Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA.
Neurofibromatosis type 1 (NF1) is one of the most common heritable autosomal dominant disorders. Alternative splicing modulates the function of neurofibromin, the NF1 gene product, by inserting the in-frame exon 23a into the region of NF1 mRNA that encodes the GTPase-activating protein-related domain. This insertion, which is predominantly skipped in neurons, reduces the ability of neurofibromin to regulate Ras by 10-fold. Here, we report that the neuron-specific Hu proteins control the production of the short protein isoform by suppressing inclusion of NF1 exon 23a, while TIA-1/TIAR proteins promote inclusion of this exon. We identify two binding sites for Hu proteins, located upstream and downstream of the regulated exon, and provide biochemical evidence that Hu proteins specifically block exon definition by preventing binding of essential splicing factors. In vitro analyses using nuclear extracts show that at the downstream site, Hu proteins prevent binding of U1 and U6 snRNPs to the 5' splice site, while TIAR increases binding. Hu proteins also decrease U2AF binding at the 3' splice site located upstream of exon 23a. In addition to providing the first mechanistic insight into tissue-specific control of NF1 splicing, these studies establish a novel strategy whereby Hu proteins regulate RNA processing.
Zhu
Hui
H
Department of Genetics, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA. hxl47@case.edu
Hinman
Melissa N
MN
Hasman
Robert A
RA
Mehta
Priyesh
P
Lou
Hua
H
D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D051959:ELAV Proteins / Q000378:metabolism; D005091:Exons / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D025542:Neurofibromin 1 / Q000235:genetics*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009928:Organ Specificity / Q000187:drug effects; D016716:PC12 Cells; D011485:Protein Binding / Q000187:drug effects; D012322:RNA Precursors / Q000235:genetics*; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D000072668:Splicing Factor U2AF
D003432:Cross-Linking Reagents; D051959:ELAV Proteins; D025542:Neurofibromin 1; D009687:Nuclear Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D017412:Ribonucleoprotein, U1 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; C000607495:Zrsr1 protein, mouse; C076751:TIAL1 protein, human