Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation

• Published in: Nucleic acids research Vol. 40; no. 16; pp. 8059 - 8071
• Main Authors: Cao, Wenguang, Razanau, Aleh, Feng, Dairong, Lobo, Vincent G, Xie, Jiuyong
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.09.2012
• Subjects: Alternative Splicing - drug effects; Amino Acid Sequence; Animals; Base Sequence; Colforsin - pharmacology; Core Binding Factor Alpha 2 Subunit - genetics; Core Binding Factor Alpha 2 Subunit - metabolism; Cyclic AMP-Dependent Protein Kinases - metabolism; Exons; HEK293 Cells; Heterogeneous-Nuclear Ribonucleoprotein K - chemistry; Heterogeneous-Nuclear Ribonucleoprotein K - metabolism; Humans; Molecular Sequence Data; Nervous System Diseases - genetics; Neurites - drug effects; Neurites - ultrastructure; Neurogenesis - genetics; Neurons - cytology; Neurons - metabolism; Nuclear Proteins - metabolism; Nucleotide Motifs; PC12 Cells; Rats; Regulatory Sequences, Ribonucleic Acid; Ribonucleoproteins - metabolism; RNA; RNA Splice Sites; Sequence Alignment; Splicing Factor U2AF; Synaptosomal-Associated Protein 25 - genetics; Synaptosomal-Associated Protein 25 - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gks504

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.