Eukaryotic cells rapidly reduce protein synthesis in response to various stress
conditions. This can be achieved by the phosphorylation-mediated inactivation of a
key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However,
the persistent translation of certain mRNAs is required for deployment of an adequate
stress response. We carried out ribosome profiling of cultured human cells under
conditions of severe stress induced with sodium arsenite. Although this led to a
5.4-fold general translational repression, the protein coding open reading frames
(ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all
resistant transcripts possess at least one efficiently translated upstream open
reading frame (uORF) that represses translation of the main coding ORF under normal
conditions. Site-specific mutagenesis of two identified stress resistant mRNAs
(PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated
translation control in both cases. Phylogenetic analysis suggests that at least two
regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein
products.DOI:
http://dx.doi.org/10.7554/eLife.03971.001
Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2-GTP-Met-tRNA(i) (Met), is used to deliver the initiator tRNA to the ribosome in all eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can direct translation without eIF2 and its GTPase-activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a bacterial-like pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. The switch from the conventional eukaryotic mode of translation initiation to the eIF2-independent mechanism occurs when eIF2 is inactivated by phosphorylation under stress conditions.
During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA i Met occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.
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 (5UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5UTR-Fluc) or bicistronic (Rluc-L1 5UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5UTR. Nevertheless, this cap-dependent initiation activity of the L1 5UTR was unexpectedly high and resembles that of the beta-actin 5UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5UTR, 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 5UTRs and call into question the conception that every long GC-rich 5UTR 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.
Many mammalian mRNAs possess long 5′ UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5′ UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5′ UTRs with so-called ‘cellular IRESes’ demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5′ UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5′ UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.
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