Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways.

Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells.

Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.

Alternative splicing modulates autoinhibition and SH3 accessibility in the Src kinase Fyn.

Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.