Caffeine Regulates Alternative Splicing in a Subset of Cancer-Associated Genes: a Role for SC35

Abstract

Alternative splicing of pre-mRNA contributes significantly to human proteomic complexity, playing a key role in development, gene expression and, when aberrant, human disease onset. Many of the factors involved in alternative splicing have been identified, but little is known about their regulation. Here we report that caffeine regulates alternative splicing of a subset of cancer-associated genes, including the tumor suppressor KLF6. This regulation is at the level of splice site selection, occurs rapidly and reversibly, and is concentration dependent. We have recapitulated caffeine-induced alternative splicing of KLF6 using a cell-based minigene assay and identified a "caffeine response element" within the KLF6 intronic sequence. Significantly, a chimeric minigene splicing assay demonstrated that this caffeine response element is functional in a heterologous context; similar elements exist within close proximity to caffeine-regulated exons of other genes in the subset. Furthermore, the SR splicing factor, SC35, was shown to be required for induction of the alternatively spliced KLF6 transcript. Importantly, SC35 is markedly induced by caffeine, and overexpression of SC35 is sufficient to mimic the effect of caffeine on KLF6 alternative splicing. Taken together, our data implicate SC35 as a key player in caffeine-mediated splicing regulation. This novel effect of caffeine provides a valuable tool for dissecting the regulation of alternative splicing of a large gene subset and may have implications with respect to splice variants associated with disease states.Alternative splicing of pre-mRNA is an essential process by which eukaryotes generate functionally diverse isoforms from a single gene through the selective joining of different exons. This process responds to developmental and environmental cues, contributing substantially to cell-specific and tissue-specific gene expression; it is estimated that over 60% of human genes are alternatively spliced (36). Given the pervasive and profound involvement of alternative splicing in multiple cellular processes, it is clear that the choice of splice site can have major phenotypic consequences, and defects in the splicing pathway are associated with a variety of disease states. Indeed, as many as 50% of human genetic diseases arise from mutations affecting splicing choices (32). For example, it has recently been demonstrated that many cancer-associated genes are regulated by alternative splicing and that a number of splice variants appear to be unique to the malignant state (53, 57). Whether these splice variants play a role in carcinogenesis or tumor maintenance remains to be determined, but their consistent association with the malignant state suggests, at the very least, a clear value as diagnostic indicators and a potential as therapeutic targets (8,22). Therefore, a better understanding of the etiology of these cancer splice variants has become an imperative.Decades of elegant studies have defined the basic splicing mechanism, which requires the interaction of spl...

Methylxanthines Increase Expression of the Splicing Factor SRSF2 by Regulating Multiple Post-transcriptional Mechanisms

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“…Real-time PCR was performed using SYBR Green PCR Master Mix (AB Applied Biosystems/ Life Technologies) and individually designed primer sets. For total SRSF2 mRNA, sense: 5Ј-CTG AGG ACG CTA TGG ATG CCA-3Ј and antisense: 5Ј-GAC TTG GAC TTG GAC CTT CGT-3Ј; for ␤ 2 M, the same as previously reported (29); for FLAG-SRSF2, sense: 5Ј-CGA CTC ACT ATA GGG AGA CC-3Ј and antisense: 5Ј-GAG GTG CGG TAG GTC AGG TT-3Ј; for GFP, sense: 5Ј-GGG TGA AGG TGA TGC AAC ATA C-3Ј and antisense: 5Ј-CTC GCA AAG CAT TGA ACA CCA-3Ј. The final C t value was an average of 3 repeats of each assay, repeated three times.…”

Estrogen receptor alpha polymorphisms: correlation with clinicopathological parameters in breast cancer

“…Accessing PUPASUITE web we observed that the polymorphic allele (A) disrupts the binding sites for the SR proteins SC35, SF2 and SRp40 (nucleolar, serine-rich protein). Overexpression of SRp40 was found during mouse mammary tumorigenesis; aberrant increases in SC35 have been associated with the cancer phenotype and SF2/ASF has been recently identified as a proto-oncogene [48][49][50].…”

Aberrant splicing of the E-cadherin transcript is a novel mechanism of gene silencing in chronic lymphocytic leukemia cells

“…SFRS2 or SC35 is another splicing factor with role in the alternative splicing of the tumor suppressor gene KLF6. 32 This splicing factor is also a transcriptional target of E2F1 and shown to change splice profiles of various apoptotic genes. 33 These data support the notion that alterations of splicing factors contribute to oncogenesis.…”