A role for polyproline motifs in the spinal muscular atrophy protein SMN -Profilins bind to and colocalize with SMN in nuclear gems

Spinal muscular atrophy (SMA) is an autosomal, recessive disorder character ized by the loss of a-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have bee n identified on human chromosome 5q. The corresponding SMN protein is expre ssed ubiquitously. It is coded by seven exons and contains conspicuous prol ine-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the contr ol of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that prof ilins bind to SMN and that this binding depends on its proline-rich motifs, These results were confirmed by coimmunoprecipitation and by in vitro bind ing studies. Two PFN isoforms, I and II, are known, of which II is characte ristic for central nervous system tissue. me show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, S MN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our fin dings suggest that the interaction of PFN II with SMN may be involved in ne uron-specific effects of SMN mutations.