The use of fluorescent proteins fused to other proteins has been very useful in revealing the location and function of many proteins. However, it is very important to show that the fusion of these reporter proteins does not impact the function of the protein of interest. Plants have 2 forms of the cap-binding protein that function in initiation of translation, eIF4E and a plant specific form, eIFiso4E. In an attempt to determine the cellular localization of eIFiso4E, fusions to GFP were made, but were found to not be competent to rescue the lethal phenotype of plants lacking eIF4E and eIFiso4E. This suggested that the GFP fusions at either the N- or C-terminus of eIFiso4E were not functional. Biochemical analysis of the fusions revealed that eIFiso4E•GFP fusions were not able to bind to m7GTP Sepharose indicating that they were not functional as cap-binding proteins. Analysis of eIF4E•GFP fusions, both in yeast and in vitro, showed that the N-terminal fusion may be functional, whereas the C-terminal fusion bound m7GTP Sepharose very poorly and functioned poorly in yeast. These results highlight the importance of verification both biochemically and in vivo that reporter fusions of proteins maintain activity and are stable in order to prevent observations that may result in artifacts.

The complexity of the eukaryotic protein synthesis machinery is partly driven by extensive and diverse modifications to associated proteins and RNAs. These modifications can have important roles in regulating translation factor activity and ribosome biogenesis and function. Further investigation of ‘translational modifications’ is warranted considering the growing evidence implicating protein synthesis as a critical point of gene expression control that is commonly deregulated in disease. New evidence suggests that translation is a major new target for oxidative modifications, specifically hydroxylations and demethylations, which generally are catalyzed by a family of emerging oxygenase enzymes that act at the interface of nutrient availability and metabolism. This review summarizes what is currently known about the role or these enzymes in targeting rRNA synthesis, protein translation and associated cellular processes.

Regulation of protein synthesis is of fundamental importance to cells. It has a critical role in the control of gene expression, and consequently cell growth and proliferation. The importance of this control is supported by the fact that protein synthesis is frequently upregulated in tumor cells. The major point at which regulation occurs is the initiation stage. Initiation of translation involves the interaction of several proteins to form the eIF4F complex, the recognition of the mRNA by this complex, and the subsequent recruitment of the 40S ribosomal subunit to the mRNA. This results in the formation of the 48S complex that then scans the mRNA for the start codon, engages the methionyl-tRNA and eventually forms the mature 80S ribosome which is elongation-competent. Formation of the 48S complex is regulated by the availability of individual initiation factors and through specific protein-protein interactions. Both of these events can be regulated by post-translational modification by ubiquitin or Ubls (ubiquitin-like modifiers) such as SUMO or ISG15. We provide here a summary of translation initiation factors that are modified by ubiquitin or Ubls and, where they have been studied in detail, describe the role of these modifications and their effects on regulating protein synthesis.

Alternative splicing of the human immunodeficiency virus 1 (HIV-1) RNA transcripts produces mRNAs encoding nine different viral proteins. The leader of each contains a common non-coding exon at the 5' end. Previous studies showed that the leaders from the common exon-containing transcripts gag, nef, vif, vpr and vpu can direct protein synthesis through internal ribosome entry sites (IRESs) with varying efficiencies. Here we explored whether the common exon acts as an IRES element in the context of all the 5' leaders or if each harbors a distinct IRES. We also explored the relationship between the IRESs and initiation codon selection. We find that the common exon adopts a similar conformation in every leader we explored and that the sequence and structure is required for IRES activity. We also find that each leader uses a scanning mechanism for start codon identification. Together, our data point to a model in which the common exon on HIV-1 transcripts acts as the ribosome landing pad, recruiting preinitiation complexes upstream of the initiation codon, followed by scanning to each transcript's initiator AUG.

Over the past 20 years, a better understanding of cancer biology, screening for early detection, improved adjuvant treatment, and targeted therapies have decreased the rate of breast cancer deaths. However, resistance to treatment is common, and new approaches are needed. Deregulation of translation initiation is associated with the commencement and progression of cancer. Often, translation initiation factors are overexpressed and the related signaling pathways activated in human tumors. Recently, a significant number of inhibitors that target translation factors and pathways have become available. These inhibitors are being tested alone or in combination with chemotherapeutic agents in clinical trials. The results are varied, and it is not yet clear which drug treatments most effectively inhibit tumor growth. This review highlights the pathways and downstream effects of the activation of translation and discusses targeting the control of translation initiation as a therapeutic approach in cancer, focusing on breast cancer clinical trials.

In yeast Saccharomyces cerevisiae, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During translation initiation, the 60S subunit joins the 40S initiation complex, producing the 80S initiation complex. During elongation, the 60S subunit binds the CCA-ends of aminoacyl- and peptidyl-tRNAs at the A-loop and P-loop, respectively, transferring the peptide onto the α-amino group of the aminoacyl-tRNA. To study the role of 25S rRNA in translation in vivo, we randomly mutated 25S rRNA and isolated and characterized seven point mutations that affected yeast cell growth and polysome profiles. Four of these mutations, G651A, A1435U, A1446G and A1587G, change a base involved in base triples crucial for structural integrity. Three other mutations change bases near the ribosomal surface: C2879U and U2408C alter the A-loop and P-loop, respectively, and G1735A maps near a Eukarya-specific bridge to the 40S subunit. By polysome profiling in mmslΔ mutants defective in nonfunctional 25S rRNA decay, we show that some of these mutations are defective in both the initiation and elongation phases of translation. Of the mutants characterized, C2879U displays the strongest defect in translation initiation. The ribosome transit-time assay directly shows that this mutation is also defective in peptide elongation/termination. Thus, our genetic analysis not only identifies bases critical for structural integrity of the 60S subunit, but also suggests a role for bases near the peptidyl transferase center in translation initiation.