Epithelial-to-mesenchymal transition (EMT) and its reversal (MET) are crucial cell plasticity programs that act during development and tumor metastasis. We have previously shown that the splicing factor and proto-oncogene SF2/ASF impacts EMT/MET through production of a constitutively active splice variant of the Ron proto-oncogene. Using an in vitro model, we now show that SF2/ASF is also regulated during EMT/MET by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD). Overexpression and small interfering RNA experiments implicate the splicing regulator Sam68 in AS-NMD of SF2/ASF transcripts and in the choice between EMT/MET programs. Moreover, Sam68 modulation of SF2/ASF splicing appears to be controlled by epithelial cell–derived soluble factors that act through the ERK1/2 signaling pathway to regulate Sam68 phosphorylation. Collectively, our results reveal a hierarchy of splicing factors that integrate splicing decisions into EMT/MET programs in response to extracellular stimuli.

Human cyclin D1 is expressed as two isoforms derived by alternate RNA splicing, termed D1a and D1b, which differ for the inclusion of intron 4 in the D1b mRNA. Both isoforms are frequently upregulated in human cancers, but cyclin D1b displays relatively higher oncogenic potential. The splicing factors that regulate alternative splicing of cyclin D1b remain unknown despite the likelihood that they contribute to cyclin D1 oncogenicity. In this study, we report that Sam68, an RNA-binding protein frequently overexpressed in prostate cancer cells, enhances splicing of cyclin D1b and supports its expression in prostate cancer cells. Chromatin immunoprecipitation and RNA coimmunoprecipitation experiments showed that Sam68 is recruited to the human CCND1 gene encoding cyclin D1 and that it binds to cyclin D1 mRNA. Transient overexpression and RNAi knockdown experiments indicated that Sam68 acts to enhance endogenous expression of cyclin D1b. Minigene reporter assays showed that Sam68 directly affected alternative splicing of CCND1 message, with a preference for the A870 allele that is known to favor cyclin D1b splicing. Sam68 interacted with the proximal region of intron 4, and its binding correlated inversely with recruitment of the spliceosomal component U1-70K. Sam68-mediated splicing was modulated by signal transduction pathways that elicit phosphorylation of Sam68 and regulate its affinity for CCND1 intron 4. Notably, Sam68 expression positively correlates with levels of cyclin D1b, but not D1a, in human prostate carcinomas. Our results identify Sam68 as the first splicing factor to affect CCND1 alternative splicing in prostate cancer cells, and suggest that increased levels of Sam68 may stimulate cyclin D1b expression in human prostate cancers. Cancer Res; 70(1); 229–39

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Long noncoding RNAs (lncRNAs) are non-protein coding RNAs regulating gene expression. Although for some lncRNAs a relevant role in hypoxic endothelium has been shown, the regulation and function of lncRNAs is still largely unknown in the vascular physio-pathology. Taking advantage of next-generation sequencing techniques, transcriptomic changes induced by endothelial cell exposure to hypoxia were investigated. Paired-end sequencing of polyadenylated RNA derived from human umbilical vein endothelial cells (HUVECs) exposed to 1% O₂ or normoxia was performed. Bioinformatics analysis identified ≈2000 differentially expressed genes, including 122 lncRNAs. Extensive validation was performed by both microarray and qPCR. Among the validated lncRNAs, H19, MIR210HG, MEG9, MALAT1 and MIR22HG were also induced in a mouse model of hindlimb ischemia. To test the functional relevance of lncRNAs in endothelial cells, knockdown of H19 expression was performed. H19 inhibition decreased HUVEC growth, inducing their accumulation in G1 phase of the cell cycle; accordingly, p21 (CDKN1A) expression was increased. Additionally, H19 knockdown also diminished HUVEC ability to form capillary like structures when plated on matrigel. In conclusion, a high-confidence signature of lncRNAs modulated by hypoxia in HUVEC was identified and a significant impact of H19 lncRNA was shown.

Meiosis requires conserved transcriptional changes, but it is not known whether there is a corresponding set of RNA splicing switches. Here, we used RNAseq of mouse testis to identify changes associated with the progression from mitotic spermatogonia to meiotic spermatocytes. We identified ∼150 splicing switches, most of which affect conserved protein-coding exons. The expression of many key splicing regulators changed in the course of meiosis, including downregulation of polypyrimidine tract binding protein (PTBP1) and heterogeneous nuclear RNP A1, and upregulation of nPTB, Tra2β, muscleblind, CELF proteins, Sam68 and T-STAR. The sequences near the regulated exons were significantly enriched in target sites for PTB, Tra2β and STAR proteins. Reporter minigene experiments investigating representative exons in transfected cells showed that PTB binding sites were critical for splicing of a cassette exon in the Ralgps2 mRNA and a shift in alternative 5′ splice site usage in the Bptf mRNA. We speculate that nPTB might functionally replace PTBP1 during meiosis for some target exons, with changes in the expression of other splicing factors helping to establish meiotic splicing patterns. Our data suggest that there are substantial changes in the determinants and patterns of alternative splicing in the mitotic-to-meiotic transition of the germ cell cycle.

The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditiselegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1^−/− muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1^−/− muscle. Moreover, muscle function was significantly impaired in Rbfox1^−/− muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology.