UTR RNA Research Articles

Translational Regulation of Acetylcholinesterase by the RNA-binding Protein Pumilio-2 at the Neuromuscular Synapse

Acetylcholinesterase (AChE) is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Our understanding of the molecular mechanisms underlying its regulation is incomplete, but they appear to involve both translational and post-translational events as well. Here, we show that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates. Immunoprecipitation of muscle cell extracts with a PUM2 specific antibody precipitated AChE mRNA, suggesting that PUM2 binds to the AChE transcripts in a complex. Gel shift assays using a bacterially expressed PUM2 RNA binding domain showed specific binding using wild type AChE 3'-UTR RNA segment that was abrogated by mutation of the consensus recognition site. Transfecting skeletal muscle cells with shRNAs specific for PUM2 up-regulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. Finally, we found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific proteins involved in modulating synaptic transmission, analogous to CNS synapses.

Far upstream element binding protein 1 activates translation of p27 Kip1 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.

Role of HIV-1 RNA and protein determinants for the selective packaging of spliced and unspliced viral RNA and host U6 and 7SL RNA in virus particles.

HIV-1 particles contain RNA species other than the unspliced viral RNA genome. For instance, viral spliced RNAs and host 7SL and U6 RNAs are natural components that are non-randomly incorporated. To understand the mechanism of packaging selectivity, we analyzed the content of a large panel of HIV-1 variants mutated either in the 5′UTR structures of the viral RNA or in the Gag-nucleocapsid protein (GagNC). In parallel, we determined whether the selection of host 7SL and U6 RNAs is dependent or not on viral RNA and/or GagNC. Our results reveal that the polyA hairpin in the 5′UTR is a major packaging determinant for both spliced and unspliced viral RNAs. In contrast, 5′UTR RNA structures have little influence on the U6 and 7SL RNAs, indicating that packaging of these host RNAs is independent of viral RNA packaging. Experiments with GagNC mutants indicated that the two zinc-fingers and N-terminal basic residues restrict the incorporation of the spliced RNAs, while favoring unspliced RNA packaging. GagNC through the zinc-finger motifs also restricts the packaging of 7SL and U6 RNAs. Thus, GagNC is a major contributor to the packaging selectivity. Altogether our results provide new molecular insight on how HIV selects distinct RNA species for incorporation into particles.

Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5′ Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome

Sequences in the 5' untranslated region (5'UTR) of hepatitis C virus (HCV) RNA is important for modulating both translation and RNA replication. The translation of the HCV genome depends on an internal ribosome entry site (IRES) located within the 341-nucleotide 5'UTR, while RNA replication requires a smaller region. A question arises whether the replication and translation functions require different regions of the 5'UTR and different sets of RNA-binding proteins. Here, we showed that the 5'-most 157 nucleotides of HCV RNA is the minimum 5'UTR for RNA replication, and it partially overlaps with the IRES. Stem-loops 1 and 2 of the 5'UTR are essential for RNA replication, whereas stem-loop 1 is not required for translation. We also found that poly(C)-binding protein 2 (PCBP2) bound to the replication region of the 5'UTR and associated with detergent-resistant membrane fractions, which are the sites of the HCV replication complex. The knockdown of PCBP2 by short hairpin RNA decreased the amounts of HCV RNA and nonstructural proteins. Antibody-mediated blocking of PCBP2 reduced HCV RNA replication in vitro, indicating that PCBP2 is directly involved in HCV RNA replication. Furthermore, PCBP2 knockdown reduced IRES-dependent translation preferentially from a dual reporter plasmid, suggesting that PCBP2 also regulated IRES activity. These findings indicate that PCBP2 participates in both HCV RNA replication and translation. Moreover, PCBP2 interacts with HCV 5'- and 3'UTR RNA fragments to form an RNA-protein complex and induces the circularization of HCV RNA, as revealed by electron microscopy. This study thus demonstrates the mechanism of the participation of PCBP2 in HCV translation and replication and provides physical evidence for HCV RNA circularization through 5'- and 3'UTR interaction.

A Simple Method to Raise Efficiency of Interaction With Nucleic Acids of Self Polymerization-prone Proteins

A basic research subject is the study on interactions between proteins, DNA and RNA. Majority of DNA and RNA-binding proteins are self polymerization-prone and may form aggregates that are difficult to bind DNA or RNA, thus hampering experiments. Labeling the proteins with fluorescein isothiocyanate significantly inhibits this tendency and markedly increases the binding efficiency of these proteins to bind nucleic acid molecules. This simple method has been utilized in the investigation on binding of C/EBP beta 3' UTR RNA by cytokeratin 18.

Tumor suppression by RNA from C/EBPβ 3'UTR through the inhibition of protein kinase Cε activity.

BackgroundSince the end of last century, RNAs from the 3′untranslated region (3′UTR) of several eukaryotic mRNAs have been found to exert tumor suppression activity when introduced into malignant cells independent of their whole mRNAs. In this study, we sought to determine the molecular mechanism of the tumor suppression activity of a short RNA from 3′UTR of C/EBPβ mRΝΑ (C/EBPβ 3′UTR RNA) in human hepatocarcinoma cells SMMC-7721.Methodology/Principal FindingsBy using Western blotting, immunocytochemistry, molecular beacon, confocal microscopy, protein kinase inhibitors and in vitro kinase assays, we found that, in the C/EBPβ 3′UTR-transfectant cells of SMMC-7721, the overexpressed C/EBPβ 3′UTR RNA induced reorganization of keratin 18 by binding to this keratin; that the C/EBPβ 3′UTR RNA also reduced phosphorylation and expression of keratin 18; and that the enzyme responsible for phosphorylating keratin 18 is protein kinase Cε. We then found that the C/EBPβ 3′UTR RNA directly inhibited the phosphorylating activity of protein kinase Cε; and that C/EBPβ 3′UTR RNA specifically bound with the protein kinase Cε-keratin 18 conjugate.Conclusion/SignificanceTogether, these facts suggest that the tumor suppression in SMMC-7721 by C/EBPβ 3′UTR RNA is due to the inhibition of protein kinase Cε activity through direct physical interaction between C/EBPβ 3′UTR RNA and protein kinase Cε. These facts indicate that the 3′UTR of some eukaryotic mRNAs may function as regulators for genes other than their own.

Alternative Ways to Think about Cellular Internal Ribosome Entry

Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of "cap-dependent" and "IRES-dependent" should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation.

Abstract 2021: MicroRNA affinity assay

Abstract Introduction: MicroRNAs (miRNAs) are small noncoding RNAs functioning in gene regulation. In animal cells, miRNAs bind to target sites in the 3’ UTR of mRNAs, causing posttranscriptional repression or degradation of mRNAs. The identification of specific miRNAs to target a 3′UTR of a given gene is essential for studies in gene regulation. However, the sequence software programs conventionally used to predict binding of individual microRNAs to a given mRNA transcript are notoriously unreliable, and specific microRNA-mRNA binding is very difficult to directly affirm using currently available techniques. We have developed a microRNA affinity assay that addresses this need. Methods: We chose a candidate gene that had been extensively studied experimentally for microRNA regulation; it is known that miR-200c and miR-200b can bind to 3′UTR RNA of TCF8 [Hurteau 2007]. Here, a 500bp fragment of the 3′UTR RNA of TCF8 was generated by IVT, and used as a bait sequence to identify miRNAs that bind a mixture of known miRNA composition, and alternately a naturally produced cellular miRNA pool. After separation, miRNA qPCR was used to check recovery rate of miRNA, and cloning and sequencing was used to identify miRNAs. Results: Specific miRNAs from a synthetic mixture miRNAs, miRNA qPCR results displayed excellent recovery rates (151.57% with pipetting error, for miR-200c), 88.88% for the closely related miR-200b, 0.31% for the sequence-unrelated miR-9, 0.67% (miR-10a), 0.1% (miR-20a), 8.78% (miR-34a with some 3′UTR homology), 0.11% (miR-100), 0.16% (miR-140-5p), 0.98% (miR-144), 0.33% (miR-802) and 1.58% (miR-1234). After cloning the captured miRNAs, there were 10 sequences from miR-200c, 7 sequences from miR-200b, and one other sequence in 18 sequenced clones. When separating specific miRNAs from naturally produced NHBE cell miRNA pool, miRNA qPCR results show that their recovery rates are 88.88% (miR-200c), 80% (miR-200b), 0.07% (miR-20a), less than 0.006% (miR-34a) and 0.01% (miR-100). Conclusions: For both simple known and complex unknown microRNA pools, this pull-down strategy is effective in determining microRNA-mRNA binding. In future work, we will use other 3′UTR RNA sequences of candidate genes as bait sequence, for microRNA-related discovery and confirmation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2021.

RNA Mapping of Mutant Myotonic Dystrophy Protein Kinase 3'-Untranslated Region Transcripts

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3’-untranslated region (3’-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans-splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3’-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a transsplicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA

BackgroundEarlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation.Methodology/Principal FindingsWe have probed the putative secondary structure of the IRF2 5′UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 3′terminus of the 5′untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2–5′UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3′end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA.Conclusion/SignificanceIt appears that PTB binding to multiple sites within IRF2 5′UTR leads to a conformational change in the RNA that facilitate binding of other trans-acting factors to mediate internal initiation of translation.

SECIS-binding protein 2, a key player in selenoprotein synthesis, is an intrinsically disordered protein

Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3′ UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca. 70% of the SBP2 sequence is disordered, whereas the RNA binding domain appears to be folded and functional. These results are consistent with a recent report on the role of the Hsp90 chaperone for the folding of SBP2 and other functionally unrelated proteins bearing an RNA binding domain homologous to SBP2.

Human initiation factor eIF3 subunit b interacts with HCV IRES RNA through its N-terminal RNA recognition motif

Many viral mRNAs contain a 5′-UTR RNA element called internal ribosome-entry site (IRES), which bypasses the requirement of some canonical initiation factors allowing cap-independent translation. The IRES of hepatitis-C virus drives translation by directly recruiting 40S ribosomal subunits and binds to eIF3 which plays a critical role in both cap-dependent and cap-independent translation. However, the molecular basis for eIF3 activity in either case remains enigmatic. Here we report that subunit b of the eIF3 complex directly binds to HCV IRES domain III via its N-terminal-RRM. Because eIF3b was previously shown to be involved in eIF3j binding, biological implications are discussed.

Germ cells in the crustacean Parhyale hawaiensis depend on Vasa protein for their maintenance but not for their formation

Germ cells are a population of cells that do not differentiate to form somatic tissue but form the egg and sperm that ensure the reproduction of the organism. To understand how germ cells form, holds a key for identifying what sets them apart from all other cells of the organism. There are large differences between embryos regarding where and when germ cells form but the expression of Vasa protein is a common trait of germ cells. We studied the role of vasa during germ cell formation in the crustacean Parhyale hawaiensis. In a striking difference to the posterior specification of the group of germ cells in the arthropod model Drosophila, all germ cells in Parhyale originate from a single germ line progenitor cell of the 8-cell stage. We found vasa RNA ubiquitously distributed from 1-cell to 16-cell stage in Parhyale and localized to the germ cells from 32-cell stage onwards. Localization of vasa RNA to the germ cells is controlled by its 3′UTR and this could be mimicked by fluorescently labeled 3′UTR RNA. Vasa protein was first detectable at the 100-cell stage. MO-mediated inhibition of vasa translation caused germ cells to die after gastrulation. This means that in Parhyale Vasa protein is not required for the initial generation of the clone of germ cells but is required for their subsequent proliferation and maintenance. It also means that the role of vasa changed substantially during an evolutionary switch in the crustaceans by Parhyale from the specification of a group of germ cells to that of a single germ line progenitor. This is the first functional study of vasa in an arthropod beyond Drosophila.

COX‐2 mRNA stability in Monocytes by Ligation of RAGE. Novel roles of hnRNPK and miR‐16

Advanced glycation end products (AGEs) act via their receptor, RAGE, and play major roles in diabetic complications. We recently showed that AGEs and a RAGE ligand, S100b, can induce the inflammatory cycoloxygenase‐2 (COX‐2) gene in monocytes by both mRNA stability and transcription mechanisms. In this study we examined the mRNA stabilizing mechanisms. S100b treatment of THP‐1 monocytes led to significant accumulation of COX‐2 mRNA at 2 hr. This was mainly due to RNA stability as also confirmed by S100b‐induced increase in luciferase activity of a COX‐2‐3’UTR‐Luc construct. Heterogenous nuclear ribonucleoprotein‐K (hnRNP K) promotes cross‐talk among multiple nuclear factors. RNA immunoprecipitation assays showed that S100b increased binding of hnRNP K to COX‐2 UTR RNA in THP‐1 cells. This was further confirmed by RNA‐EMSA and supershift with hnRNP K antibody. siRNAs to hnRNPK blocked S100b‐induced COX‐2 mRNA stability. Furthermore, micro RNA(miR)‐16 was also involved in S100b induced COX‐2 stability. In untreated cells, siRNA to miR‐16 enhanced COX‐2 3’UTR‐Luc activity and mRNA stability suggesting that miR‐16 can destabilize COX‐2 mRNA. Upon S100b treatment, miR16 levels (QPCR and N.blots) were reduced, while COX‐2 stability was enhanced. These new findings show that diabetic stimuli like AGEs can stabilize inflammatory genes such as COX‐2 by novel RNA regulatory mechanims involving hnRNP K and miR16.

An efficient RNA‐cleaving DNA enzyme can specifically target the 5′‐untranslated region of severe acute respiratory syndrome associated coronavirus (SARS‐CoV)

BackgroundThe worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS‐CoV. We report the use of DNAzyme (catalytic DNA) to target the 5′‐untranslated region (5′UTR) of a highly conserved fragment in the SARS genome as an approach to suppression of SARS‐CoV replication. A mono‐DNA enzyme (Dz‐104) possessing the 10–23 catalytic motif was synthesized and tested both in vitro and in cell culture.Materials and methodsSARS‐CoV total RNA was isolated, extracted from the SARS‐CoV‐WHU strain and converted into cDNA. We designed a RNA‐cleaving 10–23 DNAzyme targeting at the loop region of the 5′UTR of SARS‐CoV. The designed DNAzyme, Dz‐104, and its mutant version, Dz‐104 (mut), as a control consist of 9 + 9 arm sequences with a 10–23 catalytic core. In vitro cleavage was performed using an in vitro transcribed 5′UTR RNA substrate. A vector containing a fused 5′UTR and enhanced green fluorescent protein (eGFP) was co‐transfected with the DNAzyme into E6 cells and the cells expressing eGFP were visualized with fluorescence microscopy and analyzed by fluorescence‐activated cell sorting (FACS).Results and conclusionsOur results demonstrated that this DNAzyme could efficiently cleave the SARS‐CoV RNA substrate in vitro and inhibit the expression of the SARS‐CoV 5′UTR‐eGFP fusion RNA in mammalian cells. This work presents a model system to rapidly screen effective DNAzymes targeting SARS and provides a basis for potential therapeutic use of DNA enzymes to combat the SARS infection. Copyright © 2007 John Wiley & Sons, Ltd.

Specific interaction of HeLa cell proteins with coxsackievirus B3 3'UTR: La autoantigen binds the 3' and 5'UTR independently of the poly(A) tail

Coxsackievirus B3 (CVB3) is a positive, single-stranded RNA virus. The secondary structure of the 3' untranslated region (3'UTR) of CVB3 RNA consists of three stem-loops and is followed by a poly(A) tail sequence. These stem-loop structures have been suggested to participate in the regulation of viral replication through interaction with cellular proteins that are yet to be identified. In this study, by competitive UV cross-linking using mutated 3'UTR probes we have demonstrated that the poly(A) tail is essential for promoting HeLa cell protein interactions with the 3'UTR because deletion of this sequence abolished most of the protein interactions. Unexpectedly, mutations that disrupted the tertiary loop-loop interactions without affecting the stem-loops did not apparently affect these protein interactions, indicating that secondary structure rather than the high-order structure may play a major role in recruiting these RNA binding proteins. Among the observed 3'UTR RNA binding proteins, we have confirmed a 52 kDa protein as the human La autoantigen by using purified recombinant protein and a polyclonal La antibody. This protein can interact with both the 3' and 5'UTRs independently of the poly(A) tail. Further analysis by two-stage UV cross-linking, we found that the 3' and 5'UTR sequences may share the same binding site on the La protein.

PUF Protein-mediated Deadenylation Is Catalyzed by Ccr4p

PUF proteins control gene expression by binding to the 3'-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.

Opposing Polymerase-Deadenylase Activities Regulate Cytoplasmic Polyadenylation

Cytoplasmic polyadenylation is one mechanism that regulates translation in early animal development. In Xenopus oocytes, polyadenylation of dormant mRNAs, including cyclin B1, is controlled by the cis-acting cytoplasmic polyadenylation element (CPE) and hexanucleotide AAUAAA through associations with CPEB and CPSF, respectively. Previously, we demonstrated that the scaffold protein symplekin contacts CPEB and CPSF and helps them interact with Gld2, a poly(A) polymerase. Here, we report the mechanism by which poly(A) tail length is regulated. Cyclin B1 pre-mRNA acquires a long poly(A) tail in the nucleus that is subsequently shortened in the cytoplasm. The shortening is controlled by CPEB and PARN, a poly(A)-specific ribonuclease. Gld2 and PARN both reside in the CPEB-containing complex. However, because PARN is more active than Gld2, the poly(A) tail is short. When oocytes mature, CPEB phosphorylation causes PARN to be expelled from the ribonucleoprotein complex, which allows Gld2 to elongate poly(A) by default.

Cytoplasmic CstF-77 Protein Belongs to a Masking Complex with Cytoplasmic Polyadenylation Element-binding Protein in Xenopus Oocytes

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

Structural characterization of the 5′ untranslated RNA of hepatitis C virus by vibrational spectroscopy

Raman and FTIR spectroscopy have been used to characterize the structure of 5′untranslated region (5′UTR, 342-mer RNA) of the HCV genome. The study of the 750–850 cm − 1 Raman spectral domain of the ribose-phosphate backbone reveals that the percentage of nucleobases involved in double helix–loop junctions is 19 ± 1%, which is very close to that of a theoretical secondary structure model (18.7%) proposed on the basis of comparative sequence analysis and thermodynamic modelling. In addition, about 68 ± 2% of the bases are helically ordered having C(3′)-endo ribofuranose pucker. FTIR-monitored H/D exchange provides the following results: (a) base-paired guanine and cytosine nucleobases show the lowest rate of isotopic exchange, and some synchronous intensity changes of marker bands of A·U pair and single stranded adenine are consistent with the presence of A ⁎A·U triplets; (b) the vibrational coupling between the ribose ether C–O stretching and 2′OH bending motions reveals that helical regions of 5′UTR RNA are characterized by hydrogen bonding between the 2′OH ribose groups and the ether oxygen atoms of neighbouring ribose residues.