Expression of RUNX2 isoforms: involvement of cap-dependent and cap-independent mechanisms of translation

RUNX2, a major regulator of skeletogenesis, is expressed as type-I and type-II isoforms. Whereas most eukaryotic mRNAs are translated by the cap-dependent scanning mechanism, translation of many mRNAs including type-I and type-II RUNX2 mRNAs has been reported to be initiated by a cap independent internal ribosomal entry site (IRES). Since the dicistronic plasmid assay used to demonstrate IRES has been questioned, we investigated the presence of IRES in RUNX2 mRNAs using dicistronic plasmid and mRNA assays. Our results show that the dicistronic plasmid assay cannot be used to demonstrate IRES in RUNX2 mRNAs because the intercistronic region of dicistronic plasmids containing the 5'-UTRs of both RUNX2 mRNAs operates as a cryptic promoter. In dicistronic mRNA transfection studies the 5'-UTRs of both RUNX2 mRNAs exhibited no IRES activity. When transfected into osteoblastic cells, monocistronic reporter mRNA preceded by the 5'-UTR of type-II RUNX2 (Type-II-FLuc-A100) was translated to a high degree only in the presence of a functional cap (m(7)GpppG); in contrast, luciferase mRNA preceded by the 5'-UTR of type-I RUNX2 mRNA (Type-I-FLuc-A100) was translated poorly in the presence of either m(7)GpppG or a nonfunctional cap (ApppG). Notably, in transfected cells inhibitors of cap-dependent translation suppressed the translation of m(7)GpppG-capped Type-II-FLuc-A100, but not ApppG-capped reporter mRNA preceded by the IRES-containing hepatitis C virus (HCV) 5'-UTR. Our study demonstrates that type-II RUNX2 mRNA is translated by the cap-dependent mechanism. Although efficient translation of type-I RUNX2 mRNA appears to require a process other than cap-dependent, the mechanism of type-I RUNX2 mRNA translation remains to be resolved.     

Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.     

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.     

The 5' untranslated region of protein kinase Cdelta directs translation by an internal ribosome entry segment that is most active in densely growing cells and during apoptosis

Protein kinase Cdelta (PKCdelta) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5' untranslated region (5' UTR) of PKCdelta is predicted to form very stable secondary structures with free energies (deltaG values) of around -170 kcal/mol. The 5' UTR of PKCdelta can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5' UTR of PKCdelta contains a functional internal ribosome entry segment (IRES). The activity of the PKCdelta IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5' UTR of PKCdelta is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCdelta has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCdelta synthesis can be maintained under these conditions when cap-dependent translation is inhibited.     

Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs

In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.     

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.     

A novel role for Gemin5 in mRNA translation

In eukaryotic cells translation initiation occurs through two alternative mechanisms, a cap-dependent operating in the majority of mRNAs, and a 5′-end-independent driven by internal ribosome entry site (IRES) elements, specific for a subset of mRNAs. IRES elements recruit the translation machinery to an internal position in the mRNA through a mechanism involving the IRES structure and several trans-acting factors. Here, we identified Gemin5 protein bound to the foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) IRES using two independent approaches, riboproteomic analysis and immunoprecipitation of photocroslinked factors. Functional analysis performed in Gemin5 shRNA-depleted cells, or in in vitro translation reactions, revealed an unanticipated role of Gemin5 in translation control as a down-regulator of cap-dependent and IRES-driven translation initiation. Consistent with this, pull-down assays showed that Gemin5 forms part of two distinct complexes, a specific IRES-ribonucleoprotein complex and an IRES-independent protein complex containing eIF4E. Thus, beyond its role in snRNPs biogenesis, Gemin5 also functions as a modulator of translation activity.     

The Polycomb protein and E3 ubiquitin ligase Ring1B harbors an IRES in its highly conserved 5' UTR

Ring1B is an essential member of the highly conserved Polycomb group proteins, which orchestrate developmental processes, cell growth and stem cell fate by modifying local chromatin structure. Ring1B was found to be the E3 ligase that monoubiquitinates histone H2A, which adds a new level of chromatin modification to Polycomb group proteins. Here we report that Ring1B belongs to the exclusive group of proteins that for their translation depend on a stable 5' UTR sequence in their mRNA known as an Internal Ribosome Entry Site (IRES). In cell transfection assays the Ring1B IRES confers significantly higher expression levels of Ring1B than a Ring1B cDNA without the IRES. Also, dual luciferase assays show strong activity of the Ring1B IRES. Although our findings indicate Ring1B can be translated under conditions where cap-dependent translation is impaired, we found the Ring1B IRES to be cap-dependent. This raises the possibility that translational control of Ring1B is a multi-layered process and that translation of Ring1B needs to be maintained under varying conditions, which is in line with its essential role as an E3 ligase for monoubiquitination of histone H2A in the PRC1 Polycomb protein complex.     

Restoration of functional gap junctions through internal ribosome entry site-dependent synthesis of endogenous connexins in density-inhibited cancer cells

Gap junctions are composed of connexins and are critical for the maintenance of the differentiated state. Consistently, connexin expression is impaired in most cancer cells, and forced expression of connexins following cDNA transfection reverses the tumor phenotype. We have found that the restoration of density inhibition of human pancreatic cancer cells by the antiproliferative somatostatin receptor 2 (sst2) is due to overexpression of endogenous connexins Cx26 and Cx43 and consequent formation of functional gap junctions. Immunoblotting along with protein metabolic labeling and mRNA monitoring revealed that connexin expression is enhanced at the level of translation but is not sensitive to the inhibition of cap-dependent translation initiation. Furthermore, we identified a new internal ribosome entry site (IRES) in the Cx26 mRNA. The activity of Cx26 IRES and that of the previously described Cx43 IRES are enhanced in density-inhibited cells. These data indicate that the restoration of functional gap junctions is likely a critical event in the antiproliferative action of the sst2 receptor. We further suggest that the existence of IRESes in connexin mRNAs permits connexin expression in density-inhibited or differentiated cells, where cap-dependent translation is generally reduced.     

The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated

A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5′-UTR of insulin mRNA. For this purpose, human islets were incubated for 2 h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5′-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5′-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40–100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic β-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5′-UTR of the human insulin mRNA.     

Was the initiation of translation in early eukaryotes IRES-driven?

The initiation of translation in eukaryotes generally involves the recognition of a 'cap' structure at the 5' end of the mRNA. However, for some viral and cellular mRNAs, a cap-independent mechanism occurs through an mRNA structure known as the internal ribosome entry site (IRES). Here, I postulate that the first eukaryotic mRNAs were translated in a cap-independent, IRES-driven manner that was then superseded in evolution by the cap-dependent mechanism, rather than vice versa. This hypothesis is supported by the following observations: (i) IRES-dependent, but not cap-dependent, translation can take place in the absence of not only a cap, but also many initiation factors; (ii) eukaryotic initiation factor 4E (eIF4E) and eIF4G, molecules absolutely required for cap-dependent translation, are among the most recently evolved translation factors; and (iii) functional similarities suggest the evolution of IRESs from spliceosomal introns. Thus, the contemporary cellular IRESs might be relics of the past.     

c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis

Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5' untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5' untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.     

Polypyrimidine tract-binding protein enhances the internal ribosomal entry site-dependent translation of p27Kip1 mRNA and modulates transition from G1 to S phase

The p27(Kip1) protein plays a critical role in the regulation of cell proliferation through the inhibition of cyclin-dependent kinase activity. Translation of p27(Kip1) is directed by an internal ribosomal entry site (IRES) in the 5' nontranslated region of p27(Kip1) mRNA. Here, we report that polypyrimidine tract-binding protein (PTB) specifically enhances the IRES activity of p27(Kip1) mRNA through an interaction with the IRES element. We found that addition of PTB to an in vitro translation system and overexpression of PTB in 293T cells augmented the IRES activity of p27(Kip1) mRNA but that knockdown of PTB by introduction of PTB-specific small interfering RNAs (siRNAs) diminished the IRES activity of p27(Kip1) mRNA. Moreover, the G(1) phase in the cell cycle (which is maintained in part by p27(Kip1)) was shortened in cells depleted of PTB by siRNA knockdown. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation in HL60 cells was used to examine PTB-induced modulation of p27(Kip1) protein synthesis during differentiation. The IRES activity of p27(Kip1) mRNA in HL60 cells was increased by TPA treatment (with a concomitant increase in PTB protein levels), but the levels of p27(Kip1) mRNA remained unchanged. Together, these data suggest that PTB modulates cell cycle and differentiation, at least in part, by enhancing the IRES activity of p27(Kip1) mRNA.