Gene conversion facilitates the adaptive evolution of self-resistance in highly toxic newts

Tarichanewts contain high concentrations of the deadly toxin TTX as an antipredator defense, requiring them to be physiologically resistant to their own toxin. Here, we reconstruct the origins of TTX self-resistance by sequencing the voltage-gated sodium channel (SCNA) gene family, the target of TTX, in newts and related salamanders. We show that extreme resistance in newts consists of a mixture of ancient changes and lineage-specific substitutions and that the nonsynonymous substitution rate is elevated in newts, suggesting positive selection. We also identify a novel exon duplication withinSCN4Aencoding an expressed TTX-binding site. Two resistance-conferring changes within newts appear to have spread via nonallelic gene conversion: in one case, one codon was copied between paralogs, and in the second, multiple substitutions were homogenized between the duplicate exons ofSCN4A. Our results demonstrate that gene conversion can accelerate the coordinated evolution of gene families in response to selection.

Androgen receptor and its splice variant, AR-V7, differentially induce mRNA splicing in prostate cancer cells

Prostate cancer (PCa) is dependent on the androgen receptor (AR). Advanced PCa is treated with an androgen deprivation therapy-based regimen; tumors develop resistance, although they typically remain AR-dependent. Expression of constitutively active AR variants lacking the ligand-binding domain including the variant AR-V7 contributes to this resistance. AR and AR-V7, as transcription factors, regulate many of the same genes, but also have unique activities. In this study, the capacity of the two AR isoforms to regulate splicing was examined. RNA-seq data from models that endogenously express AR and express AR-V7 in response to doxycycline were used. Both AR isoforms induced multiple changes in splicing and many changes were isoform-specific. Analyses of two endogenous genes, PGAP2 and TPD52, were performed to examine differential splicing. A novel exon that appears to be a novel transcription start site was preferentially induced by AR-V7 in PGAP2 although it is induced to a lesser extent by AR. The previously described AR induced promoter 2 usage that results in a novel protein derived from TPD52 (PrLZ) was not induced by AR-V7. AR, but not AR-V7, bound to a site proximal to promoter 2, and induction was found to depend on FOXA1.

Binding of CCCTC-Binding Factor (CTCF) to the Minute Virus of Mice Genome Is Important for Proper Processing of Viral P4-Generated Pre-mRNAs

Specific chromatin immunoprecipitation of salt-fractionated infected cell extracts has demonstrated that the CCCTC-binding factor (CTCF), a highly conserved, 11-zinc-finger DNA-binding protein with known roles in cellular and viral genome organization and gene expression, specifically binds the genome of Minute Virus of Mice (MVM). Mutations that diminish binding of CTCF to MVM affect processing of the P4-generated pre-mRNAs. These RNAs are spliced less efficiently to generate the R1 mRNA, and definition of the NS2-specific exon upstream of the small intron is reduced, leading to relatively less R2 and the generation of a novel exon-skipped product. These results suggest a model in which CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of the P4-generated pre-mRNA.

Full-length-transcriptomic analysis in mice and human heart using Single-Molecule Real-time Sequencing (SMRT) identified 15 novel isoforms and a novel promoter region of PGC1-alpha

Background Alternative splicing is a process by which exons within a pre-mRNA are joined or skipped, resulting in isoforms being encoded by a single gene. Alternative Splicing affecting transcription factors may have substantial impact on cellular dynamics. The PPARG Coactivator 1 Alpha (PGC1-α), is a major modulator in energy metabolism. Data from murine skeletal muscle revealed distinctive isoform patterns giving rise to different phenotypes, i.e. mitogenesis and hypertrophy. Here, we aimed to establish a complete dataset of isoforms in murine and human heart applying single-molecule real-time (SMRT)-sequencing as novel approach to identify transcripts without need for assembly, resulting in true full-length sequences. Moreover, we aimed to unravel functional relevance of the various isoforms during experimental ischemia reperfusion (I/R). Methods RNA-Isolation was performed in murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), followed by library preparation and SMRT-Sequencing. Bioinformatic analysis was done using a modified IsoSeq3-Pipeline and OS-tools. Identification of PGC1-α isoforms was fulfilled by similarity search against exonic sequences within the full-length, non-concatemere (FLNC) reads. Isoforms with Open-Reading-Frame (ORF) were manually curated and validated by PCR and Sanger-Sequencing. I/R was induced by ligature of the LAD for 45 min in mice on standard chow as well as on high-fat-high-sucrose diet. Area At Risk (AAR) and remote tissue were collected three and 16 days after I/R or sham-surgery (n=4 per time point). Promotor patterns were analyzed by qPCR. Results Deciphering the full-length transcriptome of murine and human heart resulted in ∼60000 Isoforms with 99% accuracy on mRNA-sequence. Focusing on murine PGC1-α-isoforms we discovered and verified 15 novel transcripts generated by hitherto unknown splicing events. Additionally, we identified a novel Exon 1 originating between the known promoters followed by a valid ORF, suggesting the discovery of a novel promoter. Remarkably, we found a homologous novel Exon1 in human heart, suggesting conservation of the postulated promoter. In I/R the AAR exhibited a significant lower expression of established and novel promoters compared to remote under standard chow 3d post I/R. 16d post I/R, the difference between AAR & Remote equalized in standard chow while remaining under High-Fat-Diet. Conclusion Applying SMRT-technique, we generated the first time a complete full-length-transcriptome of the murine and human heart, identifying 15 novel potentially coding transcripts of PGC1-α and a novel exon 1. These transcripts are differentially regulated in experimental I/R in AAR and remote myocardium, suggesting transcriptional regulation and alternative splicing modulating PGC1-α function in heart. Differences between standard chow and high fat diet suggest impact of impaired glucose metabolism on regulatory processes after myocardial infarction. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

Further identification of a 140bp sequence from amid intron 9 of human FMR1 gene as a new exon

Background: the disease gene of fragile X syndrome, FMR1 gene, encodes fragile X mental retardation protein (FMRP). The alternative splicing (AS) of FMR1 can affect the structure and function of FMRP. However, the biological functions of alternatively spliced isoforms remain elusive. In a previous study, we identified a new 140 bp exon from the intron 9 of human FMR1 gene. In this study, we further examined the biological functions of this new exon and its underlying signaling pathways. Results: qRT-PCR results showed that this novel exon is commonly expressed in the peripheral blood of normal individuals. Comparative genomics showed that sequences paralogous to the 140 bp sequence only exist in the genomes of primates. To explore the biological functions of the new transcript, we constructed recombinant eukaryotic expression vectors and lentiviral overexpression vectors. Results showed that the spliced transcript encoded a truncated protein which was expressed mainly in the cell nucleus. Additionally, several genes, including the BEX1 gene involved in mGluR-LTP or mGluR-LTD signaling pathways were significantly influenced when the truncated FMRP was overexpressed. Conclusions: our work identified a new exon from amid intron 9 of human FMR1 gene with wide expression in normal healthy individuals, which emphasizes the notion that the AS of FMR1 gene is complex and may in a large part account for the multiple functions of FMRP.