1. 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

2. MicroRNA and MicroRNA Target Prediction Programs A.What are microRNAs? B.What biological function or functions do they perform? C.With which biomolecules do they interact? D.How do microRNA target detection programs predict mRNA/target interaction? E.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 RISC - RNA induced silencing complex UTR - untranslated region of an mRNA mRNA Suppression Images from Bartel. (2004) Cell, Vol 116: 281-297

4. mRNA 3’ UTR mRNA1 3’ UTR mRNA2 3’ UTR mRNA3 3’ UTR MicroRNA Target Prediction Programs rely on MicroRNA Targeting Promiscuity microRNA1 1.One microRNA can bind to the 3’ UTR of an mRNA. microRNA microRNA1 microRNA2 2. Multiple microRNAs can bind to the 3’ UTR of one mRNA. 3. A single microRNA can have many distinct mRNA targets. Known target previously unknown target

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

6. 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 NASA

7. Evidence of MicroRNA Involvement in Transcriptional Regulation of Muscle Differentiation prolif.diff. MyoblastMyotube miR-1/133 clusters miR-133miR-1 SRFHDAC4 MEF2MyoD A Model MEF2 - muscle-related transcription factor HDAC4 - inhibitor of muscle differentiation SRF - myoblast proliferation MyoD - myogenic differentiation See Chen et al. 2006 for more information MicroRNAs regulate genes which make muscle. Could microRNAs regulate genes which destroy muscle?

8. Potential MicroRNA Involvement in Muscle Degradation MyoblastMyotube miR-1/133 clusters miR-133miR-1 SRFHDAC4 MEF2 MyoD prolif.diff. MuRF-1MAFbx microRNA- regulated? Chen et al. 2006 Could microRNA target detection programs be used to identify the microRNAs regulating MuRF-1 and MAFbx?

9. migo: Identifying Genes with Multiple Common microRNA Binding Sites 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 List of Genes: Gene1 Gene2 Gene3 miRNA 1miRNA 2miRNA 3 Gene 13110 Gene 2520 Gene 31752 Microinspector - Tabler Lab XClust - Eisen Lab TreeView - Eisen Lab miRBase … miRNA 1 miRNA 3 miRNA 5 miRNA 2 …

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

12. migo Screenshot: Results Viewed Using TreeView

13. microLink - A First Addition To Migo MAFbxMuRF1 miRs 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.

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