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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 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?
MicroRNAs: Gene Regulation at the Post-transcriptional LevelMicroRNAs 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
mRNA 3’ UTR
mRNA1 3’ UTR
mRNA2 3’ UTR
mRNA3 3’ UTR
MicroRNA Target Prediction Programs rely on MicroRNA Targeting Promiscuity
microRNA1 microRNA1
microRNA1
1. One microRNA can bind to the 3’ UTR of an mRNA.
microRNA
microRNA1
microRNA1
microRNA1
microRNA2
microRNA2
2. Multiple microRNAs can bind to the 3’ UTR of one mRNA.
3. A single microRNA can have many distinct mRNA targets.
Known targetpreviously unknown target
MicroInspector: MicroRNA Target Prediction Using Databases of Known MicroRNAs
mRNA 3’UTR
miR-A
miR-B
miR-A miR-A
miR-B
miR-A
miR-B miR-BmiR-CmiR-C miR-C
microRNA AUUGCAUTGACGTAmRNA
5’3’
5’ 3’
Region of High Complementarity
1
2
U
mRNAmicroRNA
Output
Predicted Structure of mRNA:microRNA Complex
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
Evidence of MicroRNA Involvement in Transcriptional Regulation of Muscle Differentiation
prolif. diff.
Myoblast Myotube
miR-1/133 clusters
miR-133 miR-1
SRF HDAC4
MEF2 MyoD
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?
Potential MicroRNA Involvement in Muscle Degradation
Myoblast Myotube
miR-1/133 clusters
miR-133 miR-1
SRF HDAC4
MEF2MyoD
prolif. diff.
MuRF-1 MAFbx
microRNA-regulated?
Chen et al. 2006
Could microRNA target detection programs be used to identify the microRNAs regulating MuRF-1 and MAFbx?
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:Gene1Gene2Gene3
miRNA 1 miRNA 2 miRNA 3
Gene 1 3 1 10
Gene 2 5 2 0
Gene 3 17 5 2 Microinspector - Tabler Lab
XClust - Eisen Lab
TreeView - Eisen Lab
miRBase…
miRNA 1miRNA 3miRNA 5 miRNA 2
…
migo Visualization Problem
• Linkage analysis: how subtrees are combinedsingle, average and complete
• XClust bugidentical entries not grouped together immediatelyproblem avoided by using complete linkage analysis instead of average linkage analysis
• Alternative: PyClusteroffers different types of linkage analysis; user can avoid the bug associated with average linkage analysis
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)
migo Screenshot: Results Viewed Using TreeView
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.
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
migmigoo
a list of Gene IDs
a list of microRNAs shared between every two genes
Helps the user select microRNA candidates
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.
microLink Screenshot (Cont.)
Figure 3: a microLink analysis in text format (right)
Figure 4: binding site positions and mRNA:microRNA interaction free energy (below)
Future Work
• Complete the graphical user interface• Improve the visualization scheme for migo• Implement migo’s own microRNA target detection
procedures
Acknowledgments
• Caltech Wold LabFor your guidance and
encouragement
• NIH/NSFFor supporting our summer
program
• SoCalBSI2006 faculty and staff
For your guidance, encouragement, subways, brownies and much much more…