Regulation of retention of FosB intron 4 by PTB

Department of Cellular and Molecular Pharmacology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.
PLoS ONE (Impact Factor: 3.23). 02/2007; 2(9):e828. DOI: 10.1371/journal.pone.0000828
Source: PubMed


One effect of stressors such as chronic drug administration is that sequence within the terminal exon of the transcription factor FosB is recognized as intronic and removed by alternative splicing. This results in an open-reading-frame shift that produces a translation stop codon and ultimately a truncated protein, termed DeltaFosB. In vitro splicing assays with control and mutated transcripts generated from a fosB mini-gene construct indicated a CU-rich sequence at the 3' end of intron 4 (I4) plays an important role in regulating fosB pre-mRNA splicing due to its binding of polypyrimidine tract binding protein (PTB). PTB binding to this sequence is dependent upon phosphorylation by protein kinase A and is blocked if the CU-rich sequence is mutated to a U-rich region. When this mutated fosB minigene is expressed in HeLa cells, the splicing efficiency of its product is increased compared to wild type. Moreover, transient transfection of PTB-1 in HeLa cells decreased the splicing efficiency of a wild type fosB minigene transcript. Depletion of PTB from nuclear extracts facilitated U2AF65 binding to wild type sequence in vitro, suggesting these proteins function in a dynamic equilibrium to modulate fosB pre-mRNA alternative splicing. These results demonstrate for the first time that phosphorylated PTB promotes intron retention and thereby silences the splicing of fosB I4.

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    • "This intron classifies as an exitron: It is protein-coding and without stop codons, and since its length is not a multiple of three, the stop codon is introduced only upon its splicing. AS in FOSB is regulated by the splicing factor PTB1 competing with U2AF65 for binding to the 3 ′ end of the exitron described as retained intron 4 (Marinescu et al. 2007). Interestingly, EIS in Arabidopsis is suppressed by SUA that interacts with U2AF65, potentially interfering with early spliceosome formation (Sugliani et al. 2010). "
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    • "In Buckley et al. [13], we demonstrated that CIRTs can contain sub-families of SINE elements from the ID family, which confers dendritic localization. Other known case studies suggest regulation of translation efficiency [40], alternate protein isoforms in response to external stimulus [41] and splice variant diversity [29]. We noted above that genes with high intronic sequence retention in neurons as well as BAT and cardiomyocyte cells tend to be enriched for processes involved in protein and mRNA transport and localization along with other cell type specific functions. "
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    ABSTRACT: Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-β mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts). The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the cytoplasm for removing CIRT sequences before translation. We also speculate that release of CIRT sequences prior to translation may form RNA-based signals within the cell potentially comprising a novel class of signaling pathways.
    PLoS ONE 10/2013; 8(10):e76194. DOI:10.1371/journal.pone.0076194 · 3.23 Impact Factor
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    • "Therefore, the IR event was not induced by weak signals of alternative splicing sites (Figure 4). Recent studies have shown that splicing is repressed by the binding of polypyrimidine tract-binding proteins (PTB) to specific sequence motifs (CUCUCU, UUCUCU, UUCCUU, and CUUCUUC), induced by IR events in FOSB [26]. We found that the PTB-binding consensus sequence UUCUCU 16 bp upstream from the 3′ end of the 9th intron was perfectly conserved from hominoid to prosimian (Supplementary Figure  2). "
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    ABSTRACT: The Zinc finger, RAN-binding domain-containing protein 2 (ZRANB2), contains arginine/serine-rich (RS) domains that mediate its function in the regulation of alternative splicing. The ZRANB2 gene contains 2 LINE elements (L3b, Plat_L3) between the 9th and 10th exons. We identified the exonization event of a LINE element (Plat_L3). Using genomic PCR, RT-PCR amplification, and sequencing of primate DNA and RNA samples, we analyzed the evolutionary features of ZRANB2 transcripts. The results indicated that 2 of the LINE elements were integrated in human and all of the tested primate samples (hominoids: 3 species; Old World monkey: 8 species; New World monkey: 6 species; prosimian: 1 species). Human, rhesus monkey, crab-eating monkey, African-green monkey, and marmoset harbor the exon derived from LINE element (Plat_L3). RT-PCR amplification revealed the long transcripts and their differential expression patterns. Intriguingly, these long transcripts were abundantly expressed in Old World monkey lineages (rhesus, crab-eating, and African-green monkeys) and were expressed via intron retention (IR). Thus, the ZRANB2 gene produces 3 transcript variants in which the Cterminus varies by transposable elements (TEs) exonization and IR mechanisms. Therefore, ZRANB2 is valuable for investigating the evolutionary mechanisms of TE exonization and IR during primate evolution.
    Comparative and Functional Genomics 06/2012; 2012:170208. DOI:10.1155/2012/170208 · 2.03 Impact Factor
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