Oscillating kissing stem–loop interactions mediate 5′ scanning-dependent translation by a viral 3′-cap-independent translation element

Abstract

The 39-untranslated regions (UTRs) of a group of novel uncapped viral RNAs allow efficient translation initiation at the 59-proximal AUG. A well-characterized model is the Barley yellow dwarf virus class of cap-independent translation elements (BTE). It facilitates translation by forming kissing stem-loops between the BTE in the 39-UTR and a BTE-complementary loop in the 59-UTR. Here we investigate the mechanisms of the long-distance interaction and ribosome entry on the RNA. Upstream AUGs or 59-extensions of the 59-UTR inhibit translation, indicating that, unlike internal ribosome entry sites in many viral RNAs, the BTE relies on 59-end-dependent ribosome scanning. Cap-independent translation occurs when the kissing sites are moved to different regions in either UTR, including outside of the BTE. The BTE can even confer cap-independent translation when fused to the 39-UTR of a reporter RNA harboring dengue virus sequences that cause base-pairing between the 39 and 59 ends. Thus, the BTE serves as a functional sensor to detect sequences capable of long-distance base-pairing. We propose that the kissing interaction is repeatedly disrupted by the scanning ribosome and re-formed in an oscillating process that regulates ribosome entry on the RNA.

Eukaryotic Initiation Factor (eIF) 4F Binding to Barley Yellow Dwarf Virus (BYDV) 3′-Untranslated Region Correlates with Translation Efficiency

“…2B). The translational efficiency was determined by the ability of the 3Ј BTE elements to function in the 5Ј leader of a luciferase reporter or by the ability to inhibit translation as described elsewhere (2,20). It was found that the binding of eIF4F to 3Ј BTE and mutants expressed as association constants (K a values) correlated well with the core translation abilities of the mutants with one exception, BTEBF (Fig.…”

“…BYDV affects cereal crops such as wheat, barley, oats, and grasses and is spread by several aphid species severely limiting food grain production and causing yield losses resulting in food scarcity (1). BYDV belongs to the Luteoviridae family, has a genome which is a positive sense RNA Ϸ5700 nucleotides long, and is lacking both a 5Ј cap (m 7 GpppX) and a poly(A) tail (2). In eukaryotes, the 5Ј cap is the binding site for eukaryotic initiation factors (eIFs) eIF4F and eIFiso4F (isozyme form of eIF4F present in higher plants).…”

“…In this study, WT 3Ј BTE and four other mutants BTEBF, SII-m1, SL-III-3, and SL-III SWAP (the mutations/changes in bases are shown in Fig. 1) with in vitro translation efficiencies from 5 to 164% compared with WT BTE (2,20) were used. Binding of eIF4F and other initiation factors (eIF4B, 4A, and PABP) to the WT 3Ј BTE and mutants (BTEBF, SII-m1, SL-III-3, and SL-III SWAP) was characterized using fluorescence anisotropy and stopped flow kinetics, which gave equilibrium binding constant values (K d ), a measure of relative complex stability, and kinetic constants.…”

“…To further examine what, if any, role binding of eIF4F had on translation, a variety of 3Ј BTE mutants with widely different translational efficiencies were selected for binding studies. 3Ј BTE and mutants with in vitro translation efficiencies from 5 to 164% (WT ϭ 100%) were studied (2,20). All of the mutants contain a short 17-nucleotide-long sequence (located in SL-I) conserved across viral classes, which is the location for eIF4F binding and is complementary to 18S ribosomal RNA (24,33).…”

“…The predicted secondary structure of the 3 BTE and mutants. BTE structure is based on SHAPE analysis and mfold data (2,20,33). Purple (SL-I) indicates the 17-nucleotide conserved bases.…”