Quantitative interactomics to identify cellular pathways affected in spinal muscular atrophy

Abstract

Spinal Muscular Atrophy (SMA) is an inherited neuromuscular disorder that results in muscle weakness, paralysis or - in most cases - death. Caused by insufficient production of the Survival of Motor Neuron (SMN) protein, this ubiquitously expressed protein is involved in numerous cellular processes from snRNP biogenesis to pre-mRNA splicing. Motor neurons are particularly susceptible to low levels of SMN, yet the precise pathomechanism behind their selective death as occurs in SMA remains to be elucidated.

To further understand the disease process, interactomic techniques offer useful methods to identify protein interactions which are disrupted in SMA. Using cell culture models of human cells expressing either SMN or SMNΔ7 (a canonically non-functional isoform of SMN), the proximity biotinylation capabilities of FLAG-tagged TurboID were utilised to identify transient protein interactions whilst FLAG-Immunoprecipitation allowed for simultaneous identification of stronger, covalent interactors. Concurrent SILAC (Stable Isotope Labelling by Amino Acids in Cell Culture) permitted downstream quantitative analysis of proteins identified via mass spectrometry (MS), allowing differential analysis of protein interaction levels.

Utilising bioinformatics tools such as STRING-DB, novel protein interactors – such as GAIT Complex components, Amino Acyl tRNA Synthetases, protein SON and SFXN1 - and affected pathways of potential relevance were obtained from MS datasets and further examined via techniques including colocalization imaging and quantitative western blot to reveal differential interactions between the SMN protein isoforms.