Survival of motor neuron 2 (SMN2) is a modifier gene for spinal muscular atrophy (SMA), a neurode... more Survival of motor neuron 2 (SMN2) is a modifier gene for spinal muscular atrophy (SMA), a neurodegenerative disease caused by insufficient SMN protein mostly due to SMN1 defect. SMN2 is nearly identical to SMN1 but unfortunately only able to produce a small amount of SMN protein due to exon 7 skipping. The exon 7-containing SMN2 transcript (SMN2_E7+) can be increased by a dietary compound, curcumin, but the involved molecular changes are not clear. Here we have found that in fibroblast cells of a SMA type II patient, curcumin enhanced the inclusion of SMN2 exon 7. Examination of the potential splicing factors showed that curcumin specifically increased the protein and transcript levels of SRSF1. The increased SRSF1 protein was mainly nuclear and hyperphosphorylated. Interestingly, the curcumin effects on the SMN2 and SRSF1 transcripts were inhibited by a protein deacetylase inhibitor, trichostatin A. Moreover, in support of its role in the SMN2 splicing, knocking down SRSF1 reduced the inclusion of SMN2 exon 7. Thus, curcumin appears to have multiple effects on the SMN2 transcript and its splicing regulators, including the change of alternative splicing and transcript/protein level as well as phosphorylation. Protein deacetylases and phosphatases are likely involved in these effects. Interestingly, the effects all seem to favor production of the SMN2_E7+ transcript in SMA patient cells.
Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism, eliminates premature terminat... more Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism, eliminates premature termination codon-containing (PTC+) transcripts. For instance, it maintains the homeostasis of splicing factors and degrades aberrant transcripts of human genetic disease genes. Here we examine the inhibitory effect on the NMD pathway and consequent increase of PTC+ transcripts by the dietary compound curcumin. We have found that several PTC+ transcripts including that of serine/arginine-rich splicing factor 1 (SRSF1) were specifically increased in cells by curcumin. We also observed a similar curcumin effect on the PTC+ mutant transcript from a Tay–Sachs-causing HEXA allele or from a beta-globin reporter gene. The curcumin effect was accompanied by significantly reduced expression of the NMD factors UPF1, 2, 3A and 3B. Consistently, in chromatin immunoprecipitation assays, curcumin specifically reduced the occupancy of acetyl-histone H3 and RNA polymerase II at the promoter region (− 376 to − 247 nt) of human UPF1, in a time- and dosage-dependent way. Importantly, knocking down UPF1 abolished or substantially reduced the difference of PTC+ transcript levels between control and curcumin-treated cells. The disrupted curcumin effect was efficiently rescued by expression of exogenous Myc–UPF1 in the knockdown cells. Together, our data demonstrate that a group of PTC+ transcripts are stabilized by a dietary compound curcumin through the inhibition of UPF factor expression and the NMD pathway.
Splicing of precursor messenger RNA (pre-mRNA) removes the intervening sequences (introns) and jo... more Splicing of precursor messenger RNA (pre-mRNA) removes the intervening sequences (introns) and joins the expressed regions (exons) in the nucleus, before an intron-containing eukaryotic mRNA transcript can be exported and translated into proteins in the cytoplasm. While some sequences are always included or excluded (constitutive splicing), others can be selectively used (alternative splicing) in this process. Particularly by alternative splicing, up to tens of thousands of variant transcripts can be produced from a single gene, which contributes greatly to the proteomic diversity for such complex cellular functions as 'wiring' neurons in the nervous system. Disruption of this process leads to aberrant splicing, which accounts for the defects of up to 50% of mutations that cause certain human genetic diseases. In this review, we describe the different mechanisms of aberrant splicing that cause or have been associated with neurological diseases.
The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains l... more 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.
Survival of motor neuron 2 (SMN2) is a modifier gene for spinal muscular atrophy (SMA), a neurode... more Survival of motor neuron 2 (SMN2) is a modifier gene for spinal muscular atrophy (SMA), a neurodegenerative disease caused by insufficient SMN protein mostly due to SMN1 defect. SMN2 is nearly identical to SMN1 but unfortunately only able to produce a small amount of SMN protein due to exon 7 skipping. The exon 7-containing SMN2 transcript (SMN2_E7+) can be increased by a dietary compound, curcumin, but the involved molecular changes are not clear. Here we have found that in fibroblast cells of a SMA type II patient, curcumin enhanced the inclusion of SMN2 exon 7. Examination of the potential splicing factors showed that curcumin specifically increased the protein and transcript levels of SRSF1. The increased SRSF1 protein was mainly nuclear and hyperphosphorylated. Interestingly, the curcumin effects on the SMN2 and SRSF1 transcripts were inhibited by a protein deacetylase inhibitor, trichostatin A. Moreover, in support of its role in the SMN2 splicing, knocking down SRSF1 reduced the inclusion of SMN2 exon 7. Thus, curcumin appears to have multiple effects on the SMN2 transcript and its splicing regulators, including the change of alternative splicing and transcript/protein level as well as phosphorylation. Protein deacetylases and phosphatases are likely involved in these effects. Interestingly, the effects all seem to favor production of the SMN2_E7+ transcript in SMA patient cells.
Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism, eliminates premature terminat... more Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism, eliminates premature termination codon-containing (PTC+) transcripts. For instance, it maintains the homeostasis of splicing factors and degrades aberrant transcripts of human genetic disease genes. Here we examine the inhibitory effect on the NMD pathway and consequent increase of PTC+ transcripts by the dietary compound curcumin. We have found that several PTC+ transcripts including that of serine/arginine-rich splicing factor 1 (SRSF1) were specifically increased in cells by curcumin. We also observed a similar curcumin effect on the PTC+ mutant transcript from a Tay–Sachs-causing HEXA allele or from a beta-globin reporter gene. The curcumin effect was accompanied by significantly reduced expression of the NMD factors UPF1, 2, 3A and 3B. Consistently, in chromatin immunoprecipitation assays, curcumin specifically reduced the occupancy of acetyl-histone H3 and RNA polymerase II at the promoter region (− 376 to − 247 nt) of human UPF1, in a time- and dosage-dependent way. Importantly, knocking down UPF1 abolished or substantially reduced the difference of PTC+ transcript levels between control and curcumin-treated cells. The disrupted curcumin effect was efficiently rescued by expression of exogenous Myc–UPF1 in the knockdown cells. Together, our data demonstrate that a group of PTC+ transcripts are stabilized by a dietary compound curcumin through the inhibition of UPF factor expression and the NMD pathway.
Splicing of precursor messenger RNA (pre-mRNA) removes the intervening sequences (introns) and jo... more Splicing of precursor messenger RNA (pre-mRNA) removes the intervening sequences (introns) and joins the expressed regions (exons) in the nucleus, before an intron-containing eukaryotic mRNA transcript can be exported and translated into proteins in the cytoplasm. While some sequences are always included or excluded (constitutive splicing), others can be selectively used (alternative splicing) in this process. Particularly by alternative splicing, up to tens of thousands of variant transcripts can be produced from a single gene, which contributes greatly to the proteomic diversity for such complex cellular functions as 'wiring' neurons in the nervous system. Disruption of this process leads to aberrant splicing, which accounts for the defects of up to 50% of mutations that cause certain human genetic diseases. In this review, we describe the different mechanisms of aberrant splicing that cause or have been associated with neurological diseases.
The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains l... more 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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Papers by Dairong Feng