hnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing

Hum Mol Genet. 2007 Dec 15;16(24):3149-59. doi: 10.1093/hmg/ddm276. Epub 2007 Sep 19.

Abstract

Homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1) causes spinal muscular atrophy. SMN1 has been duplicated in humans to create SMN2, which produces a low level of functional SMN protein. However, most SMN2 transcripts lack exon 7, resulting in a non-functional protein. A single nucleotide difference near the 5' end of exon 7 largely accounts for SMN2 exon 7 skipping, an effect that has been attributed to loss of an exonic splicing enhancer (ESE) dependent on the SR protein splicing factor ASF/SF2 or to the creation of an exonic splicing silencer (ESS) element that functions by binding of the splicing repressor hnRNP A1. Our earlier experiments favored the latter mechanism and here we provide further evidence supporting the ESS model. We demonstrate that the striking effect of hnRNP A1 depletion on SMN2 exon 7 splicing is specific, as hnRNP A1 depletion has little or no effect on other inefficient splicing events tested, and ASF/SF2 depletion does not affect SMN1/2 splicing. By two different methods, we find a strong and specific interaction of hnRNPA1 with SMN2 exon 7 and only weak and equivalent interactions between ASF/SF2 and other SR proteins with the 5' ends of SMN1 and SMN2 exon 7. Finally, we describe two disease-related exon-skipping mutations that create hnRNP A1 binding sites, but show that splicing can be restored only modestly or not at all by hnRNP A1 depletion. Together our results provide strong support for the idea that SMN2 exon 7 splicing is repressed by an hnRNPA1-dependent ESS, but also indicate that creation of such elements is context-dependent.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alternative Splicing* / drug effects
  • Binding Sites
  • Cells, Cultured
  • Cyclic AMP Response Element-Binding Protein / genetics*
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Exons*
  • Fibrillins
  • Genes, BRCA1
  • Genetic Diseases, Inborn / genetics
  • HeLa Cells
  • Heterogeneous Nuclear Ribonucleoprotein A1
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B / metabolism
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B / physiology*
  • Humans
  • Microfilament Proteins / genetics
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / physiology
  • Point Mutation
  • Protein Binding
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / pharmacology
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • RNA-Binding Proteins / physiology
  • Regulatory Elements, Transcriptional
  • Repressor Proteins / metabolism
  • Repressor Proteins / physiology*
  • SMN Complex Proteins
  • Serine-Arginine Splicing Factors
  • Substrate Specificity
  • Survival of Motor Neuron 1 Protein
  • Survival of Motor Neuron 2 Protein

Substances

  • Cyclic AMP Response Element-Binding Protein
  • Fibrillins
  • Heterogeneous Nuclear Ribonucleoprotein A1
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B
  • Microfilament Proteins
  • Nerve Tissue Proteins
  • Nuclear Proteins
  • RNA, Messenger
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Repressor Proteins
  • SMN Complex Proteins
  • SMN1 protein, human
  • SMN2 protein, human
  • Survival of Motor Neuron 1 Protein
  • Survival of Motor Neuron 2 Protein
  • hnRNPA1 protein, human
  • Serine-Arginine Splicing Factors