We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.

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Autophagy is a widely studied self-renewal pathway that is essential for degrading damaged cellular organelles or recycling biomolecules to maintain cellular homeostasis, particularly under cellular stress. This pathway initiates with formation of an autophagosome, which is a double-membrane structure that envelopes cytosolic components and fuses with a lysosome to facilitate degradation of the contents. The endosomal sorting complexes required for transport (ESCRT) proteins play an integral role in controlling autophagosome fusion events and disruption to this machinery leads to autophagosome accumulation. Given the central role of autophagy in maintaining cellular health, it is unsurprising that dysfunction of this process is associated with many human maladies including cancer and neurodegenerative diseases. The cell can also rapidly respond to cellular stress through alternative pre-mRNA splicing that enables adaptive changes to the cell’s proteome in response to stress. Thus, alternative pre-mRNA splicing of genes that are involved in autophagy adds another layer of complexity to the cell’s stress response. Consequently, the dysregulation of alternative splicing of genes associated with autophagy and ESCRT may also precipitate disease states by either reducing the ability of the cell to respond to stress or triggering a maladaptive response that is pathogenic. In this review, we summarize the diverse roles of the ESCRT machinery and alternative splicing in regulating autophagy and how their dysfunction can have implications for human disease.

Pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential kinase first identified in the fission yeast Schizosaccharomyces pombe that is evolutionarily conserved from amoebae to animals. During spliceosomal assembly, PRP4K interacts with and phosphorylates PRPF6 and PRPF31 to facilitate the formation of the spliceosome B complex. However, over the past decade additional evidence has emerged that PRP4K has many diverse cellular roles beyond splicing that contribute to tumour suppression and chemotherapeutic responses in mammals. For example, PRP4K appears to play roles in regulating transcription and the spindle assembly checkpoint (SAC), a key pathway in maintaining chromosomes stability and the response of cancer cells to taxane-based chemotherapy. In addition, PRP4K has been revealed to be a haploinsufficient tumour suppressor that promotes aggressive cancer phenotypes when partially depleted. PRP4K is regulated by both the HER2 and estrogen receptor, and its partial loss increases resistance to the taxanes in multiple malignancies including cervical, breast and ovarian cancer. Moreover, ovarian and triple negative breast cancer patients harboring tumours with low PRP4K expression exhibit worse overall survival. The depletion of PRP4K also enhances both Yap and epidermal growth factor receptor (EGFR) signaling, the latter promoting anoikis resistance in breast and ovarian cancer. Finally, PRP4K is negatively regulated during epithelial-to-mesenchymal transition (EMT), a process that promotes increased cell motility, drug resistance and cancer metastasis. Thus, as we discuss in this review, PRP4K likely plays evolutionarily conserved roles not only in splicing but in a number of cellular pathways that together contribute to tumour suppression.

Reduced expression of haploinsufficient tumour suppressor genes is sufficient to alter cellular phenotypes towards carcinogenesis without complete loss of gene expression. As an essential gene, complete expression loss of pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is lethal. However, we demonstrate here that reduction of PRP4K levels by small interfering RNA in the mammary epithelial cell lines HMLE and MCF10A can induce partial epithelial-to-mesenchymal transition (EMT) marked by the retention of epithelial markers such as Zo-1 and E-cadherin, and upregulation of mesenchymal markers such as fibronectin and Zeb1. This partial EMT phenotype in non-transformed PRP4K-depleted cells is associated with greater invasive potential in 3D transwell assays, but either reduces or has no effect on 2D migration examined by scratch assay. This is in contrast to depletion of PRP4K in transformed triple-negative MDA-MB-231 breast cancer cells, which results in enhanced migration in 2D and invasion in 3D. Induction of EMT, using EMT-inducing media containing WNT-5a and TGF-β1 or depletion of eukaryotic translation initiation factor 3e (eIF3e) by shRNA, results in marked reduction of PRP4K expression. EMT induced by eIF3e depletion does not affect the transcription of PRP4K mRNA or turn-over of PRP4K protein, but rather reduces its protein translation. Finally, reduced PRP4K levels after eIF3e depletion correlated with increased YAP activity and nuclear localization, the latter being reversed by overexpression of exogenous PRP4K. Together, these data indicate that PRP4K is a haploinsufficient tumour suppressor negatively regulated by EMT, and that when depleted can induce partial EMT and increased cell invasion.

We have uncovered a novel role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the PML tumour suppressor protein in mammals is SUMOylated and forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish are not SUMOylated and function as cytoplasmic DEDDh exonucleases. In contrast, more closely related avian and turtle PML proteins are predicted to be SUMOylated and localized both to the cytoplasm and to nuclear bodies. We also identified PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases sharing homology to gar PML. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. We also characterized the first non-mammalian TREX1 homologs in axolotl. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus and suppressed CGAS activation. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its role in the assembly of nuclear bodies and the acquisition of SUMO-modification.