Translational Control Mechanisms in Metabolic Regulation: Critical Role of RNA binding proteins, micrornas, and Cytoplasmic RNA granules

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1 Articles in PresS. Am J Physiol Endocrinol Metab (October 4, 2011). doi: /ajpendo Translational Control Mechanisms in Metabolic Regulation: Critical Role of RNA binding proteins, micrornas, and Cytoplasmic RNA granules Khosrow Adeli Molecular Structure and Function, Research Institute, The Hospital for Sick Children, and Department of Biochemistry & Laboratory Medicine, University of Toronto s Correspondence Address: Program in Molecular Structure & Function Research Institute, The Hospital for Sick Children, Atrium 3653; 555 University Avenue, Toronto, Ontario, M5G 1X8 Phone: ; k.adeli@utoronto.ca; khosrow.adeli@sickkids.ca Abbreviations: Apolipoprotein B, apob; eif, eukaryotic initiation factor; IRES, internal ribosomal entry sites; IRE, iron-responsive element; mrna, messenger RNA; mrnp, messenger ribonucleoprotein; mirisc, RNA-induced silencing complex; mirna, microrna; ORF, open reading frame; P bodies, processing bodies; PABP, poly(a) binding protein; UPR, unfolded protein response; UTR, untranslated region 1 Copyright 2011 by the American Physiological Society.

2 Summary: Regulated cell metabolism involves acute and chronic regulation of gene expression by various nutritional and endocrine stimuli. To respond effectively to endogenous and exogenous signals, cells require rapid response mechanisms to modulate transcript expression and protein synthesis and cannot in most cases rely on control of transcriptional initiation that requires hours to take effect. Thus co- and post-translational mechanisms have been increasingly recognized as key modulators of metabolic function. This review will highlight the critical role of mrna translational control in modulation of global protein synthesis as well as specific protein factors that regulate metabolic function. First, the complex lifecycle of eukaryotic mrnas will be reviewed including our current understanding of translational control mechanisms, regulation by RNA binding proteins and micro RNAs, and the role of RNA granules, including Processing Bodies and Stress Granules in regulation of mrna translation and protein synthesis. Secondly, the current evidence linking regulation of mrna translation with normal physiological and metabolic pathways, and the associated disease states will be reviewed. A growing body of evidence supports a key role of translational control in metabolic regulation and implicates translational mechanisms in the pathogenesis of metabolic disorders such as type 2 diabetes. The review will also highlight translational control of apolipoprotein B (apob) mrna by insulin as a clear example of endocrine modulation of mrna translation to bring about changes in specific metabolic pathways. Recent findings made on the role of 5 untranslated regions (5 UTR), 3 untranslated region (3 UTR), RNA binding proteins and RNA granules in mediating insulin regulation of apob mrna translation, apob protein synthesis and hepatic lipoprotein production will be discussed

3 Translational Control as a Critical Checkpoint Governing Eukaryotic Gene Expression The expression of biologically active proteins in eukaryotic cells is controlled at multiple points during the process of gene expression including chromatin structure, transcriptional initiation, processing and modification of mrna transcripts, transport of mrna into cytoplasm, stability/decay of mrna transcripts, initiation and elongation of mrna translation, co/posttranslational modification, and intracellular transport and degradation of the expressed protein. Although regulation of transcription initiation may be the most important form of gene control in eukaryotes as well as bacteria, the efficiency of mrna translation and the rate of protein synthesis play critical roles in many fundamental biological processes. It has been ten years since the sequencing of the human genome, and of the approximately 50,000 base pairs composing the average-sized human gene, less than 2 percent appear to code for proteins (95; 162), while more than 95 percent of the bases are introns and noncoding 5 and 3 regions (61). These non-coding sequences are transcribed but not translated and may play a critical role in directing posttranscriptional control. Moreover, many mrna transcripts in eukaryotic cells have long halflives (>6 h) suggesting the larger role of translational control in eukaryotes (130). About 95% of mrna transcripts in both the liver cancer cell line HepG2, and a primary cell line Bud8 have half-lives greater than 2 h (174). The longer mrna half-life of these eukaryotic mrnas allows a greater opportunity for translational control (130; 174). Translation is the mechanism by which ribosomes read the genetic code in mrna and synthesize a protein product based on a specific genetic code. Translation occurs in the cytoplasm of eukaryotes and is not concurrent with transcription (37). Some mrnas may be sequestered in the nucleus or cytoplasm, and translation is delayed until a signal is received to turn on translation. For example, maternal mrnas are known to be stored in the oocyte and are 3

4 translated upon fertilization (99; 144) (135) an observation also made with the mrna for prostaglandin endoperoxide H synthase-1 gene induction (PGHS-1) (21; 22). Moreover, eukaryotic mrnas have structural cis-acting elements such as 5 and 3 UTR, providing more opportunity for specific regulation by trans-acting RNA binding proteins. Translational processes may also be influenced by global controls, which target the translational machinery or common elements shared by many mrnas molecules (60; 118). Growing evidence also suggest a critical role for small micro RNAs (mirnas) that hybridize to mrna sequences within the 3 UTR or other regions, and control the mrna stability and its rate of translation. Figure 1 depicts in a very simplified form the complex lifecycle of eukaryotic mrnas and some the key regulatory mechanisms. Translational control of specific mrnas is known to be dependent upon the inherent structural features of the specific transcript itself and trans-acting protein factors that bind the regulatory elements located within the UTRs. The presence of a start-site consensus sequence, secondary structure, upstream open reading frames (uorfs) or AUGs, terminal oligopyrimidine (TOP) and Internal Ribosomal Entry Sites (IRES) are all motifs within an mrna that can determine translational efficiency (171). Translational initiation depends on the binding of eukaryotic initiation factors to the 5' cap and subsequent scanning of the 40S ribosomal complex for the first AUG codon. Recognition of the AUG codon is in turn dependent on the context and position of the AUG codon and the presence of upstream AUG codons that may modify the translation process. Secondary structure and sequence elements within the UTRs may be potential binding sites for interaction with RNA binding proteins that modulate mrna stability and translational control. 4

5 RNA binding proteins are capable of regulating translation initiation and translational repression by a variety of mechanisms. The RNA recognition motifs of these regulatory proteins are wide and varied in both structure and complexity, ranging from a simple hairpin to a complex pseudoknot. RNA regulatory domains are referred to as cis-acting elements within an mrna sequence that interact with specific RNA binding proteins called trans-acting factors. The mechanism by which RNA binding proteins repress translation can be complex and may involve competition with ribosome for RNA, direct binding to the mrna promoting an RNA structure that inhibits ribosome binding, or helping translational repression by induction of ribosomal trapping (7). Mechanisms of translational control at the levels of initiation and elongation have been extensively reviewed elsewhere (10; 73) and are not the subject of the current review. Here, we will focus on recent findings related to the role of cis-trans interactions on the 5 and 3 UTR in translational control as well as the role of RNA granules in maintaining and controlling mrna translation. Specific examples of translational control via these novel mechanisms will be reviewed with a special focus on mrnas involved in metabolic control and disease Translational Control via Cis-Trans Interactions on the 5 and 3 UTR There is now ample evidence that secondary structure and sequence elements within the 5' and 3'UTR sequences mediate translational control. These structural features or elements may be potential binding sites for RNA-binding proteins; the interaction of cis-elements with transacting factors may modulate translation or alter stability of the message (53; 85; 110; 142). UTRs can either induce or inhibit translation as the nucleotides surrounding the AUG start codon can influence recognition efficiency by the 43S pre-initiation complex (92). The length of the 5 UTR leader also influences translation and increasing the 5 UTR length can increase mrna 5

6 translation (93). Moreover, secondary structure near the 5 end can cause steric hindrance and block binding of the 43S pre-initiation complex to the cap structure (37). Studies of the function of 5 -UTR binding proteins of the iron regulatory protein complex have been particularly informative. Iron regulatory proteins (IRPs) were found to act as translational repressors in response to intracellular iron concentrations. IRPs control several mrnas that contain a stem loop structure known as the iron-responsive element (IRE). Mutations within the IRE indicate the important role of this RNA protein complex plays in various genetic disorders (25; 171). While most highly structured 5 UTRs have an inhibitory effect on translation (94), there are some examples of translation enhancement by 5 UTR secondary structures such as Hsp70 (163) (177) surfactant protein A (168) (168), and GLUT1 transporter (15-17). 3 UTRs similarly contain numerous binding sites for trans-acting regulatory factors (113; 118; 169). While trans-acting regulatory factors are normally proteins, some trans-acting RNAs have also been described that appear to regulate translation at the initiation step (171). In addition, cytoplasmic changes to the poly (A)-tail length have also been correlated with changes in the translation of mrnas. Translational activation occurs with increases in poly (A)-tail length (171), a process regulated by poly(a) binding protein (PABP) (74) and interactions with other RNA binding proteins - reviewed in (171). Cytoplasmic polyadenylation-element binding proteins (CPEBs) are also involved in translational regulation (repression) (111) by binding to cytoplasmic polyadenylation-element (CPEs). CPEs repress translation in a process mediated by maskin, a protein that interacts with both CPEB and eif-4e (149). Transferrin mrna contains IREs within its 3 UTR of mrnas that mediate positive translational activation via interaction with the IRP which stabilizes the stem loop structure increasing mrna stability and translation (67). 6

7 Mutations within the 5 and 3 UTR have been implicated in pathogenesis of many disease states. Hyperferritinemia/cataract syndrome (HHCS) is caused by mutations in the 5 UTR of the L-ferritin gene. Ferritin expression is tightly regulated by iron-responsive elements (IREs) and disruption of the IRE leads to high ferritin production (25). A mutation in the 5 UTR of the connexin-32 gene causes Charcot-Marie-Tooth disease by abolishing the function of the internal ribosome entry site (IRES) (69). On the other hand, a mutation in the c-myc 5 UTR leads to increased activity of an IRES, excess c-myc production and contribution to multiple myeloma (27). Analysis of the composition of complexes containing the RBP fragile X mental retardation protein (FMRP) which causes mental retardation demonstrated the presence of approximately 430 mrnas co-precipitated with defective translational regulation and developmental disorders. More than 50% of the FMRP-associated mrnas had abnormal polysomal profiles (i.e. an altered proportion of mrna in polyribosome fractions) (20). An example of a 3 UTR variation leading to disease has been reported for human protein tyrosine phosphatase-1b (hptp1b) which increases its protein synthesis and contributes to development of insulin resistance (39). hptp1b is an important regulator of insulin signaling and directly interacts with and dephosphorylates the activated insulin receptor, thus blocking downstream insulin signaling and action (138). hptp1b overexpression can result in insulin resistance while loss of hptp1b leads to increased insulin sensitivity and resistance to diet induced obesity (1; 43; 84; 86). Translational control of hptp1b would thus have important implications in modulation of insulin sensitivity and the associated metabolic disturbances Intracellular mrna Traffic and Translational Control: Role of RNA Granules Mature mrnas in the cytoplasm of eukaryotic cells are known to be associated with a complex network of ribonucleoprotein particles (mrnps) (83; 148). Early studies of mrnps 7

8 showed the presence of two major proteins(75), a 70-kDa poly(a) binding protein (PABP)(14) and a 50-kDa protein (p50) responsible for the repressed, nonactive state of mrnas, such as globin mrna within free mrnp particles(114). More recent studies have identified a large number of other protein components of mrnps including RNA binding proteins, RNA helicases, and translational factors (5). Importantly, interaction with these and other RNA binding proteins leads to the formation of granules initially in the nucleus of somatic cells and subsequently to the formation of cytoplasmic RNA granules (82). Microtubule motor proteins like kinesin, dynein, and myosin provide a vehicle to transport RNA granules on actin filaments within the cytoplasm (104). Cytoplasmic RNA granules have been proposed as the main controllers of posttranscriptional gene regulation processes and epigenetic changes (4). They are divided into 4 groups: germinal granules, stress granules, processing bodies (P bodies), and neural granules. The former is found in germ cells and the three others exist in somatic cells. These granules are composed of various RNAs and a subset of different proteins and contribute to the localization, stability, and translation of their associated RNAs (2). Stress granules contain mrnas encoding most cellular proteins and appear following exposure to environmental stress (3). Other cytoplasmic RNA granules, called P bodies, contain an mrna decay machinery and the RNAinduced silencing complex (124). All RNA granules contain translationally-silenced mrnas. New evidence suggests a dynamic interaction between these RNA granules and translationallyactive polysomal mrnas (19; 82; 151), suggesting that the availability/activity of some mrnas could be regulated by the rate of release from translationally-silenced mrnas within RNA granules. mrnas can exit RNA granules and reenter the translational machinery under certain conditions (124). Regulated mrna traffic between polysomes and RNA granules can thus allow the cell to tailor translation to changes in environmental conditions (81). mrna 8

9 compartmentalization in the form of RNA granules enhances the ability of a cell to control protein synthesis in response to certain stimuli without the need for new gene transcription (104). The localization of some mrnas to a specific cytoplasmic site involves cis-acting elements within the mrna sequence, referred to as localization elements or zipcodes, usually located in the 3 UTR and less frequently within the 5 UTR or coding sequence in some mrnas. These elements vary in length from as few as five nucleotides to hundreds of nucleotides that are capable of forming stem loop secondary structures. Trans-acting RNA binding proteins often identify these sequences to direct message localization and control translational activity (104). Stress granules are dynamic cytoplasmic foci that host non-translating mrnas mostly found in cytoplasm of plant and mammalian cells (104). Stress granules are formed in response to stresses such as drug treatment, depletion of transcription factors, impaired translation initiation and other conditions. Stress induces dissociation of polysomal machinery and stalled translation of most mrnas except for those involved in the stress response. As a result circular polyadenylated mrnps are formed that can be assembled into either P bodies or stress granules (47; 57) which consists of translational initiation machinery components. In most cases, stress granule formation is triggered by eif2α phosphorylation leading to translational arrest and nucleation of stress granules enriched in poly A-containing mrnas and PABP (81). Under stress conditions, translational silencers TIA1, and TIAR are recruited by the mrnps and promote the aggregation of stress granules (63; 102; 105). The complete structure of stress granules is not known; nevertheless, they are composed of a series of translation initiation proteins, the small ribosomal subunit (40S), the poly A binding protein (Pab1) (3; 63; 140; 140; 154), Staufen, FMRP, and mrnas that express most cellular proteins except stress response proteins such as 9

10 heat-shock proteins (104). These granules have been predicted to act as a triage spot for mrna storage, degradation, and translation re-initiation (3). P bodies are also dynamic RNA-Protein structures found in the cytoplasm of eukaryotes from yeast to mammals. P body assembly correlates positively with the pool of un-translating mrna in a cell (101; 104; 126; 151). They contain mrna decay machinery components. If polysomes are disassembled in a CCR4-NOT1 mediated manner, poly(a)-binding protein 1(PABP1) dissociates from eif4g, the circle of the polysomal transcript breaks and a linear deadenylated mrna is formed. The CC4-NOT1 complex is involved in deadenylation of mrnas leading to polysomal disassembly and translational silencing (or mrna degradation) (51). However, polysomal disassembly may also occur independent of the CC4-NOT1 complex. Central to P body assembly are translationally-inactive mrna and the decapping factor which induces mrna decay and blocks translation by decapping and induction of 5 3 mrna decay. P bodies are composed of Dcp1/Dcp2 (decapping enzymes), Dhh1/ RCK/p54, Pat1, Scd6/RAP55, Edc3, the Lsm1-7 complex (activators of decapping), Xrn1 (5-3 exonuclease) (2; 47; 48), nonsense mediated decay machinery, mirna repression system (58; 140), staufen, and FMRP (RNA binding proteins mediating mrna transport) (104). P bodies assembly factors recruit mrna into P bodies through multimerization domains present in P body assembly factors (45). Notably, stress granules and P bodies have been shown to physically interact suggesting potential trafficking of stored mrna between these compartments (124) MicroRNAs (mirnas) and Translational Control mirnas comprise a large family of small ~21-nucleotide-long non-coding RNAs that bind to complementary sequences on target mrna transcripts and have emerged as key posttranscriptional regulators of translation and mrna decay in all eukaryotic cells. The regulatory 10

11 pathways mediated by these small RNAs are usually collectively referred to as RNA interference (RNAi) or RNA silencing. Based on bioinformatics analyses it has been suggested that up to 60% of protein-coding genes may be regulated by mirnas (52). mirnas usually function via translational repression and gene silencing (52) and/or by triggering deadenylation at the 3 UTR and subsequent degradation of mrna targets (28; 51). Less commonly, some mirnas may act positively by activating mrna translation (159; 160; 160) (121). mrnas that lack a functional 5 -cap structure, or whose translation is cap-independent, appear to be refractory to mirnamediated translational repression (52). It is now well accepted that mirnas destabilize target mrnas through deadenylation and subsequent decapping and 5-3 exonucleolytic digestion. mrna decay by mirnas require the Argonaute and GW182 components of the mirisc (RNA-induced silencing complex). The GW182-Argonaute interaction is mediated via GW repeats in the GW182 N-terminus through binding to the Argonaute MID/PIWI domain (49; 50). In addition, GW182 recruits the CCR4- NOT1 deadenylase complex to promote deadenylation of mirna-targeted mrnas, a process that also requires poly (A)-binding proteins. mirnas can also repress initiation by inhibiting 60S subunit binding to 40S ( ) and the mirisc association with eif6 disrupts polysome formation by inhibiting 80S complex assembly (26). Strikingly, mirna-mediated translational silencing coincides with trafficking of mrna transcripts into RNA granules. mirnas recruit the repression/decay machinery to mrnas and promote their repression, deadenylation, and decapping in association with components of P bodies (124). Critical components of the mirisc complex including Argonaute and GW182 family proteins are enriched in P bodies. 258 Translational Control in Metabolic Regulation: 11

12 Translational control of cellular metabolism has received little attention as there is a significant gap of knowledge in the regulatory pathways and the complex mechanisms involved. Regulation of metabolic pathways requires in many cases a rapid response of the cell to endogenous metabolites and exogenous nutrients and endocrine signals. This rapid response can be more effectively delivered via translational control of the rate of global protein synthesis or specific protein factors to regulate metabolic function. There is now ample evidence for a critical role of translational control in metabolic and physiological mechanisms. Here, we review some of the current evidence linking regulation of mrna translation within normally regulated physiological and metabolic pathways, and their associated metabolic disease states such as type 2 diabetes. Figure 2 summaries the links so far identified among factors involved in translational control with specific metabolic pathways and disorders. Translational Control in Glucose Homoeostasis and Diabetes: There is considerable evidence for the involvement of translational control in many components of type 2 diabetes and metabolic syndrome including insulin biosynthesis, hepatic and peripheral insulin sensitivity, and diabetic complications (139). Early evidence showed that glucose acutely stimulates insulin synthesis and secretion by pancreatic islets within minutes of glucose exposure via a translational mechanism (164). Proinsulin biosynthesis is determined by the cooperative interaction of the 5' and 3'UTR sequences in glucose-stimulated translation of preproinsulin mrna in the islets (170). The 5'UTR of the preproinsulin mrna contains an element that forms a stem-loop that is believed to be important for glucose-stimulated translation. The 3'UTR contains a conserved sequence UUGAA that increases the stability of the preproinsulin mrna but is believed to decrease glucose-stimulated translation (170). More recently, it was also shown that glucagonlike peptide-1 (GLP-1) mediated stimulation of beta cell proliferation and glucose competence is 12

13 mediated by translational induction of insulin-like growth factor-1 (IGF-1) receptor expression (32). A monogenetic form of diabetes, Wolcott-Rallison syndrome (WRS), appears to result from mutations in protein kinase-like ER stress kinase (PERK), a key kinase regulating eif2 phosphorylation and translational activity (24; 38; 153). PERK is activated by phosphorylation of its kinase domain triggered by accumulation of unfolded proteins that are proposed to displace the heat shock protein-70-like ER chaperone, Bip, from binding to the ER lumenal domain (9). Phosphorylation of eif2 α by PERK leads to the inhibition of general protein synthesis. Mice deficient in PERK develop diabetes and a syndrome similar to WRS within 2-4 weeks of age due to a marked decrease in insulin mrna and production in pancreatic beta cells (68). PERK s function appears to be important to normal islet function and may act as a critical checkpoint that balances the rate of insulin synthesis with the ER capacity to fold and process newly-synthesized insulin polypeptides. Interestingly, different phenotypes are observed upon knockout of PERK or homozygous or heterzygous mutation of eif2α (77). eif2 phosphorylation has also been implicated in regulation of hepatic gluconeogensis and glycogenolysis via modulation of a family of bzip transcription factors including CCAATenhancer-binding protein-α (C/EBPα) and C/EBPβ (139) which control the expression of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase, hepatocyte nuclear factor-3 (HNF-3), pyruvate kinase, and glycogen synthase. Translational regulation also involves eif2b which is activated by insulin and decreased in diabetes and with exercise (54; 91; 128). Translational regulation of C/EBP expression by eif4e has been suggested to regulate adipocyte differentiation and insulin sensitivity. Differential selection of the translation initiation site of C/EBPα and C/EBPβ leads to formation of various truncated isoforms which can regulate 13

14 metabolic function (23). C/EBPα deficiency leads to insulin resistance in vitro (in fibroblasts) whereas C/EBPα knockout results in enhanced insulin sensitivity in mice (42). Fibroblasts that do not express C/EBPa fail to develop insulin-regulated glucose transport but retroviral expression of C/EBPa restores insulin-sensitive glucose uptake (42). As C/EBPs are also important regulators of peroxisome proliferator-activated receptor-γ (PPARγ), translational control of C/EBPs can have profound effects on metabolic regulation in insulin sensitive tissues. Notably, knockout of 4E-BP1 (a key protein involved in controlling translational initiation) also increases the expression of PPARγ coactivator-1 (PGC-1) which is a major transcriptional coactivator of uncoupling protein 1 (UCP1) in white adipose tissues (155). Insulin-sensitive tissues such as fat and skeletal muscle also regulate protein synthesis via 4E-BPs, key proteins that bind to eif4e and regulate the formation of the eif4f complex and translational initiation. A number of growth factors and hormones such as insulin, cytokines and angiotensin II induce phosphorylation of 4E-BPs which causes their dissociation from eif4eactivating mrna translation. Remarkably, 4E-BP1 knockout mice exhibit increased insulin sensitivity and have lower circulating glucose in the presence of normal plasma insulin (156). Insulin may also regulate glucose uptake by controlling GLUT1 and GLUT4 mrna translation (150). Similar to 4E-BPs, phosphorylation of S6 kinase (S6K1) appears to be a critical regulator of insulin signaling pathways downstream of mammalian target of rapamycin (mtor). S6K1 is required for pancreatic beta cell development(125) and S6K1 knockout mice develop hyperglycemia in response to glucose challenge due to reduced insulin content in the islets and impaired insulin secretion (125). mtor is an important Ser/Thr kinase implicated in a number of physiological processes and pathological states including obesity (33). The mtor kinase consists of two distinct protein 14

15 complexes, mtorc1 and mtorc2 (66). mtorc1 activation is an important regulator of cell growth in diverse organisms (172) and acts via control of mrna translation. mtor phosphorylates the ribosomal protein S6 kinases (S6K1 and S6K2) and the eukaryotic initiation factor 4E (eif4e)-binding proteins (4E-BP1 and 4E-BP2), thus controlling cap-dependent translation initiation(103). mtorc1 activation is sufficient to stimulate specific metabolic pathways, notably glycolysis, de novo lipogenesis, and the oxidative arm of the pentose phosphate pathway (41). mtorc1 regulation is mediated by activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1α) and SREBP1 and SREBP2 (41). mtorc1 signaling promotes HIF1α mrna translation via its 5 UTR, increasing cellular protein levels (96), in a process involving the inhibitory phosphorylation of 4E-BP1 (41). In brief, a number of metabolic pathways appear to be regulated at the translational level principally via modulation of PERK, eif2, 4E-BP1, and S6K. It is important to note that some of the metabolic effects of the altered amounts or activity of these various mrna are due to developmental defects rather than a dysfunctional metabolic regulatory pathway per se. Polymorphisms within the genes encoding these translational modulators can have a profound influence on protein synthesis and metabolic function in insulin sensitive tissues including the liver, adipose, and skeletal muscle. Translational Control in Lipid Homeostasis: There is also evidence for translational control of key factors involved in lipogenesis and lipid metabolism, including SREBP-1, SREBP-2, and apolipoprotein B (apob). SREBP-1a expression was recently shown to be regulated at the level of mrna translation via a mechanism involving 5 UTR and IRES-mediated translation (161). Interestingly, a DNA polymorphism in the 5 UTR of SREBP-1a had been previously reported to increase the risk for the development of atherosclerosis (161). The 5 UTR of SREBP-1a contains 15

16 an IRES that allows cap-independent translation to be maintained during ER stress and unfolded protein response (UPR), conditions that are inhibitory to cap-dependent translation. Recent studies have also implicated a number of mirnas in the regulation of lipid metabolism and insulin signaling (117). Intronic mirnas, mir-33a and mir-33b, which are located within the genes encoding SREBP-1 and SREBP-2 have been shown to contribute to regulation of cholesterol homeostasis (119; 132). mir-33a/b regulate cholesterol metabolism by controlling the stability and expression of a number of genes involved in cholesterol metabolism including ABCA1, ABCG1, and NPC1 (132). More recently, additional roles in regulation of fatty acid metabolism and insulin signaling were revealed for these mirnas which target the 3 UTR of peroxisomal carnitine o-octanoyltransferase (CROT), carnitine palmitoyltransferase-1a (CPT1a), hydroxyacyl-coa dehydrogenase/3-ketoacyl-coa thiolase/enoyl-coa hydratase (HADHB), insulin receptor substrate-2 (IRS2), and 5 AMP-activated protein kinase a (AMPKa) (35). The functional consequence of anti-mir33 oligonucleotide treatment was enhanced reverse cholesterol transport and atherosclerotic plaque regression in LDL receptor-deficient mice (131). In addition, mir-122, mir-370, mir-335, mir378, mir-27, and mir-125a-5p have also been found to regulate cholesterol and fatty acid metabolism as well as lipogenesis via control of mrna stability and/or translation (55; 116). mir-122 regulates mrnas expressing a number of key enzymes involved in fatty acid metabolism including fatty acid synthase (FAS), acetyl-coa carboxylase 1 (ACC1), and ACC2 (44; 46). mir-122 knockdown reduces cholesterol synthesis, increases fatty acid oxidation, and reduces hepatic steatosis in high-fat fed mice (46). Remarkably, mir-122 is itself regulated by another microrna, mir-370 whose overexpession can lead to increased mir-122 and upregulation of SREBP-1c and acyl-coa:diacylglycerol acyltransferase 2 (DGAT2) (71). 16

17 CD36, a key scavenger receptor implicated in development of atherosclerosis in vascular tissues as well as lipid absorption in the intestinal tract, is also regulated at the translational level. Glucose can acutely induce CD36 expression via a translational control mechanism involving ribosomal reinitiation (65). Hyperglycemic conditions can lead to CD36 overexpression via enhanced translation involving three upstream ORFs in the 5 UTR of CD36 mrna (65). Although the underlying mechanisms remain to be fully elucidated, this is a striking example of direct modulation of translational initiation by glucose via a 5 upstream ORF. There are also recent examples of metabolically-important proteins regulated at the level of mrna stability. The rhythmically expressed mammalian deadenylase Nocturnin is sensitive to acute extracellular stimuli, such as induction by insulin in 3T3-L1 preadipocytes (79; 80) and fasting in white adipose tissue (62). Nocturnin -./- mice are resistant to diet-induced weight gain and hepatosteatosis, although the direct mrna targets of this deadenylase that confer this phenotype are not yet known (64). Nocturnin however has been shown to deadenylate Igf1 transcripts by binding the 3 UTR (78). Interestingly, the Nocturnin mrna itself contains a mir-122 recognition site in the 3 UTR (89). MTP expression is also regulated by the degradation of its mrna. Inositol-requiring enzyme 1β (IRE1β) expression is lower in high-fat and high cholesterol diets, and IRE1β-deficient mice exhibit enhanced intestinal lipid absorption. It turns out that IRE1β possesses endonuclease activity that cleaves between exons 2 and 7, thereby decreasing MTP mrna levels in the enterocyte and inhibiting chylomicron secretion (72). Translational Control in Inflammation: Inflammatory pathways are also subject to translational regulation with a significant impact on metabolism. Tumour necrosis factor-α (TNFα) mrna translation is modulated by steroid receptor co-activator (SRC-3)-mediated translational 17

18 silencing in association with the poly(a) binding protein (176) thus contributing to regulation of the inflammatory process. TNFα mrna translation appears to involve regulation by mir369-3 which triggers its translation (159). Immune cell function and inflammation have also been shown to be regulated by stress granule formation which store inflammatory factor mrnas (such as IL-4 and IL-13 mrnas). Stimulation by T cell receptor signaling triggers the release of these stored mrnas leading to cytokine synthesis and secretion (137). Additionally, the proinflammatory induction of IL-8 in cystic fibrosis appears to be mediated by mirna mediated mechanisms. mir-155 was recently shown to specifically reduce SHIP1 expression leading to activation of PI-3 kinase/akt pathway and increased expression of IL-8 in CF lung epithelial cells (11; 12). Translational Control in Copper and Iron Metabolism: Translational control mechanisms are also involved in both iron and copper metabolism. Cellular iron homeostasis is controlled largely at the translational level via coordinated expression of the ferritin and transferrin receptor, which modulate iron uptake and storage (123). Translational control is accomplished by two cytoplasmic iron regulatory proteins, IRP1 and IRP2 that interact with specific IREs, located in the 3' or 5' UTR of ferritin and transferrin receptor mrnas. At low iron concentrations, IRPs stabilize the transferrin receptor mrna while inhibiting the translation of ferritin mrna in numerous types of mammalian cells including duodenal enterocytes, reticuloendothelial macrophages, hepatocytes, and bone marrow precursors of red blood cells. With high iron levels, there is a lack of binding of IRPs to IRE, resulting in reduced transferrin receptor mrna and enhancing the expression of ferritin mrna. Remarkably, the expression of other IREcontaining mrnas, encoding proteins of iron and energy metabolism are also controlled by the IRE/IRP system. In addition, IRP1 and IRP2 are also sensitive to iron-independent signals, such 18

19 as hydrogen peroxide, hypoxia, or nitric oxide, and can be induced to regulate many other metabolic pathways. A very recent study implicates translational control by IRP1 and IRP2 in regulating mitochondrial iron supply and function. Mice deficient in hepatic IRP1 and IRP2 show evidence of mitochondrial iron deficiency and dysfunction, eventually leading to liver failure and death (59). It has also been shown that gamma interferon (IFN-γ) regulates expression of ceruloplasmin via its 3 -UTR. Ceruloplasmin is a 132-kDa, copper-containing glycoprotein secreted by the liver and monocyte/macrophages ( ). Translational repression by IFN-γ was mediated by the binding of a cytosolic factor to the ceruloplasmin mrna 3 UTR (106; 108) Translational Control of Apolipoprotein B mrna and Insulin Modulation Another example of translational control in lipid metabolism is the insulin regulation of apolipoprotein B (apob) mrna translation and protein synthesis. Physiologically, constitutive apob synthesis in the liver is essential for rapid assembly of lipids (both exogenous lipid influx as well as endogenously synthesized) into lipoprotein particles for export. Translational control of apob mrna can allow the cell to rapidly supply freshly-synthesized apob molecules for assembly of cellular lipids in the form of lipoprotein particles (134). Insulin inhibits lipoprotein assembly at least partially by blocking apob mrna translation (134; 146). Here, the evidence for apob mrna translational control is reviewed with a particular focus on recent findings in our laboratory that have shed new light on molecular factors and signaling mechanisms that govern translational control. Early Evidence for ApoB mrna Translational Control: Biosynthesis of apob and its assembly and secretion as a very low density lipoprotein (VLDL) particle is a highly regulated process, 19

20 critical to maintaining the circulating plasma levels of lipids and lipoprotein particles. The full length apob (apob100) expressed in the liver, is a glycoprotein with a molecular mass of about 550 kda (composed of a single chain of 4536 amino acids) containing 16 N-glycans. It is one of the largest eukaryotic proteins and subject to multiple steps of co- and post-translational regulation at the levels of mrna translation, ER translocation, degradation, and assembly with lipids to form lipoprotein particles. Although most attention has been given in recent years to apob degradative mechanisms, work in several laboratories including ours point to importance of translational control of apob mrna. ApoB is constitutively expressed in the liver and usually its mrna level remains constant under most metabolic stimuli (34; 100; 115; 120; 129; 147). ApoB mrna is nucleotides long with a half life of approximately 16 hours and encodes a 4563 amino acid protein with a molecular weight of about 512 kda (87). The apolipoprotein B mrna contains 5 and 3 UTRs which are 128 and 304 nucleotides in length respectively (88). The 5 UTR is GC-rich with a GC content of about 76%. Analysis of the 5 UTR revealed the presence of two GC boxes with a typical CCGCCC sequence similar to promoter sequences of eukaryotic genes. The two GC boxes are located at nucleotides +20 and +81 upstream of the translational start site. The 3 UTR sequence is AU-rich and contains many AU repeats (13). Evidence for translational control comes from studies of apob synthesis in HepG2 cells(152), primary rat hepatocytes (30; 145), streptozotocin-induced diabetic rats (147), cultured human fetal intestinal cells (97), and in hepatocytes treated with CP-10447, an inhibitor of microsomal triglyceride transfer protein (MTP) (a protein that facilitates lipidation of apoblipoproteins) (122). Interestingly, apob mrna is edited in the human intestine (C-to-U RNA editing in the nucleus by an editing factor, apobec-1) leading to the introduction of a stop codon, 20

21 premature termination of translation, and the synthesis of a shorter form of the apob (apob48, which is about 48% of the apob100)(36). Transfection of human apob48 in liver cells increases synthesis of apob48 but reduces the synthesis of endogenous full-length protein (70), suggesting competition between the translation of apob48 (edited) and apob100 (unedited) mrnas. Role of UTR sequences in ApoB mrna Translational Control: Although there is ample evidence for translational control of apob synthesis, the molecular mechanisms or factors that mediate translational control of apob mrna had not been elucidated until our recent studies on the role of 5 and 3 UTRs (127; ) and intracellular traffic into RNA granules (76). Recent investigation of the 5' and 3'UTR sequences of apob mrna has revealed elements within the 5 UTR with the potential to form stable secondary structures and stimulate apob mrna translation. The 5'UTR sequence is unusually GC rich, comprising of 76% G+C nucleotides (two GC boxes with the typical CCGCCC sequence and a GAGGCC doublet). G+C-rich regions have a high potential for forming stable secondary structure (94). Studies have shown that highly structured 5'UTR sequences tend to inhibit efficient translation (93; 94). Results obtained from analysis of the 5'UTR using the computer program M-fold (127) predict a free energy of formation of specific structure in the range of to kcal/mol. Of the five predictions provided by the program, all possess stable stem-loop structures. A total ΔG value in the range of >-50 kcal/mol is indicative of the potential to form very stable secondary structures and would be expected to result in inefficient translation due to the inability of the ribosomal complex to unwind the secondary structure and reach the AUG codon (94). This suggests that the 5'UTR should confer an inhibition of translation. However, we observed considerable stimulation of apob synthesis in both in vivo and in vitro translation studies (127; 142). It is possible that the secondary structure present in the 5'UTR of apob mrna is capable of being unwound by the 21

22 presence of initiation factor, eif4a. Initiation factor eif4a, upon activation by eif4b, may unwind highly structured 5'UTR s through its ATP-dependent RNA helicase activity and thus decrease the repression imposed by secondary structure (133; 157; 158). Deletion analysis of 5 UTR suggests that the first 96 nucleotides are essential to the translational effect of the 5 UTR. Secondary structure analysis of the 5'UTR 1-96 mutant construct predicted two structures both capable of forming the Y shaped structure containing stem loops I, II, and III of the full-length 5'UTR. Importantly, electrophoretic mobility shift assays (EMSA) showed the binding of a transacting p110 protein factor to the 5 UTR of the apob mrna (142), which appeared to mediate translational stimulation. Insulin was found to down-regulate apob mrna translation through inhibition of the interaction of the p110 factor with the apob 5 UTR (142). Using dual (bicistronic) luciferase constructs, we also examined the role of internal ribosomal entry (IRES) with respect to the 5'UTR of the apob mrna and found that the apob 5 UTR possesses IRES activity and basal translational activity of the apob mrna may be partly cap independent (141). These observation will however require further confirmation as recent studies suggest that some putative IRES elements, when inserted into a bicistronic construct, can act as cryptic promoter elements, generating monocistronic mrnas highlighting the limitation of this type of analysis (8; 175) Signaling Modulation of ApoB mrna Translation: Interestingly, although insulin normally activates global translation of cellular protein synthesis in mammalian cells (128), it has a specific inhibitory effect on apob mrna translation. This suggests that insulin induces a unique signaling cascade that leads to specific inhibition of apob mrna translation despite global translational stimulation. Recently, translational control of apob mrna via the 5 UTR and the binding of the 110 kda protein factor was found to be regulated by protein kinase C (PKC) 22

23 signaling cascade (143). The mechanisms mediating PKC regulation of apob mrna translation are unknown. PKCs are known to activate phosphorylation of eif-4e (136; 178). eif4e is known to play an important role in mrna translation by binding the 5 -cap structure of mrnas and facilitating the recruitment of other translation factors and the 40S ribosomal subunit (18; 157). Furthermore, phosphorylation of eif-4e has been shown to decrease its affinity for the 5 cap (178). It has also been proposed that eif-4e phosphorylation at serine 209 may reprogram the translational machinery by releasing the translational apparatus from existing complexes, allowing other mrnas to be translated. These mrnas could be highly structured mrnas, such as the apob mrna. mrnas with significant 5 -UTR secondary structure are normally poorly translated, but can be significantly activated by increasing eif4e activity (40; 90). Finally, other reports of PKC-mediated control of eukaryotic mrna translation via cell signalling have emerged such as the insulin modulation rates of muscle protein synthesis in Zucker rats (56). Translational Control via Intracellular Traffic into RNA Granules: It is currently unknown whether apob mrna translation can be controlled by the release of translatable apob mrna transcripts from cytoplasmic stores of mrnps that are translationally repressed. However, the long half life of apob mrna (16 h) clearly suggests that a significant proportion of the message may be stored in the cell prior to translation. Interestingly, ApoB mrna polysome complexes have been reported to show unusual physical properties and exhibit unique sedimentation behaviors more characteristic of nonpolysomal mrnps (29) which further suggests existence of apob mrna in RNA granules. Very recent studies in our laboratory indicate that apob mrna is localized with markers of cytoplasmic P bodies, suggesting intracellular storage in RNA granules (76). Under certain conditons, the rate of apob mrna translation may be controlled via traffic from cytoplasmic P bodies and the release of translatable mrna transcripts from stored 23

24 mrnps. We found that insulin pretreatment of cultured hepatocytes significantly increased colocalization of apob mrna with P body markers, suggesting insulin-induced silencing of apob mrna. This increased P body localization coincided with reduced polysomal association confirming insulin-stimulated traffic of apob mrna away from translationally-active polysomes and into translationally-silent pools associated with cytoplasmic P bodies (76). Collectively, our data suggest that insulin-mediated translational silencing of apob mrna is at least partially mediated via intracellular traffic into cytoplasmic RNA granules. The physiological significance of this mode of translational regulation is currently unknown but we postulate that it may play an important role in controlling the rate of apob synthesis and hepatic lipoprotein (VLDL) production under certain metabolic conditions. It is important to note that although overproduction of apob-containing VLDL particles is commonly observed in hyperinsulinemic states (and is a major contributor to hypertriglyceridemia seen in diabetic states)(31; 98; 173), insulin has been shown in numerous studies to acutely suppress apob and VLDL secretion (31; 112; 146). The increased VLDL production in hyperinsulinemic states is likely due to hepatic insulin resistance and resistance of hepatocytes to insulin s suppressive action on VLDL production (6; 31). The studies discussed above suggest that insulin-mediated suppression of apob mrna translation may be a key mechanism in control of VLDL secretion. Figure 3 depicts our current understanding of the mechanisms mediating translational control of apob mrna by insulin. 554 Concluding Remarks: Regulating metabolic pathways via modulation of mrna translation and protein synthesis makes sense physiologically as it allows the cell to rapidly alter its proteome in response to various nutritional, endocrine, and neuronal signals. The recent discovery of mirnas and RNA 24

25 granules and their critical role in determining the fate and activity of mrna transcripts has brought the importance of translational control into sharp focus. The complex interaction of mirnas, RNA binding proteins, and cytoplasmic RNA granules controls the stability and translational activity of freshly-transcribed mrnas, and plays a critical role in fine-tuning protein expression. Of interest to the field of endocrinology and metabolism, there is now substantial evidence pointing to the role of translational control in regulating metabolic function and determining a cell s response to various endocrine and metabolic signals. Defects in pathways controlling mrna translation and protein synthesis are now implicated in the pathogenesis of metabolic disorders such as type 2 diabetes at multiple levels including insulin synthesis and secretion, hepatic glucose uptake and gluconeogenesis, hepatic lipogenesis, as well as lipid and lipoprotein production. With the recent explosion of interest in the role of mirnas in regulating metabolic pathways, we are likely to gain over the coming years a much better understanding of the role of translational control mechanisms in modulating metabolic function and to witness many new exciting discoveries in how complex regulation of mrna transcripts leads to modulation of specific metabolic pathways in health and disease Acknowledgements: This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). I would also like to thank my administrative assistant, Catherine Lim-Shue, who provided considerable help with compiling and formatting most of the cited articles

26 578 Figure Legends: Figure 1: Complex mrna Lifecycle. This diagram depicts in a very simplified form the complex life of mrna transcripts in eukaryotic cells and the involvement of multiple nodes of regulation before translation and protein synthesis can occur. Upon gene transcription, mrna is synthesized as precursor heteronuclear RNA (hnrna) transcripts that require complex processing to form mature mrna molecules. hnrna processing occurs largely in the nucleus and is accompanied by the binding of mrna with numerous RNA binding proteins that form mrnps. Nuclear export of mrnps is itself a complex process that ensures delivery of fresh mrna transcripts to cytoplasm for translation and protein synthesis. mrnps are however frequently stored in cytoplasmic RNA granules which act as regulated reservoirs for translatable mrna and can dictate, depending on metabolic conditions, either translational activation or degradation via mrna decay mechanisms. Increasingly, evidence also points to mirnas as key regulators of mrna translational efficiency and decay Figure 2: Translational control mechanisms in cellular metabolism. Available evidence supports a critical role for translational control mechanisms in numerous metabolic pathways and the potential involvement of defects in translational control in the development of metabolic disorders such as type 2 diabetes. The diagram summaries some of the metabolic targets of translational control involving regulation by 5 and 3 UTRs and mirnas. Further details are given in the text Figure 3: Insulin modulation of apob mrna translation. Recent evidence suggest that insulin suppresses apob mrna translation and protein synthesis via mechanisms involving cis- trans interactions at the 5 UTR as well as mrna traffic into cytoplasmic P bodies. Insulin 26

27 signaling blocks the binding of an RNA binding protein (p110, also referred to as the insulin sensitive factor) to the 5 UTR of apob mrna resulting in translational repression. Insulin may also modulate the expression level or activity of the p110 factor although this has not been shown experimentally. This process is subject to regulation by protein kinase C signaling and may be dysregulated in conditions such as insulin resistance. In addition, apob mrna has a long half-life of 16 hours and thus traffics to cytoplasmic RNA granules for storage providing a constant supply of mrna for constitutive synthesis of apob protein. ApoB mrna traffic into RNA granules away from ribosomes and translational machinery is yet another site of insulinmediated translational repression. X indicates a yet to be identified putative RNA binding protein that may regulate IRES-mediated apob mrna translation

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51

52 Figure 1 Gene Transcription hnrna Synthesis Protein Synthesis Metabolic Response hnrna Processing mrna maturation ti Translational Activation Ribosome Recruitment mrna Translation Translation Complex mrna Lifecycle Degradation mrna Transport & Nuclear Exit RNA granules mrnps mirnas mrna storage or decay mrnp Formation RNA Granule Assembly

53 Figure 2 Energy Balance Glucose Homeostasis Insulin Sensitivity GLUT1 and GLUT4 PPARγ, PGC-1, UCP1 Liver PEPCK,,glucose-6- phosphatase, HNF-3 HIF-1alpha Lipid metabolism Lipogenesis Inflammation Glycolysis Gluconeogenesis Insulin resistance mtorc-1 C/EBPα C/EBPβ 4E-BPs S6K-1 Translational l Control in Cell Metabolism mirnas SREBP-1 & SREBP-2 FAS, ACC1, CROT, CPT1α, HADHB IRS2, AMPKα Lipid metabolism Insulin Sensitivity Cholesterol Homeostasis Mitochondrial Function eif2α PERK Pancreatic β Cells Insulin Biosynthesis GLP-1 action Glucose Homeostasis Type 2 diabetes

54 Figure 3 Insulin PI-3 Kinase PKCα/β??? + Insulin Resistance _ Insulin sensitive Factor (p110) 5 UTR Protein Complexes AUG 5'UTR Apo B Gene CPEB 3'UTR P bodies 3 UTR Protein Complexes 40S Ribosomal Subunit ApoB Protein Synthesis