A Novel Alternative Exon of the Caenorhabditis Elegans Lev‐11 Tropomyosin Gene is used to Express a Head‐Muscle‐Specific Isoform

Tropomyosin (TM) is a coiled‐coil dimer that binds along actin filaments in both muscle and non‐muscle cells. TM is involved in cellular processes including muscle contraction, cell motility, and cell division. In mammals, more than 40 isoforms of TM are expressed from four genes through extensive alternative splicing. Isoform‐specific actin cytoskeletal regulation is also achieved by tissue‐specific expression and subcellular localization. We utilized the nematode Caenorhabditis elegans to study functional diversity of TM isoforms. In C. elegans , previous studies reported four TM isoforms from a single gene, lev‐11 , by alternative promotors and splicing. Two high‐molecular‐weight (HMW) isoforms are expressed by use of the upstream promoter and encoded by nine exons; whereas two low‐molecular‐weight isoforms are expressed from the downstream promoter and encoded by seven exons. In these four isoforms, exons 6, 7, and 8 were constitutive. Through analysis of Expressed Sequence Tag sequences and RT‐PCR, we identified a novel exon (Exon 7a) that is alternative to Exon 7b (previously reported as exon 7) and cloned a full‐length cDNA encoding a third HMW isoform containing Exon 7a, which is termed LEV‐11O. We utilized fluorescent reporter mini‐genes and found favored inclusion of exon 7b in the body‐wall muscles of the main body as well as pharyngeal muscles. Exon 7a was alternatively spliced in the body‐wall muscles in the head, with slight overlap where splice variants containing either exon 7a or 7b were both detected. In striated muscles, TM regulates muscle contractility together with the troponin complex. The inhibitory region of troponin I (TNI) is sufficient to stabilize TM in an inhibitory state for actomyosin interaction. Interestingly, although TNI expresses four isoforms from four genes, LEV‐11O and TNI‐3 are both head‐specific isoforms. Mutations in lev‐11 confer resistance to levamisole, an acetylcholine receptor agonist that induces hyper‐contracted muscle paralysis. We examined levamisole sensitivity in nematode strains with a mutation in either lev‐11 exon 7a or 7b, both resulting in amino acid charge reversals. In the presence of levamisole, wild‐type nematodes exhibited muscle hyper‐contraction in the entire body. An amino acid change from within exon 7b (E234K in LEV‐11A) prevented levamisole‐induced hyper‐contraction in the main body but not the head, whereas an amino acid change in exon 7a (E196K in LEV‐11O) prevented levamisole‐induced hyper‐contraction in the head but not in the main body. Sarcomere localization of LEV‐11 proteins was similar in the head and main body and these mutations did not alter localization patterns. These data indicate that exons 7a and 7b have non‐redundant functions for muscle regulation in the head or main body, respectively. Bacterially‐expressed LEV‐11A and LEV‐11O bound actin with similar affinity and mediated inhibition of actomyosin ATPase by a TNI inhibitory peptide. LEV‐11A(E234K) had similar biochemical properties to wild‐type LEV‐11A; however, LEV‐11O(E196K) strongly inhibited actomyosin ATPase even in the absence of the TNI inhibitory peptide. This inhibitory activity was still slightly‐enhanced with the inhibitory peptide, suggesting that enhanced inhibitory effects on actomyosin ATPase is the basis of levamisole resistance. These results suggest that C. elegans utilizes muscle‐type‐specific alternative splicing to produce functionally distinct TM isoforms. Support or Funding Information NIH 2R01AR048615‐11A1