A translation enhancer element from black beetle virus engages yeast eIF4G1 to drive cap-independent translation initiation

Cap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions. Translation of uncapped or de-capped transcripts can be stimulated by Cap-independent translation enhancer (CITE) elements, but the mechanisms of CITE-mediated translation initiation remain understudied. Here, we characterized a short 5ʹ-UTR RNA sequence from black beetle virus, BBV-seq. Mutational analysis indicates that the entire BBV-seq is required for efficient translation initiation, but this sequence does not operate as an IRES-type module. In yeast cell-free translation extracts, BBV-seq promoted efficient initiation on cap-free mRNA using a scanning mechanism. Moreover, BBV-seq can increase translation efficiency resulting from conventional cap-dependent translation initiation. Using genetic approaches, we found that BBV-seq exploits RNA-binding properties of eIF4G1 to promote initiation. Thus, BBV-seq constitutes a previously uncharacterized short, linear CITE that influences eIF4G1 to initiate 5′ end-dependent, cap-independent translation. These findings bring new insights into CITE-mediated translational control of gene expression.


Growth medium and yeast strains.
We used standard YPD medium (1% yeast extract, 2% peptone and 2% dextrose) sterilized by filtration through 0.2 m PES filters (Thermo Scientific). Wild-type S. cerevisiae BY4741 (MATa his3-1 leu2-0 met15-0 ura3-0) was obtained from Open Biosystems. To generate the PTEF-6×HIS-TIF4631 strain, a KanMX-PTEF-6×HIS-TIF4631-TCYC1 cassette was generated by PCR with primers designed to add flanking HO homology arms from the integration plasmid construct pTEF:6×HisTIF4631 (see below). Genomic DNA was isolated from G418-resistant colonies of BY4741 cells appearing after transformation with the above cassette and verified for the correct integration into the HO locus by PCR using a primer annealing ∼100 bp upstream of the integration site and a second primer annealing within the KanMX cassette. To generate mutant derivatives of the PTEF-6×HIS-TIF4631 strain, designated as PTEF-6×HIS-TIF46313 and PTEF-6×HIS-TIF463123, we used similar manipulations as for PTEF-6×HIS-TIF4631 strain generation.
G418 was purchased from Thermo Fisher Scientific, TRI REAGENT-LS was from Molecular Research Center (cat # TS 120), ECL substrate was from Millipore; DTT was purchased from Sigma, PMSF was from Amresco; creatine phosphate was from Alfa Aesar.
RiboLock was purchased from Thermo Fisher; SIGMAFAST™ Protease Inhibitor Cocktail was from Sigma; all the restriction enzymes and polynucleotide kinase (PNK) were obtained from Thermo Fisher. Creatine phosphokinase was purchased from BioVision.

Plasmids.
pYes was purchased from Invitrogen. To generate the initial construct pYes-TAP-RLuc, we used a two-step cloning strategy. First, we amplified the TAP sequence using the pBS1761 plasmid as a template with the forward primer containing a BamHI site and the reverse primer containing an XhoI site, whereby the stop codon was omitted from the coding sequence on the reverse primer. The amplified TAP sequence was cloned into pYes between BamHI and XhoI sites. The Renilla luciferase gene amplified by PCR from pJD375 using a forward primer containing an XhoI site and a reverse primer containing an XbaI site was cloned into pYes-TAP between XhoI and XbaI sites. The generated pYes-TAP-RLuc construct was used as the "linker sequence" control in this study, that we termed "(-1) pyrimidine linker".
To generate pYes-CrPV-TAP-RLuc, the CrPV sequence was amplified by PCR from the dicistronic Rluc-CrPV IGR-FLuc (Wilson et al., 2000) using a HindIII-containing forward primer and a BamHI-containing reverse primer and cloned between HindIII and BamHI sites of pYes-TAP-RLuc. Sequences for URE2, NCE102 and YMR181c were generated by Genewiz and contained HindIII and BamHI sites at the 5'-and 3'-ends, respectively. These double-stranded DNA fragments were individually ligated with pYes-TAP-RLuc digested with HindIII and BamHI.
The original pYes-BBV-seq-TAP-RLuc construct was generated as follows: two BBV sequence oligonucleotides (top and complementary bottom strands) were synthesized by Sigma-Genosys; 100 pmoles of each oligo were phosphorylated by PNK in the presence of 10 mM ATP for 30 min at 30°C, oligos were combined, heated at 80°C for 10 min and slowly cooled down. After annealing, HindIII and BamHI sticky ends were formed at the 5'-end and 3'-end of the BBV-seq insert, respectively. This insert was ligated into pYes-TAP-RLuc that had been digested with HindIII and BamHI enzymes.
We also used a different strategy to generate the pYes-BBV-seq-TAP-RLuc* construct (shown in Figure 1C) and its derivatives, based on incorporation of BBV-seq wild-type or mutant sequence(s) into the forward primer. This primer also contained a BamHI site immediately upstream of the BBV-seq sequence or its derivatives and a region complementary to the first 28 nucleotides within the TAP gene sequence (see Table S3). The reverse primer contained an XbaI site and a region complementary to the 3'-end of the Renilla luciferase gene. Thus, the PCR products carried BBV-seq (wild-type or mutant) followed by the TAP-RLuc gene sequence; they were digested with BamHI and XbaI and ligated with pYes cleaved with BamHI/XbaI. The resulting pYes-BBV-seq-TAP-RLuc* construct has HindIII, KpnI and BamHI sites available. The pYes-(-1) purine-TAP-RLuc and pYes-unstructured linker-TAP-RLuc constructs were generated using similar strategy, wherein we amplified TAP-RLuc sequence using the forward primer that contained either sequence of (-1) purine linker or sequence of unstructured linker (see Table S3) and HindIII site, while the reverse primer contained XbaI site and a region complementary to the 3'-end of the Renilla luciferase gene. PCR products were digested with HindIII and Xba1 and ligated with pYes between HindIII/Xba1 sites.
To generate bicistronic constructs, we used three-step strategy. First, we cloned TAP sequence containing stop codon into pYes between HindIII and BamHI. TAP was amplified using primers annotated in Table S3. In parallel, we generated pYes-BBV-seq-RLuc plasmid: the BBV-seq-RLuc was amplified using the forward primer that contained BamHI site followed by BBV-seq sequence and sequence complementary to 5'-end of Renilla luciferase gene, while the reverse primer contained XbaI site and a region complementary to the 3'-end of the Renilla luciferase gene (see Table S3). PCR product was cloned into pYes between BamHI and XbaI sites. Next, we amplified 15xC-BBV-seq-RLuc using the forward primer that contained BamHI site followed by 15xC and a region complementary to the 5'-end of BBV-seq, while the reverse primer containing XbaI site and a region complementary to the 3'-end of the Renilla luciferase gene. As a template, we used pYes-BBV-seq-RLuc plasmid. Resulting PCR products were cloned into pYes-TAP plasmid between BamHI and XbaI sites, resulting in pYes-TAP-spacer-BBV-seq-RLuc. To generate pYes-TAP-spacer-RLuc plasmid, we amplified RLuc using the forward primer containing BamHI followed by 15xC sequence and a region complementary to 5'-end of Renilla luciferase gene, while the reverse primer contained XbaI site and a region complementary to the 3'-end of the Renilla luciferase gene (see Table S3). Finally, the hairpin oligonucleotide  pYes-TAP-RLuc Figure S1 (-1) purine linker pYes-TAP-RLuc Figure S1 (top) was used to calculate TAP-RLuc normalized to Rpl3 present in the same reaction. TAP-RLuc/Rpl3 ratios are presented in the graph. TAP-RLuc/Rpl3 ratio corresponding to the reaction charged with m7G-capped BBV-seq-TAP-RLuc RNA is set as 100% (shown in red) and used to calculate percentage of the reporter protein in other samples (shown in black). Translationallyactive lysates were prepared from wild-type strain BY4741 or from PTEF6xHIS-TIF4631 that expresses extra-copies of eIF4G1. (b&d). Northern hybridization signal corresponding to TAP-RLuc RNA was normalized by a signal derived from 25S rRNA present is the same sample (shown in Figure 7a&b, bottom), TAP-RLuc/25S rRNA ratios are presented as a bar graph.

Supplementary Figure S6
Supplementary Figure