Poster abstracts
Poster number 21 submitted by Anton Blatnik
Sm-mRNP: Sm-protein rings can assemble on more than snRNAs
Anton J. Blatnik III (Department of Biological Chemistry and Pharmacology, The Ohio State University Wexner Medical Center, Columbus OH), Jacob Slivka, Benjamin Pastore, Wen Tang (Department of Biological Chemistry and Pharmacology, The Ohio State University Wexner Medical Center, Columbus OH), Manu Sanjeev, Caleb Embree (Department of Molecular Genetics, The Ohio State University, Columbus OH), Guramrit Singh (Department of Molecular Genetics, The Ohio State University, Columbus OH), Arthur HM Burghes (Department of Biological Chemistry and Pharmacology, The Ohio State University Wexner Medical Center, Columbus OH)
Abstract:
Spinal muscular atrophy (SMA) is caused by Survival motor neuron (SMN) protein deficiency, resulting from a loss of function mutation or deletion in SMN1 gene and reliance on the less productive SMN2 gene(1). SMN is essential, ubiquitous, and necessary for assembling the Sm-protein rings onto the spliceosomal small nuclear ribonucleic acids (snRNAs) and implicated in assembly of other ribonucleoprotein complexes (RNPs). Thus, why SMN deficiency results in an SMA phenotype is perplexing. We recently identified a mutation in SmF—SmFD37N—that rescues survival in a cell line expressing the SMA-causing SMNE134K mutation, suggesting the lost function of SMNE134K is Sm-ring assembly. Sm-ring assembly is dependent on ATP, SMN, and the presence of an Sm-binding sequence in the snRNA(2). Several groups have determined several classes of RNAs, other than snRNAs, associate with Sm-proteins, but suggest these are nonspecific without testing ATP, SMN, or Sm-site dependence(3). We performed RNA immunoprecipitation followed by next generation sequencing experiments to identify RNAs that co-purify with Sm-proteins in an ATP-dependent manner. Interestingly, several snoRNAs, scaRNAs, and mRNAs are enriched when reactions are supplemented with ATP, suggesting Sm-rings are assembled on these RNAs. In parallel, we identified mRNAs with putative Sm-sites in silico, and found considerable overlap with those mRNAs enriched in the ATP supplemented reactions. Furthermore, the level of enrichment increases with the number of putative Sm-sites indentified within a given mRNA. We are now directly confirming that Sm-rings are assembled on these mRNAs using the methods previously described for snRNAs, testing for ATP, SMN, and Sm-site dependence. These experiments provide direct evidence Sm-rings are not only assembled on spliceosomal and U7 snRNAs, but are also involved in some, currently unknown, mRNA-associated function. This opens the possibility that disruption of both ring-assemby functions—snRNPs and mRNPs—are critical to SMA pathogenesis. This work was supported by CureSMA, The Marshall Heritage Foundation, NICHD, NINDS. All mice were generated at the Ohio State University Transgenic Core and the Transgenic Animal Model Core at the University of Michigan.
References:
(1) Burghes AH, Beattie CE. Nat Rev Neurosci. 2009 Aug;10(8):597-609. (2) Wan L, Battle DJ, Yong J, Gubitz AK, Kolb SJ, Wang J, Dreyfuss G. Mol Cell Biol. 2005 Jul;25(13):5543-51. (3) Lu Z, Guan X, Schmidt CA, Matera AG. Genome Biol. 2014 Jan 7;15(1):R7.
Keywords: SMN, RIP-seq, mRNP