Insulin silences apolipoprotein B mRNA translation by inducing intracellular traffic into cytoplasmic RNA granules

Insulin is a potent inducer of global mRNA translation and protein synthesis, yet it negatively regulates apolipoprotein B (apoB) mRNA translation, via an unknown mechanism. ApoB mRNA has a long half-life of 16 h, suggesting intracellular storage as mRNPs likely in the form of RNA granules. The availability of apoB mRNA for translation may be regulated by the rate of release from translationally silenced mRNPs within cytoplasmic foci called processing bodies (P bodies). In this report, we directly imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by entering cytoplasmic P bodies. We assessed the colocalization of apoB mRNA and β-globin mRNA (as a control) with P body (PB) markers using a strong interaction between the bacteriophage capsid protein MS2 and a sequence specific RNA stem-loop structure. We observed statistically significant increases in the localization of apoB mRNA into P bodies 4-16 h after insulin treatment (by 72-89%). The movement of apoB mRNA into cytoplasmic P bodies correlated with reduced translational efficiency as assessed by polysomal profiling and measurement of apoB mRNA abundance. PB localization of β-globin mRNA was insensitive to insulin treatment, suggesting selective regulation of apoB mRNA by insulin. Overall, our data suggest that insulin may specifically silence apoB mRNA translation by reprogramming its mRNA into P bodies and reducing the size of translationally competent mRNA pools. Translational control via traffic into cytoplasmic RNA granules may be an important mechanism for controlling the rate of apoB synthesis and hepatic lipoprotein production.     

Translational control of apolipoprotein B mRNA via insulin and the protein kinase C signaling cascades: evidence for modulation of RNA-protein interactions at the 5'UTR

The link between hepatic insulin signaling and apolipoprotein B (apoB) production has important implications in understanding the etiology of metabolic dyslipidemia commonly observed in insulin-resistant states. Recent studies have revealed important translational mechanisms of apoB mRNA involving the 5' untranslated region (5'UTR) and insulin-mediated translational suppression via an insulin-sensitive RNA binding protein. Here, we have investigated the role of the protein kinase C (PKCs) signaling cascade in the regulation of apoB mRNA translation, using a series of chimeric apoB UTR-luciferase constructs, in vitro translation of UTR-luciferase cRNAs, and metabolic labeling of intact HepG2 cells. The PKC activator, phorbol 12-myristate 13-acetate (PMA), increased luciferase expression of constructs containing the apoB 5' UTR whereas treatment with Bis-I, a general PKC inhibitor or Go6976, a more specific PKC alpha/beta inhibitor, decreased expression, under both basal and insulin-treated conditions. These effects were confirmed to be translational in nature based on in vitro translation studies of T7 apoB UTR-luciferase constructs transcribed and translated in vitro in the presence of HepG2 cytosol treated with insulin or signaling modulators. Mobility shift experiments using cytosol treated with either PKC inhibitor (Bis-I) or activator (PMA) showed parallel changes between translation of apoB 5'UTR-luciferase constructs and the binding of a protein(s) complex migrating around 110 kDa to the apoB 5' UTR. ApoB mRNA levels were unaltered under these conditions based on real-time PCR analysis. Bis-I and Go6976 were both able to significantly decrease newly synthesized apoB100 protein in the presence or absence of insulin. Overall, the data suggests that PKC activation may induce increased mRNA translation and synthesis of apoB100 protein through a mechanism involving the interaction of trans-acting factors with the apoB 5'UTR. We postulate potential links between PKC activation as seen in insulin-resistant/diabetic states, enhanced translation of apoB mRNA, and hepatic VLDL-apoB overproduction.     

Regulation of RNA granules by FMRP and implications for neurological diseases

RNA granule formation, which can be regulated by RNA‐binding proteins (RBPs) such as fragile X mental retardation protein (FMRP), acts as a mechanism to control both the repression and subcellular localization of translation. Dysregulated assembly of RNA granules has been implicated in multiple neurological disorders, such as amyotrophic lateral sclerosis. Thus, it is crucial to understand the cellular pathways impinging upon granule assembly or disassembly. The goal of this review is to summarize recent advances in our understanding of the role of the RBP, FMRP, in translational repression underlying RNA granule dynamics, mRNA transport and localized. We summarize the known mechanisms of translational regulation by FMRP, the role of FMRP in RNA transport granules, fragile X granules and stress granules. Focusing on the emerging link between FMRP and stress granules, we propose a model for how hyperassembly and hypoassembly of RNA granules may contribute to neurological diseases.     

Insulin-mediated suppression of apolipoprotein B mRNA translation requires the 5' UTR and is characterized by decreased binding of an insulin-sensitive 110-kDa 5' UTR RNA-binding protein

Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels; however, mechanisms of apoB mRNA translational control are largely unknown. Recent studies of apoB untranslated regions (UTRs) revealed a potentially important role for cis-trans interactions at the 5' and 3' UTRs. In the present paper, deletion constructs of the UTR regions of apoB revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB15. Metabolic radiolabeling and in vitro translation experiments in the presence of protease inhibitors confirmed that the effect of insulin on the apoB 5' UTR was translational in nature. Using the nondenaturing electrophoretic mobility shift assay (EMSA), protein-RNA complexes were detected binding to the apoB 5' and 3' UTRs. Denaturing EMSA identified a 110-kDa protein interacting at the 5' UTR. Nondenaturing EMSA determined that insulin altered binding of large protein complexes to the 5' UTR. Binding specificity was determined by competition with both specific and nonspecific competitors. Insulin treatment decreased binding of the 110-kDa protein to the 5' UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 5' UTR by 2-fold. Analysis of the 3' UTR showed no significant insulin-mediated changes in binding of trans-acting factors. We thus propose the existence of a novel RNA-binding insulin-sensitive factor that binds to the 5' UTR and may regulate apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin-resistant states may alter cis-trans interactions at the 5' UTR, leading to alterations in the rate of apoB mRNA translation, thus contributing to apoB-lipoprotein overproduction.     

Cyclin B1 mRNA translation is temporally controlled through formation and disassembly of RNA granules

Temporal control of messenger RNA (mRNA) translation is an important mechanism for regulating cellular, neuronal, and developmental processes. However, mechanisms that coordinate timing of translational activation remain largely unresolved. Full-grown oocytes arrest meiosis at prophase I and deposit dormant mRNAs. Of these, translational control of cyclin B1 mRNA in response to maturation-inducing hormone is important for normal progression of oocyte maturation, through which oocytes acquire fertility. In this study, we found that dormant cyclin B1 mRNA forms granules in the cytoplasm of zebrafish and mouse oocytes. Real-time imaging of translation revealed that the granules disassemble at the time of translational activation during maturation. Formation of cyclin B1 RNA granules requires binding of the mRNA to Pumilio1 protein and depends on actin filaments. Disruption of cyclin B1 RNA granules accelerated the timing of their translational activation after induction of maturation, whereas stabilization hindered translational activation. Thus, our results suggest that RNA granule formation is critical for the regulation of timing of translational activation.     

RNA Granule Assembly and Disassembly Modulated by Nuclear Factor Associated with Double-stranded RNA 2 and Nuclear Factor 45

RNA granules are large messenger ribonucleoprotein complexes that regulate translation and mRNA translocation to control the timing and location of protein synthesis. The regulation of RNA granule assembly and disassembly is a structural basis of translational control, and its disorder is implicated in degenerative disease. Here, we used proteomic analysis to identify proteins associated with RNA granule protein 105 (RNG105)/caprin1, an RNA-binding protein in RNA granules. Among the identified proteins, we focused on nuclear factor (NF) 45 and its binding partner, nuclear factor associated with dsRNA 2 (NFAR2), and we demonstrated that NF45 promotes disassembly of RNA granules, whereas NFAR2 enhances the assembly of RNA granules in cultured cells. The GQSY domain of NFAR2 was required to associate with messenger ribonucleoprotein complexes containing RNG105/caprin1, and it was structurally and functionally related to the low complexity sequence domain of the fused in sarcoma protein, which drives the assembly of RNA granules. Another domain of NFAR2, the DZF domain, was dispensable for association with the RNG105 complex, but it was involved in positive and negative regulation of RNA granule assembly by being phosphorylated at double-stranded RNA-activated kinase sites and by association with NF45, respectively. These results suggest a novel molecular mechanism for the modulation of RNA granule assembly and disassembly by NFAR2, NF45, and phosphorylation at double-stranded RNA-activated kinase PKR sites.     

Characterizing mRNA interactions with RNA granules during translation initiation inhibition

When cells experience environmental stresses, global translational arrest is often accompanied by the formation of stress granules (SG) and an increase in the number of p-bodies (PBs), which are thought to play a crucial role in the regulation of eukaryotic gene expression through the control of mRNA translation and degradation. SGs and PBs have been extensively studied from the perspective of their protein content and dynamics but, to date, there have not been systematic studies on how they interact with native mRNA granules. Here, we demonstrate the use of live-cell hybridization assays with multiply-labeled tetravalent RNA imaging probes (MTRIPs) combined with immunofluorescence, as a tool to characterize the polyA+ and β-actin mRNA distributions within the cytoplasm of epithelial cell lines, and the changes in their colocalization with native RNA granules including SGs, PBs and the RNA exosome during the inhibition of translational initiation. Translation initiation inhibition was achieved via the induction of oxidative stress using sodium arsenite, as well as through the use of Pateamine A, puromycin and cycloheximide. This methodology represents a valuable tool for future studies of mRNA trafficking and regulation within living cells.     

Insulin inhibition of apolipoprotein B mRNA translation is mediated via the PI-3 kinase/mTOR signaling cascade but does not involve internal ribosomal entry site (IRES) initiation

Although insulin normally activates global mRNA translation, it has a specific inhibitory effect on translation of apolipoprotein B (apoB) mRNA. This suggests that insulin induces a unique signaling cascade that leads to specific inhibition of apoB mRNA translation despite global translational stimulation. Recent studies have revealed that insulin functions to regulate apoB mRNA translation through a mechanism involving the apoB mRNA 5' untranslated region (5' UTR). Here, we further investigate the role of downstream insulin signaling molecules on apoB mRNA translation, and the mechanism of apoB mRNA translation itself. Transfection studies in HepG2 cells expressing deletion constructs of the apoB 5' UTR showed that the cis-acting region responding to insulin was localized within the first 64 nucleotides. Experiments using chimeric apoB UTR-luciferase constructs transfected into HepG2 cells followed by treatment with wortmannin, a PI-3K inhibitor, and rapamycin, an mTOR inhibitor, showed that signaling via PI-3K and mTOR pathways is necessary for insulin-mediated inhibition of chimeric 5' UTR-luciferase expression. In vitro translation of chimeric cRNA confirmed that the effects observed were translational in nature. Furthermore, using RNA-EMSA we found that wortmannin pretreatment blocked insulin-mediated inhibition of the binding of RNA-binding factor(s), migrating near the 110 kDa marker, to the 5' UTR. Radiolabeling studies in HepG2 cells also showed that insulin-mediated control of the synthesis of endogenously expressed full length apoB100 is mediated via the PI-3K and mTOR pathways. Finally, using dual-cistronic luciferase constructs we demonstrate that apoB 5' UTR may have weak internal ribosomal entry (IRES) translation which is not affected by insulin stimulation, and may function to stimulate basal levels of apoB mRNA translation.     

Role of RNA secondary structure of the iron-responsive element in translational regulation of ferritin synthesis.

Iron regulates synthesis of the iron storage protein ferritin at the translational level through interaction between a stem-loop structure, the iron-responsive element (IRE), located in the 5'-untranslated region (5'-UTR) of ferritin mRNAs, and a protein, the iron regulatory protein (IRP). The role of IRE secondary structure in translational regulation of ferritin synthesis was explored by introducing ferritin constructs containing mutations in the IRE into Rat-2 fibroblasts. Our in vivo studies demonstrate that size and sequence of the loop within the IRE and the distance and/or spatial relationship of this loop to the bulged nucleotide region closest to the loop must be preserved in order to observe iron-dependent translation of ferritin mRNA. In contrast, changes in nucleotide sequence of the upper stem can be introduced without affecting translational regulation in vivo, as long as a stem can be formed. Our in vivo results suggest that only a very small variation in the affinity of interaction of IRP with IRE can be tolerated in order to maintain iron-dependent regulation of translation.     

Translational control by a small RNA: dendritic BC1 RNA targets the eukaryotic initiation factor 4A helicase mechanism

Translational repressors, increasing evidence suggests, participate in the regulation of protein synthesis at the synapse, thus providing a basis for the long-term plastic modulation of synaptic strength. Dendritic BC1 RNA is a non-protein-coding RNA that represses translation at the level of initiation. However, the molecular mechanism of BC1 repression has remained unknown. Here we identify the catalytic activity of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase, as a target of BC1-mediated translational control. BC1 RNA specifically blocks the RNA duplex unwinding activity of eIF4A but, at the same time, stimulates its ATPase activity. BC200 RNA, the primate-specific BC1 counterpart, targets eIF4A activity in identical fashion, as a result decoupling ATP hydrolysis from RNA duplex unwinding. In vivo, BC1 RNA represses translation of a reporter mRNA with 5' secondary structure. The eIF4A mechanism places BC RNAs in a central position to modulate protein synthesis in neurons.     

Differences in the translation efficiency and mRNA stability mediated by 5'-UTR splice variants of human SP-A1 and SP-A2 genes

Surfactant protein A (SP-A) plays an important role in host defense, modulation of inflammatory processes, and surfactant-related functions of the lung. The human SP-A (hSP-A) locus consists of two functional genes, SP-A1 and SP-A2. Several hSP-A 5'-untranslated region (UTR) splice variants for each gene have been characterized and shown to be translated in vitro and in vivo. In this report, we investigated the role of hSP-A 5'-UTR splice variants on SP-A production and molecular mechanisms involved. We used in vitro transient expression of hSP-A 5'-UTR constructs containing luciferase as the reporter gene and quantitative real-time PCR to study hSP-A 5'-UTR-mediated gene expression. We found that 1) the four (A'D', ABD, AB'D', and A'CD') 5'-UTR splice variants under study enhanced gene expression, by increasing luciferase activity from 2.5- to 19.5-fold and luciferase mRNA from 4.3- to 8.8-fold compared with the control vector that lacked hSP-A 5'-UTR; 2) all four 5'-UTR splice variants studied regulated mRNA stability. The ABD variant exhibited the lowest rate of mRNA decay compared with the other three constructs (A'D', AB'D', and A'CD'). These three constructs also exhibited significantly lower rate of mRNA decay compared with the control vector; 3) based on the indexes of translational efficiency (luciferase activity/mRNA), ABD and AB'D' exhibited higher translational efficiency compared with the control vector, whereas the translational efficiency of each A'D' and A'CD' was lower than that of the control vector. These findings indicate that the hSP-A 5'-UTR splice variants play an important role in both SP-A translation and mRNA stability.     

Neuronal RNA granules: movers and makers

RNA localization contributes to cell polarity and synaptic plasticity. Evidence will be discussed that RNA transport and local translation in neurons may be more intimately linked than originally thought. Second, neuronal RNA granules, originally defined as intermediates involved in mRNA transport, are much more diverse in their composition and functions than previously anticipated. We focus on three classes of RNA granules that include transport RNPs, stress granules, and P bodies and discuss their potential functions in RNA localization, microRNA-mediated translational regulation, and mRNA degradation.