Programmed translational bypassing is a process whereby ribosomes "ignore" a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a "takeoff codon" immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching "landing triplet" 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions.ribosome hopping | mitochondrial genome | proteome analysis | heterologous expression T he traditional view of translation is that mRNA is read sequentially, one codon at a time. However, low-level nonprogrammed translational bypassing (i.e., the occasional skipping of a few nucleotides) can be triggered by various factors, including tRNA paucity, unusual codons, and homo-polymer sequence tracts (1). In addition, programmed translational bypassing of 50 nt has been demonstrated for the gene 60 transcript of bacteriophage T4 (2-4). In vitro mutagenesis experiments showed that efficient translational "jumping" or "hopping" in T4 requires matching takeoff and landing codons (most effective is the wild-type GGA), a stop codon, and both a hairpin RNA secondary structure directly downstream of the takeoff site, and a Shine-Dalgarno (SD) sequence a few nucleotides upstream of the landing codon. Finally, a particular amino acid sequence in the nascent peptide encoded upstream of the takeoff site confers highest jumping efficiency. Additional cases of programmed bypassing have been postulated but currently lack supporting evidence (e.g., ref. 5), making the T4 gene 60 expression the only confirmed instance.Here, we report the massive occurrence of translational bypassing elements in mitochondria of the opportunistic human pathogen Magnusiomyces (also known as Blastoschizomyces or Geotrichum) capitatus (6), which belongs to a deeply branching lineage of Saccharomycetales (Fig. 1A). Our findings suggest that translational bypassing might be more widespread than previously thought. ResultsProtein-Coding Genes Interrupted by Dozens of In...

Aims/hypothesis We previously used an integrative genetics approach to demonstrate that 5-lipoxygenase (5-LO) deficiency in mice (Alox5 −/− ) protects against atherosclerosis despite increasing lipid levels and fat mass. In the present study, we sought to further examine the role of 5-LO in adiposity and pancreatic function. Methods Alox5 −/− and wild-type (WT) mice were characterised with respect to adiposity and glucose/insulin metabolism using in vivo and in vitro approaches. The role of ALOX5 in pancreatic function in human islets was assessed through short interfering RNA (siRNA) knockdown experiments. Results Beginning at 12 weeks of age, Alox5 −/− mice had significantly increased fat mass, plasma leptin levels and fasting glucose levels, but lower fasting insulin levels (p< 0.05). Although Alox5 −/− mice did not exhibit insulin resistance, they had impaired insulin secretion in response to a bolus glucose injection. Histological analyses revealed Diabetologia (2008) that Alox5 −/− mice had increased islet area, beta cell nuclear size, and numbers of beta cells/mm 2 islet (p < 0.05), indicative of both hyperplasia and hypertrophy. Basal and stimulated insulin secretion in isolated Alox5 −/− islets were significantly lower than in WT islets (p <0.05) and accompanied by a three-to fivefold decrease in the expression of the genes encoding insulin and pancreatic duodenal homeobox 1 (Pdx1). Direct perturbation of ALOX5 in isolated human islets with siRNA decreased insulin and PDX1 gene expression by 50% and insulin secretion by threefold (p<0.05). Conclusions/interpretation These results provide strong evidence for pleiotropic metabolic effects of 5-LO on adiposity and pancreatic function and may have important implications for therapeutic strategies targeting this pathway for the treatment of cardiovascular disease.

The de novo synthesis of cytochrome c oxidase subunits I, II (COI and COII), and apocytochrome b (Cyb) was investigated in kinetoplast-mitochondria of Leishmania. The organelles were isolated after breaking whole cells with nitrogen cavitation. Individual COI, COII, and Cyb polypeptides were identified by fractionation of the kinetoplast membranes, labeled with [ 35 S]methionine and cysteine, using two-dimensional (9 versus 14% and 20 versus 11%) denaturing gel electrophoresis. The reaction did not require exogenous energy sources or amino acids. On the contrary, the presence of amino acids other than methionine somewhat inhibited the labeling reaction probably by competing with the uptake of labeled amino acids. The synthesis reaction was insensitive to 100 g/ml chloramphenicol, gentamycin, paromomycin, lincomycin, hygromycin, and tetracycline, as well as cycloheximide. The process showed a linear increase in the amount of synthesized polypeptides during the first 2 h of incubation, followed by a slower accumulation of products for up to 4 h. The de novo synthesized polypeptides were stable for several additional hours. Their assembly into respiratory complexes, investigated using two-dimensional Blue Native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-SDS gels, began early during the incubation and continued throughout the course of the synthesis. This work represents the first unequivocal identification of the polypeptide synthesis in kinetoplasts.The kinetoplast-mitochondrial genetic system of trypanosomes has revealed features that clearly distinguish it from animal, fungal, or plant mitochondria. The maxicircle component of kinetoplast DNA networks contains cryptic genes whose expression depends on post-transcriptional mRNA editing by uridylate insertions and deletions (1-3). This process is mediated by guide RNAs encoded in the kinetoplast DNA minicircles, which account for the bulk of DNA in these mitochondria. After the discoveries of RNA editing in 1986 (4) and guide RNAs in 1990 (5), there has been a steady progress toward elucidating the mechanism of this process, which has now been demonstrated to involve several enzymes and protein factors (6 -10).At the same time very little is known about other aspects of kinetoplast gene expression, especially at the level of translation. The very existence of a functional translation system remained unproven for almost 3 decades (summarized in Ref. 11). This question is important because kinetoplast translation must utilize templates modified or created by editing, and it is interesting to investigate interactions of these two systems. Early works demonstrated that cycloheximide at low concentrations effectively inhibited cytosolic translation in Crithidia luciliae and Trypanosoma brucei in vivo. A small fraction of the total cell protein synthesis was resistant to cycloheximide and at the same time insensitive to D-chloramphenicol and other inhibitors at concentrations that inhibit mitochondrial translation in other organisms (12, 13). However, high conce...

Kinetoplast maxicircle DNA sequence organization was investigated in Leishmania donovani, strain 1S LdBob. Gene arrangement in the coding (conserved) region of the maxicircle is collinear with that of most trypanosomatids, with individual genes showing 80-90% nucleotide identity to Leishmania tarentolae, strain UC. The notable exception was an integration of a full-size minicircle sequence in the ND1 gene coding region found in L. donovani. Editing patterns of the mitochondrial mRNAs investigated also followed L. tarentolae UC patterns, including productive editing of the components of respiratory complexes III, IV and V, and ribosomal protein S12 (RPS12), as well as the lack of productive editing in five out of six pan-edited cryptogenes (ND3, ND8, ND9, G3, G4) found in these species. Several guide RNAs for the editing events were localized in minicircles and maxicircles in the locations that are conserved between the species. Mitochondrial activity, including rates of oxygen consumption, the presence and the levels of respiratory complexes and their individual subunits and the steady-state levels of several mitochondrial-encoded mRNAs were essentially the same in axenically grown amastigotes and in promastigotes of L. donovani. However, some modulation of mitochondrial activity between these developmental stages was suggested by the finding of an amastigote-specific component in Complex IV, a down-regulation of mitochondrial RNA-binding proteins (MRP) [define] and MRP-associated protein (MRP-AP) in amastigotes, and by variations in the levels of RPS12, ND3, ND9, G3 and G4 pre-edited transcripts.

Telomeric sequences constitute only a small fraction of the whole genome yet they are crucial for ensuring genomic stability. This function is in large part mediated by protein complexes recruited to telomeric sequences by specific telomere-binding proteins (TBPs). Although the principal tasks of nuclear telomeres are the same in all eukaryotes, TBPs in various taxa exhibit a surprising diversity indicating their distinct evolutionary origin. This diversity is especially pronounced in ascomycetous yeasts where they must have co-evolved with rapidly diversifying sequences of telomeric repeats. In this article we (i) provide a historical overview of the discoveries leading to the current list of TBPs binding to double-stranded (ds) regions of telomeres, (ii) describe examples of dsTBPs highlighting their diversity in even closely related species, and (iii) speculate about possible evolutionary trajectories leading to a long list of various dsTBPs fulfilling the same general role(s) in their own unique ways.