Ribosome profiling of SMN-primed ribosomes and active ribosome profiling from brains of a mouse model of severe SMA

Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality, with an incidence of around 1 in 6,000-10,000 live births. SMA is caused by low levels of full-length survival of motor neuron protein (SMN). We demonstrate that SMN binds to ribosomes and that this interaction is tissue-dependent. We performed ribosome profiling analyses on lysates from P5 wild-type mouse brains exposed to RNase I, from which we isolated SMN-primed ribosomes by immunoprecipitation. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs which are enriched for translational enhancer sequences in the 5'UTR and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Additionally, we analyzed the positioning of active ribosomes using the Active-RiboSeq method based on RiboLace and compared early symptomatic SMA mouse brains to age-matched controls. We found that loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins involved in motor neuron function and stability. Keywords: motor neuron disease, spinal muscular atrophy, SMA, SMN, SMN1, survival of motor neuron, SMN-primed ribosome profiling, translation, RiboLace, Ribo-Seq, active translation, ribosome heterogeneity Overall design: We performed ribosome profiling of SMN-primed ribosomes in brain extracts from control mice at P5 (Smn+/-; SMN2tg/0). Experiments were performed in biological triplicate. In parallel, samples from immunoprecipitation with IgG were used as controls for possible unspecific isolation of ribosome protected fragments. Additionally, we performed active ribosome profiling with RiboLace (PMID: 30355487) and ribosome profiling in SMA early symptomatic mouse brains (Smn-/-; SMN2tg/0). Experiments were performed in biological duplicate. Ribosome profiling of age-matched controls were deposited under accessions GSE102354 (RiboLace) and GSE102318 (ribosome profiling).