Poster abstracts
Poster number 12 submitted by Anton Blatnik
Using SMN mutants to determine what functions SMN performs and which are critical to SMA
Anton J. Blatnik III (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center), Vicki L. McGovern, Corey Ruhno, Arthur H.M. Burghes (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center), Veronica Pessino, Shawn Driscoll, Samuel Pfaff (Gene Expression Laboratory, The Salk Institute for Biological Studies), Utz Fischer (Department of Biochemistry, The University of Wurzburg), Shibi B. Likhite, Kathrin C. Meyer (Department of Gene Therapy, The Abigail Wexner Research Institute at Nationwide Childrens Hospital)
Abstract:
How survival motor neuron (SMN) protein deficiency results in motor neuron loss in spinal muscular atrophy (SMA) is currently unknown. SMN is an essential protein, whose canonical function is assembling the Sm-ring onto the spliceosomal small nuclear ribonucleic acids (snRNAs), but other functions have been proposed.
We have developed a cell assay that can conditionally delete wild-type SMN production using Cre-recombinase, resulting in cell death. Using these cells, we have determined that all disease causing SMN missense mutations in are incapable of essential function on their own. Additionally, an SMN lacking exon 2B is capable of essential function and snRNP assembly. Residues in exon 2B are reported to bind the COPI subunit alpha-COP, important for transmembrane protein shuttling and a suggested mechanism of RNP trafficking down motor neuron axons. SMA mice injected with an AAV9 expressing the SMN lacking exon 2B show rescued motor units and increased survival, suggesting the alpha-COP association is not critical to SMA.
Also, we have performed a suppressor screen to determine the lost function of the SMNE134K mutation and confirmed a mutant Sm protein as a suppressor. This implicates Sm-assembly as the essential function of SMN and in vitro snRNP assembly assays indicate, together these mutants perform assembly more efficiently than wild-type conditions. We have created SMA mice that transgenically express SMNE134K and will treat them with an AAV9 expressing the Sm-suppressor to determine if Sm-assembly is the critical function of SMN pertaining to SMA. We are also investigating whether the SMNE134K and Sm suppressor combinantion retains fidelity of snRNP assembly.
These experiments will determine what functions SMN performs and whether they play a critical role in the development of SMA. This may reveal a mechanism by which mutations in a ubiquitous RNP assembler gives rise to a cell specific disease phenotype.
References:
Blatnik, A.J., McGovern, V.L., Le, T.T., Iyer, C.C., Kaspar, B.K. and Burghes, A.H.M. (2020) Conditional deletion of SMN in cell culture identifies functional SMN alleles. Hum Mol Genet, 29, 3477-3492.
Custer, S.K., Todd, A.G., Singh, N.N. and Androphy, E.J. (2013) Dilysine motifs in exon 2b of SMN protein mediate binding to the COPI vesicle protein alpha-COP and neurite outgrowth in a cell culture model of spinal muscular atrophy. Hum Mol Genet, 22, 4043-4052.
Li, H., Custer, S.K., Gilson, T., Hao le, T., Beattie, C.E. and Androphy, E.J. (2015) alpha-COP binding to the survival motor neuron protein SMN is required for neuronal process outgrowth. Hum Mol Genet, 24, 7295-7307.
Keywords: SMN, Sm-assembly, suppressor screen