The serine/threonine kinase pim-1 mRNA contains a long and G/C rich 5′-untranslated region (5′-UTR). Previous work suggested that the pim-1 5′-UTR harbors an internal ribosomal entry site (IRES) allowing for internal initiation of translation. However, several previously reported eukaryotic IRES elements actually contain cryptic promoter activity. To test whether an IRES or a cryptic promoter is present in the pim-1 5′-UTR, the 5′-UTR was re-examined using stringent test procedures. Our results show the presence of strong promoter activity in the DNA sequence corresponding to the pim-1 5′-UTR. Both promoterless dicistronic test and northern blot analysis show transcripts being derived from the cryptic promoter in the pim-1 5′-UTR sequence. This cryptic promoter is active in all cell types tested, including Cos-7, NIH3T3, HEK293, Jurkat and K562 cells. When a dicistronic mRNA containing the pim-1 5′-UTR was translated in vitro or in vivo, no IRES activity could be detected. However, the control IRESs from both human rhinovirus and encephalomyocarditis virus exhibited strong IRES activities. In addition, both the RNase protection assay and the 5′-RACE assay detected endogenous pim-1 transcripts with shorter 5′-UTRs. Our data strongly suggest that the IRES activity reported earlier for the pim-1 5′-UTR sequence is due to cryptic promoter activity.
As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5′ untranslated region (5′-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5′-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5′-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPβ. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5′-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5′-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs.
The p27Kip1 protein plays a critical role in the regulation of cell proliferation through the inhibition of cyclin-dependent kinase activity. Translation of p27Kip1 is directed by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of p27Kip1 mRNA. Here, we report that polypyrimidine tract-binding protein (PTB) specifically enhances the IRES activity of p27Kip1 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 p27Kip1 mRNA but that knockdown of PTB by introduction of PTB-specific small interfering RNAs (siRNAs) diminished the IRES activity of p27Kip1 mRNA. Moreover, the G1 phase in the cell cycle (which is maintained in part by p27Kip1) 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 p27Kip1 protein synthesis during differentiation. The IRES activity of p27Kip1 mRNA in HL60 cells was increased by TPA treatment (with a concomitant increase in PTB protein levels), but the levels of p27Kip1 mRNA remained unchanged. Together, these data suggest that PTB modulates cell cycle and differentiation, at least in part, by enhancing the IRES activity of p27Kip1 mRNA.
Upstream of N-ras (Unr) has been described as an internal initiation trans-acting factor (ITAF) in the cap-independent translation of some particular viral and cellular mRNAs. Two factors led us to hypothesize that the UNR 5′-untranslated region (5′-UTR) may contain an internal ribosome entry site (IRES). The first was the requirement for persisting Unr expression under conditions that correlate with cap-independent translation. The other was the observation that the primary UNR transcript contains a 447 nt long 5′-UTR including two upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Here we report that the UNR 5′-UTR allows IRES-dependent translation, as revealed by a dicistronic reporter assay. Various controls ruled out the contribution of leaky scanning, cryptic promoter sequences or RNA processing events to the ability of the UNR 5′-UTR to mediate internal initiation of translation. Ultraviolet cross-linking analysis and RNA affinity chromatography revealed the binding of polypyrimidine tract binding protein (PTB) to the UNR IRES, requiring a pyrimidine-rich region (nucleotides 335–355). Whereas overexpression of PTB in several cell lines inhibited UNR IRES activity and UNR protein expression, depletion of endogenous PTB using RNAi increased UNR IRES activity. Moreover, a mutant version of the UNR IRES lacking the PTB binding site was more efficient at directing IRES-mediated translation. In conclusion, our results demonstrate that translation of the ITAF Unr can itself be regulated by an IRES that is downregulated by PTB.
Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5′ untranslated region (5′UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5′UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5′UTR-Fluc) or bicistronic (Rluc-L1 5′UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5′UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5′UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5′UTR. Nevertheless, this cap-dependent initiation activity of the L1 5′UTR was unexpectedly high and resembles that of the beta-actin 5′UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5′UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5′UTRs and call into question the conception that every long GC-rich 5′UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.
A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3′ splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3′ ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3′ ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.
We constructed a dual regulated expression vector cassette (pDuoRex)
whereby two heterologous genes can be independently regulated via
streptogramin- and tetracycline-responsive promoters. Two different
constructs containing growth-promoting and growth-inhibiting genes
were stably transfected in recombinant Chinese hamster ovary (CHO)
cells that express the streptogramin- and tetracycline-dependent
transactivators in a dicistronic configuration. An optimally balanced
heterologous growth control scenario was achieved by reciprocal
expression of the growth-inhibiting human cyclin-dependent kinase
inhibitor p27Kip1 in sense (p27Kip1S) and
antisense (p27Kip1AS) orientation. Exclusive expression
of p27Kip1S resulted in complete G1-phase-specific
growth arrest, while expression of only p27Kip1AS showed
significantly increased proliferation compared to control cultures
(both antibiotics present), presumably by decreasing host cell p27Kip1 expression.
In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression
of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated
expression of p27Kip1 was stably transfected into CHO
cells. Expression of sT alone resulted in an increase in cell proliferation,
but the expression of p27Kip1 failed to provide the expected
G1-specific growth arrest despite having demonstrated
expression of the protein. This illustrates the difficulty in balancing
the complex pathways underlying cell proliferation control through
the expression of two functionally distinct genes involved in those
pathways, and how a single-gene sense/antisense approach
using pDuoRex can overcome this barrier to complete metabolic engineering
The ubiquitous transcription factor NRF (NF-κB repressing factor) is a constitutive transcriptional silencer of the multifunctional cytokine interferon-β. NRF mRNA contains a long 5′ untranslated region (5′UTR) predicted to fold into a strong secondary structure. The presence of stable hairpins is known to be incompatible with efficient translation by ribosomal scanning. Using dicistronic reporter gene constructs, we show that the NRF 5′UTR acts as an internal ribosome entry site (IRES) which directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES in various cell lines is at least 30-fold higher than that of picornaviral IRESs. The NRF 5′UTR also functions as a translational enhancer in the context of monocistronic mRNAs. Our results indicate that the NRF 5′UTR contains a highly potent IRES, which may allow for an alternate mode of translation under physiological conditions in which cap-dependent translation is inhibited.
This review takes a second look at a set of mRNAs that purportedly employ an alternative mechanism of initiation when cap-dependent translation is reduced during mitosis or stress conditions. A closer look is necessary because evidence cited in support of the internal initiation hypothesis is often flawed. When putative internal ribosome entry sequences (IRESs) are examined more carefully, they often turn out to harbor cryptic promoters or splice sites. This undermines the dicistronic assay, wherein IRES activity is measured by the ability to support translation of the 3′ cistron. Most putative IRESs still have not been checked carefully to determine whether the dicistronic vector produces only the intended dicistronic mRNA. The widespread use of the pRF vector is a major problem because this vector, which has Renilla luciferase as the 5′ cistron and firefly luciferase as the 3′ cistron, has been found to generate spliced transcripts. RNA transfection assays could theoretically circumvent these problems, but most candidate IRESs score very weakly in that test. The practice of calling even very weak results ‘positive’ is one of the problems discussed herein. The extremely low efficiency of putative IRESs is inconsistent with their postulated biological roles.
‘…if it is a Miracle, any sort of evidence will answer, but if it is a Fact, proof is necessary’
—Mark Twain, Letters from the Earth
Translation initiation in Hepatitis C Virus is controlled by the presence of an internal ribosome entry site element (IRES) principally located in its 5' untranslated region (UTR). Mutation/deletion analyses have shown that the integrity of this structure is essential for initiation of cap-independent protein synthesis. We have developed a strategy to swap the position of the two major domains (II and III) on the 5'UTR sequence. The aim was to further characterize this mechanism by preserving domain-specific interactions but possibly losing contacts that require any interdomain geometry. The expression of dicistronic mRNAs containing these different UTRs showed that the positioning of the different domains on the 5'UTR is essential for efficient IRES functioning. We then used these mutants to identify cellular factors implicated in IRES activity. Using UV crosslinking assays we found that domain III makes direct contact with two proteins (p170/p120) which can be associated with efficient IRES activity. In particular, we have mapped the binding sites of these proteins and shown that p120 binds to the apical loop segment of domain III, whilst p170 binds in the stem portion, independently of domain III position or context. Finally, we provide evidence showing that p170 and p120 represent two subunits of eukaryotic initiation factor eIF3: p170 and p116/p110.
The cyclin-dependent kinase inhibitor p27Kip1 is essential for proper control of cell cycle progression. The levels of p27Kip1 are regulated by several mechanisms including transcriptional and translational controls. In order to delineate the molecular details of these regulatory mechanisms it is important to identify the transcription initiation site within the p27Kip1 gene, thereby defining the promoter region of the gene and the 5'-untranslated region of the p27Kip1 mRNA. Although several previous studies have attempted to map p27Kip1 transcription start sites, the results vary widely for both the mouse and human genes. In addition, even though the mouse and human p27Kip1 gene sequences are very highly conserved, the reported start sites are notably different.
In this report, using a method that identifies capped ends of mRNA molecules together with RNase protection assays, we demonstrate that p27Kip1 transcription is initiated predominantly from a single site which is conserved in the human and mouse genes. Initiation at this site produces a 5'-untranslated region of 472 nucleotides in the human p27Kip1 mRNA and 502 nucleotides in the mouse p27Kip1 mRNA. In addition, several minor transcription start sites were identified for both the mouse and human genes.
These results demonstrate that the major transcription initiation sites in the mouse and human p27Kip1 genes are conserved and that the 5'-UTR of the p27Kip1 mRNA is much longer than generally believed. It will be important to consider these findings when designing experiments to identify elements that are involved in regulating the cellular levels of p27Kip1.
Translation of uncapped giardiavirus (GLV) mRNA in Giardia lamblia requires the presence of a 5′-untranslated region (5′-UTR) and a viral capsid coding region. We used dicistronic viral constructs to show that the downstream 253 nucleotides (nt) of the 5′-UTR plus the initial 264-nt capsid coding region constitute an internal ribosome entry site (IRES). Predicted secondary structures in the 253-nt 5′-UTR include stem-loops U3, U4a, U4b, U4c, and U5. Chemical and enzymatic probing analysis confirmed the presence of all predicted stem-loops except U4a. Disruption of stem-loop structures U3 and U5 by site-directed mutagenesis resulted in a drastic reduction in translation of a monocistronic viral transcript, which could be restored by compensatory sequence changes. Mutations disrupting stem-loops U4b and U4c do not exert an appreciable effect on translation, but certain sequences in the U4a region and in U4b do appear to play important roles in the IRES. Structural analysis also suggests that an 8-nt U3 loop sequence (nt 147 to 154) pairs with an 8-nt downstream sequence (nt 168 to 175) to form a pseudoknot. Disruption of this pseudoknot by mutagenesis resulted in a drastic reduction in translation, which could be restored by compensatory sequence changes. This study has defined the secondary structure in the 5′-UTR of the IRES. Together with the previous results, we have now completed analysis of the entire structure of GLV IRES and fully defined the functionally essential structural elements in it.
The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.
We have made a dicistronic construct where the picornaviral internal ribosome-entry site (IRES) driving the expression of the beta-geo gene has been inserted into the 3'untranslated region of the human CFTR gene present in a YAC. When introduced into the human cell line Caco-2 expressing the CFTR gene, the expression of the dicistronic gene can be detected by lacZ staining and follows the accumulation of the endogenous CFTR mRNA upon differentiation of the cells. These data demonstrate that this IRES-based approach presents an alternative to mRNA in situ hybridisation and allows detection of expression in an autologous system.
Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5′ untranslated region (5′UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5′UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5′UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.
p27kip1 (p27) is a cell cycle inhibitor and tumor suppressor whose expression is tightly regulated in the cell. Translational control of p27 mRNA has emerged as a prominent mechanism to regulate p27 expression during differentiation, quiescence, and cancer progression. The microRNAs miR-221 and miR-222 repress p27 expression in various cancer cells, and this repression promotes tumor cell proliferation. In addition, the presence of an internal ribosome entry site in the 5′ untranslated region (UTR) of p27 mRNA has been reported. Here, we show that p27 mRNA is translated via a cap-dependent mechanism in HeLa and HL60 cells and that the previously reported IRES activity can be attributed to cryptic promoters in the sequence corresponding to the p27 5′ UTR. Furthermore, cap-dependent translation of p27 mRNA is repressed by miR-181a in undifferentiated HL60 cells. Repression by miR-181a is relieved during differentiation of HL60 into monocyte-like cells, allowing the accumulation of p27, which is necessary to fully block cell cycle progression and reach terminal differentiation. These results identify miR-181a as a regulator of p27 mRNA translation during myeloid cell differentiation.
SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5′-end of a reporter mRNA having a short 5′ untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5′ untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5′ cap structure and 3′ poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5′-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.
Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is the causative agent of SARS. The nsp1 protein of SCoV blocks host protein synthesis, including type I interferon, a general inhibitor of virus replication, in infected cells. This finding suggests that SCoV nsp1 protein plays a key role in the severe symptoms that accompany SARS infection. Nsp1 binds to the 40S ribosome subunit, which is an essential component for protein synthesis, and inactivates the translation activity of the ribosome. Furthermore, nsp1 binding to the 40S ribosome induces the modification of host mRNAs, leading to the accelerated decay of these RNAs in SCoV-infected cells. We found that the nature of nsp1-induced RNA modification was RNA cleavage and that nsp1 did not recognize specific nucleotides in host mRNAs to induce this cleavage. Interestingly, nsp1 did not induce RNA cleavage in SCoV mRNAs. These data indicate that nsp1 induces RNA cleavage of host mRNAs to suppress the expression of host genes, including those having antiviral functions; yet viral mRNAs are spared from such cleavage events, which, most likely, facilitate efficient SCoV protein synthesis and virus replication in infected cells.
Recently, a group of related Leishmania RNA viruses (Leishmania RNA virus 1 [LRV1]) has been isolated from Leishmania guyanensis and L. brasiliensis. These viruses persist in the cytoplasm and contain double-stranded RNA genomes. Miniexon sequences are absent from the 5' end of the viral RNA, and the 5' end of the viral RNA lacks a cap structure, suggesting that LRV1 has evolved a cap-independent mechanism of translation. Cap-independent translation of picornavirus genomic RNA requires a cis element, within the 5' untranslated region (UTR), referred to as an internal ribosome entry site (IRES). In order to find out if the 5' UTR of LRV1 possessed IRES activity, we modified a Leishmania expression vector, pX63NEO-GUS, so that it would produce a dicistronic transcript in which the neomycin phosphotransferase gene was separated from the downstream beta-glucuronidase (GUS) gene by the LRV1 5' UTR. High levels of GUS activity were detected in L. major stably transformed with this plasmid. Elimination of the first 120 nucleotides of the viral 5' UTR lowered GUS activity 10-fold. Furthermore, when the entire 5' UTR was eliminated, GUS activity was undetectable. These results, together with the absence of trans-spliced GUS transcripts, are consistent with the hypothesis that the 5' UTR of LRV1 functions as an IRES element. The ability to couple expression of genes via an IRES element should prove useful in genetic experiments with Leishmania spp.
Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h.
Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1.
Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH.
Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.
cell cycle regulator; cyclin-dependent kinase inhibitor; Kip1; compensatory growth; liver regeneration; rat hepatocytes; cell proliferation
The 5' untranslated regions (UTRs) of the Drosophila Ubx and Antp genes were tested for their ability to promote cap-independent translation initiation. The Ubx and the Antp 5' UTR were inserted between the CAT and lacZ coding sequences in a dicistronic gene and tested for IRES activity in transgenic Drosophila. Northern analysis of the mRNAs showed the presence of the predicted full-length dicistronic mRNAs. High CAT activity was expressed from the first cistron from all of the dicistronic constructs introduced into the fly genome. The dicistronic transgenic strains bearing the Ubx and Antp IRES elements expressed significant levels of beta-galactosidase (betaGAL) from the second cistron whereas little or no betaGAL was expressed in the controls lacking the IRESs. In situ analysis of betaGAL expression in the transgenic strains indicates that expression of the second cistron is spatially and temporally regulated. Although the developmental patterns of expression directed by the Antp and Ubx IRESs overlap, they exhibit several differences indicating that these IRESs are not functionally equivalent.
The mRNA of Scamper, a putative intracellular calcium channel activated by sphingosylphosphocholine, contains a long 5′ transcript leader with several upstream AUGs. In this work we have investigated the role this sequence plays in the translational control of Scamper expression. The cytosolic transcription machinery of a T7 RNA polymerase recombinant vaccinia virus was used to avoid artifacts arising from cryptic promoters or mRNA processing. Based on transient transfection experiments of dicistronic and bi-monocistronic plasmids expressing reporter genes, we present evidence that the 5′ transcript leader of Scamper contains a functional internal ribosome entry site (IRES). Our data indicate that Scamper translation in Madin-Darby canine kidney cells is driven by a cap-independent mechanism supported by the IRES activity of its mRNA. Finally, the Scamper IRES appears to be the first IRES with specificity for kidney epithelial cells.
Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (Family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced > 95 % compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in-vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.
Alphavirus; replicon; IRES; dicistronic vector; promoterless; VEE; EMCV; EV71
HIF-1α is the regulated subunit of the HIF-1 transcription factor, which induces transcription of a number of genes involved in the cellular response to hypoxia. The HIF-1α protein is rapidly degraded in cells supplied with adequate oxygen but is stabilized in hypoxic cells. Using polysome profile analysis, we found that translation of HIF-1α mRNA in NIH3T3 cells is spared the general reduction in translation rate that occurs during hypoxia. To assess whether the 5′UTR of the HIF-1α mRNA contains an internal ribosome entry site (IRES), we constructed a dicistronic reporter with the HIF-1α 5′UTR inserted between two reporter coding regions. We found that the HIF-1α 5′UTR promoted translation of the downstream reporter, indicating the presence of an IRES. The IRES had activity comparable to that of the well-characterized c-myc IRES. IRES activity was not affected by hypoxic conditions that caused a reduction in cap-dependent translation, and IRES activity was less affected by serum-starvation than was cap-dependent translation. These data indicate that the presence of an IRES in the HIF-1α 5′UTR allows translation to be maintained under conditions that are inhibitory to cap-dependent translation.
The cyclin dependent kinase inhibitor p27Kip1 plays an important role in controlling the eukaryotic cell cycle. The 5′-untranslated region of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, the RNA-associated protein CUGBP1 was shown to interact with the human p27 5′-untranslated region. Overexpression of CUGBP1 inhibited endogenous p27 expression and reduced translation initiation through the p27 IRES. In contrast, repression of CUGBP1 by siRNA transfection enhanced p27 protein levels and stimulated p27 IRES activity. Addition of recombinant CUGBP1 repressed p27 IRES reporter mRNA translation in vitro. At last, our finding showed that cytosolic form of CUGBP1 binds efficiently to the p27 5′-untranslated region.
p27Kip1; cell cycle; IRES; CUGBP1; 5′-untranslated region
K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.