Abstract
The repressor element silencing transcription factor (REST) is a coordinate transcriptional and epigenetic regulator which functions as a tumor suppressor or an oncogene depending on cellular context, and a truncated splice variant REST4 has been linked to various types of cancer. We performed a comprehensive analysis of alternative splicing (AS) of REST by rapid amplification of cDNA ends and PCR amplification of cDNAs from various tissues and cell lines with specific primers. We identified 8 novel alternative exons including an alternate last exon which doubles the REST gene boundary, along with numerous 5′/3′ splice sites and ends in the constitutive exons. With the combination of various splicing patterns (e.g. exon skipping and alternative usage of the first and last exons) that are predictive of altered REST activity, at least 45 alternatively spliced variants of coding and non-coding mRNA were expressed in a species- and cell-type/tissue-specific manner with individual differences. By examining the repertoire of REST pre-mRNA splicing in 27 patients with kidney, liver and lung cancer, we found that all patients without exception showed differential expression of various REST splice variants between paired tumor and adjacent normal tissues, with striking cell-type/tissue and individual differences. Moreover, we revealed that exon 3 skipping, which causes no frame shift but loss of a domain essential for nuclear translocation, was affected by pioglitazone, a highly selective activator of the peroxisome proliferator-activated receptor gamma (PPARγ) which contributes to cell differentiation and tumorigenesis besides its metabolic actions. Accordingly, this study demonstrates an extensive AS of REST pre-mRNA which redefines REST gene boundary and structure, along with a general but differential link between REST pre-mRNA splicing and various types of cancer. These findings advance our understanding of the complex, context-dependent regulation of REST gene expression and function, and provide potential biomarkers and therapeutic targets for cancer.
Highlights
Alternative splicing (AS), a process to differentially link exons in a single precursor mRNA to produce two or more different mature mRNAs, is a major contributor to transcriptome and proteome diversity, with .90% of human genes undergoing AS in a tissue- and developmental stage-specific manner [1,2]
We demonstrate that: 1) repressor element silencing transcription factor (REST) undergoes extensive AS across a gene boundary doubled by a novel last exon (E5), with numerous coding and non-coding mRNAs being formed with a species- and cell-type/tissue-specific expression; 2) numerous REST splice variants, which are caused by various splicing patterns predictive of altered REST activity, are generally but differentially linked to various types of cancer; and 3) exon 3 (E3) skipping, which causes no frame shift but loss of zinc finger motifs (ZFMs)-5 essential for nuclear translocation, is remarkably affected by pioglitazone, a highly selective agonist for peroxisome proliferator-activated receptor gamma (PPARc) which modulates cell differentiation and tumorigenesis besides its metabolic actions
Using a reverse primer E4R1 targeting the proximal exon 4 (E4) paired with the forward primers E1aF1 and E2F1 targeting exons 1a (E1a) and 2 (E2) (Figure 1A and Table 2), respectively, we identified several REST splice variants with E2 and/or E3 skipped (Figure 1B and 1C)
Summary
Alternative splicing (AS), a process to differentially link exons in a single precursor mRNA (pre-mRNA) to produce two or more different mature mRNAs, is a major contributor to transcriptome and proteome diversity, with .90% of human genes undergoing AS in a tissue- and developmental stage-specific manner [1,2]. It is recognized that the coupling between transcription and splicing is crucial for AS regulation [3,4], and that exon-intron junctions or splice sites (SS) are specified by epigenetic modifications dependent on cellular context [4,5]. REST binds to widely distributed genomic regulatory sequences including the repressor element-1 (RE1) by a DNA-binding domain (DBD) which comprises 8 zinc finger motifs (ZFMs), while its effect on gene expression is mediated by two independent repression domains (RD1 and RD2) which directly or indirectly recruit numerous transcriptional and epigenetic cofactors. By recruiting numerous cofactors to target gene loci, REST promotes dynamic, context-dependent chromatin organization and repression/activation of thousands of genes involved in many cellular processes including tumorigenesis, for which it functions as a tumor suppressor or an oncogene depending on cellular context [18,19]. The diverse, contextdependent function of REST is specified by multiple mechanisms including proteasomal degradation, nuclear translocation and premRNA splicing [20,21,22,23], as well as the modulation by non-coding RNAs (ncRNAs) and binding affinity of REST to diverse RE1 and non-RE1 sites [24,25]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
