High-affinity RNA targets of the Survival Motor Neuron protein reveal diverse preferences for sequence and structural motifs

• Published in: Nucleic acids research 2018-11, Vol.46 (20), p.10983-11001
• Main Authors: Ottesen, Eric W, Singh, Natalia N, Luo, Diou, Singh, Ravindra N
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Binding Sites - genetics; Binding, Competitive; Cell Line, Tumor; Humans; Neurons - metabolism; Nucleotide Motifs; Protein Binding; Protein Domains; RNA - chemistry; RNA - genetics; RNA - metabolism; RNA and RNA-protein complexes; Sequence Homology, Nucleic Acid; Survival of Motor Neuron 1 Protein - chemistry; Survival of Motor Neuron 1 Protein - genetics; Survival of Motor Neuron 1 Protein - metabolism; Survival of Motor Neuron 2 Protein - chemistry; Survival of Motor Neuron 2 Protein - genetics; Survival of Motor Neuron 2 Protein - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gky770

Abstract The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA-SMN interactions. Our results reveal that motifs that drive RNA-SMN interactions are diverse and suggest that tight RNA-SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA-SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions.