Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.
The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage.
Department of Physiology, University of Manitoba, Winnipeg, MB R3E 0J9, Canada.
Wenguang
W
Cao
Aleh
A
Razanau
Dairong
D
Feng
Vincent G
VG
Lobo
Jiuyong
J
Xie
D017398:Alternative Splicing / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology*; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005091:Exons; D057809:HEK293 Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009422:Nervous System Diseases / Q000235:genetics; D016501:Neurites / Q000187:drug effects / Q000648:ultrastructure; D055495:Neurogenesis / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D059372:Nucleotide Motifs; D016716:PC12 Cells; D022821:RNA Splice Sites; D051381:Rats; D038621:Regulatory Sequences, Ribonucleic Acid; D012261:Ribonucleoproteins / Q000378:metabolism; D016415:Sequence Alignment; D000072668:Splicing Factor U2AF; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism
D050676:Core Binding Factor Alpha 2 Subunit; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D009687:Nuclear Proteins; D022821:RNA Splice Sites; D038621:Regulatory Sequences, Ribonucleic Acid; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; D050825:Synaptosomal-Associated Protein 25; C000607494:U2AF2 protein, human; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases