SMN2 exon 7 splicing is inhibited by binding of hnRNP A1 to a common ESS motif that spans the 3' splice site.
Spinal Muscular Atrophy is caused by homozygous loss of SMN1 with phenotypic modulation by SMN2. SMN2 expresses only limited amounts of full-length transcript due to skipping of exon 7 caused by disruption of an SF2/ASF binding ESE. Additionally, hnRNP A1 has been reported to inhibit inclusion of SMN2 exon 7. We previously reported high similarity between the sequence spanning the 3' ss of SMN1 and SMN2 exon 7 and an hnRNP A1 binding ESS, which regulates MCAD exon 5 splicing. We show here that this 3' ss motif indeed functions as a crucial hnRNP A1 binding ESS, which inhibits inclusion of SMN1/2 exon 7 and is antagonized by the SMN1 ESE, but not by the inactive SMN2 sequence. Pull-down experiments revealed a specific interaction between hnRNP A1 and the 3' ss AG-dinucleotide, which could be disrupted by mutations shown to improve splicing in reporter minigenes. Genomic analyses revealed that in the human genome, 3' ss matching the SMN1/2 ESS motif region are much less abundant than 3' ss with a disrupted ESS motif. This indicates that this ESS may be a general splicing inhibitory motif, which binds hnRNP A1 and inhibits exon inclusion by binding to 3' ss harboring this ESS motif.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
Thomas Koed
TK
Doktor
Lisbeth Dahl
LD
Schroeder
Anne
A
Vested
Johan
J
Palmfeldt
Henriette Skovgaard
HS
Andersen
Niels
N
Gregersen
Brage Storstein
BS
Andresen
D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D022821:RNA Splice Sites; D012326:RNA Splicing; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D055540:Survival of Motor Neuron 2 Protein / Q000378:metabolism*
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D022821:RNA Splice Sites; D055540:Survival of Motor Neuron 2 Protein