procedures

  18. Acknowledgments • Caltech Wold Lab For your guidance and encouragement • NIH/NSF For supporting our summer program • SoCalBSI2006 faculty and staff For your guidance, encouragement, subways, brownies and much much more…

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