Characterization of functional domains of the SMN protein in vivo.
The Survival of Motor Neurons (SMN) is the disease gene of spinal muscular atrophy. We have previously established a genetic system based on the chicken pre-B cell line DT40, in which expression of SMN protein is regulated by tetracycline, to study the function of SMN in vivo. Depletion of SMN protein is lethal to these cells. Here we tested the functionality of mutant SMN proteins by determining their capacity to rescue the cells after depletion of wild-type SMN. Surprisingly, all of the spinal muscular atrophy-associated missense mutations tested were able to support cell viability and proliferation. Deletion of the amino acids encoded by exon 7 of the SMN gene resulted in a partial loss of function. A mutant SMN protein lacking both the tyrosine/glycine repeat (in exon 6) and exon 7 failed to sustain viability, indicating that the C terminus of the protein is critical for SMN activity. Interestingly, the Tudor domain of SMN, encoded by exon 3, does not appear to be essential for SMN function since a mutant deleted of this domain restored cell viability. Unexpectedly, a chicken SMN mutant (DeltaN39) lacking the N-terminal 39 amino acids that encompass the Gemin2-binding domain also rescued the lethal phenotype. Moreover, the level of Gemin2 in DeltaN39-rescued cells was significantly reduced, indicating that Gemin2 is not required for DeltaN39 to perform the essential function of SMN in DT40 cells. These findings suggest that SMN may perform a novel function in DT40 cells.
Wang
J
J
Howard Hughes Medical Institute and the Department of Biochemistry, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Dreyfuss
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002470:Cell Survival; D002645:Chickens / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein; D018076:DNA, Complementary / Q000378:metabolism; D000939:Epitopes; D005091:Exons; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D012190:Retroviridae / Q000235:genetics; D055532:SMN Complex Proteins; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014443:Tyrosine / Q000737:chemistry
D017362:Cyclic AMP Response Element-Binding Protein; D018076:DNA, Complementary; D000939:Epitopes; C108381:GEMIN2 protein, human; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; D014443:Tyrosine; D005998:Glycine