Poster abstracts
Poster number 16 submitted by Anton Blatnik
Identification of Functional SMNs and Suppressors of the Nonfunctional SMNE134K
Anton J Blatnik III (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center), Vicki L. McGovern, Corey Ruhno, Thanh T. Le (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center), Veronica Pessino, Shawn Driscoll, Sam Pfaff (Salk Institute for Biological Studies, Howard Hughes Medical Institute, La Jolla CA), Shibi Likhite, Brian Kaspar (Department of Gene Therapy, The Research Institute at Nationwide Childrens Hospital), Arthur HM Burghes (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center)
Abstract:
Spinal muscular atrophy (SMA) is caused by loss of SMN1 and retention of SMN2, leading to SMN deficiency. SMN functions in assembly of spliceosomal snRNPs and U7 snRNP, however other functions have been proposed. How deficiency of SMN results in SMA is unclear. To determine which functions are important to SMA, we have developed a cell line which can be conditionally deleted for SMN via Cre expression. In these lines, removal of SMN always results in lethality, thus they can be used to which SMN alleles are functional. Bernabo et al reported a cell line in which we determined that one mutation results in exclusion of exon 2B. Surprisingly, this results in a functional protein, as this allele rescues lethality of our cells upon SMN deletion. This cell line was used to characterize the role of SMN in translation, yet does produce functional SMN. We have used our cell lines to assay whether SMN missense mutations have any partial function in the absence of SMN2. We report the missense mutants SMNA2G, SMND44V, SMNA111G, SMNE134K, and SMNT274I are all nonfunctional in cells lacking SMN. To date no missense alleles have partial function in mammals, but instead work in concert with SMN2. Given SMN forms an oligomeric complex, we asked whether two different mutant alleles interact to produce a functional complex via allelic complementation. We have utilized our cell line to analyze this effect in more detail finding complementation between different SMN alleles to occur between several combinations. For example SMNE134K complements SMNA2G, indicating different functional domains between the N-terminus and Tudor domain. Complementation has been analyzed in mice, in which SMNA111G complements SMNT274I and SMNQ282A, rescuing survival, electrophysiology, and snRNP assembly. Furthermore, we have used the SMNE134K mutation in a screen for suppressors. While the SMNE134K mutation never rescues deleted SMN cell lines, ENU mutagenesis yielded five lines which do survive. 4 of these have alterations in SMN associated proteins and one has a mutation in an Sm protein. We reintroduced this Sm mutation into the SMNE134K cell line and confirmed its ability to suppress lethality when SMN is deleted. This implicates Sm assembly as the critical function affected by the SMNE134K mutation.
Keywords: SMN, allelic complementation, suppressor screen