The impact of the phosphomimetic eIF2αS/D on global translation, reinitiation and the integrated stress response is attenuated in N2a cells

A plethora of stresses trigger a rapid downregulation of protein synthesis. However, a fraction of mRNAs continue to be recruited onto polysomes and their protein products play a key role in deciding cell fate. These transcripts are characterized by the presence of uORFs within their 5′ TL coupling protein expression to reinitiation. The translational brake arises due to the activation of a family of kinases targeting the α subunit of the trimolecular eIF2(αβγ) initiation factor. Phosphorylation of eIF2αSer51 inhibits ternary complex regeneration reducing the pool of 43S ribosomes. It is popular to mimic this event, and hence the integrated stress response (ISR), by the expression of the phosphomimetic eIF2αS51D. However, we report that whereas the ISR is reproduced by eIF2αS51D expression in human HEK293T cells this is not the case in N2a mouse neuroblastoma cells. With regards to translational downregulation, this arises due to the failure of the phosphomimetic protein to assemble an eIF2 complex with endogenous eIF2β/γ. This can be compensated for by the transient co-expression of all three subunits. Curiously, these conditions do not modulate reinitiation and consequently fail to trigger the ISR. This is the first demonstration that the inhibitory and reinitiation functions of eIF2αS/D can be separate

The translational response of the human mdm2 gene in HEK293T cells exposed to rapamycin: a role for the 5′-UTRs

Polysomal messenger RNA (mRNA) populations change rapidly in response to alterations in the physiological status of the cell. For this reason, translational regulation, mediated principally at the level of initiation, plays a key role in the maintenance of cellular homeostasis. In an earlier translational profiling study, we followed the impact of rapamycin on polysome re-seeding. Despite the overall negative effect on transcript recruitment, we nonetheless observed that some mRNAs were significantly less affected. Consequently, their relative polysomal occupancy increased in the rapamycin-treated cells. The behaviour of one of these genes, mdm2, has been further analysed. Despite the absence of internal ribosome entry site activity we demonstrate, using a dual reporter assay, that both the reported mdm2 5′-UTRs confer resistance to rapamycin relative to the 5′-UTR of β-actin. This relative resistance is responsive to the downstream targets mTORC1 but did not respond to changes in the La protein, a reported factor acting positively on MDM2 translational expression. Furthermore, extended exposure to rapamycin in the presence of serum increased the steady-state level of the endogenous MDM2 protein. However, this response was effectively reversed when serum levels were reduced. Taken globally, these studies suggest that experimental conditions can dramatically modulate the expressional output during rapamycin exposur

Alternatively spliced isoforms of the human elk-1 mRNA within the 5′ UTR: implications for ELK-1 expression

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5′ UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5′ UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5′ UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological condition

Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity

FEN-1 and XPG are members of the FEN-1 family of structure-specific nucleases, which share a conserved active site. FEN-1 plays a central role in DNA replication, whereas XPG is involved in nucleotide excision repair (NER). Both FEN-1 and XPG are active on flap structures, but only XPG cleaves bubble substrates. The spacer region of XPG is dispensable for nuclease activity on flap substrates but is required for NER activity and for efficient processing of bubble substrates. Here, we inserted the spacer region of XPG between the nuclease domains of FEN-1 to test whether this domain would be sufficient to confer XPG-like substrate specificity and NER activity on a related nuclease. The resulting FEN-1-XPG hybrid protein is active on flap and, albeit at low levels, on bubble substrates. Like FEN-1, the activity of FEN-1-XPG was stimulated by a double-flap substrate containing a 1-nt 3′ flap, whereas XPG does not show this substrate preference. Although no NER activity was detected in vitro, the FEN-1-XPG hybrid displays substantial NER activity in vivo. Hence, insertion of the XPG spacer region into FEN-1 results in a hybrid protein with biochemical properties reminiscent of both nucleases, including partial NER activity

An approach to analyse the specific impact of rapamycin on mRNA-ribosome association

<p>Abstract</p> <p>Background</p> <p>Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background.</p> <p>Methods</p> <p>We describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out). For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation.</p> <p>Results</p> <p>High throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug.</p> <p>Conclusion</p> <p>The technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of a particular drug in a living cell.</p

The translational response of the human mdm2 gene in HEK293T cells exposed to rapamycin: a role for the 5′-UTRs

Polysomal messenger RNA (mRNA) populations change rapidly in response to alterations in the physiological status of the cell. For this reason, translational regulation, mediated principally at the level of initiation, plays a key role in the maintenance of cellular homeostasis. In an earlier translational profiling study, we followed the impact of rapamycin on polysome re-seeding. Despite the overall negative effect on transcript recruitment, we nonetheless observed that some mRNAs were significantly less affected. Consequently, their relative polysomal occupancy increased in the rapamycin-treated cells. The behaviour of one of these genes, mdm2, has been further analysed. Despite the absence of internal ribosome entry site activity we demonstrate, using a dual reporter assay, that both the reported mdm2 5′-UTRs confer resistance to rapamycin relative to the 5′-UTR of β-actin. This relative resistance is responsive to the downstream targets mTORC1 but did not respond to changes in the La protein, a reported factor acting positively on MDM2 translational expression. Furthermore, extended exposure to rapamycin in the presence of serum increased the steady-state level of the endogenous MDM2 protein. However, this response was effectively reversed when serum levels were reduced. Taken globally, these studies suggest that experimental conditions can dramatically modulate the expressional output during rapamycin exposure

Alternatively spliced isoforms of the human elk-1 mRNA within the 5′ UTR: implications for ELK-1 expression

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5′ UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5′ UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5′ UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions

Preinitiation complex loading onto mRNAs with long versus short 5′ TLs

The first step in translation initiation consists in the recruitment of the small ribosome onto the mRNA. This preinitiation complex (PIC) loads via interactions with eIF4F that has assembled on the 5′ cap. It then scans the 5′ TL (transcript leader) to locate a start site. The molecular architecture of the PIC-mRNA complex over the cap is beginning to be resolved. As part of this, we have been examining the role of the 5′ TL length. We observed in vivo initiation events on AUG codons positioned within 3 nts of the 5′ cap and robust initiation in vitro at start sites immediately downstream of the 5′ end. Ribosomal toe-printing confirmed the positioning of these codons within the P site, indicating that the ribosome reads from the +1 position. To explore differences in the eIF4E-5′ cap interaction in the context of long versus short TL, we followed the fate of the eIF4E-cap interaction using a novel solid phase in vitro expression assay. We observed that ribosome recruitment onto a short TL disrupts the eIF4E-cap contact releasing all the mRNA from the solid phase, whereas with a long the mRNA distributes between both phases. These results are discussed in the context of current recruitment models.</p

The impact of the phosphomimetic eIF2αS/D on global translation, reinitiation and the integrated stress response is attenuated in N2a cells

A plethora of stresses trigger a rapid downregulation of protein synthesis. However, a fraction of mRNAs continue to be recruited onto polysomes and their protein products play a key role in deciding cell fate. These transcripts are characterized by the presence of uORFs within their 5′ TL coupling protein expression to reinitiation. The translational brake arises due to the activation of a family of kinases targeting the α subunit of the trimolecular eIF2(αβγ) initiation factor. Phosphorylation of eIF2αSer51 inhibits ternary complex regeneration reducing the pool of 43S ribosomes. It is popular to mimic this event, and hence the integrated stress response (ISR), by the expression of the phosphomimetic eIF2αS51D. However, we report that whereas the ISR is reproduced by eIF2αS51D expression in human HEK293T cells this is not the case in N2a mouse neuroblastoma cells. With regards to translational downregulation, this arises due to the failure of the phosphomimetic protein to assemble an eIF2 complex with endogenous eIF2β/γ. This can be compensated for by the transient co-expression of all three subunits. Curiously, these conditions do not modulate reinitiation and consequently fail to trigger the ISR. This is the first demonstration that the inhibitory and reinitiation functions of eIF2αS/D can be separated