In Vivo Selection of Kinase-responsive RNA Elements Controlling Alternative Splicing

Li, Hongzhao; Liu, Guodong; Yu, Jiankun; Cao, Wenguang; Lobo, Vincent G.; Xie, Jiuyong

doi:10.1074/jbc.m900393200

Cited by 19 publications

(24 citation statements)

References 50 publications

(72 reference statements)

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“…These reporters were transfected into HEK293T cells, and their responses to coexpressed Flag-PKA or its kinase-dead mutant Flag-PKAm, as used previously (32), were examined by semiquantitative RT-PCR. From these preliminary tests, the only observed effect that is consistent with the endogenous exon changes in PC12 cells was a repression by PKA in several reporters containing exon 5a only (Figure 1D).…”

Section: Resultsmentioning

confidence: 99%

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

Cao

Razanau

Feng

et al. 2012

Nucleic Acids Research

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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.

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Section: Resultsmentioning

confidence: 99%

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

Cao

Razanau

Feng

et al. 2012

Nucleic Acids Research

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“…Apart from regulation of transcription, little is known about the function of the C subunit in the nucleus. It is known that PKA phosphorylates several splicing factors and is involved in the pre-mRNA splicing (30,38,39). We recently found that in AD brain, the activity of PKA is down-regulated as a result of proteolysis of the regulatory subunit by over-activated calpain I (40).…”

mentioning

confidence: 99%

Cyclic AMP-dependent Protein Kinase Regulates the Alternative Splicing of Tau Exon 10

Shi

Qian

Yin

et al. 2011

Journal of Biological Chemistry

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Hyperphosphorylation and deposition of tau into neurofibrillary tangles is a hallmark of Alzheimer disease (AD).Alternative splicing of tau exon 10 generates tau isoforms containing three or four microtubule binding repeats (3R-tau and 4R-tau), which are equally expressed in adult human brain. Dysregulation of exon 10 causes neurofibrillary degeneration. Here, we report that cyclic AMP-dependent protein kinase, PKA, phosphorylates splicing factor SRSF1, modulates its binding to tau pre-mRNA, and promotes tau exon 10 inclusion in cultured cells and in vivo in rat brain. PKA-C␣, but not PKA-C␤, interacts with SRSF1 and elevates SRSF1-mediated tau exon 10 inclusion. In AD brain, the decreased level of PKA-C␣ correlates with the increased level of 3R-tau. These findings suggest that a downregulation of PKA dysregulates the alternative splicing of tau exon 10 and contributes to neurofibrillary degeneration in AD by causing an imbalance in 3R-tau and 4R-tau expression.Tau is a neuronal microtubule-associated protein, the function of which is to stimulate microtubule assembly and stabilize microtubules. Hyperphosphorylation of tau leads to its aggregation into neurofibrillary tangles, a hallmark of Alzheimer disease (AD) 2 and related neurodegenerative diseases called tauopathies (1-4). Adult human brain expresses six different tau isoforms from a single gene by alternative splicing of exons 2, 3, and 10 of its pre-mRNA (5). The exon 10 encodes the second microtubule binding repeat (6). Alternative splicing of exon 10 generates tau with three or four microtubule binding repeats (3R-tau or 4R-tau), which is under developmental and cell type-specific regulation. Only 3R-tau is expressed during embryogenesis, whereas 3R-tau and 4R-tau are expressed in approximately equal amounts in adult human brain (6, 7). Several mutations in tau gene result in either an increase or a decrease in 4R-tau expression and cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), one of the tauopathies (8). Thus, alteration in the 3R-tau/ 4R-tau ratio is sufficient to trigger neurodegeneration in frontotemporal dementia and might also play a role in other neurodegenerative disorders such as Pick's disease, progressive nuclear palsy, or corticobasal degeneration in which the 3R-tau/4R-tau ratio is markedly altered (9 -12). Thus, the regulation of alternative splicing of human tau exon 10 has been of critical interest. However, results of studies of the alternative splicing of tau exon 10 in AD brain have been contradictory (13)(14)(15). Recent studies have shown that aggregation and deposition of 3R-tau may be associated with more advanced stages (16,17).Alternative splicing of tau exon 10 is regulated by several trans-acting factors, including serine-and arginine-rich (SR) proteins, and their phosphorylation (18 -24). Splicing factor 2/alternative splicing factor (ASF/SF2), now named SRSF1 (serine/arginine-rich splicing factor 1) (25), is a prototypical SR protein that participates in both constitutive and alternativ...

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“…Furthermore, coexpression of PKA dramatically elevated phosphorylation levels of these two transcriptional activators. In contrast, the PKA dead kinase (K73E and K169E) (46) failed to promote phosphorylation of both CLOCK and BMAL1 in the same procedure of experiments ( Fig. 3B).…”

Section: Both Clock and Bmal1 Are Pka Targets And Cnot1 Promotes Assomentioning

confidence: 72%

CNOT1 mediates phosphorylation via Protein kinase A on the circadian clock

Zhang

Qin

Feng

et al. 2019

Preprint

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At the core of the mammalian circadian feedback loop, CLOCK (NPAS2)-BMAL1 is the positive element to activate transcription of downstream genes encoding the negative elements PERs and CRYs. Here we show that CNOT1 associates with both CLOCK and BMAL1, promotes their phosphorylation and increases their protein stability, and in turn inhibits the transcriptional activity of CLOCK-BMAL1. Expression of either CLOCK, BMAL1 or CNOT1 could interact with endogenous Protein Kinase A (PKA) as assessed by co-immunoprecipitation (Co-IP) and kinase assays. PKA could phosphorylate CLOCK and BMAL1 and this was promoted by CNOT1. Genetic deletion of PKA-Cα by CRISPR-Cas9 results in a longer period of the circadian rhythm; while overexpression of PKA-Cα induces a shorter period. Furthermore, we demonstrate that CNOT1 associates with CLOCK and BMAL1 in the mouse liver and promotes their phosphorylation. PER2, but not CRY2, is also a PKA target. Our results suggest that CNOT1 and PKA play a critical role in the mammalian circadian clock, revealing a conserved function in eukaryotic circadian regulations.

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In Vivo Selection of Kinase-responsive RNA Elements Controlling Alternative Splicing

Cited by 19 publications

References 50 publications

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

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

Cyclic AMP-dependent Protein Kinase Regulates the Alternative Splicing of Tau Exon 10

CNOT1 mediates phosphorylation via Protein kinase A on the circadian clock

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