Eukaryotic initiation factor (eIF) 3 mediates Barley Yellow Dwarf Viral mRNA 3′–5′ UTR interactions and 40S ribosomal subunit binding to facilitate cap-independent translation

Figures

• 

  Figure 1.. RNA structural elements involved in 3′BTE-mediated translation initiation (BYDV 5′UTR and 3′BTE). The 5′UTR consists of four stem loops (SL-A, B, C and D). The 3′BTE has a cruciform secondary structure with three stem loops (SL-I, II, III) and a base loop, SL-IV. Positions of the five complementary bases involved in long distance kissing loop interaction are shown in green (on 3′BTE SL-III and 5′UTR SL-D). The six-base tract complementary to 18S rRNA in the 3′BTE is shown in red.

• 

  Figure 2.. Schematics showing the experiments performed to isolate and identify protein/ribosomal complexes bound to the 3′BTE. The 3′BTE bound proteins/complexes were purified using a streptotagged 3′BTE RNA and were fractionated using sucrose gradients. The fractionated complexes were pooled and analyzed on 1% native agarose gel (lane 1: purified 40S and 60S ribosomal subunits for reference, lane 2: 3′BTE-ribosome complex, lane 3: the tagged 3′BTE RNA used for affinity purification) and the protein components were identified using LC–MS.

• 

  Figure 3.. (A) A 10% SDS-PAGE gel showing the purified eIF3. The subunits are labeled according to their approximate mobilities. (B) A western blot probed with anti-eIF4G antibodies showing the absence of copurified eIF4G in the purified eIF3 preparation. Crude wheat germ lysate was used as a control. (C and D) show fluorescence anisotropy for the binding of 5′ fluorescein labeled BYDV UTRs to native wheat eIF3 (excitation, 495 nM and emission, 520 nM). The titrations were performed at 25°C for 30 min and 100 data points were collected. (C) The binding of the 3′BTE to eIF3 was non-specific in the absence of the helicase complex, but increased significantly in the presence of the helicase complex (Kd = 29.6 ± 1). The 3′BTE showed tight binding to eIF3 even in the absence of eIF4F (Kd = 19 ± 0.9). The negative control 18S rRNA, polyUC RNA and MNeSV ISS RNA showed no binding to eIF3, even in the presence of the helicase complex. (D) The binding of the 5′UTR to eIF3 was not significant in the absence of the helicase complex but increased significantly in presence of the helicase complex (Kd = 39.4 ± 2.9). The binding was abolished if no ATP was added to the helicase complex or if it was…

• 

  Figure 4.. (A) Gel shift assay showing simultaneous binding of BYDV UTRs to eIF3. Both the RNAs were pre-incubated with the helicase complex and then eIF3 was added. The MNeSV ISS RNA, polyUC RNA and 18S rRNA derived oligonucleotide were also assessed for binding to eIF3. Additional details are described in Materials and Methods. (B) For competition binding assays, 50 nM of labeled 5′UTR was pre-incubated with the helicase complex either with or without added 150 nM unlabeled 3′BTE for 20 min at 25°C. After the incubations, the sample was titrated with eIF3. The assays were performed in 20 mM HEPES pH 7.6, 5 mM MgCl2, 200 mM KCl.

• 

  Figure 5.. Fluorescence anisotropy binding of BYDV UTRs to wheat 40S ribosomal subunit. The fluorescein labeled RNAs (50 nM) were pre-incubated with the helicase complex, eIF3 (150 nM) and ATP (5 mM) at 25°C for 20 min, and then titrated with 40S ribosomal subunits (2 μM). Both the UTRs showed very tight binding to 40S subunits in the presence of the helicase complex and eIF3 (BTE-40S, Kd = 4.9 ± 0.4 nM, and 5′UTR-40S, Kd = 4.2 ± 0.9 nM). The 5′UTR showed no binding to 40S subunits in the absence of eIF3, but in the presence of eIF3, the 5′UTR showed very tight binding to 40S ribosomal subunits even in the presence of a 3-fold molar excess of unlabeled 3′BTE RNA (Kd = 2.7 ± 0.6 nM).

• 

  Figure 6.. Summary of the binding affinities of BYDV UTRs for different eIFs (eIF4A, eIF4B, eIF4F) and 40S including eIF3. In the presence of eIF3, both the UTRs showed very tight binding to 40S subunits within the physiological range reported for these complexes.

• 

  Figure 7.. Model for recruitment of the pre-initiation complex to the 5′UTR via 3′BTE. The eIF4F complex binds directly to SLI of the 3′BTE, and recruits eIF4A and eIF4B via the conventional factor-factor interactions. In the presence of ATP, eIF3 (possibly as a part of the 43S pre-initiation complex) binds to the 3′BTE (via direct binding and through interaction of eIF3 with eIF4G) and the 40S/PIC is recruited. Subsequently (or simultaneously), the kissing loop interaction between SLIII (of the 3′BTE) and SL-D (of the 5′UTR, BCL) brings the UTRs together in appropriate alignment enabling eIF3 to stabilize the interaction through direct binding to the 5′ UTR. This stable complex, with the assistance of the helicase complex is able to transfer the 43S PIC to the 5′UTR for translation to begin. Additionally, eIF4A, eIF4B and ATP unwinding of the sequence GAUCCU in the 3′BTE, which is complementary to a stretch of bases in 18S rRNA may also act as a lynchpin to recruit the complex at the 3′BTE.