A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis

The upstream of N-Ras (Unr) protein is involved in translational regulation of specific genes. For example, the Unr protein contributes to translation mediated by several viral and cellular internal ribosome entry sites (IRESs), including the PITSLRE IRES, which is activated at mitosis. Previously, we have shown that translation of the Unr mRNA itself can be initiated through an IRES. Here, we show that UNR mRNA translation and UNR IRES activity are significantly increased during mitosis. Functional analysis identified hnRNP C1/C2 proteins as UNR IRES stimulatory factors, whereas both polypyrimidine tract-binding protein (PTB) and Unr were found to function as inhibitors of UNR IRES-mediated translation. The increased UNR IRES activity during mitosis results from enhanced binding of the stimulatory hnRNP C1/C2 proteins and concomitant dissociation of PTB and Unr from the UNR IRES RNA. Our data suggest the existence of an IRES-dependent cascade in mitosis comprising hnRNP C1/C2 proteins that stimulate Unr expression, and Unr, in turn, contributes to PITSLRE IRES activity. The observation that RNA interference-mediated knockdown of hnRNP C1/C2 and Unr, respectively, abrogates and retards mitosis points out that regulation of IRES-mediated translation by hnRNP C1/C2 and Unr might be important in mitosis.     

IRES-mediated pathways to polysomes: nuclear versus cytoplasmic routes

Eukaryotic mRNA initiates translation by cap-dependent scanning, ribosome shunting and cap-independent internal ribosome entry. Internal ribosome entry was first discovered for cytoplasmic RNA viruses but has also been identified for DNA viruses and cellular mRNAs. An internal ribosome entry site (IRES) directs internal binding of ribosomes and nucleates the formation of a translation initiation complex. Current research is aimed at identifying interactions between IRES elements and RNA-binding proteins known as ITAFs (IRES trans-acting factors). Here we compare IRES elements from cytoplasmic RNA viruses with those of cellular mRNAs and DNA viruses with nuclear mRNA synthesis, and suggest that ITAF composition and IRES function directly reflect the site of synthesis of mRNA and the history of its pathway to polysomes.     

Translational control of the picornavirus phenotype

Picornaviruses are small animal viruses with positive-stranded genomic RNA, which is translated using cap-independent internal translation initiation. The key role in this is played by cis elements of the 5'-untranslated region (5'-UTR) and, in particular, by the internal ribosome entry site (IRES). The function of translational cis elements requires both canonical translation initiation factors (eIFs) and additional IRES trans-acting factors (ITAFs). All known ITAFs are cell RNA-binding proteins which play a variety of functions in noninfected cells. Specific features of translational cis elements substantially affect the phenotype and, in particular, tissue tropism and pathogenic properties of picornaviruses. It is clear that, in some cases, the molecular mechanism of this is a change in interactions between viral cis elements and ITAFs. The properties and tissue distribution of ITAFs may determine the biological properties of other viruses that also use the IRES-dependent translation initiation. Since this mechanism is also involved in translation of several cell mRNAs, ITAF may contribute to the regulation of the most important aspects of the living activity in noninfected cells.     

Identification of the TXNIP IRES and characterization of the impact of regulatory IRES trans-acting factors

Translation is a tightly regulated process and is predominantly controlled at the level of its initiation. Translation initiation mostly occurs in a cap-dependent manner. Under stress conditions when cap-dependent translation is hampered, internal ribosome entry sites (IRESes) allow for cap-independent translation of certain mRNAs. IRES-dependent translation is commonly regulated by RNA-interacting proteins, known as IRES trans-acting factors (ITAFs). In the present study, we found the 5′ untranslated region (UTR) of the thioredoxin-interacting protein (TXNIP) mRNA to be bound by the ITAF hnRNPA1. Upon verification of an IRES element within the 5′UTR of TXNIP, we determined additional interacting proteins, which predominantly appeared to interact with the IRES-regulatory second half of the 5′UTR. Amongst these PTB emerged as an inhibitory ITAF, whereas FBP3 and GEMIN5 appeared to contain TXNIP IRES-enhancing properties. In summary, we identified and characterized a novel IRES within the 5′UTR of TXNIP, which is regulated by the ITAFs PTB, FBP3, and GEMIN5.     

The different pathways of HIV genomic RNA translation

Lentiviruses, the prototype of which is HIV-1, can initiate translation either by the classical cap-dependent mechanism or by internal recruitment of the ribosome through RNA domains called IRESs (internal ribosome entry sites). Depending on the virus considered, the mechanism of IRES-dependent translation differs widely. It can occur by direct binding of the 40S subunit to the mRNA, necessitating a subset or most of the canonical initiation factors and/or ITAF (IRES trans-acting factors). Nonetheless, a common feature of IRESs is that ribosomal recruitment relies, at least in part, on IRES structural determinants. Lentiviral genomic RNAs present an additional level of complexity, as, in addition to the 5'-UTR (untranslated region) IRES, the presence of a new type of IRES, embedded within Gag coding region was described recently. This IRES, conserved in all three lentiviruses examined, presents conserved structural motifs that are crucial for its activity, thus reinforcing the link between RNA structure and function. However, there are still important gaps in our understanding of the molecular mechanism underlying IRES-dependent translation initiation of HIV, including the determination of the initiation factors required, the dynamics of initiation complex assembly and the dynamics of the RNA structure during initiation complex formation. Finally, the ability of HIV genomic RNA to initiate translation through different pathways questions the possible mechanisms of regulation and their correlation to the viral paradigm, i.e. translation versus encapsidation of its genomic RNA.     

Cell proteins bind to a linear polypyrimidine-rich sequence within the 5'-untranslated region of rhinovirus 14 RNA.

Members of the picornavirus family initiate translation of their RNA genomes by a cap-independent mechanism in which ribosomes bind to an internal site in the 5' untranslated region (5'-UTR). This unique process requires an internal ribosome entry site (IRES), a highly structured RNA whose function is mediated in part by interactions with cell proteins. The IRES element of human rhinovirus 2 (HRV-2) extends from nucleotide (nt) 10 to between nt 544 and 568 and has been shown to interact with two cell proteins, pyrimidine tract-binding protein (pPTB) and p97. To map the specific regions of HRV-14 RNA that bind cell proteins, mobility shift, UV cross-linking and Western immunoblot analyses were performed. The results indicate that an RNA sequence from nt 538 to 591 interacts with pPTB and La, two proteins previously shown to functionally interact with the IRES elements of several picornaviruses. Two additional proteins, p97 and p68, were also cross-linked to nt 538 to 591 RNA. These four proteins interact with a putatively unstructured portion of the 5'-UTR that contains a polypyrimidine tract and has been shown to be present at the 3' border of sequences that are essential for IRES function of HRV-2. These protein-RNA interactions are likely to play a role in internal initiation of translation.     

Oscillating kissing stem-loop interactions mediate 5' scanning-dependent translation by a viral 3'-cap-independent translation element

The 3'-untranslated regions (UTRs) of a group of novel uncapped viral RNAs allow efficient translation initiation at the 5'-proximal AUG. A well-characterized model is the Barley yellow dwarf virus class of cap-independent translation elements (BTE). It facilitates translation by forming kissing stem-loops between the BTE in the 3'-UTR and a BTE-complementary loop in the 5'-UTR. Here we investigate the mechanisms of the long-distance interaction and ribosome entry on the RNA. Upstream AUGs or 5'-extensions of the 5'-UTR inhibit translation, indicating that, unlike internal ribosome entry sites in many viral RNAs, the BTE relies on 5'-end-dependent ribosome scanning. Cap-independent translation occurs when the kissing sites are moved to different regions in either UTR, including outside of the BTE. The BTE can even confer cap-independent translation when fused to the 3'-UTR of a reporter RNA harboring dengue virus sequences that cause base-pairing between the 3'- and 5'-ends. Thus, the BTE serves as a functional sensor to detect sequences capable of long-distance base-pairing. We propose that the kissing interaction is repeatedly disrupted by the scanning ribosome and re-formed in an oscillating process that regulates ribosome entry on the RNA.     

An internal ribosome entry site mediates translation of lymphoid enhancer factor-1

The lymphoid enhancer factor-1 LEF1 locus produces multiple mRNAs via alternative promoters. Full-length LEF-1 protein is produced via translation of an mRNA with a 1.2-kb, GC-rich 5'-untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5'-UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator beta-catenin. Truncated LEF-1 lacks the beta-catenin binding domain and opposes WNT signaling as a competitive inhibitor for WNT response elements. In this study we tested the hypothesis that the long, GC-rich 5'-UTR within the full-length LEF1 mRNA contains an internal ribosome entry site (IRES). Using a dicistronic vector in transient DNA transfections, we show that the LEF1 5'-UTR mediates cap-independent translation. Additional experiments involving a promoter-less dicistronic vector, Northern blot analysis, and transient transfections of dicistronic mRNAs into cultured mammalian cells compromised for cap-dependent translation demonstrate that the 5'-UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5'-UTR shows that maximal IRES activity requires the majority of the 5'-UTR, consistent with the notion that cellular IRESs require multiple modules for efficient activity. This study demonstrates that full-length LEF1 mRNA has evolved to utilize a cap-independent mechanism for translation of full-length LEF-1, whereas the truncated isoform is produced via the canonical cap-dependent ribosome scanning mechanism.     

Functional involvement of polypyrimidine tract-binding protein in translation initiation complexes with the internal ribosome entry site of foot-and-mouth disease virus.

The synthesis of picornavirus polyproteins is initiated cap independently far downstream from the 5' end of the viral RNA at the internal ribosome entry site (IRES). The cellular polypyrimidine tract-binding protein (PTB) binds to the IRES of foot-and-mouth disease virus (FMDV). In this study, we demonstrate that PTB is a component of 48S and 80S ribosomal initiation complexes formed with FMDV IRES RNA. The incorporation of PTB into these initiation complexes is dependent on the entry of the IRES RNA, since PTB and IRES RNA can be enriched in parallel either in 48S or 80S ribosomal complexes by stage-specific inhibitors of translation initiation. The formation of the ribosomal initiation complexes with the IRES occurs slowly, is temperature dependent, and correlates with the incorporation of PTB into these complexes. In a first step, PTB binds to the IRES, and then the small ribosomal subunit encounters this PTB-IRES complex. Mutations in the major PTB-binding site interfere simultaneously with the formation of initiation complexes, translation efficiency, and PTB cross-linking. PTB stimulates translation directed by the FMDV IRES in a rabbit reticulocyte lysate depleted of internal PTB, and the efficiency of translation can be restored to the original level by the addition of PTB. These results indicate that PTB plays an important role in the formation of initiation complexes with FMDV IRES RNA and in stimulation of internal translation initiation with this picornavirus.     

Heterogeneous Nuclear Ribonucleoprotein L Interacts with the 3′ Border of the Internal Ribosomal Entry Site of Hepatitis C Virus

Translation initiation of hepatitis C virus (HCV) RNA occurs by internal entry of a ribosome into the 5′ nontranslated region in a cap-independent manner. The HCV RNA sequence from about nucleotide 40 up to the N terminus of the coding sequence of the core protein is required for efficient internal initiation of translation, though the precise border of the HCV internal ribosomal entry site (IRES) has yet to be determined. Several cellular proteins have been proposed to direct HCV IRES-dependent translation by binding to the HCV IRES. Here we report on a novel cellular protein that specifically interacts with the 3′ border of the HCV IRES in the core-coding sequence. This protein with an apparent molecular mass of 68 kDa turned out to be heterogeneous nuclear ribonucleoprotein L (hnRNP L). The binding of hnRNP L to the HCV IRES correlates with the translational efficiencies of corresponding mRNAs. This finding suggests that hnRNP L may play an important role in the translation of HCV mRNA through the IRES element.     

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.     

A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by cap-independent internal ribosome binding to the 5' noncoding region (NCR). To identify the sequences and structural elements within the 5' NCR of HCV RNA that contribute to the initiation of translation, a series of point mutations was introduced within this sequence. Since the pyrimidine-rich tract is considered a characteristic feature of picornavirus internal ribosome entry site (IRES) elements, our mutational analysis focused on two putative pyrimidine tracts (Py-I and Py-II) within the HCV 5' NCR. Translational efficiency of these mutant RNAs was examined by in vitro translation and after RNA transfection into liver-derived cells. Mutational analysis of Py-I (nucleotides 120 to 130), supported by compensatory mutants, demonstrates that the primary sequence of this motif is not important but that a helical structural element associated with this region is critical for HCV IRES function. Mutations in Py-II (nucleotides 191 to 199) show that this motif is dispensable for IRES function as well. Thus, the pyrimidine-rich tract motif, which is considered as an essential element of the picornavirus IRES elements, does not appear to be a functional component of the HCV IRES. Further, the insertional mutagenesis study suggests a requirement for proper spacing between the initiator AUG and the upstream structures of the HCV IRES element for internal initiation of translation.     

The Apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with PTB and unr

We have shown previously that polypyrimidine tract binding protein 1 (PTB) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr) is prebound. Here we show that the Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation.     

Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation

The cellular polypyrimidine tract-binding protein (PTB) is recruited by the genomic RNAs of picornaviruses to stimulate translation initiation at their internal ribosome entry site (IRES) elements. We investigated the contribution of the individual RNA recognition motif (RRM) domains of PTB to its interaction with the IRES of foot-and-mouth disease virus (FMDV). Using a native gel system, we found that PTB is a monomer, confirming recent reports that challenged the previous view that PTB is a dimer. Mapping the spatial orientation of PTB relative to the bound IRES RNA, we found that the two C-terminal RRM domains III and IV of PTB bind in an oriented way to the IRES. Domain III contacts the IRES stem-loop 2, while domain IV contacts the separate IRES 3' region. PTB domain I appears not to be involved directly in RNA binding, but domain II stabilizes the RNA binding conferred by domains III and IV. A PTB protein containing only these two C-terminal PTB domains is sufficient to enhance the entry of initiation factor eIF4G to the IRES and stimulate IRES activity, and the long-lived PTB-IRES interaction stabilized by domain II is not a prerequisite for this function. Thus, PTB most likely acts as an RNA chaperone to stabilize IRES structure and, in that way, augment IRES activity.     

Translational Control of the Picornavirus Phenotype

Picornaviruses are small animal viruses with positive-strand genomic RNA, which is translated using cap-independent internal translation initiation. The key role in this is played by ciselements of the 5"-untranslated region (5"-UTR) and, in particular, by the internal ribosome entry site (IRES). The function of translational ciselements requires both canonical translation initiation factors (eIFs) and additional IRES trans-acting factors (ITAFs). All known ITAFs are cell RNA-binding proteins which play a variety of functions in noninfected cells. Specific features of translational ciselements substantially affect the phenotype and, in particular, tissue tropism and pathogenic properties of picornaviruses. It is clear that, in some cases, the molecular mechanism involved is a change in interactions between viral ciselements and ITAFs. The properties and tissue distribution of ITAFs may determine the biological properties of other viruses that also use the IRES-dependent translation initiation. Since this mechanism is also involved in translation of several cell mRNAs, ITAF may contribute to the regulation of the most important aspects of the living activity in noninfected cells.     

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.     

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.     

The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute.

Picornavirus RNAs are translated by an unusual mechanism of internal ribosome entry that requires a substantial segment of the viral 5'-untranslated region, generally known as the internal ribosome entry segment (IRES), and in some circumstances may require cellular trans-acting proteins, particularly polypyrimidine tract binding protein (PTB). It is shown here that for encephalomyocarditis virus (EMCV), the PTB dependence of IRES function in vitro is determined partly by the nature of the reporter cistron, and more especially by the size of an A-rich bulge in the IRES. With a wild-type EMCV IRES (which has a bulge of 6 As), translation is effectively independent of PTB provided the IRES is driving the synthesis of EMCV viral polyprotein. With an enlarged (7A) bulge and heterologous reporters, translation is highly dependent on PTB. Intermediate levels of PTB dependence are seen with a 7A bulge IRES driving viral polyprotein synthesis or a wild-type (6A) bulge IRES linked to a heterologous reporter. None of these parameters influenced the binding of PTB to the high-affinity site in the IRES. These results argue that PTB is not an essential and universal internal initiation factor, but, rather, that when it is required, its binding to the IRES helps to maintain the appropriate higher-order structure and to reverse distortions caused, for example, by an enlarged A-rich bulge.     

Mechanistic Role of Structurally Dynamic Regions in Dicistroviridae IGR IRESs

Dicistroviridae intergenic region (IGR) internal ribosome entry site(s) (IRES) RNAs drive a cap-independent pathway of translation initiation, recruiting both small and large ribosomal subunits to viral RNA without the use of any canonical translation initiation factors. This ability is conferred by the folded three-dimensional structure of the IRES RNA, which has been solved by X-ray crystallography. Here, we report the chemical probing of Plautia stali intestine virus IGR IRES in the unbound form, in the 40S-subunit-bound form, and in the 80S-ribosome-bound form. The results, when combined with an analysis of crystal structures, suggest that parts of the IRES RNA change structure as the preinitiation complex forms. Using mutagenesis coupled with native gel electrophoresis, preinitiation complex assembly assays, and translation initiation assays, we show that these potentially structurally dynamic elements of the IRES are involved in different steps in the pathway of ribosome recruitment and translation initiation. Like tRNAs, it appears that the IGR IRES undergoes local structural changes that are coordinated with structural changes in the ribosome, and these are critical for the IRES mechanism of action.