Muscular Dystrophy
RNA Interference in Muscle
Muscle Cell Biology

Muscular Dystrophy

My research program focuses on determining the pathogenetic mechanisms of muscular dystrophy. Members of the lab are examining the muscle function using biochemical, molecular, and cellular approaches.

There are many forms of muscular dystrophy caused by primary genetic mutations within different muscle genes. For instance, mutations in dystrophin are responsible for causing Duchenne muscular dystrophy (DMD) while mutations in any of the four sarcoglycan genes cause limb-girdle muscular dystrophy.

Congenital and Emery-Dreyfus muscular dystrophies are caused by mutations in laminin and emerin, respectively. These muscle diseases differ based on the types of muscles that are affected, clinical progress of the disease, and mode of inheritance.

My lab is interested in 1) determining why these proteins are critical for normal muscle function 2) creating animal models for specific forms of muscular dystrophy using transgenic and knockout mouse technology and 3) developing new therapeutic agents.

We are in the process of developing novel animal models for muscle proteins that are known to cause muscular dystrophy. By this method, we can examine the progress of muscle disease in a mouse at the molecular, cellular, and tissue levels.

Finally, we can determine how molecular changes within myofibers result in aberrant muscle function. We will compare our murine models of muscular dystrophy with current mouse models for Duchenne muscular dystrophy and congenital muscular dystrophy.

RNA Interference in Muscle

Double-stranded RNA interference (RNAi) is a potent mechanism for sequence-specific silencing of gene expression and represents an invaluable approach for investigating gene function in normal and diseased states, as well as for drug target validation.

We have reported that skeletal muscle myoblasts and terminally-differentiated myotubes are susceptible to RNAi (Yi et al., 2003). We employed an approach in which dsRNA is generated by cellular transcription from plasmids containing long (1 kb) inverted DNA repeats of the target gene, rather than using dsRNA synthesized in vitro.

We found that gene silencing by this method is effective for endogenously expressed genes, as well as for exogenous reporter genes. Analysis of the expression of several endogenous genes and exogenous reporters demonstrates that the silencing effect is specific for the target gene containing sequences within the inverted repeat.

Our method eliminates the need to chemically synthesize double-stranded RNA and is not accompanied by global repression of gene expression. Furthermore, we show that sequence-specific dsRNA-mediated gene silencing is possible in differentiated, multinucleated skeletal muscle myotubes.

We are currently using this approach as a tool for the examination of protein function in terminally differentiated muscle cells and for generating disease models.

Muscle Cell Biology

The structural design of a cell is composed of three key cytoskeletal networks: actin filaments, intermediate filaments, and microtubules. Traditionally, these major cytoskeletal networks have been thought to play distinct roles in cytoskeletal function.

Actin filaments are responsible for cell polarity and contraction; intermediate filaments give cells mechanical strength; and microtubules traffic organelles, proteins and vesicles through the cytoplasm, as well as organize chromosomes during mitosis.

Recently, a number of novel proteins have been found to physically link two or more cytoskeletal networks, highlighting the importance of cross talk and communication within the cytoskeleton. Preliminary data suggests that GAS11, a novel cytoskeletal protein, may function to cross-link actin and microtubule networks in mammalian muscle.

The mammalian GAS11 gene was first identified as one of several genes upregulated by growth arrest in cultured murine cells. Subsequently, GAS11 was found to be part of a large family of cytoskeleton-associated proteins present in several diverse eukaryotic organisms such as protozoa, algae, drosophila, fish, mice and humans.

The best-characterized member of this family is trypanin from Trypanosoma brucei, a protozoan parasite that causes African sleeping sickness. Trypanin links the trypanosome’s flagellum to the microtubule cytoskeleton and is required for flagellum based cell motility (Hutchings et al., 2002). However, although trypanin has been characterized in African trypanosomes, the function of trypanin related proteins (e.g. GAS11) in other organisms has yet to be investigated.

Mammalian GAS11 contains a well-conserved 143 amino acid domain that localizes to the plus ends of microtubules when expressed as a GFP fusion protein in trypanosomes. This same fusion protein co-localizes with cytoplasmic microtubules in cultured mammalian cells.

Several independent lines of evidence suggest that GAS11 protein plays an important role in mammalian cell physiology. i) Expression of the mouse GAS11 gene is upregulated in quiescent cells; ii) The human GAS11 gene has been identified as a candidate tumor suppressor that is located on a region of chromosome 16 (16q24.3) that is commonly deleted in breast and prostate cancer; iii) The human GAS11 protein contains a novel microtubule-binding domain that co-localizes with microtubules in vivo6.

GAS11 may be particularly important in cardiac and skeletal muscle, as indicated by abundant GAS11 mRNA levels in these tissues. Northern blot analysis reveals two GAS11 transcripts (1.8 kb and 3.4 kb) that are expressed in virtually all human tissues, and a 7.3 kb GAS11 transcript that is highly expressed in skeletal and cardiac muscle.

A fourth 1.4 kb transcript is uniquely expressed in skeletal muscle. The expression of muscle specific GAS11 transcripts suggests that there may be multiple GAS11 isoforms with distinct roles in muscle function.

We are currently investigating the association of GAS11 with actin and microtubules. This work is being performed in collaboration with Dr. Kent L. Hill, Department of Microbiology, Immunology, and Molecular Genetics, UCLA.