Structure and function analysis of Sam68 and hnRNP A1 synergy in the exclusion of exon 7 from SMN2 transcripts

Protein Sci. 2023 Apr;32(4):e4553. doi: 10.1002/pro.4553.

Abstract

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the absence of a functional copy of the Survival of Motor Neuron 1 gene (SMN1). The nearly identical paralog, SMN2, cannot compensate for the loss of SMN1 because exon 7 is aberrantly skipped from most SMN2 transcripts, a process mediated by synergistic activities of Src-associated during mitosis, 68 kDa (Sam68/KHDRBS1) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1. This results in the production of a truncated, nonfunctional protein that is rapidly degraded. Here, we present several crystal structures of Sam68 RNA-binding domain (RBD). Sam68-RBD forms stable symmetric homodimers by antiparallel association of helices α3 from two monomers. However, the details of domain organization and the dimerization interface differ significantly from previously characterized homologs. We demonstrate that Sam68 and hnRNP A1 can simultaneously bind proximal motifs within the central region of SMN2 (ex7). Furthermore, we show that the RNA-binding pockets of the two proteins are close to each other in their heterodimeric complex and identify contact residues using crosslinking-mass spectrometry. We present a model of the ternary Sam68·SMN2 (ex7)·hnRNP A1 complex that reconciles all available information on SMN1/2 splicing. Our findings have important implications for the etiology of SMA and open new avenues for the design of novel therapeutics to treat splicing diseases.

Keywords: RNA binding proteins; STAR family members; Sam68; alternative splicing; hnRNP A1; homodimer; protein-RNA interactions; spinal muscular atrophy; x-ray crystal structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Exons / genetics
  • Heterogeneous Nuclear Ribonucleoprotein A1 / genetics
  • Humans
  • Muscular Atrophy, Spinal* / genetics
  • Muscular Atrophy, Spinal* / metabolism
  • Neurodegenerative Diseases* / genetics
  • RNA Splicing
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Survival of Motor Neuron 2 Protein / genetics
  • Survival of Motor Neuron 2 Protein / metabolism

Substances

  • Heterogeneous Nuclear Ribonucleoprotein A1
  • RNA-Binding Proteins
  • KHDRBS1 protein, human
  • DNA-Binding Proteins
  • Adaptor Proteins, Signal Transducing
  • SMN2 protein, human
  • Survival of Motor Neuron 2 Protein