Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome.

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding the cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes, but strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at internal in-frame and out-of-frame start sites, can be functionally important and contribute to the "alternative" bacterial proteome. The internal start sites may also play regulatory roles in gene expression.

PTENε suppresses tumor metastasis through regulation of filopodia formation.

PTEN is one of the most frequently mutated genes in malignancies and acts as a powerful tumor suppressor. Tumorigenesis is involved in multiple and complex processes including initiation, invasion, and metastasis. The complexity of PTEN function is partially attributed to PTEN family members such as PTENα and PTENß. Here, we report the identification of PTENε (also named as PTEN5), a novel N-terminal-extended PTEN isoform that suppresses tumor invasion and metastasis. We show that the translation of PTENε/PTEN5 is initiated from the CUG816 codon within the 5'UTR region of PTEN mRNA. PTENε/PTEN5 mainly localizes in the cell membrane and physically associates with and dephosphorylates VASP and ACTR2, which govern filopodia formation and cell motility. We found that endogenous depletion of PTENε/PTEN5 promotes filopodia formation and enhances the metastasis capacity of tumor cells. Overall, we identify a new isoform of PTEN with distinct subcellular localization and molecular function compared to the known members of the PTEN family. These findings advance our current understanding of the importance and diversity of PTEN functions.

A Retrospective on eIF2A-and Not the Alpha Subunit of eIF2.

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action is still not well understood. Eukaryotic initiation factor 2A (eIF2A) is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e., they stimulate initiator Met-tRNAi binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2 (α,ß,γ) showed that this factor, and not eIF2A, was primarily responsible for the binding of Met-tRNAi to 40S subunit in eukaryotes. It was found however, that eIF2A can promote recruitment of Met-tRNAi to 40S/mRNA complexes under conditions of inhibition of eIF2 activity (eIF2α-phosphorylation), or its absence. eIF2A does not function in major steps in the initiation process, but is suggested to act at some minor/alternative initiation events such as re-initiation, internal initiation, or non-AUG initiation, important for translational control of specific mRNAs. This review summarizes our current understanding of the eIF2A structure and function.

Translation of Senecavirus A polyprotein is initiated from the IRES-proximal initiation codon.

To clarify whether Senecavirus A (SVA) has the potential of alternative translation, an extra G residue was inserted into an SVA cDNA clone, resultantly generating an "AUGAUG" motif. The second AUG is the authentic SVA initiation codon, whereas the first AUG is a putative one. Subsequently, eighteen nucleotides were inserted one by one between AUG and AUG for reconstructing cDNA clones. The test of virus recovery showed that three replication-competent SVAs, whose AUG/AUG-flanked sequences were not multiples of three nucleotides, were successfully rescued from their individual cDNA clones. The wild-type SVA possesses a UUUUU motif within the polyprotein-encoding region. Sanger sequencing showed that these three replication-competent SVAs harbored one or two extra U residues in the UUUUU motif, implying that polyprotein translation was initiated from the putative AUG, and the authentic AUG would be inactivated. This is probably attributed to the lack of ribosome scanning along an SVA genome.

Translational Control of Serrate Expression in Drosophila Cells.

BACKGROUND/AIM: The DSL proteins, Serrate and Delta, which act as Notch receptor ligands, mediate signalling between adjacent cells, when a ligand-expressing cell binds to Notch on an adjacent receiving cell. Notch is ubiquitously expressed and DSL protein mis-expression can have devastating developmental consequences. Although transcriptional regulation of Delta and Serrate has been amply documented, we examined whether they are also regulated at the level of translation. MATERIALS AND METHODS: We generated a series of deletions to investigate the initiation codon usage for Serrate using Drosophila S2 cells. RESULTS: Serrate mRNA contains three putative ATG initiation codons spanning a 60-codon region upstream of its signal peptide; we found that each one can act as an initiation codon, however, with a different translational efficiency. CONCLUSION: Serrate expression is strictly regulated at the translational level.

Negative charge in the RACK1 loop broadens the translational capacity of the human ribosome.

Although the roles of initiation factors, RNA binding proteins, and RNA elements in regulating translation are well defined, how the ribosome functionally diversifies remains poorly understood. In their human hosts, poxviruses phosphorylate serine 278 (S278) at the tip of a loop domain in the small subunit ribosomal protein RACK1, thereby mimicking negatively charged residues in the RACK1 loops of dicot plants and protists to stimulate translation of transcripts with 5' poly(A) leaders. However, how a negatively charged RACK1 loop affects ribosome structure and its broader translational output is not known. Here, we show that although ribotoxin-induced stress signaling and stalling on poly(A) sequences are unaffected, negative charge in the RACK1 loop alters the swivel motion of the 40S head domain in a manner similar to several internal ribosome entry sites (IRESs), confers resistance to various protein synthesis inhibitors, and broadly supports noncanonical modes of translation.

Alternative Translation Initiation at a UUG Codon Gives Rise to Two Functional Variants of the Mitochondrial Protein Kgd4.

Kgd4 is a novel subunit of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDH). In yeast, the protein is present in two forms of unknown origin, as there is only one open reading frame and no alternative splicing. Here, we show that the two forms of Kgd4 derive from one mRNA that is translated by employing two alternative start sites. The standard, annotated AUG codon gives rise to the short form of the protein, while an upstream UUG codon is utilized to generate the larger form. However, both forms can be efficiently imported into mitochondria and stably incorporate into KGDH to support its activity. Translation of the long variant depends on sequences directly upstream of the alternative initiation site, demonstrating that translation initiation and its efficiency are dictated by the sequence context surrounding a specific codon. In summary, the two forms of Kgd4 follow a very unusual biogenesis pathway, supporting the notion that translation initiation in yeast is more flexible than it is widely recognized.

The eIF2A knockout mouse.

Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNAMeti) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNAMeti binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNAMeti to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.

RUNX1T1/MTG8/ETO gene expression status in human t(8;21)(q22;q22)-positive acute myeloid leukemia cells.

The RUNX1-RUNX1T1 fusion gene, a product of the nonhomologous balanced translocation t(8;21)(q22;q22), is a complex genetic locus. We performed extensive bioinformatic analysis of transcription initiation as well as transcription termination sites in this locus and predicted a number of different RUNX1T1 transcripts. To confirm and quantify the RUNX1T1 gene expression, we analyzed samples from seven acute myeloid leukemia (AML) patients and from the Kasumi-1 cell line. We found variable activity of the four predicted RUNX1T1 promoters located downstream of the chromosome breakpoint. Nineteen alternative RUNX1T1 transcripts were identified by sequencing at least seventeen of which predictably can be translated into functional proteins. While the RUNX1T1 gene is not expressed in normal hematopoietic cells, it may participate in t(8;21)(q22;q22)-dependent leukemic transformation due to its multiple interactions in cell regulatory network particularly through synergistic or antagonistic effects in relation to activity of RUNX1-RUNX1T1 fusion gene.