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…