After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA and release factor eRF1, and must be recycled by dissociating these ligands and separating ribosomes into subunits. Although recycling of eukaryotic post-termination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1 and eIF1A (Pisarev et al., 2007), this energy-free mechanism can function only in a narrow range of low Mg2+ concentrations. Here we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg2+ concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1's absence.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

The RNAs of many plant viruses lack a 5′ cap and must be translated by a cap-independent mechanism. Here, we discuss the remarkably diverse cap-independent translation elements that have been identified in members of the Potyviridae, Luteoviridae, and Tombusviridae families, and genus Tobamovirus. Many other plant viruses have uncapped RNAs but their translation control elements are uncharacterized. Cap-independent translation elements of plant viruses differ strikingly from those of animal viruses: they are smaller (<200 nt), some are located in the 3′ untranslated region, some require ribosome scanning from the 5′ end of the mRNA, and the 5′ UTR elements are much less structured than those of animal viruses. We discuss how these elements may interact with host translation factors, and speculate on their mechanism of action and their roles in the virus replication cycle. Much remains to be learned about how these elements enable plant viruses to usurp the host translational machinery.

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.

Much of the regulation of gene expression occurs at the level of protein synthesis. In addition to the canonical translation factors, a multitude of proteins and microRNAs (miRNAs) act as regulatory trans-acting factors. Mechanistic analysis of translational control benefits from functional cell-free systems that can be depleted of the responsible regulatory factors. Although antisense oligonucleotides facilitate the functional sequestration of the regulatory RNAs, immunodepletion of protein factors is technically challenging. Here we describe a simple and robust alternative protocol for the preparation of factor-depleted in vitro translation system derived from HeLa cells. The procedure relies on RNA interference-mediated knockdown of the factor of interest prior to extract preparation, and it overcomes problems with the availability and specificity of antibodies, as well as with the co-depletion of proteins associated with the factor under study. The complete procedure can normally be conducted within 1 week and carried out in parallel for multiple (candidate) factors.