Modulation and function of Pumilio proteins in cancer.

Post-transcriptional regulation is involved in tumorigenesis, and in this control, RNA-binding proteins are the main protagonists. Pumilio proteins are highly conserved RNA-binding proteins that regulate many aspects of RNA processing. The dysregulation of Pumilio expression is associated with different types of cancer. This review summarizes the roles of Pumilio 1 and Pumilio 2 in cancer and discusses the factors that account for their distinct biological functions. Pumilio levels seem to be related to tumor progression and poor prognoses in some kinds of tumors, such as lung, pancreatic, prostate, and cervical cancers. Pumilio 1 is associated with cancer proliferation, migration, and invasion, and so is Pumilio 2, although there are contradictory reports regarding the latter. Furthermore, the circular RNA, circPUM1, has been described as a miRNAs sponge, regulating miRNA involved in the cell cycle. The expression and function of Pumilio proteins depend on the fine adjustment of a set of modulators, including miRNAs, lncRNAs, and circRNAs; this demonstrates that Pumilio plays an important role in tumorigenesis through a variety of regulatory axes.

Downregulation of the RNA-binding protein PUM2 facilitates MSC-driven bone regeneration and prevents OVX-induced bone loss.

BACKGROUND: Although mRNA dysregulation can induce changes in mesenchymal stem cell (MSC) homeostasis, the mechanisms by which post-transcriptional regulation influences MSC differentiation potential remain understudied. PUMILIO2 (PUM2) represses translation by binding target mRNAs in a sequence-specific manner. METHODS: In vitro osteogenic differentiation assays were conducted using human bone marrow-derived MSCs. Alkaline phosphatase and alizarin red S staining were used to evaluate the osteogenic potential of MSCs. A rat xenograft model featuring a calvarial defect to examine effects of MSC-driven bone regeneration. RNA-immunoprecipitation (RNA-IP) assay was used to determine the interaction between PUM2 protein and Distal-Less Homeobox 5 (DLX5) mRNA. Ovariectomized (OVX) mice were employed to evaluate the effect of gene therapy for postmenopausal osteoporosis. RESULTS: Here, we elucidated the molecular mechanism of PUM2 in MSC osteogenesis and evaluated the applicability of PUM2 knockdown (KD) as a potential cell-based or gene therapy. PUM2 level was downregulated during MSC osteogenic differentiation, and PUM2 KD enhanced MSC osteogenic potential. Following PUM2 KD, MSCs were transplanted onto calvarial defects in 12-week-old rats; after 8 weeks, transplanted MSCs promoted bone regeneration. PUM2 KD upregulated the expression of DLX5 mRNA and protein and the reporter activity of its 3'-untranslated region. RNA-IP revealed direct binding of PUM2 to DLX5 mRNA. We then evaluated the potential of adeno-associated virus serotype 9 (AAV9)-siPum2 as a gene therapy for osteoporosis in OVX mice. CONCLUSION: Our findings suggest a novel role for PUM2 in MSC osteogenesis and highlight the potential of PUM2 KD-MSCs in bone regeneration. Additionally, we showed that AAV9-siPum2 is a potential gene therapy for osteoporosis.

RNA-binding protein PUM2 promotes T-cell acute lymphoblastic leukemia via competitively binding to RBM5 3'UTR with miR-28-5p.

OBJECTIVE: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy, and T-ALL patients are prone to early disease relapse and suffer from poor outcomes. The crucial function of RNA-binding proteins (RBPs) has been reported in the progression of cancers by regulating the expression of transcripts. This study aimed to reveal the role and molecular regulatory mechanism of RBP Pumilio2 (PUM2) in T-ALL. METHODS: The expression of genes was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The viability, proliferation, and apoptosis of T-ALL cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine, and flow cytometry analysis. Luciferase reporter, RNA pulldown, and RNA immunoprecipitation assays were performed to confirm the binding of PUM2 to RBM5. The combination between RNA-binding motif protein 5 (RBM5) and microRNA (miR)-28-5p was validated using luciferase reporter assay. RESULTS: Our data revealed that PUM2 was highly expressed in T-ALL blood samples and cell lines. PUM2 knockdown suppressed the proliferation but accelerated the apoptosis of T-ALL cells in vitro. Additionally, RBM5 exhibited a low expression level in T-ALL samples and cells. PUM2 negatively regulated RBM5 via targeting its 3'untranslated region (3'UTR). Moreover, PUM2 competitively bound to RBM5 3'UTR with miR-28-5p. Rescue experiments showed that RBM5 knockdown reversed the anti-tumor effects mediated by PUM2 knockdown in T-ALL cells. CONCLUSION: PUM2 plays as a novel oncogenic RBP in T-ALL by competitively binding to RBM5 mRNA with miR-28-5p.

Pumilio2 Promotes Growth of Mature Neurons.

RNA-binding proteins (RBPs) are essential regulators controlling both the cellular transcriptome and translatome. These processes enable cellular plasticity, an important prerequisite for growth. Cellular growth is a complex, tightly controlled process. Using cancer cells as model, we looked for RBPs displaying strong expression in published transcriptome datasets. Interestingly, we found the Pumilio (Pum) protein family to be highly expressed in all these cells. Moreover, we observed that Pum2 is regulated by basic fibroblast growth factor (bFGF). bFGF selectively enhances protein levels of Pum2 and the eukaryotic initiation factor 4E (eIF4E). Exploiting atomic force microscopy and in vitro pulldown assays, we show that Pum2 selects for eIF4E mRNA binding. Loss of Pum2 reduces eIF4E translation. Accordingly, depletion of Pum2 led to decreased soma size and dendritic branching of mature neurons, which was accompanied by a reduction in essential growth factors. In conclusion, we identify Pum2 as an important growth factor for mature neurons. Consequently, it is tempting to speculate that Pum2 may promote cancer growth.

RNA-binding protein PUM2 regulates mesenchymal stem cell fate via repression of JAK2 and RUNX2 mRNAs.

The differentiation of mesenchymal stem cells (MSCs) into unwanted lineages can generate potential problems in clinical trials. Thus, understanding the molecular mechanisms, involved in this process, would help prevent unexpected complications. Regulation of gene expression, at the posttranscriptional level, is a new approach in cell therapies. PUMILIO is a conserved posttranscriptional regulator. However, the underlying mechanisms of PUMILIO, in vertebrate stem cells, remain elusive. Here, we show that depletion of PUMILIO2 (PUM2) blocks MSC adipogenesis and enhances osteogenesis. We also demonstrate that PUM2 works as a negative regulator on the 3'-untranslated regions of JAK2 and RUNX2 via direct binding. CRISPR/Cas9-mediated gene silencing of Pum2 inhibited lipid accumulation and induced excessive bone formation in zebrafish larvae. Our findings reveal novel roles of PUM2 in MSCs and provide potential therapeutic targets for related diseases.

Reduced Pumilio-2 expression in patients with temporal lobe epilepsy and in the lithium-pilocarpine induced epilepsy rat model.

OBJECTIVE: Drosophila Pumilio (Pum), a homolog of mammalian Pum2, plays an important role in translational regulation in the central nervous system (CNS), particularly for dendrite outgrowth and neuronal excitability. We investigated the expression pattern and cellular distribution of Pum2 in patients with drug-refractory temporal lobe epilepsy (TLE) and rats with lithium chloride-pilocarpine-induced epilepsy. METHODS: Real-time quantitative PCR (RT-qPCR), Western blot, immunohistochemistry, and double-labeled immunofluorescence were utilized to determine the expression level and distribution of Pum2 in temporal neocortex tissues from patients with intractable TLE (n=20) and patients with severe head trauma (n=20) in addition to the hippocampus and adjacent cortex of rats with lithium chloride-pilocarpine-induced TLE and controls. RESULTS: Pum2 was expressed in the cell bodies and dendrites of neurons but did not colocalize with glial fibrillary acidic protein-positive astrocytes or propidium iodide (PI) in nuclei. The expression of Pum2 was significantly reduced in patients and rats with TLE in comparison to controls (P<0.05). CONCLUSION: Pum2 expression was less in patients with TLE and a rodent model of epilepsy, suggesting that decreased expression of Pum2 may be involved in the pathogenesis of TLE.

Pumilio2 and Staufen2 selectively balance the synaptic proteome.

Neurons have the capacity to adapt to environmental stimuli, a phenomenon termed cellular plasticity. The underlying processes are controlled by a network of RNA-binding proteins (RBPs). Their precise impact, however, is largely unknown. To address this important question, we chose Pumilio2 (Pum2) and Staufen2 (Stau2), which both regulate synaptic transmission. Surprisingly, even though both RBPs dynamically interact with each other in neurons, their respective impact on the transcriptome and proteome is highly selective. Although Pum2 deficiency leads to reduced translation and protein expression, Stau2 depletion preferentially impacts RNA levels and increases protein abundance. Furthermore, we show that Pum2 activates expression of key GABAergic synaptic components, e.g., the GABAA receptor scaffold protein Gephyrin. Consequently, Pum2 depletion selectively reduced the amplitude of miniature inhibitory postsynaptic currents. Together, our data argue for an important role of RBPs to maintain proteostasis in order to control distinct aspects of synaptic transmission.

Pumilio2-deficient mice show a predisposition for epilepsy.

Epilepsy is a neurological disease that is caused by abnormal hypersynchronous activities of neuronal ensembles leading to recurrent and spontaneous seizures in human patients. Enhanced neuronal excitability and a high level of synchrony between neurons seem to trigger these spontaneous seizures. The molecular mechanisms, however, regarding the development of neuronal hyperexcitability and maintenance of epilepsy are still poorly understood. Here, we show that pumilio RNA-binding family member 2 (Pumilio2; Pum2) plays a role in the regulation of excitability in hippocampal neurons of weaned and 5-month-old male mice. Almost complete deficiency of Pum2 in adult Pum2 gene-trap mice (Pum2 GT) causes misregulation of genes involved in neuronal excitability control. Interestingly, this finding is accompanied by the development of spontaneous epileptic seizures in Pum2 GT mice. Furthermore, we detect an age-dependent increase in Scn1a (Nav1.1) and Scn8a (Nav1.6) mRNA levels together with a decrease in Scn2a (Nav1.2) transcript levels in weaned Pum2 GT that is absent in older mice. Moreover, field recordings of CA1 pyramidal neurons show a tendency towards a reduced paired-pulse inhibition after stimulation of the Schaffer-collateral-commissural pathway in Pum2 GT mice, indicating a predisposition to the development of spontaneous seizures at later stages. With the onset of spontaneous seizures at the age of 5 months, we detect increased protein levels of Nav1.1 and Nav1.2 as well as decreased protein levels of Nav1.6 in those mice. In addition, GABA receptor subunit alpha-2 (Gabra2) mRNA levels are increased in weaned and adult mice. Furthermore, we observe an enhanced GABRA2 protein level in the dendritic field of the CA1 subregion in the Pum2 GT hippocampus. We conclude that altered expression levels of known epileptic risk factors such as Nav1.1, Nav1.2, Nav1.6 and GABRA2 result in enhanced seizure susceptibility and manifestation of epilepsy in the hippocampus. Thus, our results argue for a role of Pum2 in epileptogenesis and the maintenance of epilepsy.

BDNF-induced local protein synthesis and synaptic plasticity.

Brain-derived neurotrophic factor (BDNF) is an important regulator of synaptic transmission and long-term potentiation (LTP) in the hippocampus and in other brain regions, playing a role in the formation of certain forms of memory. The effects of BDNF in LTP are mediated by TrkB (tropomyosin-related kinase B) receptors, which are known to be coupled to the activation of the Ras/ERK, phosphatidylinositol 3-kinase/Akt and phospholipase C-γ (PLC-γ) pathways. The role of BDNF in LTP is best studied in the hippocampus, where the neurotrophin acts at pre- and post-synaptic levels. Recent studies have shown that BDNF regulates the transport of mRNAs along dendrites and their translation at the synapse, by modulating the initiation and elongation phases of protein synthesis, and by acting on specific miRNAs. Furthermore, the effect of BDNF on transcription regulation may further contribute to long-term changes in the synaptic proteome. In this review we discuss the recent progress in understanding the mechanisms contributing to the short- and long-term regulation of the synaptic proteome by BDNF, and the role in synaptic plasticity, which is likely to influence learning and memory formation. This article is part of the Special Issue entitled 'BDNF Regulation of Synaptic Structure, Function, and Plasticity'.