The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7—the logistical centerpiece of LE biology—as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system.

3′-Untranslated region (UTR) shortening of mRNAs via alternative polyadenylation (APA) has important ramifications for gene expression. By using proximal APA sites and switching to shorter 3′-UTRs, proliferating cells avoid miRNA-mediated repression. Such APA and 3′-UTR shortening events may explain the basis of some of the proto-oncogene activation cases observed in cancer cells. In this study, we investigated whether 17 β-estradiol (E2), a potent proliferation signal, induces APA and 3′-UTR shortening to activate proto-oncogenes in estrogen receptor positive (ER+) breast cancers. Our initial probe based screen of independent expression arrays suggested upregulation and 3′-UTR shortening of an essential regulator of DNA replication, CDC6 (cell division cycle 6), upon E2 treatment. We further confirmed the E2- and ER-dependent upregulation and 3′UTR shortening of CDC6, which lead to increased CDC6 protein levels and higher BrdU incorporation. Consequently, miRNA binding predictions and dual luciferase assays suggested that 3′-UTR shortening of CDC6 was a mechanism to avoid 3′-UTR-dependent negative regulations. Hence, we demonstrated CDC6 APA induction by the proliferative effect of E2 in ER+ cells and provided new insights into the complex regulation of APA. E2-induced APA is likely to be an important but previously overlooked mechanism of E2-responsive gene expression.

Alternative polyadenylation (APA) plays a role in gene expression regulation generally by shortening of 3'UTRs (untranslated regions) upon proliferative signals and relieving microRNA-mediated repression. Owing to high proliferative indices of triple negative breast cancers (TNBCs), we hypothesized APA to cause 3'UTR length changes in this aggressive subgroup of breast cancers. Our probe-based meta-analysis approach identified 3'UTR length alterations where the significant majority was shortening events (∼70%, 113 of 165) of mostly proliferation-related transcripts in 520 TNBC patients compared with controls. Representative shortening events were further investigated for their microRNA binding potentials by computational predictions and dual-luciferase assay. In silico-predicted 3'UTR shortening events were experimentally confirmed in patient and cell line samples. To begin addressing the underlying mechanisms, we found CSTF2 (cleavage stimulation factor 2), a major regulator of 3'UTR shortening to be up-regulated in response to epidermal growth factor (EGF). EGF treatment also resulted with further shortening of the 3'UTRs. To investigate the contribution of CSTF2 and 3'UTR length alterations to the proliferative phenotype, we showed pharmacological inhibition of the EGF pathway to lead to a reduction in CSTF2 levels. Accordingly, RNAi-induced silencing of CSTF2 decreased the proliferative rate of cancer cells. Therefore, our computational and experimental approach revealed a pattern of 3'UTR length changes in TNBC patients and a potential link between APA and EGF signaling. Overall, detection of 3'UTR length alterations of various genes may help the discovery of new cancer-related genes, which may have been overlooked in conventional microarray gene expression analyses.

Understanding the extent and functions of m6A is likely to improve our understanding of the complexities of the transcriptome regulation in normal and in disease states.

Advancements in sequencing and transcriptome analysis methods have led to seminal discoveries that have begun to unravel the complexity of cancer. These studies are paving the way toward the development of improved diagnostics, prognostic predictions, and targeted treatment options. However, it is clear that pieces of the cancer puzzle are still missing. In an effort to have a more comprehensive understanding of the development and progression of cancer, we have come to appreciate the value of the noncoding regions of our genomes, partly due to the discovery of miRNAs and their significance in gene regulation. Interestingly, the miRNA-mRNA interactions are not solely dependent on variations in miRNA levels. Instead, the majority of genes harbor multiple polyadenylation signals on their 3 0 UTRs (untranslated regions) that can be differentially selected on the basis of the physiologic state of cells, resulting in alternative 3 0 UTR isoforms. Deregulation of alternative polyadenylation (APA) has increasing interest in cancer research, because APA generates mRNA 3 0 UTR isoforms with potentially different stabilities, subcellular localizations, translation efficiencies, and functions. This review focuses on the link between APA and cancer and discusses the mechanisms as well as the tools available for investigating APA events in cancer. Overall, detection of deregulated APA-generated isoforms in cancer may implicate some proto-oncogene activation cases of unknown causes and may help the discovery of novel cases; thus, contributing to a better understanding of molecular mechanisms of cancer. Mol Cancer Res; 14(6); 507-17. Ó2016 AACR.

MicroRNAs (miRNAs), are ~22 nucleotides long, non-coding RNAs that control gene expression post-transcriptionally by binding to their target mRNA's 3'UTRs (untranslated regions). Due to their roles in various important regulatory processes and pathways, miRNAs have been implicated in disease mechanisms such as tumorigenesis when their expression is deregulated. To date, a significant number of miRNAs and their target messenger RNAs (mRNAs) have been identified and verified. It is generally accepted that miRNAs can potentially bind to many mRNAs, which brings the requirement of validation of these interactions. While understanding that such individual interactions is crucial to delineate the role of a specific miRNA, we took a holistic approach and analyzed global changes in the cell due to expression of a miRNA in a model cell line system. Our model consisted of MCF7 cells stably transfected with miR-125b (MCF7-125b) and empty vector (MCF7-EV). MiR-125b is one of the known down-regulated miRNAs in breast cancers. In this study we examined the global structural changes in MCF7 cells lacking and expressing miR-125b by Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy and investigated the dynamic changes by more sensitive spin-labelling Electron Spin Resonance (ESR) spectroscopy. Our results revealed less RNA, protein, lipid, and glycogen content in MCF7-125b compared to MCF7-EV cells. Membrane fluidity and proliferation rate were shown to be lower in MCF7-125b cells. Based on these changes, MCF7-125b and MCF7-EV cells were discriminated successfully by cluster analysis. Here, we provide a novel means to understand the global effects of miRNAs in cells. Potential applications of this approach are not only limited to research purposes. Such a strategy is also promising to pioneer the development of future diagnostic tools for deregulated miRNA expression in patient samples.

ABSTRACT. Mounting evidence suggests involvement of deregulated microRNA (miRNA) expression during the complex events of tumorigenesis. Among such deregulated miRNAs in cancer, miR-125b expression is reported to be consistently low in breast cancers. In this study, we screened a panel of breast cancer cell lines (BCCLs) for miR-125b expression and detected decreased expression in 14 of 19 BCCLs. Due to the heterogeneity of breast cancers, MCF7 cells were chosen as a model system for ERBB2 independent breast cancers to restore miR-125b expression (MCF7-125b) to investigate the phenotypical and related functional changes. Earlier, miR-125b was shown to regulate cell motility by targeting ERBB2 in ERBB2 overexpressing breast cancer cells. Here we showed decreased motility and migration in miR-125b expressing MCF7 cells, independent of ERBB2. MCF7-125b cells demonstrated profoundly decreased cytoplasmic protrusions detected by phalloidin staining of filamentous actin along with decreased motility and migration behaviors detected by in vitro wound closure and transwell migration assays compared to empty vector transfected cells (MCF7-EV). Among possible numerous targets of miR-125b, we showed ARID3B (AT-rich interactive domain 3B) to be a novel target with roles in cell motility in breast cancer cells. When ARID3B was transiently silenced, the decreased cell migration was also observed. In light of these findings, miR-125b continues to emerge as an interesting regulator of cancer related phenotypes.

MicroRNAs are 20-24-nucleotide-long noncoding RNAs that bind to the 3' UTR (untranslated region) of target mRNAs. Since their discovery, microRNAs have been gaining attention for their ability to contribute to gene expression regulation under various physiological conditions. Consequently, deregulated expression of microRNAs has been linked to different disease states. Here, a brief overview of the canonical and alternative microRNA biogenesis pathways and microRNA functions in biological systems is given based on recent developments. In addition, newly emerging regulatory mechanisms, such as alternative polyadenylation, in connection with microRNA-dependent gene expression regulation are discussed.