mPer2 antisense oligonucleotides inhibit mPER2 expression but not circadian rhythms of physiological activity in cultured suprachiasmatic nucleus neurons

Various day-night rhythms, observed at molecular, cellular, and behavioral levels, are governed by an endogenous circadian clock, predominantly functioning in the hypothalamic suprachiasmatic nucleus (SCN). A class of clock genes, mammalian Period (mPer), is known to be rhythmically expressed in SCN neurons, but the correlation between mPER protein levels and autonomous rhythmic activity in SCN neurons is not well understood. Therefore, we blocked mPer translation using antisense phosphothioate oligonucleotides (ODNs) for mPer1 and mPer2 mRNAs and examined the effects on the circadian rhythm of cytosolic Ca2+ concentration and action potentials in SCN slice cultures. Treatment with mPer2 ODNs (20microM for 3 days) but not randomized control ODNs significantly reduced mPER2 immunoreactivity (-63%) in the SCN. Nevertheless, mPer1/2 ODNs treatment inhibited neither action potential firing rhythms nor cytosolic Ca2+ rhythms. These suggest that circadian rhythms in mPER protein levels are not necessarily coupled to autonomous rhythmic activity in SCN neurons.     

Direct association between mouse PERIOD and CKIepsilon is critical for a functioning circadian clock

The mPER1 and mPER2 proteins have important roles in the circadian clock mechanism, whereas mPER3 is expendable. Here we examine the posttranslational regulation of mPER3 in vivo in mouse liver and compare it to the other mPER proteins to define the salient features required for clock function. Like mPER1 and mPER2, mPER3 is phosphorylated, changes cellular location, and interacts with other clock proteins in a time-dependent manner. Consistent with behavioral data from mPer2/3 and mPer1/3 double-mutant mice, either mPER1 or mPER2 alone can sustain rhythmic posttranslational events. However, mPER3 is unable to sustain molecular rhythmicity in mPer1/2 double-mutant mice. Indeed, mPER3 is always cytoplasmic and is not phosphorylated in the livers of mPer1-deficient mice, suggesting that mPER3 is regulated by mPER1 at a posttranslational level. In vitro studies with chimeric proteins suggest that the inability of mPER3 to support circadian clock function results in part from lack of direct and stable interaction with casein kinase Iepsilon (CKIepsilon). We thus propose that the CKIepsilon-binding domain is critical not only for mPER phosphorylation but also for a functioning circadian clock.     

Light-induced phase shifts in mice lacking mPER1 or mPER2

Three homologs of the Drosophila Period gene have been identified in mammals. In mice, these three genes (mPer1, mPer2, and mPer3) have distinct roles in the circadian clockwork. While products of mPer1 and mPer2 play important roles in the maintenance of circadian rhythmicity, mPer3 gene products are dispensable for rhythmicity. Several studies also implicate mPER1 and mPER2 in transduction of photic information to the core circadian clockwork. The phase-shifting effects of light were examined in mPER1-deficient and mPER2-deficient mice using T cycle paradigms, in which mice received 1 h of light per day at an interval of T hours. To assess phase delays, repeated exposure to 1 h of light per day at T = 24 was used. To assess phase advances, exposure to 1-h light pulses at T = 22-h intervals was used. The degeneration of rhythmicity in the mutant mice prevented assessment of a response in most cases. Nevertheless, clear examples of phase delays and phase advances were observed in both mPer1 and mPer2 mutant mice. These results are not consistent with the hypothesis that mPER1 and mPER2 play necessary and nonoverlapping roles in mediating the effects of light on the circadian dock.     

Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 had severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms were blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not of mPER1-deficient mice. Peak mPER and mCRY1 protein levels were reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resembled rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice were immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes.     

Oculopharyngeal muscular dystrophy mutations link the RNA-binding protein HNRNPQ to autophagosome biogenesis

Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear ribonucleoprotein Q (HNRNPQ)/SYNCRIP is required to stimulate early events in autophagosome biogenesis, in particular the induction of VPS34 kinase by ULK1-mediated beclin 1 phosphorylation. The RBPs HNRNPQ and poly(A) binding protein nuclear 1 (PABPN1) form a regulatory network that controls the turnover of distinct autophagy-related (ATG) proteins. We also show that oculopharyngeal muscular dystrophy (OPMD) mutations engender a switch from autophagosome stimulation to autophagosome inhibition by impairing PABPN1 and HNRNPQ control of the level of ULK1. The overexpression of HNRNPQ in OPMD patient-derived cells rescues the defective autophagy in these cells. Our data reveal a regulatory mechanism of autophagy induction that is compromised by PABPN1 disease mutations, and may thus further contribute to their deleterious effects.     

The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element

Initiation of translation of the full-length messenger RNA of HIV-1, which generates the viral structural proteins and enzymes, is cap-dependent but can also use an internal ribosome entry site (IRES) located in the 5' untranslated region. Our aim was to define, through a mutational analysis, regions of HIV-1 IRES that are important for its activity. A dual-luciferase reporter construct where the Renilla luciferase (Rluc) translation is cap-dependent while the firefly luciferase (Fluc) translation depends on HIV-1 IRES was used. The Fluc/Rluc ratio was measured in lysates of Jurkat T cells transfected with the dual-luciferase plasmid bearing either the wild-type or a mutated IRES. Deletions or mutations in three regions decreased the IRES activity but deletion or mutations of a stem-loop preceding the primer binding site increased the IRES activity. The wild-type IRES activity, but not that of an IRES with a mutated stem-loop, was increased when cells were treated with agents that induce oxidative stress. Such stress is known to be caused by HIV-1 infection and we propose that this stem-loop is involved in a switch that stimulates the IRES activity in cells infected with HIV-1, supporting the suggestion that the IRES activity is up-regulated in the course of HIV-1 replication cycle.     

miR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components

In animals, microRNAs (miRNAs) generally repress gene expression by binding to sites in the 3'-untranslated region (UTR) of target mRNAs. miRNAs have also been reported to repress or activate gene expression by binding to 5'-UTR sites, but the extent of such regulation and the factors that govern these different responses are unknown. Liver-specific miR-122 binds to sites in the 5'-UTR of hepatitis C virus (HCV) RNA and positively regulates the viral life cycle, in part by stimulating HCV translation. Here, we characterize the features that allow miR-122 to activate translation via the HCV 5'-UTR. We find that this regulation is a highly specialized process that requires uncapped RNA, the HCV internal ribosome entry site (IRES) and the 3' region of miR-122. Translation activation does not involve a previously proposed structural transition in the HCV IRES and is mediated by Argonaute proteins. This study provides an important insight into the requirements for the miR-122-HCV interaction, and the broader consequences of miRNAs binding to 5'-UTR sites.     

The sequence element of the internal ribosome entry site and a 25-kilodalton cellular protein contribute to efficient internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by internal entry of ribosomes into the 5' noncoding region (NCR). This process depends on genomic elements within the 5' NCR called the internal ribosome entry site (IRES) and may involve host factors. The alpha-branch structure (nucleotides 47 to 67) of the HCV IRES is considered a cis-acting element critical for translation initiation because it is indispensable for translation in vitro (S. Fukushi, K. Katayama, C. Kurihara, N. Ishiyama, F. B. Hoshino, T. Ando, and A. Oya, Biochem. Biophys. Res. Commun. 199:425-432, 1994). In order to further characterize the function of the alpha-branch, we determined whether sequence exchange within the alpha-branch had any effect on translation initiation. An in vitro translation study revealed that the stem sequences of this region played an important role in efficient IRES function. In addition to several HeLa cell proteins, which had a binding affinity for the 5' NCR, a novel 25-kDa protein that specifically interacted with the HCV IRES was discovered. The binding affinity of the 25-kDa protein for the 5' NCR was correlated with the efficiency of translation initiation of HCV RNA, indicating a critical role for the 25-kDa protein in HCV translation.     

Far upstream element binding protein 1 activates translation of p27Kip1 mRNA through its internal ribosomal entry site

The cyclin dependent kinase inhibitor p27 plays an important role in controlling the eukaryotic cell cycle by regulating progression through G1 and entry into S phase. It is often elevated during differentiation and under conditions of cellular stress. In contrast, it is commonly downregulated in cancer cells and its levels are generally inversely correlated with favorable prognosis. The cellular levels of p27 are regulated, in part, by translational control mechanisms. The 5′-untranslated region (5′-UTR) of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, Far Upstream Element (FUSE) Binding Protein 1 (FBP1) was shown to directly bind to the human p27 5′-UTR and to promote IRES activity. An eight-nucleotide element downstream of a U-rich region within the 5′-UTR was important for FBP1 binding and p27 IRES activity. Overexpression of FBP1 enhanced endogenous p27 levels and stimulated translation initiation. In contrast, repression of FBP1 by siRNA transfection downregulated endogenous p27 protein levels. Using rabbit reticulocyte lysates, FBP1 stimulated p27 mRNA translation in vitro. The central domain of FBP1, containing four K homology motifs, was required for p27 5′-UTR RNA binding and the N terminal domain was important for translational activation. These findings indicate that FBP1 is a novel activator of p27 translation upon binding to the 5′-UTR.     

microRNA-122 Dependent Binding of Ago2 Protein to Hepatitis C Virus RNA Is Associated with Enhanced RNA Stability and Translation Stimulation

Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.     

Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.     

La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro

Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5' untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.