Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development

Abstract

We studied differential splicing of nebulin, a giant filamentous F-actin binding protein (M r ~700-800 kDa) that is found in skeletal muscle. Nebulin spans the thin filament length, its C-terminus is anchored in the Z-disc, and its N-terminal region is located toward the thin filament pointed end. Various lines of evidence indicate that nebulin plays important roles in thin filament and Z-disc structure in skeletal muscle. In the present work we studied nebulin in a range of muscle types during postnatal development and performed transcript studies with a mouse nebulin exon microarray, developed by us, whose results were confirmed by RT-PCR. We also performed protein studies with high-resolution SDS-agarose gels and Western blots, and structural studies with electron microscopy. We found during postnatal development of the soleus muscle major changes in splicing in both the super-repeat region and the Z-disc region of nebulin; interestingly, these changes were absent in other muscle types. Three novel Z-disc exons, previously described in the mouse gene, were upregulated during postnatal development of soleus muscle and this was correlated with a significant increase in Z-disc width. These findings support the view that nebulin plays an important role in Z-disc width regulation. In summary, we discovered changes in both the super-repeat region and the Z-disc region of nebulin, that these changes are muscle-type specific, and that they correlate with differences in sarcomere structure.

Hyperphosphorylation of Mouse Cardiac Titin Contributes to Transverse Aortic Constriction-Induced Diastolic Dysfunction

“…The titin exon microarray experiments were performed as described previously 20 . Details in Supplement.…”

A two-segment model for thin filament architecture in skeletal muscle

“…Indeed, investigations of chicken, mouse, rabbit, and human skeletal muscles have revealed that the nebulin N-terminus is a relatively constant distance (~0.95 µm) from the Z-line in all skeletal muscles, while both Tmod and the actin filament pointed ends are located at diverse distances from the Z-line (ranging from ~1.00 µm to ~1.40 µm, but always further than the nebulin N-terminus) 24, 25, 45–47 . Moreover, the variation in nebulin isoform sizes across muscles appears to be due to variable-length insertions in the nebulin C-terminus region within the Z-line 45, 49, 50 .…”

The Nebulin family: an actin support group

“…Nebulin not only maintains the structure of the Z-disc but also may specify its width. In the mouse soleus muscle, the number of C-terminal nebulin exons expressed increases during development and correlates with an increase in Z-disc width [26]. Nebulin is also essential for the proper contractile function of skeletal muscle; the muscles of nebulin knockout mice produce significantly less force than their wild-type counterparts, which results in death shortly after birth [3, 15, 27].…”