Regulation of fungal gene expression via short open reading frames in the mRNA 5'untranslated region

We review how the expression of fungal mRNAs can be controlled by ribosome interactions with short upstream open reading frames (uORFs) within the 5'untranslated region. The efficiency of uAUG recognition modulates the impact of a uORF but steps during and after translation of the uORF also influence uORF function. The post-termination behaviour of ribosomes, therefore, plays a major role in determining the expression level of these main ORFs. Translation of a uORF can produce a cis-acting peptide that causes effector molecule-dependent stalling of the ribosomes at the end of the uORF. In other cases it is the length or position, or other features of the uORF, rather than the peptide it encodes, that determine the efficiency with which ribosomes reinitiate translation downstream of it. Whether the form of the ribosome that resumes scanning after termination is the 40S subunit alone or the entire 80S ribosome is not known. Translation of the uORF can also control gene expression by affecting the stability of the mRNA. Finally, trans-acting factors may participate in the regulatory mechanisms. Future work will need not only to provide more information on the mechanisms underlying the known cases of uORF-mediated control but also to define the full complement of uORF-containing mRNAs in at least one fungal organism.     

Efficient cap-dependent translation of mammalian mRNAs with long and highly structured 5′-untranslated regions in vitro and in vivo

According to the generally accepted scanning model proposed by M. Kozak, the secondary structure of the 5′-untranslated region (5′-UTR) of eukaryotic mRNA can only inhibit the translation initiation by counteracting migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. The existence of efficiently translatable mRNAs with long and highly structured 5′-UTRs is incompatible with the cap-dependent scanning mechanism. Such mRNAs are expected to use alternative ways of translation initiation to be efficiently translated, primarily the mechanism of internal ribosome entry mediated by internal ribosome entry sites (IRESs), special RNA structures that reside in the 5′-UTR. The paper shows that this viewpoint is incorrect and is probably based on experiments with mRNA translation in rabbit reticulocyte lysate. This cell-free system fails to adequately reflect the relative translation efficiencies observed for different mRNAs in vivo. Five structurally similar mRNAs with either short leaders of the β-globin and β-actin mRNAs or long and highly structured 5′-UTRs of the c-myc, LINE-1, and Apaf-1 mRNAs displayed comparable translation activities in transfected cells and an entire cytoplasmic extract of cultivated cells. Translation activity proved to strongly depend on the presence of a cap at the 5′ end.     

SF2/ASF regulates proteomic diversity by affecting the balance between translation initiation mechanisms

Post‐splicing activities have been described for a subset of shuttling serine/arginine‐rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras‐PI 3‐kinase‐Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co‐regulated levels of isoform‐specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5′‐terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap‐independent manner by the action of cis‐acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap‐dependent manner while the other by internal ribosome entry site‐dependent initiation, to show that in vivo over‐expression of SF2/ASF increases the ratio between cap‐dependent and internal ribosome entry site‐dependent translation. Consistently, knocking‐down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor‐2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field. J. Cell. Biochem. 107: 826–833, 2009. © 2009 Wiley‐Liss, Inc.     

Translational regulation of the immunoglobulin heavy-chain binding protein mRNA

Translation of the mRNA encoding the immunoglobulin heavy-chain binding protein (BiP) is enhanced in poliovirus-infected cells at a time when translation of host cell mRNAs is inhibited. To test whether the mRNA of BiP is translated by internal ribosome binding, like picornaviral RNAs, we constructed plasmids for the expression of dicistronic hybrid RNAs containing the 5' noncoding region (5'NCR) of BiP as an intercistronic spacer element between two cistrons. Expression of these dicistronic mRNAs in mammalian cells resulted in efficient translation of both cistrons, demonstrating that the 5'NCR of BiP can confer internal ribosome binding to a heterologous RNA. This result suggests that the mRNA encoding BiP is bifunctional and can be translated by an internal ribosome-binding mechanism, in addition to the conventional cap-dependent scanning mechanism. This is the first demonstration of a cellular mRNA that can be translated by internal ribosome binding, and implies that this may be a mechanism for cellular translational regulation.     

Translational control of growth factor and proto-oncogene expression

Control of translation is now understood to be one of the major regulatory events in eukaryotic gene expression. Moreover there is evidence which suggests that aberrant expression of growth-related genes by translational mechanisms makes a significant contribution to cell transformation. However, the mechanisms which regulate translation of specific growth-related mRNAs have yet to be fully elucidated. The majority of these mRNAs have long 5' untranslated regions (UTRs) and three features which are important in translational control have been identified, namely (i) structured regions which inhibit the scanning mechanisms of translation, (ii) regulatory upstream open reading frames and (iii) internal ribosome entry segments which are capable of initiating cap-independent translation. In this review the translational regulation of specific mRNAs encoding growth factors and proto-oncogenes by these three mechanisms will be discussed, together with examples of altered translational regulation in neoplasia.     

Leaky scanning and scanning-independent ribosome migration on the tricistronic S1 mRNA of avian reovirus

The S1 genome segments of avian and Nelson Bay reovirus encode tricistronic mRNAs containing three sequential partially overlapping open reading frames (ORFs). The translation start site of the 3'-proximal ORF encoding the sigmaC protein lies downstream of two ORFs encoding the unrelated p10 and p17 proteins and more than 600 nucleotides distal from the 5'-end of the mRNA. It is unclear how translation of this remarkable tricistronic mRNA is regulated. We now show that the p10 and p17 ORFs are coordinately expressed by leaky scanning. Translation initiation events at these 5'-proximal ORFs, however, have little to no effect on translation of the 3'-proximal sigmaC ORF. Northern blotting, insertion of upstream stop codons or optimized translation start sites, 5'-truncation analysis, and poliovirus 2A protease-mediated cleavage of eIF4G indicated sigmaC translation derives from a full-length tricistronic mRNA using a mechanism that is eIF4G-dependent but leaky scanning- and translation reinitiation-independent. Further analysis of artificial bicistronic mRNAs failed to provide any evidence that sigmaC translation derives from an internal ribosome entry site. Additional features of the S1 mRNA and the mechanism of sigmaC translation also differ from current models of ribosomal shunting. Translation of the tricistronic reovirus S1 mRNA, therefore, is dependent both on leaky scanning and on a novel scanning-independent mechanism that allows translation initiation complexes to efficiently bypass two functional upstream ORFs.     

Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts

The intergenic region (IGR) located upstream of the capsid protein gene in dicistroviruses contains an internal ribosome entry site (IRES). Translation initiation mediated by the IRES does not require initiator methionine tRNA. Comparison of the IGRs among dicistroviruses suggested that Taura syndrome virus (TSV) and acute bee paralysis virus have an extra side stem loop in the predicted IRES. We examined whether the side stem is responsible for translation activity mediated by the IGR using constructs with compensatory mutations. In vitro translation analysis showed that TSV has an IGR-IRES that is structurally distinct from those previously described. Because IGR-IRES elements determine the translation initiation site by virtue of their own tertiary structure formation, the discovery of this initiation mechanism suggests the possibility that eukaryotic mRNAs might have more extensive coding regions than previously predicted. To test this hypothesis, we searched full-length cDNA databases and whole genome sequences of eukaryotes using the pattern matching program, Scan For Matches, with parameters that can extract sequences containing secondary structure elements resembling those of IGR-IRES. Our search yielded several sequences, but their predicted secondary structures were suggested to be unstable in comparison to those of dicistroviruses. These results suggest that RNAs structurally similar to dicistroviruses are not common. If some eukaryotic mRNAs are translated independently of an initiator methionine tRNA, their structures are likely to be significantly distinct from those of dicistroviruses.     

5'-untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation

Upstream AUGs (uAUGs) and upstream open reading frames (uORFs) are common features of mRNAs that encode regulatory proteins and have been shown to profoundly influence translation of the main ORF. In this study, we employed a series of artificial 5′-untranslated regions (5′-UTRs) containing one or more uAUGs/uORFs to systematically assess translation initiation at the main AUG by leaky scanning and reinitiation mechanisms. Constructs containing either one or two uAUGs in varying contexts but without an in-frame stop codon upstream of the main AUG were used to analyse the leaky scanning mechanism. This analysis largely confirmed the ranking of different AUG contextual sequences that was determined previously by Kozak. In addition, this ranking was the same for both the first and second uAUGs, although the magnitude of initiation efficiency differed. Moreover, ~10% of ribosomes exhibited leaky scanning at uAUGs in the most favourable context and initiated at a downstream AUG. A second group of constructs containing different numbers of uORFs, each with optimal uAUGs, were used to measure the capacity for reinitiation. We found significant levels of initiation at the main ORF even in constructs containing four uORFs, with nearly 10% of ribosomes capable of reinitiating five times. This study shows that for mRNAs containing multiple uORFs/uAUGs, ribosome reinitiation and leaky scanning are efficient mechanisms for initiation at their main AUGs.     

核糖体图谱技术在植物学研究中的应用

随着高通量测序技术的持续发展和进步,开发出很多新颖的测序技术,为诸多悬而未决的生物学难题提供了解决方案。其中,核糖体图谱技术能够在全基因组水平和单核苷酸分辨率上监控细胞内的翻译事件,填补了转录组学和蛋白质组学研究之间的空隙。核糖体图谱技术不仅能够鉴定处于翻译状态的RNA分子,还能够精确定位RNA分子上正在翻译的核苷酸,进而准确描绘RNA分子上的开放阅读框。此外,结合转录组测序数据,核糖体图谱技术还可以确定每个转录本上的核糖体数量,从而计算每个转录本的翻译效率。目前,核糖体图谱技术已成功应用于动物、植物和微生物等研究领域,加深了人们对翻译调控机制的认识。然而,由于植物细胞和组织的特性,核糖体图谱技术在植物学研究中的应用仍然存在局限。该文综述了核糖体图谱技术的实验原理,以及在植物学研究中的相关进展。     

Cytoplasmic expression of mRNAs containing the internal ribosome entry site and 3' noncoding region of hepatitis C virus: effects of the 3' leader on mRNA translation and mRNA stability

Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5' end of the mRNA, and the polyadenosine binding protein, bound to the 3'-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3'-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3'-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5' ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3' end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3' noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3'-end sequences of these chimeric RNAs were examined. It was found that the HCV 3' noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.     

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.     

Localized IRES-Dependent Translation of ER Chaperone Protein mRNA in Sensory Axons

Transport of neuronal mRNAs into distal nerve terminals and growth cones allows axonal processes to generate proteins autonomous from the cell body. While the mechanisms for targeting mRNAs for transport into axons has received much attention, how specificity is provided to the localized translational apparatus remains largely unknown. In other cellular systems, protein synthesis can be regulated by both cap-dependent and cap-independent mechanisms. The possibility that these mechanisms are used by axons has not been tested. Here, we have used expression constructs encoding axonally targeted bicistronic reporter mRNAs to determine if sensory axons can translate mRNAs through cap-independent mechanisms. Our data show that the well-defined IRES element of encephalomyocarditis virus (EMCV) can drive internal translational initiation of a bicistronic reporter mRNA in distal DRG axons. To test the potential for cap-independent translation of cellular mRNAs, we asked if calreticulin or grp78/BiP mRNA 5'UTRs might have IRES activity in axons. Only grp78/BiP mRNA 5'UTR showed clear IRES activity in axons when placed between the open reading frames of diffusion limited fluorescent reporters. Indeed, calreticulin's 5'UTR provided an excellent control for potential read through by ribosomes, since there was no evidence of internal initiation when this UTR was placed between reporter ORFs in a bicistronic mRNA. This study shows that axons have the capacity to translate through internal ribosome entry sites, but a simple binary choice between cap-dependent and cap-independent translation cannot explain the specificity for translation of individual mRNAs in distal axons.     

Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3'' noncoding region is mediated by a cotranslational mechanism.

The 3' noncoding region element (AUUUA)n specifically targets many short-lived mRNAs for degradation. Although the mechanism by which this sequence functions is not yet understood, a potential link between facilitated mRNA turnover and translation has been implied by the stabilization of cellular mRNAs in the presence of protein synthesis inhibitors. We therefore directly investigated the role of translation on mRNA stability. We demonstrate that mRNAs which are poorly translated through the introduction of stable secondary structure in the 5' noncoding region are not efficiently targeted for selective destabilization by the (AUUUA)n element. These results suggest that AUUUA-mediated degradation involves either a 5'-->3' exonuclease or is coupled to ongoing translation of the mRNA. To distinguish between these two possibilities, we inserted the poliovirus internal ribosome entry site, which promotes internal ribosome initiation, downstream of the 5' secondary structure. Translation directed by internal ribosome binding was found to fully restore targeted destabilization of AUUUA-containing mRNAs despite the presence of 5' secondary structure. This study therefore demonstrates that selective degradation mediated by the (AUUUA)n element is coupled to ribosome binding or ongoing translation of the mRNA and does not involve 5'-to-3' exonuclease activity.     

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.     

Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria

In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA.70 S.fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.