Increase of a group of PTC+ transcripts by curcumin through inhibition of the NMD pathway

https://doi.org/10.1016/j.bbagrm.2015.04.002Get rights and content

Highlights

  • Curcumin increases the level of a group of NMD transcripts including SRSF1 and HEXA.

  • Curcumin inhibits the transcript and protein levels of NMD factors including UPF1.

  • Curcumin inhibits acetyl-histone and RNA polymerase II occupancy at hUPF1 promoter.

  • Curcumin effect on NMD transcripts is verified by hUPF1 knockdown/rescue assay.

Abstract

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.

Introduction

Nonsense-mediated mRNA decay (NMD) eliminates transcripts with a premature termination codon (PTC) that is located more than 50–55 nucleotides upstream of the last exon–exon junction [1], [2]. The PTC is recognized in a translation-coupled surveillance to induce the assembly of NMD factors including UPF1–3 proteins to the target mRNA [3], [4], [5]. PTC-containing (PTC+) transcripts thus marked by the NMD factors decay through SMG6-mediated endonucleolytic cleavage or SMG5–SMG7-mediated deadenylation and/or decapping pathway [5], [6], [7], [8], [9], with the former pathway appearing to be predominant in mammalian cells [10], [11].

In addition to transcriptome-wide quality control [12], [13], NMD also regulates gene expression by targeting RNA transcripts that encode full-length functional proteins, thereby influencing various biological or disease processes [14], [15]. Large-scale analyses revealed that one-third of splice variants contain PTCs and are likely targets of NMD [16]. Particularly, a group of splicing factors autoregulate their homeostasis through alternative splicing-coupled NMD [15], [17], [18], [19], [20], [21], including SRSF1 [19].

NMD also appears to play an important role in the pathogenesis of many genetic diseases. It is estimated that about a third of disease-causing mutations result in PTC+ transcripts [14], [22], [23], [24], including the HEXA (hexosaminidase A) gene of the Tay–Sachs disease [25], [26]. Besides these cis-acting mutation-induced PTCs of disease-causing genes, autoregulated splicing-induced PTCs of splicing factors likely also modulate human genetic diseases, such as that of SRSF1 on the SMN2 exon 7 in Spinal Muscular Atrophy (SMA) [19], [27], [28]. For promoting the translation-read-through of PTCs, chemicals have been identified to enhance translation of mutant transcripts of disease genes [29], [30], but dietary compounds that could increase the levels of these transcripts have not been reported.

Curcumin is a dietary polyphenolic compound enriched in the turmeric root with therapeutic potential for a number of diseases [31]. It specifically inhibits histone acetyltransferase (HAT) p300/CBP to regulate gene transcription [32]. Curcumin also regulates the expression of splicing factors in SMA [33], [34], but the underlying molecular basis is unknown. We have been mainly studying the regulation of alternative splicing by extracellular factors [35]. In investigating the effect of curcumin on splicing factors, we have found that curcumin stabilizes PTC+ transcripts by inhibiting the expression of NMD factors and NMD. Here we report the findings from this study.

Section snippets

Curcumin increases the PTC+ variant transcripts of SRSF1

In studying the changes of splicing factors by extracellular factors, we observed that curcumin specifically increased all or some of the PTC+ transcripts of SRSF1 in several cell lines, including BJ301J dermal fibroblast cells, NSC-34 and HEK293T cells, in a time-dependent manner (Supplementary Fig. S1). This change was accompanied by a decrease in the full-length SRSF1 variant 1, likely due to the autoregulated splicing and conversion of this SRSF1 variant to shorter ones as previously

Discussion

There have been no dietary compounds shown to inhibit the expression of NMD factors. CHX, emetine and puromycin can prevent NMD [46], but they are global inhibitors of translation. Wortmannin and caffeine have also been reported to inhibit NMD but through inhibiting UPF1 phosphorylation as inhibitors of PI3K-related protein kinases [47], [48]. The curcumin inhibition of the expression of NMD factors allows both the elevation of target mRNA levels and protein translation that are impossible by

Plasmids, lentivirus production and transduction

NMD reporter plasmid pmCMV-Gl (Norm or 39Ter) and the reference plasmid phCMV-MUP were kindly provided by Dr. Lynne E. Maquat, University of Rochester, Rochester, NY [39], [40], [59]. Plasmids for shRNA expression against 3′UTR of the UPF1 gene (pLKO.1-shUPF1, RHS3979-9589662, mature antisense: 5′-TTA TTA CCC AGA ATA AGA TGC-3′) and human UPF1 cDNA (MHS6278-202758993) were obtained from GE Dharmacon. Human UPF1 ORF with N-terminal Myc tag (Myc–UPF1) was created by PCR amplification and was

Statistical analysis

Data were analyzed by two-tailed Student's t-test. A p value of less than 0.05 was considered significant.

Conflict of interest statement

None declared.

Acknowledgements

We sincerely thank the patient and his family for donating the SMA skin biopsy sample and Ms. Weimin Zhang for help in establishing the primary culture of BJ301J; Dr. Lynne E. Maquat for plasmids pmCMV-Gl (Norm and 39Ter) and phCMV-MUP; Dr. Neil Cashman for the NSC-34 cells, Dr. Suresh Mishra for the C2C12 cells; Drs. Aleh Razanau, Wenguang Cao and James Davie for helpful discussions. This work was supported by a Manitoba Research Chair Fund and in part by a Canadian Institutes of Health

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