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. 2012 Jul;40(12):5602-14.
doi: 10.1093/nar/gks182. Epub 2012 Feb 28.

Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

Dmitri E Andreev  1 , Juliane Hirnet, Ilya M Terenin, Sergey E Dmitriev, Michael Niepmann, Ivan N Shatsky
Affiliations

Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

Dmitri E Andreev et al. Nucleic Acids Res. 2012 Jul.

Abstract

Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5' end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNA(Gly) anticodon stem-loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus-host cell interaction are discussed.

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Figures

Figure 1.
Figure 1.
Glycyl-tRNA synthetase (GARS) binds the IRES element of poliovirus RNA and stimulates its activity. (A) The proteins bound to PV IRES in comparison with those bound to the EMCV IRES. The position of the 74-kDa band specific for the PV IRES is indicated by an arrow. (B) GARS domain organization. ABD, anticodon binding domain. (C) Effect of addition of the recombinant GARS and its two deletion versions (ΔWHEP and ΔABD) on translation of the PV IRES-Fluc RNA and the control capped Rluc RNA in RRL. The PV IRES-Fluc RNA and the control mRNA were co-translated in the same samples. The amounts of synthesized Fluc and Rluc were normalized to those produced in the control sample, i.e. without addition of GARS or its derivatives. (D) The same for the EMCV-Fluc RNA, the luciferase level of the control without GARS was set as 100% (Rluc values are not showed for space limitations since there is no significant change.).
Figure 2.
Figure 2.
GARS binds to domain V of poliovirus IRES RNA. (A) The secondary structure of the entire 5′-UTR of poliovirus RNA. Stem–loop domains are numbered by Roman numbers. The PV IRES covers the region from domII through domVI. The positions of the non-initiator (AUG586) and authentic (AUG743) start codons (see text) as well as of a polypyrimidine tract characteristic of picornavirus IRES elements are also shown. (B) Proteins interacting with the wt 5′-UTR of poliovirus RNA and the 5′-UTRs with deleted individual domains (ΔI–III, ΔIV, ΔV or ΔVI). The position of GARS is indicated by an arrow.
Figure 3.
Figure 3.
Mapping of the GARS binding site in the PV IRES by primer extension inhibition and enzymatic foot-printing. (A) The secondary structure of domV of PV IRES. Positions of toeprint stops and the sites of protection by GARS from nuclease attacks are indicated. (B) Primer extension inhibition (toeprinting) performed for the complexes of GARS with the PV IRES wt and PV IRES mut (the IRES in which ACC ‘anticodon’ of domV was mutated to UAG). The stops of reverse transcription (‘toe’) are denoted by arrows. The sequencing lanes obtained using the same oligodeoxynucleotide primer are shown on the right. (C) Foot-printing of the complexes of recombinant GARS with the wt and mutant PV IRES. The nucleotide positions protected by GARS from the attack by nucleases V1 and T2 are marked by asterisks. The sequencing lanes obtained for the mutant IRES using the same primer are shown on the left. (D) Sequence logo generated from aligned sequences of anticodon loop plus flanked residues corresponding to nucleotides 488–503 of PV IRES. The alignment was obtained from Rfam database (ID of alignment—RF00229).
Figure 4.
Figure 4.
The GARS-mediated stimulation of translation of PV IRES-Fluc mRNA is accounted for by the interaction of the enzyme with the ‘anticodon’ of domV. (A) Effect of addition of tRNAGly, tRNAPhe, individual domV wt and domV mut (ACC → UAG) on the translation of PV IRES-Fluc in the RRL + HeLa cell-free system. All values for Fluc were normalized to Rluc synthesized from a co-translated capped Rluc mRNA. The Fluc/Rluc value for the PV IRES-Fluc without addition of competitor RNAs was set to 100%. (B) Inhibition of the PV IRES-Fluc translation by the isolated domV wt (15-fold molar excess) in the RRL + HeLa system and its relief by addition of GARS. The Fluc/Rluc value for the PV IRES-Fluc without addition of the competitor RNA and GARS was set to 100% (control).
Figure 5.
Figure 5.
Effect of the ‘Gly-anticodon’ mutation of domV (ACC → UAG) on the translation of PV IRES-Fluc in cell-free systems and in cultured cells. (A) Translation in a nuclease untreated RRL. The Fluc values were normalized to those for Rluc. The Rluc values did not vary significantly in parallel assays. (B) Translation in RRL supplemented with 20% (v/v) of cytoplasmic HeLa extract. (C) Expression of Fluc in three human cell lines transfected with the PV IRES-Fluc RNAs harboring either the domV wt or domV mut. The Fluc/Rluc value for the wt RNA construct was set to 100% for each cell line.
Figure 6.
Figure 6.
RelE-printing of 48S initiation complexes formed in the RRL + HeLa with the wt and mutated PV IRES. The 48S complexes were formed in the presence of GMPPNP. (A) Positions of RelE prints are shown on the left (for AUG586) and right (for AUG743) of the gel. Two different primers were used to detect RelE prints for the AUG586 and AUG743 and hence two separate dideoxynucleotide sequences generated with the corresponding primers were run in parallel. (B) Inhibition of RelE cleavages in the PV IRES-Fluc by addition of the isolated domV wt and its partial relief by the addition of GARS. A 15-fold molar excess of domV wt transcript over the mRNA was added to the RRL + HeLa translation system (see Figure 4B). Intensities of RelE-prints were quantified using Aida Image Analyzer. The intensity of each RelE print was normalized to the overall intensity in the corresponding lane, the RelE print for the control lane without addition of GARS and domain V was set to 100%.
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